EP1159431A2 - Human kallikrein-like genes - Google Patents

Human kallikrein-like genes

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Publication number
EP1159431A2
EP1159431A2 EP00908878A EP00908878A EP1159431A2 EP 1159431 A2 EP1159431 A2 EP 1159431A2 EP 00908878 A EP00908878 A EP 00908878A EP 00908878 A EP00908878 A EP 00908878A EP 1159431 A2 EP1159431 A2 EP 1159431A2
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EP
European Patent Office
Prior art keywords
klk
protein
gene
nucleic acid
genes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00908878A
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German (de)
French (fr)
Inventor
George M. Yousef
Eleftherios P. Diamandis
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Mount Sinai Hospital Corp
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Mount Sinai Hospital Corp
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Publication date
Application filed by Mount Sinai Hospital Corp filed Critical Mount Sinai Hospital Corp
Priority to EP06076156A priority Critical patent/EP1724351A3/en
Publication of EP1159431A2 publication Critical patent/EP1159431A2/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to nucleic acid molecules, proteins encoded by such nucleic acid molecules, and use of the proteins and nucleic acid molecules BACKGROUND OF THE INVENTION
  • Kailikreins and kallikrein- ke proteins are a subgroup of the serine protease enzyme family and exhibit a high degree of substrate specificity (1)
  • the biological role of these kailikreins is the selective cleavage of specific polypeptide precursors (substrates) to release peptides with potent biological activity (2)
  • kailikreins are encoded by large multigene families In the mouse genome, at least 24 genes have been identified (3) Expression of 11 of these genes has been confirmed, the rest are presumed to be pseudogenes (4)
  • a similar family of 15-20 kailikreins has been found in the rat genome (5) where at least 4 of these are known to be expressed (6)
  • PSA prostatic specific antigen
  • KLK2 human glandular kallikrem
  • KLKl tissue (pancreatic-renal) kalhkrem
  • the mouse kal krem genes are clustered in groups of up to 11 genes on chromosome 7 and the distance between the genes in the various clusters can be as small as 3-7 Kb (3) All three human kalhkrein genes have been assigned to chromosome 19q 13 2 - 19q 13 4 and the distance between PSA and KLK2 has been estimated to be 12 Kb (9)
  • KLK-L3, KLK-L4, KLK-L5, or KLK-L6 and collectively as “kallikrein-hke proteins” oi "KLK-L Proteins”
  • the genes encoding the proteins are referred to as " Uk-ll klk-12 lclk-13 klk-14 klk-1 oi klk 16 ", and collectively as “kallikrein-hke genes” or "klk-I genes"
  • the present invention relates to an isolated nucleic acid molecule which comprises (l) a nucleic acid sequence encoding a protein having substantial sequence identity with an amino acid sequence of KLK-L 1, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 as shown m SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively, (u) a nucleic acid sequence encoding a protein comprising an ammo acid sequence of KLK-
  • KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively,
  • a nucleic acid sequence capable of hybridizing under stringent conditions to a nucleic acid sequence in (I), (n) or (in), (vi) a nucleic acid sequence encoding a truncation, an analog, an allelic or species variation of a protein comprising an amino acid sequence of KLK-L1, KLK-L2, KLK-L3, KLK- L4, KLK-L5, or KLK-L6 as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively, or (vn) a fragment, or allelic or species variation of (l), (n) or (in)
  • a purified and isolated nucleic acid molecule of the invention comprises
  • nucleic acid sequence comprising the sequence of SEQ ID NO 1, 13, 21, 43, 56, or 65 wherein T can also be U
  • nucleic acid sequences complementary to (l) preferably complementary to the full nucleic acid sequence of SEQ ID NO 1, 13, 21, 43, 56, or 65
  • a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid of (I) or (n) and preferably having at least 18 nucleotides
  • a nucleic acid molecule differing from any of the nucleic acids of (I) to (in) in codon sequences due to the degeneracy of the genetic code
  • the invention also contemplates a nucleic acid molecule comprising a sequence encoding a truncation of a KLK-L protein, an analog, or a homolog of a KLK-L Protein or a truncation thereof (KLK-
  • KLK-L Related Proteins L Proteins and truncations, analogs and homologs of KLK-L Proteins are also collectively referred to herein as "KLK-L Related Proteins"
  • nucleic acid molecules of the invention may be inserted into an appropriate expression vector, l e a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence
  • recombinant expression vectors adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements linked to the nucleic acid molecule
  • the recombinant expression vector can be used to prepare transformed host cells expressing KLK- L Related Proteins Therefore, the invention further provides host cells containing a recombinant molecule of the invention
  • the invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a recombinant molecule comprising a nucleic acid molecule of the invention, in particular one which encodes an analog of a KLK-L Protein, or a truncation of a KLK-L Protein
  • the invention further provides a method for preparing KLK-L Related Proteins utilizing the purified and isolated nucleic acid molecules of the invention
  • a method for preparing a KLK-L Related Protein comprising (a) transferring a recombinant expression vector of the invention into a host cell, (b) selecting transformed host cells from untransformed host cells, (c) culturing a selected transformed host cell under conditions which allow expression of the KLK-L Related Protein, and (d) isolating the KLK-
  • the invention further broadly contemplates an isolated KLK-L Protein comprising an amino acid sequence as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • the KLK-L Related Proteins of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins This may be accomplished, for example, by the synthesis of N-terrrunal or C-terminal fusion proteins
  • the invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the invention and/or to proteins of the invention Therefore, the invention also relates to a probe comprising a nucleic acid sequence of the invention, or a nucleic acid sequence encoding a protein of the invention, or a part thereof
  • the probe may be labeled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleic acid molecule of the invention including nucleic acid molecules coding for a protein which displays one or more of the properties of a protein of the invention
  • the invention still further provides a method for identifying a substance which binds to a protein of the invention comprising reacting the protein with at least one substance which potentially can bind with the protein, under conditions which permit the formation of complexes between the substance and protein and detecting binding Binding may be detected by assaying for complexes, for free substance, or for non- complexed protein
  • the invention also contemplates methods for identifying substances that bind to other intracellular proteins that interact with a KLK-L Related Protein Methods can also be utilized which identify compounds which bind to KLK-L gene regulatory sequences (e g promoter sequences)
  • the invention provides a method for evaluating a compound for its ability to modulate the biological activity of a KLK-L Related Protein of the invention
  • a substance which inhibits or enhances the interaction of the protein and a substance which binds to the protein may be evaluated
  • the method comprises providing a known concentration of a KLK-L Related Protein, with a substance which binds to the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and removing and/or detecting complexes
  • Compounds which modulate the biological activity ot a protein ot the invention may also be identified using the methods of the invention by comparing the pattern and level of expression of the protein of the invention in tissues and cells, in the presence, and in the absence of the compounds
  • proteins of the invention and substances and compounds identified using the methods of the invention, and peptides of the invention may be used to modulate the biological activity of a KLK-L
  • the substances and compounds may be formulated into compositions for administration to individuals suffering from cancer
  • the present invention also relates to a composition
  • a composition comprising one or more of a protein of the invention, a peptide of the invention, or a substance or compound identified using the methods of the invention, and a pharmaceutically acceptable carrier, exc ⁇ ient or diluent
  • a method for treating or preventing cancer comprising administering to a patient m need thereof, a KLK-L Related
  • Protein of the invention or a composition of the invention
  • Figure 1 shows an approximate 300 Kb of contiguous genomic sequence around chromosome 19ql3 3 - ql3 4 represented by 8 contigs, each one shown with its length in Kb
  • the contig numbers refer to those reported in the Lawrence Livermore National Laboratory website Note the localization of the seven known genes (PSA, KLK2, Zyme, NES1, HSCCE, neuropsin and TLSP) (see abbreviations for full names of these genes) All genes are represented with arrows denoting the direction of transcription
  • the gene with no homology to human kailikreins is termed UG (unknown gene)
  • the five new kallikrein-hke genes (KLK-L 1 to KLK-L5) were numbered from the most centrome ⁇ c to the most telome ⁇ c Numbers just below or just above the arrows indicate appropriate Kb lengths in each contig Gene lengths and distances between genes are rounded to the nearest 6 5 kb
  • Figure 2 shows a contiguous
  • FIG. 3 shows tissue expression of the prostase/KLK-Ll gene as determined by RT-PCR Actin and PSA are control genes Interpretations are presented in Table 9
  • Figure 4 shows the sequence of PCR product obtained with cDNA from female breast tissue using prostase/KLK-Ll primers Primer sequences are underlined The sequence is identical to the sequence obtained from prostatic tissue
  • Figure 6 is a schematic diagram showing comparison of the genomic structure of PSA, KLKl,
  • KLK2, zyme, neuropsin and prostase/KLK-Ll genes Exons are shown by open boxes and introns by the connecting lines Arrow head shows the start codons and the vertical arrow represents stop codons Letters above boxes indicate relative positions of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases
  • the intron phase refers to the location of the intron within the codon, I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons Numbers inside boxes indicate exon lengths in base pairs
  • Figure 7 shows the genomic organization and partial genomic sequence of the KLK-L2 gene Intronic sequences are not shown except for the splice junctions. Introns are shown with lower case letters and exons with capital letters. The start and stop codons are encircled and the exon -intron junctions are boxed The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are inside triangles Putative polyadenylation signal is underlined
  • Figure 8 shows an approximate 300 Kb region of almost contiguous genomic sequence around chromosome 19ql3.3- ql3.4. Genes are represented by horizontal arrows denoting the direction of the coding sequence Distances between genes are mentioned in base pairs
  • Figure 9 shows the alignment of the deduced amino acid sequence of KLK-L2 with members of the kal krein multi-gene family.
  • Genes are (from top to bottom) • Prostase/KLK-Ll, enamel matrix serine proteinase 1 (EMSPl) (GenBank accession # NP_004908), KLK-L2, zyme (GenBank accession # Q92876), neuropsin (GenBank accession # BAA28673), trypsin-hke serine protease (TLSP) (GenBank accession # B AA33404), PSA (GenBank accession # P07288), KLK2 (GenBank accession # P20151),
  • KLKl GenBank accession # NP_0022478
  • trypsinogen GenBank accession # P07477)
  • Dashes represent gaps to bring the sequences to better alignment
  • the residues of the catalytic triad are represented by (•---•) and the 29 invariant serine protease residues by (I or •---•)
  • conserveed areas around the catalytic triad are boxed
  • the predicted cleavage sites are indicated by ( )
  • the dotted area represents the kalhkrein loop sequence
  • the trypsin like cleavage pattern is indicated by ( ⁇ )
  • Figure 10(A) shows a dendrogram of the predicted phylogenetic tree for some kal krein genes
  • KLKl, KLK2, and PSA together and aligned the KLK-L2 gene in one group with EMSP, prostase, and TLSP (B) Plot of hydrophobicity and hydrophilicity of KLK-L2
  • Figure 11 is a blot showing tissue expression of KLK-L2 gene as determined by RT-PCR Actin and PSA are control genes Interpretations are presented in Table 12
  • Figure 13 are blots of EtBr-stained agarose gels Total RNA was extracted from normal, benign, and cancer tissues and used to generate cDNA PCR was performed on cDNA
  • Figure 14 shows an approximate 300 Kb region of almost contiguous genomic sequence around chromosome 19q I3 3- ql3 4 Genes are represented by horizontal arrows denoting the direction of the coding sequence Gene lengths and distances between genes are rounded to the nearest 05 kb The site of the gap is marked with an asterisk Telome ⁇ c to TLSP there are likely another three kallikrein-hke genes
  • Figure 15 shows the genomic organization and partial genomic sequence of the KLK-L3 gene
  • Intronic sequences are not shown except for the splice junctions Introns are shown with lower case letters and exons with capital letters For the full sequence, see SEQ ID NO 21 The start and stop codons are encircled and the exon -intron junctions are boxed The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are inside triangles Putative polyadenylation signal is underlined
  • Figure 16 is a plot of hydrophobicity and hydrophilicity, comparing the pattern of the KLK-L3 with that of the zyme gene Note the hydrophobic region around the first twenty amino acids, likely representing the signal peptide
  • Figure 17 is an alignment of the deduced amino acid sequence of KLK-L3 with members of the kalhkrein multi-gene family Genes are (from top to bottom and in brackets is the GenBank accession #) PSA (P07288), KLK2 (P20151), KLKl (NP002248), trypsinogen (P07477), KLK-L3 (AF135026), trypsin- like serine protease (TLSP) (BAA33404), neuropsin (BAA28673), zyme (Q92876), human stratum corneum chymotryptic enzyme (HSCCE) (AAD49718), and prostase/KLK-Ll (AAD21581) (See SEQ ID NOs 78 to 84) Dashes represent gaps to bring the sequences to better alignment The residues of the catalytic triad are bold and in italics, and the 29 invariant serine protease residues are denoted by ( ⁇
  • Cysteine residues are marked by (•) conserveed areas around the catalytic triad are highlited in black The arrow heads ( "* ⁇ ) represent the potential cleavage sites The dotted area represents the kalhkrein loop sequence
  • Figure 18 is a dendrogram of the predicted phylogenetic tree for some serine proteases and kal krein genes Neighbor-joimng/UPGMA method was used to align KLK-L3 with other members of the kalhkrein gene family Gene names and accession numbers are listed in Figure 17 The tree grouped the classical kailikreins (KLKl , KLK2, and PSA) together and aligned the KLK-L3 gene in one group with TLSP, neuropsin, and NES 1 genes KLK-L4 (SEQ ID NO 43) lies further telome ⁇ c to TLSP (21)
  • Figure 19 is a blot showing tissue expression of the KLK-L3 gene as determined by RT-PCR Actin and PSA are control genes
  • Figure 21 is a schematic diagram showing the comparison of the genomic structure of PSA, KLK2, neuropsin, NES1, and KLK-L3 genes Exons are shown by black boxes and introns by the connecting lines Arrowheads show the start codon, and arrows show the stop codon Letters above boxes indicate relative positions of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases
  • the intron phase refers to the location of the intron within the codon, I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons
  • Numbers inside boxes indicate exon lengths base pairs
  • Figure 22 shows a comparative genomic structure of the ESTs (Table 16), the clone from The German Genome Project, and the long form of KLK-L4 Exons are represented by solid bars and introns by the connecting lines Exon numbers on top of solid bars
  • Figure 24 in the Upper Panel is a Diagram showing the comparative genomic structure of the long KLK-L4 form and the short KLK-L4 variant Exons are represented by boxes and introns by the connecting lines Exon numbers refer to SEQ ID NO 43 and GenBank Accession No AF135024
  • the black region indicates the extra fragment (214 bp) that is found in the long, but not in the short form of the gene
  • the positions of the stop codons of the two forms are marked with asterisks Frame shifting occurs as a result of utilization of an alternative splice site, and a stop codon is generated at the beginning of exon 4 in the short form
  • the Lower Panel shows PCR products of the amplification of the KLK-L4 gene using L4-R1 and L4-X1 primers ( Figure 22 and Table 15) Note the predominant long form and a minor band representing the short form of KLK-L4 mRNA (M), Markers with sizes in bp shown on the left Tissues used (1), salivary gland, (2), mamm
  • Figure 25 shows the genomic organization and partial genomic sequence of the KLK-L4 gene Intronic sequences are not shown except for the splice junction areas Introns are shown with lower case letters and exons with capital letters For full sequence, see SEQ ID NO 43 or GenBank Accession
  • Figure 26 is a plot of hydrophobicity and hydrophilicity of the KLK-L4 protein, as compared with the glandular kal krein gene 2 (KLK2) Note the hydrophobic region at the amino terminus, suggesting presence of a signal peptide
  • Figure 27 shows an alignment of the deduced am o acid sequence of KLK-L4 with members of the kalhkrein multi-gene family Genes are (from top to bottom, and in brackets are the GenBank accession #) KLK-L 1/prostdse (AAD21581).
  • Figure 31 is a schematic diagram showing the comparison of the genomic structure of PSA, KLK2, neuropsin, NES 1, and KLK-L4 genes Exons are shown by black boxes and introns by the connecting lines
  • the arrowhead shows the start codons and the arrow the stop codons
  • Letters above boxes indicate the relative positions of the amino acids of the catalytic triad
  • H denotes histidine
  • D aspartic acid and S serine Roman numbers indicate intron phases
  • the intron phase refers to the location of the intron within the codon, I, the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons
  • Numbers inside boxes indicate exon lengths in base pairs The question mark indicates the possibility of more untranslated bases
  • Figure 32 is a diagram showing the comparative genomic structure of the three splice forms of KLK-L5, the classical kalhkrein form, related protein- 1, and related prote ⁇ n-2 Exons are represented by solid bars and introns by the connecting lines Exon numbers refer to SEQ ID NO 56 and GenBank
  • Figure 33 shows the genomic organization and partial genomic sequence of the KLK-L5 gene
  • Intronic sequences are not shown except for short sequences around the splice junctions Introns are shown with lower case letters and exons with capital letters For full sequence, see SEQ ID NO 56 The start and stop codons are encircled and the exon -intron junctions are underlined The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are boxed
  • Figure 34 is a schematic diagram showing the comparison of the genomic structure of PSA, KLK2, neuropsin, NES1, KLK-L4 and KLK-L5 genes Exons are shown by solid bars and introns by the connecting lines Arrowhead marks the site of the start codon, and the arrow represents the stop codon Letters above boxes indicate relative positions of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases The intron phase refers to the location of the intron with the codon, I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons Numbers inside boxes indicate exon lengths in base pairs Question marks indicate that exact length is not accurately known
  • Figure 35 is a plot of hydrophobicity and hydrophilicity of KLK-L5 protein compared to prostate specific antigen (PSA)
  • PSA prostate specific antigen
  • Figure 36 shows an alignment of the deduced ammo acid sequence of KLK-L5 with members of the kalhkrein multigene family (See SEQ ID NOs 78-81, 83, 84) Dashes represent gaps to bring the sequences to better alignment
  • the residues of the catalytic triad are represented by bold letters, and the 29 invariant serine protease residues are marked with (•)
  • the cysteine residues are marked by ( ⁇ )
  • conserveed areas are highlighted in grey
  • the predicted cleavage sites in signal peptide are indicated by (4)
  • the dotted area represents the kalhkrein loop sequence
  • a vertical arrow marks the trypsin like cleavage site
  • Figure 37 is a dendrogram of the predicted phylogenetic tree for some serine proteases and other kalhkrein proteins Neighbor-joining/UPGMA method was used to align KLK-L5 with other serine proteases and members of the kalhkrein gene family The tree grouped the classical kailikreins (hKl, hK2, and PSA) together and aligned the KLK-L5 protein in one group with NES 1 and neuropsin Other serine proteases were aligned in different groups
  • Figure 38 shows tissue expression of the KLK-L5 gene as determined by RT-PCR
  • the upper band (905 base pairs, bp) is the classical form (see Figure 32, the middle (776 bp) the related protein- 1 , and the lower band (644 bp) the related prote ⁇ n-2
  • the primers used were L5-F2 and L5-R2, as shown in Table 17
  • Figure 39 shows hormonal regulation of the KLK-L5 gene in the LnCaP prostatic carcinoma cell line, BT-474 and T-47D breast carcinoma cell lines Steroids were at 10 8 M final concentration Actin (not regulated by steroid hoi mones) was used as a control gene Note detection ot three isotorms only in LNCaP
  • Figure 40 shows the expression of the KLK-L5 gene in breast cancer ( 1-17) and normal ( 18) tissues Note complete absence of expression in 12 cancer tissues.
  • Figure 38 Figure 41 shows the full structure of a KLK-L6 nucleic acid molecule
  • Figure 42 is a plot of hydrophobicity and hydrophilicity of KLK-L6 protein compared to prostate specific antigen (PSA).
  • Figure 43 shows an alignment of the deduced amino acid sequence of KLK-L6 with members of the kallikrem multigene family. (See SEQ. ID. NOs. 78-81, 83, 84). Dashes represent gaps to bring the sequences to better alignment
  • Figure 44 is a dendrogram of the predicted phylogenetic tree for some serine proteases and other kalhkrein proteins. Neighbor-joining UPGMA method was used to align KLK-L6 with other serine proteases and members of the kalhkrein gene family. DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature.
  • nucleic Acid Molecules of the invention provides an isolated nucleic acid molecule having a sequence encoding a KLK-L Protein.
  • isolated refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical reactants, or other chemicals when chemically synthesized.
  • An "isolated” nucleic acid may also be free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) from which the nucleic acid is derived
  • nucleic acid is intended to include DNA and
  • RNA can be either double stranded or single stranded
  • a nucleic acid molecule encodes a KLK-L Protein comprising an amino acid sequence as shown in SEQ.ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, preferably a nucleic acid molecule comprising a nucleic acid sequence as shown in SEQ.ID.NO. 1, 13, 21, 43, 56, or 65.
  • the invention includes nucleic acid sequences complementary to a nucleic acid encoding a KLK-L
  • Protein comprising an amino acid sequence as shown in SEQ.ID.NO. 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, preferably the nucleic acid sequences complementary to a full nucleic acid sequence shown in SEQ.ID NO.
  • the invention includes nucleic acid molecules having substantial sequence identity or homology to nucleic acid sequences of the invention or encoding proteins having substantial identity or similarity to the amino acid sequence shown in in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • the nucleic acids have substantial sequence identity for example at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% nucleic acid identity, more preferably 90% nucleic acid identity, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity "Identity" as known in the art and used herein, is a relationship between two or more amino acid sequences or two or more nucleic acid sequences, as determined by comparing the sequences It also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequence
  • nucleic acid molecules encoding a KLK-L Protein and having a sequence which differs from a nucleic acid sequence of the invention due to degeneracy in the genetic code are also within the scope of the invention
  • nucleic acids encode functionally equivalent proteins (e g , a KLK-L Protein) but differ in sequence from the sequence of a KLK-L Protein due to degeneracy in the genetic code
  • DNA sequence polymorphisms within the nucleotide sequence of a KLK-L Protein may result in silent mutations which do not affect the ammo acid sequence Variations in one or more nucleotides may exist among individuals within a population due to natural allelic variation Any and all such nucleic acid variations are within the scope of the invention
  • DNA sequence polymorphisms may also occur which lead to changes in the amino acid sequence of a KLK-L Protein These amino acid polymorphisms are also within the scope of the present invention
  • nucleic acid molecule which hybridizes under stringent conditions, preferably high stringency conditions to a nucleic acid molecule which comprises a sequence which encodes a KLK-L Protein having an amino acid sequence shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1989), 6 3 1-6 3 6
  • 6 0 x sodium chloride/sodium citrate (SSC) at about 45°C, tollowed by a wash of 2 0 x SSC at 50°C may be employed
  • the stringency may be selected based on the conditions used in the wash step
  • the salt concentration in the wash step can be selected from a high stringency of about 0 2 x SSC at 50°C
  • the temperature in the wash step can be at
  • nucleic acid molecules of the invention which comprises DNA can be isolated by preparing a labelled nucleic acid probe based on all or part of a nucleic acid sequence of the invention The labeled nucleic acid probe is used to screen an appropriate DNA library (e g a cDNA or genomic DNA library)
  • a cDNA library can be used to isolate a cDNA encoding a KLK-L Related Protein by screening the library with the labeled probe using standard techniques
  • a genomic DNA library can be similarly screened to isolate a genomic clone
  • An isolated nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid encoding a KLK-L Related Protein using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA It is possible to design synthetic oligonucleotide primers from the nucleotide sequence of the invention for use in PCR
  • a nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques
  • the nucleic acid so amplified can be cloned into an appropriate vector and characte ⁇ zed by DNA sequence analysis cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al , Biochemistry,
  • cDNA is then synthesized from the mRNA using reverse transc ⁇ ptase (for example, Moloney MLV reverse transc ⁇ ptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transc ⁇ ptase available from Seikagaku America, Inc , St Louis, FL)
  • reverse transc ⁇ ptase for example, Moloney MLV reverse transc ⁇ ptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transc ⁇ ptase available from Seikagaku America, Inc , St Louis, FL
  • RNA nucleic acid molecule of the invention which is RNA
  • a cDNA can be cloned downstream of a bacteriophage promoter, (e g a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by conventional techniques
  • Nucleic acid molecules of the invention may be chemically synthesized using standard techniques Methods of chemically synthesizing polydeoxynucleotides are known, including but not limited to solid- phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e g , Itakura et al U S Patent No 4,598,049, Caruthers et al U S Patent No 4,
  • the initiation codon and untranslated sequences of a KLK-L Related Protein may be determined using computer software designed for the purpose, such as PC/Gene (IntelhGenetics Inc , Calif )
  • the intron-exon structure and the transcription regulatory sequences of a gene encoding a KLK-L Related Protein may be confirmed by using a nucleic acid molecule of the invention encoding a KLK-L Related Protein to probe a genomic DNA clone library
  • Regulatory elements can be identified using standard techniques
  • the function of the elements can be confirmed by using these elements to express a reporter gene such as the lacZ gene which is operatively linked to the elements
  • These constructs may be introduced into cultured cells using conventional procedures or into non-human transgenic animal models In addition to identifying regulatory elements in DNA, such constructs may also be used to identify nuclear proteins interacting with the elements, using techniques known in the art
  • the nucleic acid molecules isolated using the methods described herein are mutant klk-l gene alleles
  • the mutant alleles may be isolated from individuals either known or proposed to have a genotype which contributes to the symptoms of for example, cancer (e g breast, testicular, brain, colon, and prostate cancer)
  • Mutant alleles and mutant allele products may be used in therapeutic and diagnostic methods described herein
  • a cDNA of a mutant klk-l gene may be isolated using PCR as desc ⁇ bed herein, and the DNA sequence of the mutant allele may be compared to the normal allele to ascertain the mutat ⁇ on(s) responsible for the loss or alteration of function of the mutant gene product
  • a genomic library can also be constructed using DNA from an individual suspected of or known to carry a mutant allele, or a cDNA library can be constructed using RNA from tissue known, or suspected to express the mutant allele
  • a nucleic acid encoding a normal klk-l gene or any suitable fragment thereof may
  • sequence of a nucleic acid molecule of the invention, or a fragment of the molecule may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule
  • An antisense nucleic acid molecule may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art 2.
  • Proteins of the Invention An amino acid sequence ot a KLK L Protein comprises a sequence as shown in Tables 1 to 5 or SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • the proteins of the present invention include truncations of a KLK-L Protein, analogs of a KLK-L Protein, and proteins having sequence identity or similarity to a KLK-L Protein, and truncations thereof as described herein (I e included in KLK-L Related Proteins)
  • Truncated proteins may comprise peptides of between 3 and 70 amino acid residues, ranging in size from a t ⁇ peptide to a 70 mer polypeptide
  • the truncated proteins may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end
  • the truncated proteins may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end
  • the proteins of the invention may also include analogs of a KLK-L Protein, and/or truncations thereof as described herein, which may include, but are not limited to a KLK-L Protein, containing one or more amino acid substitutions, insertions, and/or deletions Amino acid substitutions
  • One or more amino acid insertions may be introduced into a KLK-L Protein Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length
  • Deletions may consist of the removal of one or more amino acids, or discrete portions from a KLK-L Protein sequence
  • the deleted amino acids may or may not be contiguous
  • the lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 20 to 40 amino acids
  • the proteins of the invention include proteins with sequence identity or similarity to a KLK-L
  • KLK-L Proteins include proteins whose amino acid sequences are comprised of the amino acid sequences of KLK-L Protein regions from other species that hybridize under selected hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain a KLK-L Protein These proteins will generally have the same regions which are characteristic of a KLK-L Protein
  • a protein will have substantial sequence identity for example, about 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% identity, preferably 90% identity, more preferably at least 95%, 96%, 97%, 98%, or 99% identity, and most preferably 98% identity with an amino acid sequence shown in Tables 1 to 5 or SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • a percent amino acid sequence homology, similarity or identity is calculated as the percentage ot aligned amino acids that match the reference sequence using known methods as described herein
  • the invention also contemplates isoforms of the proteins of the invention
  • An isoform contains the same number and kinds of amino acids as a protein of the invention, but the isoform has a different molecular structure
  • Isoforms contemplated by the present invention preferably have the same properties as a protein of the invention as described herein
  • the present invention also includes KLK-L Related Proteins conjugated with a selected protein, or a marker protein (see below) to produce fusion proteins Additionally, immunogenic portions of a KLK- L Protein and a KLK-L Protein Related Protein are within the scope of the invention
  • a KLK-L Related Protein of the invention may be prepared using recombinant DNA methods Accordingly, the nucleic acid molecules of the present invention having a sequence which encodes a KLK- L Related Protein of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e g replication defective retroviruses, adenoviruses and adeno- associated viruses), so long as the vector is compatible with the host cell used
  • the invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence
  • Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology Methods m Enzymology 185, Academic Press, San Diego, CA (1990) Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art
  • the necessary regulatory sequences may be supplied by the native KLK-L Protein and/or its flanking regions
  • the invention further provides a recombinant expression vector comprising a DNA nucleic acid molecule of the invention cloned into the expression vector in an antisense orientation That is, the DNA molecule is linked to a regulatory sequence in a manner which allows for expression, by transcription of
  • the recombinant expression vectors of the invention may also contain a marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention
  • marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, ⁇ -galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG
  • the markers can be introduced on a separate vector from the nucleic acid of interest
  • the lecombinant expiession vectors may also contain genes which encode a fusion moiety which provides increased expression ot the recombinant protein, increased solubility of the recombinant protein, and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification
  • a proteolytic cleavage site may be added to the
  • Transformant host cells include host cells which have been transformed or transfected with a recombinant expression vector of the invention
  • the terms "transformed with”, “transfected with”, “transformation” and “transfection” encompass the introduction of a nucleic acid (e g a vector) into a cell by one of many standard techniques
  • Prokaryotic cells can be transformed with a nucleic acid by, for example, electroporation or calcium-chloride mediated transformation
  • a nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, pofectin, electroporation or microinjection Suitable methods for transforming and transfecting host cells can be found in Sambrook et al (Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks Suitable host cells include a wide variety of prokaryotic
  • a host cell may also be chosen which modulates the expression of an inserted nucleic acid sequence, or modifies (e g glycosylation or phosphorylation) and processes (e g cleaves) the protein in a desired fashion
  • Host systems or cell lines may be selected which have specific and characteristic mechanisms for post-translational processing and modification of proteins
  • eukaryotic host cells including CHO, VERO, BHK, HeLA, COS, MDCK, 293, 3T3, and WI38 may be used for long-term high-yield stable expression of the protein, cell lines and host systems which stably express the gene product may be engineered
  • Host cells and in particular cell lines produced using the methods desc ⁇ bed herein may be particularly useful in screening and evaluating compounds that modulate the activity of a KLK-L Related Protein
  • the proteins of the invention may also be expressed in non-human transgenic animals including but not limited to mice, rats, rabbits, guinea pigs, micro-pigs, goats, sheep, pigs, non-human primates (e g baboons, monkeys, and chimpanzees) [see Hammer et al (Nature 315 680-683, 1985), Palmiter et al
  • the transgene may be integrated as a single transgene or in concatamers
  • the transgene may be selectively introduced into and activated in specific cell types (See for example, Lasko et al, 1992 Proc. Natl. Acad. Sci. USA 89: 6236).
  • the transgene may be integrated into the chromosomal site of the endogenous gene by gene targeting.
  • the transgene may be selectively introduced into a particular cell type inactivating the endogenous gene in that cell type (See Gu et al Science 265: 103-106).
  • the expression of a recombinant KLK-L Related Protein in a transgenic animal may be assayed using standard techniques. Initial screening may be conducted by Southern Blot analysis, or PCR methods to analyze whether the transgene has been integrated. The level of mRNA expression in the tissues of transgenic animals may also be assessed using techniques including Northern blot analysis of tissue samples, in situ hybridization, and RT-PCR. Tissue may also be evaluated lmmunocytochemically using antibodies against KLK-L Protein.
  • Proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Mer ⁇ field, 1964, J. Am. Chem. Assoc. 85:2149- 2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed E Wansch, Vol. 15 I and II, Thieme, Stuttgart).
  • N-terminal or C-terminal fusion proteins comprising a KLK-L Related Protein of the invention conjugated with other molecules, such as proteins, may be prepared by fusing, through recombinant techniques, the N-terminal or C-termmal of a KLK-L Related Protein, and the sequence of a selected protein or marker protein with a desired biological function.
  • the resultant fusion proteins contain KLK-L
  • proteins which may be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc 3.
  • Antibodies KLK-L Related Proteins of the invention can be used to prepare antibodies specific for the proteins. Antibodies can be prepared which bind a distinct epitope in an unconserved region of the protein An unconserved region of the protein is one that does not have substantial sequence homology to other proteins. A region from a conserved region such as a well-characte ⁇ zed domain can also be used to prepare an antibody to a conserved region of a KLK-L Related Protein. Antibodies having specificity for a KLK-L Related Protein may also be raised from fusion proteins created by expressing fusion proteins in bacteria as described herein
  • the invention can employ intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g. a Fab, (Fab) 2 fragment, or Fab expression library fragments and epitope-binding fragments theieof), an antibody heavy chain, and antibody light chain, a genetically engineered single chain Fv molecule (Ladner et al, U S Pat No 4,946,778), or a chimenc antibody, for example, an antibody which contains the binding specificity ot a mu ⁇ ne antibody, but in which the remaining portions are of human origin
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras may be prepared using methods known to those skilled in the art
  • the nucleic acid molecules, KLK-L Related Proteins, and antibodies of the invention may be used in the prognostic and diagnostic evaluation of cancer (e g breast, testicular, and prostate cancer) or other conditions, and the identification of subjects with a predisposition to cancer (Section 4 1 1 and 4 1 2)
  • Methods for detecting nucleic acid molecules and KLK-L Related Proteins of the invention can be used to monitor conditions including cancer, by detecting KLK-L Related Proteins and nucleic acid molecules encoding KLK-L Related Proteins
  • the applications of the present invention also include methods for the identification of compounds that modulate the biological activity of KLK-L or KLK-L Related Proteins (Section 4 2)
  • the compounds, antibodies etc may be used for the treatment of cancer (Section 4 3) 4.1 Diagnostic Methods
  • a variety of methods can be used to monitor conditions including cancer, by detecting KLK-L Related Proteins and nucleic acid molecules encoding KLK-L Related Proteins.
  • the methods desc ⁇ bed herein may be performed by utilizing pre-packaged diagnostic kits comprising at least one specific KLK-L nucleic acid or antibody described herein, which may be conveniently used, e g , in clinical settings, to screen and diagnose patients and to screen and identify those individuals exhibiting a predisposition to developing a disorder
  • Nucleic acid-based detection techniques are described, below, in Section 4 1 1
  • Peptide detection techniques are described, below, in Section 4 1 2
  • the samples that may be analyzed using the methods of the invention include those which are known or suspected to express KLK-L or contain KLK-L Related Proteins
  • the samples may be derived from a patient or a cell culture, and include but are not limited to biological fluids, tissue extracts, treshly harvested cells, and lysates ot cells which have been incubated in cell cultures
  • Oligonucleotides or longer fragments derived trom any of the nucleic acid molecules of the invention may be used as targets in a microarray
  • the microarray can be used to simultaneously monitor the expression levels of large numbers of genes and to identify genetic variants, mutations, and polymorphisms
  • the information from the microarray may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents
  • nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleic acid sequences of the invention in samples Suitable probes include nucleic acid molecules based on nucleic acid sequences encoding at least
  • the nucleic acid probes may be used to detect genes, preferably in human cells, that encode KLK-L Related Proteins
  • the nucleotide probes may also be useful in the diagnosis of cancer, in monitoring the progression of cancer, or monitoring a therapeutic treatment
  • the probe may be used in hybridization techniques to detect genes that encode KLK-L Related
  • the technique generally involves contacting and incubating nucleic acids (e g recombinant DNA molecules, cloned genes) obtained from a sample from a patient or other cellular source with a probe of the present invention under conditions favorable for the specific annealing of the probes to complementary sequences in the nucleic acids After incubation, the non-annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected
  • nucleic acids e g recombinant DNA molecules, cloned genes
  • the detection of nucleic acid molecules of the invention may involve the amplification of specific gene sequences using an amplification method such as PCR, followed by the analysis of the amplified molecules using techniques known to those skilled in the art Suitable primers can be routinely designed by one ot skill in the art
  • Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities involving klk I structure, including point mutations, insertions, deletions, and chromosomal rearrangements
  • direct sequencing single stranded conformational polymorphism analyses, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical mismatch cleavage, and oligonucleotide hybridization may be utilized
  • Genotyping techniques known to one skilled in the art can be used to type polymorphisms that are in close proximity to the mutations in a klk-l gene
  • the polymorphisms may be used to identify individuals in families that are likely to carry mutations If a polymorphism exhibits linkage disequalib ⁇ um with mutations in a klk-l gene, it can also be used to screen for individuals in the general population likely to carry mutations
  • Polymorphisms which may be used include restriction fragment length polymorphisms (RFLPs), single-base polymorphisms, and simple sequence repeat polymorphisms (SSLPs)
  • a probe of the invention may be used to directly identify RFLPs
  • a probe or primer of the invention can additionally be used to isolate genomic clones such as YACs, BACs, PACs, cosmids, phage or plasmids
  • genomic clones such as YACs, BACs, PACs, cosmids, phage or plasmids
  • the DNA in the clones can be screened for SSLPs using hybridization or sequencing procedures
  • Hybridization and amplification techniques described herein may be used to assay qualitative and quantitative aspects of klk-l expression
  • RNA may be isolated from a cell type or tissue known to express klk-l and tested utilizing the hybridization (e g standard Northern analyses) or PCR techniques referred to herein
  • the techniques may be used to detect differences in transcript size which may be due to normal or abnormal alternative splicing
  • the techniques may be used to detect quantitative differences between levels of full length and/or alternatively splice transcripts detected in normal individuals relative to those individuals exhibiting cancer symptoms or other disease conditions
  • the primers and probes may be used in the above desc ⁇ bed methods in situ l e directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections
  • Antibodies specifically reactive with a KLK-L Related Protein, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect KLK-L Related Proteins in various samples (e g biological materials) They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of KLK-L Related Proteins expression, or abnormalities in the structure, and/or temporal, tissue, cellular, or subcellular location of a KLK-L Related Protein Antibodies may also be used to screen potentially therapeutic compounds in vitro to determine their effects on cancer, and other conditions In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies The antibodies of the invention may also be used in vitro to determine the level of KLK-L expression in cells genetically engineered to produce a KLK-L Related Protein
  • the antibodies may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of a KLK-L Related Protein and the antibodies Examples of such assays are radioimmunoassays, enzyme immunoassays (e g ELISA), lmmunofluorescence, immunoprecipitation, latex agglutination, hemagglutmation, and histochemi il tests
  • the antibodies may be used to detect and quantity KLK-L Related Proteins in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states
  • the antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect a KLK-L Related Protein, to localize it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a KLK-L Related Protein
  • an antibody of the invention may be labeled with a detectable substance and a KLK-L Related Protein may be localised m tissues and cells based upon the presence of the detectable substance
  • detectable substances include, but are not limited to, the following radioisotopes (e g , 3 H, 14 C, 35 S, 125 I, 131 I), fluorescent labels (e g , FITC, rhodamine, lanthanide phosphors), luminescent labels such as lurrunol, enzymatic labels (e g , horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylchohnesterase), biotinyl groups (which can be detected by marked avidin e g , streptavidin containing a fluorescent marker or
  • a KLK-L Related Protein may be localized by radioautography
  • the results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains 4.2 Methods for Identifying or Evaluating Substances/Compounds
  • the methods described herein are designed to identify substances that modulate the biological activity of a KLK-L Related Protein including substances that bind to KLK-L Related Proteins, or bind to other proteins that interact with a KLK-L Related Protein, to compounds that interfere with, or enhance the interaction of a KLK-L Related Protein and substances that bind to the KLK-L Related Protein or other proteins that interact with a KLK-L Related Protein Methods are also utilized that identify compounds that bind to KLK L regulatory sequences
  • the substances and compounds identified using the methods ot the invention include but aie not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members ot random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e g polyclonal, monoclonal, humanized, anti-idiotypic,
  • a substance that associates with a polypeptide of the invention may be an agonist or antagonist of the biological or immunological activity of a polypeptide of the invention
  • the term "agonist' refers to a molecule that increases the amount of, or prolongs the duration of, the activity of the polypeptide
  • the term "antagonist” refers to a molecule which decreases the biological or immunological activity of the polypeptide
  • Agonists and antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules that associate with a polypeptide of the invention
  • Substances which can bind with a KLK-L Related Protein may be identified by reacting a KLK-L Related Protein with a test substance which potentially binds to a KLK-L Related Protein, under conditions which permit the formation of substance-KLK-L Related Protein complexes and removing and/or detecting the complexes
  • the substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof
  • isolation techniques for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof
  • antibody against KLK-L Related Protein or the substance, or labeled KLK-L Related Protein, or a labeled substance may be utilized.
  • the antibodies, proteins, or substances may be labeled with a detectable substance as described above
  • a KLK-L Related Protein, or the substance used in the method of the invention may be insolubilized
  • a KLK-L Related Protein, or substance may be bound to a suitable carrier such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, lon- exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc
  • the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc
  • the insolubilized protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical ethods, toi example, cyanogen bromide coupling
  • the invention also contemplates a method tor evaluating a compound for its ability to modulate the biological activity of a KLK-L Related Protein ot the invention, by assaying for an agonist or antagonist (1 e enhancer or inhibitor) of the binding of a KLK-L Related Protein with a substance which binds with a KLK-L Related Protein
  • the basic method for evaluating if a compound is an agonist or antagonist of the binding of a KLK-L Related Protein and a substance that binds to the protein is to prepare a reaction mixture containing the KLK-L Related Protein and the substance under conditions which permit the formation of substance-KLK-L Related Protein complexes, in the presence of a test compound
  • the test compound may be initially added to the mixture, or may be added subsequent to the addition of the KLK-L Related Protein and substance Control reaction mixtures without the test compound or with a placebo are also prepared
  • the formation of complexes is detected and the formation of complexes in the control reaction but not in the reaction mixture indicates that the test compound
  • agonists and antagonists l e inhibitors and enhancers that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or alloste ⁇ c sites
  • the invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of KLK-L Related Protein with a substance which is capable of binding to the KLK-L Related Protein
  • the invention may be used to assay for a compound that competes for the same binding site of a KLK-L Related Protein
  • the invention also contemplates methods for identifying compounds that bind to proteins that interact with a KLK-L Related Protein Protein-protein interactions may be identified using conventional methods such as co-immunoprecipitation, crosshnking and co-purification through gradients or chromatographic columns Methods may also be employed that result in the simultaneous identification of genes which encode proteins interacting with a KLK-L Related Protein These methods include probing expression libraries with labeled KLK-L Related Protein
  • Two-hybrid systems may also be used to detect protein interactions in vivo
  • plasmids are constructed that encode two hybrid proteins
  • a first hybrid protein consists of the DNA-binding domain of a transcription activator protein fused to a KLK-L Related Protein
  • the second hybrid protein consists of the transcription activator protein's activator domain fused to an unknown protein encoded by a cDNA which has been recombined into the plasmid as part of a cDNA library
  • the plasmids are transformed into a strain of yeast (e g S cerevisiae) that contains a reporter gene (e g lacZ, luciferase, alkaline phosphatase, horseradish peroxidase) whose regulatory region contains the transcription activator's binding site
  • yeast e g S cerevisiae
  • a reporter gene e g lacZ, luciferase, alkaline phosphatase, horseradish peroxidase
  • fusion proteins may be used in the above-described methods
  • KLK-L Related Proteins fused to a glutathione S-transferase may be used in the methods
  • the reagents suitable for applying the methods of the invention to evaluate compounds that modulate a KLK-L Related Protein may be packaged into convenient kits providing the necessary materials packaged into suitable containers
  • the kits may also include suitable supports useful in performing the methods of the invention 4.3 Compositions and Treatments
  • the proteins of the invention, substances or compounds identified by the methods desc ⁇ bed herein, antibodies, and antisense nucleic acid molecules of the invention may be used for modulating the biological activity of a KLK-L Related Protein, and they may be used in the treatment of conditions such as cancer (e g prostate, testicular, brain, uterine, thymus, ovarian, colon, ovarian, or breast cancer)
  • the substances, antibodies, peptides, and compounds may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in viv
  • the active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc ), oral administration, inhalation, transdermal application, or rectal administration Depending on the route of administration, the active substance may be coated in a material to protect the substance from the action of enzymes, acids and other natural conditions that may inactivate the substance
  • compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa , USA 1985) On this basis, the compositions include, albeit not exclusively, solutions of the active substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and lso-osmotic with the physiological fluids Based upon their homology to genes encoding kalhkrein, nucleic acid molecules ot the invention may be also useful in the treatment ot conditions such as hypertension, cardiac hypertrophy, arthritis, inflammatory disorders, neurological disorders, and blood clotting disorders
  • Vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used to deliver nucleic acid molecules to a targeted organ, tissue, or cell population Methods well known to those skilled in the art may be used to construct recombinant vectors which will express antisense nucleic acid molecules of the invention (See, for example, the techniques described in Sambrook et al (supra) and Ausubel et al (supra))
  • nucleic acid molecules comprising full length cDNA sequences and/or their regulatory elements enable a skilled artisan to use sequences encoding a protein of the invention as an investigative tool in sense (Youssoufian H and H F Lodish 1993 Mol Cell Biol 13 98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem 60 631-652) regulation of gene function
  • sense or antisense o gomers, or larger fragments can be designed from various locations along the coding or control regions
  • Genes encoding a protein of the invention can be turned off by transfectmg a cell or tissue with vectors which express high levels of a desired KLK-L-encoding fragment
  • Such constructs can inundate cells with untranslatable sense or antisense sequences Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases Modifications of gene expression can be obtained by designing antisense molecules,
  • Ribozymes are enzymatic RNA molecules that catalyze the specific cleavage of RNA Ribozymes act by sequence-specific hybridization of the ⁇ bozyme molecule to complementary target RNA, followed by endonucleolytic cleavage
  • the invention therefore contemplates engineered hammerhead motif ⁇ bozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a protein of the invention
  • ⁇ bozyme cleavage sites within any potential RNA target may initially be identified by scanning the target molecule for ⁇ bozyme cleavage sites which include the following sequences, GUA,
  • RNA sequences of between 15 and 20 ⁇ bonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable
  • the suitability of candidate targets may also be detei mined by testing accessibility to hybridization with complementary oligonucleotides using ⁇ bonuclease piotection assays
  • Methods tor introducing vectors into cells or tissues include those methods discussed herein and which are suitable tor m vivo, m vitro and ex vivo therapy
  • vectors may be introduced into stem cells obtained from a patient and clonally propagated for autologous transplant into the same patient (See U S Pat Nos 5,399,493 and 5,437,994) Delivery by transfection and by liposome are well known in the art
  • nucleic acid molecules disclosed herein may also be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including but not limited to such properties as the triplet genetic code and specific base pair interactions
  • the invention also provides methods for studying the function of a polypeptide of the invention
  • Cells, tissues, and non-human animals lacking in expression or partially lacking in expression of a nucleic acid molecule or gene of the invention may be developed using recombinant expression vectors of the invention having specific deletion or insertion mutations in the gene
  • a recombinant expression vector may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a deficient cell, tissue, or animal
  • Null alleles may be generated in cells, such as embryonic stem cells by deletion mutation
  • a recombinant gene may also be engineered to contain an insertion mutation that inactivates the gene
  • Such a construct may then be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, electroporation, injection etc
  • Cells lacking an intact gene may then be identified, for example by Southern blotting, Northern Blotting, or by assaying for expression of the encoded polypeptide using the methods described herein
  • Such cells may then be fused to embryonic stem cells to generate transgenic non-human animals deficient in a polypeptide of the invention
  • Germlme transmission of the mutation may be achieved, for example, by aggregating the embryonic stem cells with early stage embryos, such as 8 cell embryos, in vitro, transferring the resulting blastocysts into recipient females and, generating germhne transmission of the resulting aggregation chimeras
  • Such a mutant animal may be used to define specific
  • the invention provides a transgenic non-human mammal all of whose germ cells and somatic cells contain a recombinant expression vector that inactivates or alters a gene encoding a KLK- L Related Protein resulting in a KLK-L Related Protein associated pathology Further the invention provides a transgenic non-human mammal which doe not express a KLK-L Related Protein of the invention. In an embodiment, the invention provides a transgenic non-human mammal which doe not express a KLK-L Related Protein of the invention resulting in a KLK-L Related Protein associated pathology
  • a KLK-L Related Protein pathology refers to a phenotype observed for a KLK-L Related Protein homozygous mutant
  • a transgenic non-human animal includes but is not limited to mouse, rat, rabbit, sheep, hamster, dog, guinea pig, micro-pig, pig, cat, goat, and non-human primates, preferably mouse
  • the invention also provides a transgenic non-human animal assay system which provides a model system for testing for an agent that reduces or inhibits a pathology associated with an KLK-L Related Protein, preferably a KLK-L Related Protein associated pathology, comprising
  • step (b) determining whether said agent reduces or inhibits the pathology (e g KLK-L Related Protein associated pathology) in the transgenic non-human animal relative to a transgenic non-human animal of step (a) which has not been administered the agent
  • the agent may be useful in the treatment and prophylaxis of conditions such as cancer as discussed herein
  • the agents may also be incorporated in a pharmaceutical composition as described herein
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics
  • the therapeutic index is the dose ratio of therapeutic to toxic effects and it can be expressed as the ED 50 /LD 50 ratio
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred
  • the Lawrence Livermore National Laboratory participates in the sequencing of the human genome project and focuses on sequencing chromosome 19 Large sequencing information on this chromosome is available at the website of the Lawrence Livermore National Laboratory (http //www- bio llnl gov/genome/gemnome html)
  • BAC 288H1 and BAC 76F7 Two clones which were positive for the Zyme gene (clones BAC 288H1 and BAC 76F7) These BACs were further analyzed by PCR and primers specific for PSA, NESl, KLKl and KLK2 These analyses indicated that both BACs were positive for Zyme, PSA and
  • KLKl, KLK2 and KLK3 are considered to represent the human kalhkrein gene family (9)
  • the work described herein provides strong evidence that a large number of kallikrein-hke genes are clustered within a 300Kb region around chromosome 19q l3 2 - ql3 4
  • the three established human kailikreins KLKl, KLK2, KLK3), Zyme and NESl, as well as the stratum corneum chymotrypticn enzyme, neuropsin, and TLSP (trypsin-like serine protease) and another five new genes , KLK-Ll to KLK-L5, may constitute a large gene family This will bring the total number of kalhkrein or kallikrein-hke genes in this region of chromosome 19 to thirteen
  • the human stratum corneum chymotryptic enzyme (19), neuropsin (20) and trypsin-hke serine protease (TLSP) (21) are three previously characterized genes which have many structural similarities with the kailikreins and other members of the serine protease family However, they have not been mapped in the past Their precise mapping in the region ot the kalhkrein gene family indicates that these three genes, along with the ones that were newly identified, or are already known, constitute a family that likely originated by duplication of an ancestral gene The relative localization of all these genes is depicted in Figure 1
  • Kalhkrem genes are a subfamily of serine proteases, traditionally characterized by their ability to liberate lysyl-bradykinin (kalhdin) from kininogen (18) More recently, however, a new, structural concept has emerged to describe kailikreins From accumulated sequence data, it is now clear that the mouse has many genes with high homology to kalhkrein coding sequences (19-20) Richard and co-workers have contributed to the concept of a " kal krem multigene family" to refer to these genes (21-22) This definition is not based much on specific enzymatic function of the gene product, but more on its sequence homology and their close linkage on mouse chromosome 7 In humans, only KLKl meets the functional definition of a kalhkrein KLK2 has trypsin-hke enzymatic activity and KLK3 (PSA) has very weak chymotrypsin-Iike enzymatic activity These activities of KLK2 and KLK3 are not known to
  • the breast cancer cell line BT-474 was purchased from the American Type Culture Collection (ATCC), Rockville, MD. BT-474 cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic tlasks, to near contluency The cells were then ahquoted into 24-well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 10 8 M Cells stimulated with 100% ethanol were included as controls The cells were cultured for 24 hours, then harvested for mRNA extraction Reverse transcriptase polymerase chain reaction
  • RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as desc ⁇ bed above for the tissue culture experiments and used for PCR reactions with the primers described in Table 8 (SEQ ID Nos 5-12) Tissue cDNAs were amplified at various dilutions Cloning and sequencing of the PCR products
  • the exon prediction strategy of the 300Kb DNA sequences around chromosome 19ql3 3 - ql3 4 identified a novel gene with a structure reminiscent of a serine protease
  • the major features of this gene were its homology, at the amino acid and DNA level, with other human kalhkrein genes, the conservation of the catalytic triad (histidine, aspartic acid, and serine), the number of exons and the complete conservation of the intron phases EST sequence homology search
  • the coding sequence of the klk-L2 gene is shown in SEQ ID NO 1 and GenBAnk Accession # AF135023
  • the exons of the gene are as follows exon 1 (939-999), exon 2 (2263-2425), exon 3 (2847- 3097), exon 4 (3181-3317), and exon 5 (4588-4740)
  • the amino acid sequence of KLK-L2 proteins are shown in SEQ ID Nos 2 and 3 Mapping and chromosomal localization of prostase /KLK-Ll gene
  • the tissue expression of prostase/KLK-Ll is summarized in Table 9 and Figure 3
  • This protease is primarily expressed in the prostate, testis, adrenals, uterus, thyroid, colon, central nervous system and mammary tissues, and, at much lower levels in other tissues
  • the specificity of the RT-PCR procedure was verified for prostase/KLK-Ll by cloning the PCR products from mammary, testicular and prostate tissues and sequencing them
  • One example with mammary tissue is shown in Figure 4 All cloned PCR products were identical in sequence to the cDNA sequence reported for the prostase/KLK-Ll Hormonal regulation of the prostase/KLK-Ll gene
  • the steroid hormone receptor-positive breast carcinoma cell line BT-474 was used as a model system to evaluate whether prostase/KLK-Ll expression is under steroid hormone regulation
  • the controls worked as expected I e , actin positivity without hormonal regulation in all cDNAs, only
  • the KLK3 gene encodes for PSA, a protein that currently represents the best tumor marker available (24) Since in rodents there are so many kalhkrein genes, the restriction of this family to only 3 genes in humans was somewhat surprising More recently, new candidate kalhkrein genes in humans have been discovered, including NESl (13) and zyme/protease M neurosin (10-12)
  • the known kailikreins and the newly discovered kalhkrein-hke genes share the following similarities (a) they encode serine proteases (b) they have five coding exons (c) they share significant DNA and protein homologies with each other (d) they map in the same locus on chromosome 19ql3 3-ql3 4, a region that is structurally similar to an area on mouse chromosome 7, where all the mouse kalhkrein genes are localized (e) they appear to be regulated by steroid hormones Prostase/KLK-Ll is a member of the same family since these common characteristics are also
  • the sensitive RT-PCR protocol reveals that the KLK-Ll enzyme is expressed m prostatic tissue and it is also expressed in significant amounts in other tissues, including testis, female mammary gland, adrenals, uterus, thyroid, colon, brain, lung and salivary glands ( Figure 3 and Table 9)
  • the specificity of the RT-PCR primers was verified by sequencing the obtained PCR products, with one example shown in Figure 4 (SEQ ID NO 4)
  • Tissue culture studies with the breast carcinoma cell line BT-474 further confirm not only the ability of these cells to produce prostase/KLK-Ll but also its hormonal regulation (Figure 5)
  • An interesting theme is now developing involving the group of homologous genes on chromosome 19ql3 3(PSA, KLK2, prostase, zyme, and NESl)
  • the combined data suggest that all of them are expressed in prostate and breast tissues, and all of them are hormonally regulated All these genes may be part of a cascade pathway that plays a role in cell proliferation, differentiation or
  • prostase/KLK-Ll can be produced by these cells and that its expression is significantly up-regulated by androgens and progestins Based on information for other homologous genes in the area ( PSA, zyme, and NESl), prostase/KLK-Ll may be involved in the pathogenesis and/or progression of prostate, breast and possibly other cancers
  • PSA prostate carcinoma cell lines
  • Sequencing data of approximately 300Kb of nucleotides on chromosome 19ql3.3-ql3 4 was obtained from the web site of the Lawrence Livermore National Laboratory (LLNL) (http://www- bio.llnl.gov/genome/genome.html). This sequence was in the form of 9 contigs of different lengths.
  • a restriction analysis study of the available sequences was performed using the "WebCutter" computer program (httpV/www.firstmarket. com/cutter/cut2.html) and with the aid of the EcoRI restriction map of this area (also available from the LLNL web site) an almost contiguous stretch of genomic sequences was constructed.
  • the KLK-L2 gene is formed of 5 coding exons and 4 intervening introns, spanning an area of 9,349 bp of genomic sequence on chromosome 19ql3 3-ql3 4
  • the lengths of the exons are 73, 262, 257,134, and 156 bp, respectively
  • the intron/exon splice sites (mGT AGm) and their flanking sequences are closely related to the consensus splicing sites (-mGTAAGT CAGm-) (32)
  • the presumptive protein coding region of the KLK-L2 gene is formed of 879 bp nucleotide sequence encoding a deduced 293-am ⁇ no acid polypeptide with a predicted molecular weight of 32 KDa
  • There are two potential translation initiation codons (ATG) at positions 1 and 25 of the predicted first exon (numbers refer to SEQ ID NO .
  • KLK-L2 will possess a trypsin-like cleavage pattern like most of the other kailikreins (e g , KLKl , KLK2, TLSP, neuropsin, zyme, prostase, and EMSP) but different from PSA which has a serine (S) residue in the corresponding position, and is known to have a chymotrypsin like activity ( Figure 9)
  • the dotted region in Figure 9 indicates an 11-am ⁇ no acid loop characteristic of the classical kailikreins (PSA, KLKl, and KLK2) but not found in KLK-L2 or other members of the kallikrein-hke gene family (11) Homology with the kalhkrein multi-gene family
  • the mature 227-am ⁇ no acid sequence of the predicted protein was aligned against the GenBank database and the known kailikreins using the "BLASTP” and "BLAST 2 sequence” programs KLK-L2 is found to have 54% amino acid sequence identity and 68% similarity with the enamel matrix serine proteinase 1 (EMSPl ) gene, 50% identity with both trypsin like serine protease (TLSP) and neuropsin genes and 47%, 46%, and 42% identity with trypsinogen, zyme, and PSA genes, respectively
  • the multiple alignment study shows that the typical catalytic triad of serine proteases is conserved in the KLK-L2 gene (H 108 , D 153 , and S 245 ) and, as the case with all other kailikreins, a well conserved peptide motif is found around the amino acid residues of the catalytic triad [I e , histidine (WLLTAA/7C), se ⁇ ne(GD
  • the KLK-L2 gene is primarily expressed in the brain, mammary gland, and testis but lower levels of expression are found in many other tissues
  • the PCR products were cloned and sequenced Hormonal regulation of the KLK-L2 gene
  • a steroid hormone receptor positive breast cancer cell line (BT-474) was used as a model to verify whether the KLK-L2 gene is under steroid hormone regulation PSA was used as a control known to be upregulated by androgens and progestins and pS2 as an estrogen upregulated control
  • PSA steroid hormone receptor positive breast cancer cell line
  • KLK-L2 for kallikrem like gene 2
  • the KLK-L2 gene is composed of 5 coding exons and 4 intervening introns and, except for the second coding exon, the exon lengths are comparable to those of other members of the kalhkrein gene family ( Figure 6)
  • the exon-intron splice junctions were identified by comparing the genomic sequence with the EST sequence and were further confirmed by the conservation of the consensus splice sequence (-mGT AGm-) (32), and the fully conserved intron phases, as shown in Figure 6. Furthermore, the position of the catalytic triad residues in relation to the different exons is also conserved (Figure 6).
  • KLK-L2 is more functionally related to trypsin than to chymotrypsin (34).
  • the wide range of tissue expression of KLK-L2 should not be surprising since, by using the more sensitive RT-PCR technique instead of Northern blot analysis, many kalhkre genes were found to be expressed in a wide variety of tissues including salivary gland, kidney, pancreas, brain, and tissues of the reproductive system (uterus, mammary gland, ovary, and testis) (34). KLK-L2 is highly expressed in the brain.
  • each kalhkrein enzyme has one specific physiological substrate
  • Serine proteases encode protein cleaving enzymes that are involved in digestion, tissue remodeling, blood clotting etc., and many of the kalhkrein genes are synthesized as precursor proteins that must be activated by cleavage of the propeptide.
  • KLK-L2 The predicted trypsin-hke cleavage specificity of KLK-L2 makes it a candidate activator of other kailikreins or it may be involved in a "cascade" of enzymatic reactions similar to those found in fib ⁇ nolysis and blood clotting (36)
  • KLK-L2 a new member of the human kalhkrein gene family, KLK-L2 was characterized This gene is hormonally regulated and it is mostly expressed in the brain, mammary gland and testis. KLK- L2 may be useful as a tumor marker Example 4
  • RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as desu ibed below and used tor PCR amplification A pinner set (L3 FI and L3-R1) was used to identity the presence of the gene in tissues, and the reverse primer (L -R1) was used with another primer (L3-F2) to amplify and clone the full cDNA of the gene
  • L3 FI and L3-R1 was used to identity the presence of the gene in tissues
  • L3-R1 was used with another primer (L3-F2) to amplify and clone the full cDNA of the gene
  • RNA 2 ⁇ g was reverse-transcribed into first strand cDNA using the SuperscriptTM preamplification system (Gibco BRL, Gaithersburg, MD) The final volume was 20 ⁇ l
  • two gene-specific primers (L3-F1 and L3-R1) were designed (Table 13, SEQ ID Nos 9-12, 24- 26) and PCR was carried out in a reaction mixture containing 1 ⁇ l of cDNA, 10 mM T ⁇ s-HCl (pH 8 3), 50 mM KCl, 1 5 mM MgCl 2 , 200 ⁇ M dNTPs (deoxynucleoside triphosphates), 150 ng of primers and 2 5 units of AmphTaq Gold DNA polymerase (Roche Molecular Systems, Branchburg, NJ, USA) on a Perkin- Elmer 9600 thermal cycler The cycling conditions were 94°C for 9 minutes, followed by 43 cycles of 94°C for
  • the breast cancer cell line BT-474 was purchased from the American Type Culture Collection (ATCC), Rockville, MD Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency The cells were then a quoted into 24-well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 10 8 M Cells stimulated with 100% ethanol were included as controls
  • the cells were cultured for 24 hours, then harvested for total RNA extraction by the T ⁇ zol method (Gibco- BRL) cDNA was prepared and amplified as described above Control genes (PSA, pS2, and actin) were amplified as previously described herein Cloning and sequencing of the PCR products. To verify the identity of the PCR products, they were cloned into the pCR 2 1-TOPO vector
  • the PCR product generated with primer set Z1S and Zl AS (Table 14, SEQ ID NOS 27-42), was purified and then labeled with 32 P by the random primer method (Sambrook, supra) and used as a probe to screen a human genomic DNA BAC library, spotted in duplicate on nylon membranes, for identification of positive clones
  • the filters were hybridized in 15% formamide, 500 mM Na 2 HP0 4 , 7% SDS, 1% BSA
  • KLKl human glandular kallikrem
  • KLK2 human glandular kallikrem
  • NESl normal epithelial cell-specific lgene
  • KLK-Ll KLK- L2 and zyme genes
  • gene-specific primers were designed for each of these genes (Table 14) and developed polymerase chain reaction (PCR)-based amplification protocols which allowed us to generate specific PCR products with genomic DNA as a template
  • PCR reactions were carried out as described above but by using an annealing/extension temperature of 65°C. Structure analysis studies.
  • the KLK-L3 gene is formed of 5 coding exons and 4 intervening introns, although, as with other kailikreins, the presence of further upstream untranslated exon(s) could not be ruled out (14, 28) All of the exon /intron splice sites conform to the consensus sequence for eukaryotic splice sites (32) The gene further follows strictly the common structural features of the human kalhkrein multigene family, as described below
  • the predicted protein-coding region of the gene is formed of 753 bp, encoding a deduced ammo acid polypeptide with a predicted molecular weight of 27 5 kDa
  • a potential translation initiation codon is found at position 28 of the predicted first exon (numbers of nucleotides refer to SEQ ID NO 21 or GenBAnk Accession # API 35026
  • This codon does not match well with the consensus Kozak sequence (33), however, it has a pu ⁇ ne at position (-3) which occurs in 97% of vertebrate mRNAs (43), and it is almost identical to the sequence of the zyme gene flanking the start codon
  • Nucleotides 6803-6808 (AGTAAA) closely resemble a consensus polyadenylation signal (44) and are followed by a stretch of 19 poly A nucleotides not found in genomic DNA, after a space of 14 nucleotides
  • the KLK-L3 gene is primarily expressed in thymus, testis, spinal cord, cerebellum, trachea, mammary gland, prostate, brain, salivary gland, ovary and skin (the latter two tissues are not shown in the figure) Lower levels of expression are seen m fetal brain, stomach, lung, thyroid, placenta, liver, small intestine, and bone marrow No expression was seen in uterus, heart, fetal liver, adrenal gland, colon, spleen, skeletal muscle, pancreas, and kidney In order to verify the RT-PCR specificity, representative PCR products were cloned and sequenced Figure 20 shows that KLK-L3 gene is regulated by steroid hormones in the human breast cancer cell line BT-474 DISCUSSION
  • kailikreins are characterized by their ability to liberate lysyl-bradykinin (kal din) tiom kininogen (2)
  • KLK2 and KLK3 are assigned to the same family based on the strong structural similarities of the genes and proteins and the close localization of these genes on the same chromosomal region (20)
  • Richards and co-workers introduced the concept of a "kalhkrein multigene family" in mice, to refer to these genes (20, 21)
  • This definition is not based much on the specific enzymatic function of the gene product, but more on its sequence homology and its close linkage on mouse chromosome 7 Irwin et al (27) proposed that the serine protease genes can be classified into five different groups according to intron position as discussed above The results indicate the presence of some more common structural features that are found in all kailikreins (including the
  • the chromosomal band 19ql3 is nonrandomly rearranged in a variety of human solid tumors including ovarian cancers (46), and the currently available data indicate that the kalhkrein gene locus is related to many malignancies At least three kalhkrein genes (PSA, zyme and NESl) are down regulated in breast cancer (10, 13, 47, 48), and NESl appears to be a novel tumor suppressor gene (29) Furthermore, PSA exhibits potent antiangiogenic activity (49) It is possible that some of these kailikreins are involved in a cascade pathway, similar to the coagulation or apoptotic process, whereby pro-forms of proteolytic enzymes are activated and then act upon downstream substrates Such activity was found for the KLK2 gene product which acts upon and activates pro PSA (50, 51)
  • the expanded human kalhkrein gene family has similar number of members as the rodent family of genes Some new compelling data have raised the possibility that at least some of these genes behave as tumor suppressors (29), as negative regulators of cell growth (52), as antiangiogenic (49) and apoptotic (53) molecules
  • the paramount diagnostic value of some members is also well-established (24, 54) For these reasons, it is important to check all members of this family of genes as potential diagnostic or prognostic markers or as candidate therapeutic targets
  • the newly identified KLK-L3 gene is expressed in many tissues, including skin, thymus, central nervous system, breast, prostate, and testis
  • the wide range of tissue expression of KLK-L3 should not be surprising since, by using the more sensitive RT-PCR technique, many kalhkrein genes were found to be expressed in a wide variety of tissues For example, PSA, KLK2, prostase/KLK-Ll, and KLK-L2 are now known to be expiessed in breast and many other tissues (30, 54)
  • KLK-L3 is regulated by steroid hormones but in a more complex fashion than PSA and KLK2 which are up-regulated by androgens and progestins (71).
  • PSA serotonins
  • KLK-L3 appears to be up-regulated by progestins > estrogens > androgens (Figure 20)
  • LLNL Lawrence Livermore National Laboratory
  • RNA isolated from 26 different human tissues was purchased from Clontech.
  • cDNA was prepared as described below, and used for PCR reactions with different sets of primers (Table 15, SEQ.IDNOs 46-55, 9-12) Tissue cD ⁇ As were amplified at various dilutions Breast cancer cell line and hormonal stimulation experiments
  • the breast cancer cell line BT-474 was purchased from the American Type Culture Collection (ATCC), Rockville, MD. Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency. The cells were then a quoted into 24- well tissue culture plates and cultured to 50% confluency.
  • the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum
  • various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 10 "8 M Cells stimulated with 100% ethanol were included as controls
  • the cells were cultured tot 24 houis, then harvested for mRNA extraction Reverse transcriptase polymerase chain reaction
  • PCR was carried out in a reaction mixture containing 1 ⁇ l of cD ⁇ A, 10 mM T ⁇ s-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 200 ⁇ M d ⁇ TPs (deoxynucleoside triphosphates), 150 ng of primers and 2.5 units of AmpliTaq Gold D ⁇ A polymerase (Roche Molecular Systems, Branchburg, ⁇ J, USA) on a Perkin-Elmer 9600 thermal cycler.
  • the cycling conditions were 94°C for 9 minutes to activate the Taq Gold D ⁇ A polymerase, followed by 43 cycles of 94°C for 30 s, 63°C for 1 minute and a final extension at 63°C for 10 min
  • Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. All primers for RT-PCR spanned at least 2 exons to avoid contamination by genomic D ⁇ A.
  • Normal breast tissues were obtained from women undergoing reduction mammoplasties.
  • Breast tumor tissues were obtained from female patients at participating hospitals of the Ontario Provincial Steroid Hormone Receptor Program The normal and tumor tissues were immediately frozen in liquid nitrogen after surgical resection and stored in this manner until extracted The tissues were pulverized with a hammer at dry ice temperature and RNA was extracted as described above, using T ⁇ zol reagent.
  • KLK-L4 is formed of five coding exons and four intervening introns, spanning an area of 8,905 bp of genomic sequence on chromosome 19ql3 3-ql3 4
  • the lengths of the coding regions are 52, 187, 269,137 and 189 bp, respectively
  • the predicted protein coding region of the gene is formed of 831 bp, encoding a deduced
  • Kb area of interest [the human kalhkrein gene family locus], enabled precise localization of all known genes and to determine the direction of transcription, as shown by the arrows in Figure 28
  • the PSA gene lies between KLKl and KLK2 genes and is separated by 13, 319 base pairs (bp) from KLK2, and both genes are transcribed in the same direction (centromere to telomere) All other kallikrein-hke genes are transcribed in the opposite direction
  • KLK-L4 is 13 kb centromeric from KLK-L6 [SEQ ID NO 65], and
  • the KLK-L4 gene is primarily expressed in mammary gland, prostate, salivary gland and testis, but, as is the case with other kailikreins, lower levels of expression are found in many other tissues
  • the PCR products were cloned and sequenced
  • a steroid hormone receptor-positive breast cancer cell line (BT-474) was used as a model, to verify whether the KLK-L4 gene is under steroid hormone regulation PSA was used as a control gene, known to be up-regulated by androgens and progestins and pS2 as an estrogen up-regulated control gene in the same cell line
  • Preliminary results indicate that KLK-L4 is up-regulated by progestins and androgens and to a lower extent by estrogens (Figure 30) Expression of KLK-L4 in breast cancer tissues and cell lines
  • RNA splicing provides a mechanism whereby protein isoform diversity can be generated and the expression of particular proteins with specialized functions can be restricted to certain cell or tissue types during development (60)
  • the sequence elements in the pre-mRNA at the 5' and 3' splice sites in metazoans have very loose consensus sequence, only the first and the last two bases (GT AG) of the introns are highly conserved (Sambrook, supra)
  • GT AG first and the last two bases
  • the predicted exons of the putative new gene were subjected to homology search using the BLASTN algorithm (37) on the National Center for Biotechnology Information web server (http://www ncbi.nlm.nih.gov/BLAST/) against the human EST database (dbEST).
  • a clone with > 95% homology was obtained from the I.M.A G.E consortium (38) through Research Genetics Inc, Huntsville, AL
  • This clone was propagated, purified and sequenced from both directions with an automated sequencer, using msert- flanking vector primers Rapid amplification of cDNA ends (RACE) According to the EST sequence and the predicted structure of the gene, two sets of gene-specific primers were designed for 5' and 3' RACE reactions.
  • RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as described below for the tissue culture experiments and used for PCR reactions After aligning all known kalhkrein genes, two primers (KLK-L5-R1 and KLK-L5-F1) (Table 17, SEQ ID NOs 61-64, 9-12, and Figure 32) were designed from areas with relatively low homology Tissue cDNAs were amplified at various dilutions Due to the high degree of homology between kailikreins, and to exclude non-specific amplification, PCR products were cloned and sequenced Normal and malignant breast tissues
  • the breast cancer cell lines BT-474 and T-47D, and the LNCaP prostate cancer cell line were purchased from the American Type Culture Collection (ATCC), Rockville, MD Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovme insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency The cells were then ahquoted into 24- well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal- stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media at a final concentration of 10 8 M Cells stimulated with 100% ethanol were included as controls The cells were cultured for 24 hours, then harvested for mRNA extraction
  • RNA was extracted from the cell lines or tissues using T ⁇ zol reagent (Gibco BRL) following the manufacturer's instructions RNA concentration was determined spectrophotomet ⁇ cally 2 ⁇ g of total RNA was reverse-transcribed into first strand cDNA using the SuperscriptTM preamplification system (Gibco BRL) The final volume was 20 ⁇ l Based on the combined information obtained from the predicted genomic structure of the new gene and the EST sequences, two gene-specific primers were designed (KLK-L5-F1 and KLK-L5-R1) (Table 17) and PCR was carried out in a reaction mixture containing 1 ⁇ l of cDNA, 10 M T ⁇ s-HCl (pH 8 3), 50 mM KCl, 1 5 mM MgCl 2 , 200 ⁇ M dNTPs (deoxynucleoside triphosphates), 150 ng of primers and 2 5 units of AmpliTaq Gold DNA poly
  • Signal peptide was predicted using the "SignalP” server (http://www.cbs.dtu.dk/services/ signal) Protein structure analysis was performed by "SAPS” (structural analysis of protein sequence) program (http://dot. ⁇ mgen.bcm.tmc.edu:9331/seq- search struc-predict.html) RESULTS
  • the stop codon is located in the sixth exon, followed by a 3' untranslated region, and a typical polyadenylation signal (AATAAA) is located 16 bp before the poly-A tail ( Figure 33).
  • AATAAA polyadenylation signal
  • This form of KLK-L5 spans a genomic length of 5,801 bp on chromosome 19ql3.3-q l3.4
  • the lengths of the coding regions of the exons are 37, 160, 260, 134, and 156 bp, respectively ( Figures 33 and 34).
  • the predicted protein-coding region is formed of 747 bp, encoding a deduced 248-am ⁇ no acid protein with a predicted molecular mass of 26 7 kDa.
  • the intron/exon splice sites (GT....AG) and their flanking sequences are in agreement with the consensus splice site sequence.
  • the second mRNA form, encoding the KLK-L5-related protein- 1 is an alternatively spliced form in which the last exon is split into two separate exons with an additional intervening intron ( Figure
  • the third mRNA form, encoding for KLK-L5-related prote ⁇ n-2, is similar to the classical form except that the fourth exon is missing ( Figure 32) This leads to frame shifting of the coding region, and an earlier m-frame stop codon will be encountered at position 9,180
  • the protein-coding region of this form consists of 336 bp, encoding toi a pasicted 1 1 1-am ⁇ no acid piotein with a molecular mass of 12 kDa This protein will lack both the serine and aspartate residues characteristic of serine proteases
  • KLK-L5 will possess a trypsin-hke cleavage pattern, similarly to most of the other kailikreins (e g , hKl, hK2, TLSP, neuropsin, zyme, prostase, and EMSP) but different from PSA, which has a serine (S) residue in the corresponding position, and is known to have chymotrypsin like activity (Figure 36) (54) Homology with other members of the kalhkrein multigene family
  • KLK-L5 protein encoded by the KLK-L5 gene is unique, it has a high degree of homology with the other kalhkrein-like genes
  • the KLK-L5 protein (classical form) has 48% amino acid sequence identity and 57% overall similarity with neuropsin, 46% identity with the normal epithelial cell-specific 1 gene product (NESl) and 38% identity with both PSA and hK2 proteins
  • NESl normal epithelial cell-specific 1 gene product
  • a well-conserved peptide motif is found around the amino acid residues of the catalytic triad as is the case with other serine proteases [1 e , histidine (VLTAA /C), serine
  • Figure 36 shows other amino acid residues that are completely conserved between kailikreins and kalhkrein-like proteins
  • Figure 37 shows the phylogenetic analysis which separated the classical kailikreins (hKl, hK2, and PSA) and clustered KLK-L5 with NES 1 and neuropsin proteins in a separate group away from other serine proteases, consistent with previously published studies (27, 41) and indicating that this group of genes probably arose from a common ancestral gene, by gene duplication
  • the KLK-L5 gene is primarily expressed in the salivary gland, stomach, uterus, trachea, prostate, thymus, lung, colon, brain, breast and thyroid gland, but, as is the case with other kailikreins, lower levels of expression are found in some other tissues (testis, pancreas, small intestine, spinal cord).
  • the PCR products were cloned and sequenced The three splice forms of the gene were expressed in most of these tissues However, the relative abundance of each form was different among tissues ( Figure 38).
  • KLK-L5 is under steroid hormone regulation
  • two breast cancer cell lines (BT-474 and T-47D) and a prostate cancer cell line (LNCaP) were used as models.
  • LNCaP cells the gene was up-regulated only by androgen and progestin. Only in this cell line all 3 isoforms were expressed.
  • KLK-L5 was found to be up-regulated, at the mRNA level, by estrogen and androgen, and to a lesser extent by the progestin
  • the rank of potency was estrogen>androgen>progest ⁇ n
  • the rank of potency for the T-47D cell line was androgen>progest ⁇ n>estrogen .
  • the short isoform (related prote ⁇ n-2) was present (Figure 39).
  • KLK-L5 is down regulated in breast cancer
  • KLK-L5 Mapping and chromosomal localization of the KLK-L5 gene
  • PSA and KLK2 transcribe from centromere to telomere; the rest of the genes are transcribed in the reverse direction.
  • the KLKl gene was found to be the most centromeric, and the KLK-L6 gene the most telome ⁇ c (KLK-L6; SEQ.ID. N0.65).
  • KLK-L5 is 21.3 Kb centromeric to KLK- L4 (SEQ.ID.NO. 43) and 1.6 kb more telomeric to the trypsin-hke serine protease gene (TLSP) ( Figure 28)
  • kailikreins are characterized by the following common structural features' (a) All genes are formed of 5 coding exons and 4 intervening introns [some genes may have extra 5( untranslated exon(s)] (14, 35) (b) The exon lengths are usually comparable (c) The intron phases are always conserved (I-II-I-0) (see legend of Figure 34 for definition of intron phases), (d) These genes are clustered in the same chromosomal region, without any intervening non-kal krein-hke genes ( Figure 28) (e) The histidine residue of the catalytic triad of serine proteases is located near the end of the second coding exon; the aspartate residue in the middle of the third coding exon; and serine at the beginning of the fitth coding exon As shown in Figure 34, all these criteria apply to the newly identified KLK-L5 gene Thus, KLK-L5 should be considered a new member of the kalhkre
  • Preproenzymes that contain an N-terminal signal peptide (pre-zymogen), followed by a short activation peptide and the enzymatic domain (41, 54)
  • PreproPSA has 24 additional residues that constitute the pre-region (signal peptide, 17 residues), and the propeptide (7 residues) (67)
  • the signal peptide directs the protein to and across the endoplasmic reticulum (ER) In the ER, the signal peptide is removed and the resulting proPSA is transported to the plasma membrane, where it is secreted
  • the hydrophobicity study ( Figure 35) indicates that the amino terminal region of the KLK-L5 protein is harboring a signal peptide
  • computer analysis of the amino acid sequence of KLK-L5 predicted a cleavage site between ammo acids 17 and 18 (SQA-AT)
  • SQA-AT computer analysis of the amino acid sequence of KLK-L5 predicted a cleavage site between ammo acids 17 and 18
  • KLK-L5 a new member of the human kalhkrein gene family, KLK-L5, has been characterized which maps to the human kalhkrein locus (chromosome 19ql3 3-ql3 4)
  • This gene has two related splice forms in addition to the main form KLK-L5 is expressed in a variety of tissues, appears to be down-regulated in breast cancer and its expression is influenced by steroid hormones Since a few other kailikreins are already used as valuable tumour markers, KLK-L5 may also find a similar clinical application
  • Example 7 Using the Materials and Methods substantially as set out in Example 6, the present inventors identified another novel gene ot the kalhkrein multigen family referred to as KLK-L6
  • KLK-L6 The full structure ot the KLK-L6 gene is shown in Figure 41 Exons 1, 2, 3, 4, 5, 6, and 7 are at nucleic acids 1172-1281, 2561-2695, 2781-2842, 3714-3885, 5715-5968, 6466-6602, and 7258-7520
  • the nucleic acid sequence ot the KLK-L6 gene is also shown in SEQ ID NO 65 and amino acid sequences for the KLK-L6 protein are shown in SEQ ID Nos 66 and 67. (See also GenBank Accession # AF161221)
  • Figure 42 shows a comparative hydrophobicity analysis of KLK-L6 and the prostate-specific antigen (PSA)
  • PSA prostate-specific antigen
  • Figure 44 shows the phylogenetic analysis which separated the classical kailikreins (hKl, hK2, and PSA) and placed KLK-L6 in a separate group
  • (+) >95% homology with published human EST sequences.

Abstract

The invention relates to nucleic acid molecules, kallikrein-like proteins encoded by such nucleic acid molecules; and use of the proteins and nucleic acid molecules

Description

TITLE: Novel Human Kallikrein-Like Genes FIELD OF THE INVENTION
The invention relates to nucleic acid molecules, proteins encoded by such nucleic acid molecules, and use of the proteins and nucleic acid molecules BACKGROUND OF THE INVENTION
Kailikreins and kallikrein- ke proteins are a subgroup of the serine protease enzyme family and exhibit a high degree of substrate specificity (1) The biological role of these kailikreins is the selective cleavage of specific polypeptide precursors (substrates) to release peptides with potent biological activity (2) In mouse and rat, kailikreins are encoded by large multigene families In the mouse genome, at least 24 genes have been identified (3) Expression of 11 of these genes has been confirmed, the rest are presumed to be pseudogenes (4) A similar family of 15-20 kailikreins has been found in the rat genome (5) where at least 4 of these are known to be expressed (6)
Three human kalhkrein genes have been described, I e prostatic specific antigen (PSA or KLK3) (7), human glandular kallikrem (KLK2) (8) and tissue (pancreatic-renal) kalhkrem (KLKl) (9) The PSA gene spans 5 8 Kb of sequence which has been published (7), the KLK2 gene has a size of 5 2 Kb and its complete structure has also been elucidated (8) The KLKl gene is approximately 45 Kb long and the exon sequences and the exon/mtron junctions of this gene have been determined (9)
The mouse kal krem genes are clustered in groups of up to 11 genes on chromosome 7 and the distance between the genes in the various clusters can be as small as 3-7 Kb (3) All three human kalhkrein genes have been assigned to chromosome 19q 13 2 - 19q 13 4 and the distance between PSA and KLK2 has been estimated to be 12 Kb (9)
A major difference between mouse and human kailikreins is that two of the human kailikreins (KLK2 and KLK3) are expressed almost exclusively in the prostate while in animals none of the kailikreins is localized in this organ Other candidate new members of the human kallikrem gene family include protease M (10) (also named Zyme (11) or neurosm (12) and the normal epithelial cell-specific gene-1 (NES1) (13) Both genes have been assigned to chromosome 19ql3 3 (10,14) and show structural homology with other serine proteases and the kalhkrein gene family (10-14) SUMMARY OF THE INVENTION In efforts to precisely define the relative genomic location of PSA, KLK2, Zyme and NES1 genes, an area spanning approximately 300 Kb of contiguous sequence on human chromosome 19 (19ql3 3 — ql3 4) was examined The present inventors were able to identify the relative location of the known kal krein genes and, in addition, they identified other kal krein- like genes which exhibit both location proximity and structural similarity with the known members of the human kalhkrein family The novel genes exhibit homology with the currently known members of the kallikrem family and they are co- localized in the same genomic region These new genes, like the already known kailikreins have utility in various cancers including those of the breast, testicular, and prostate
The kallikrein-hke proteins descπbed herein are individually referred to as "KLK-L1, KLK-L2, _ 9 _
KLK-L3, KLK-L4, KLK-L5, or KLK-L6", and collectively as "kallikrein-hke proteins" oi "KLK-L Proteins" The genes encoding the proteins are referred to as " Uk-ll klk-12 lclk-13 klk-14 klk-1 oi klk 16 ", and collectively as "kallikrein-hke genes" or "klk-I genes"
Broadly stated the present invention relates to an isolated nucleic acid molecule which comprises (l) a nucleic acid sequence encoding a protein having substantial sequence identity with an amino acid sequence of KLK-L 1, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 as shown m SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively, (u) a nucleic acid sequence encoding a protein comprising an ammo acid sequence of KLK-
Ll, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively,
(in) nucleic acid sequences complementary to (l), (iv) a degenerate form of a nucleic acid sequence of (I),
(v) a nucleic acid sequence capable of hybridizing under stringent conditions to a nucleic acid sequence in (I), (n) or (in), (vi) a nucleic acid sequence encoding a truncation, an analog, an allelic or species variation of a protein comprising an amino acid sequence of KLK-L1, KLK-L2, KLK-L3, KLK- L4, KLK-L5, or KLK-L6 as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively, or (vn) a fragment, or allelic or species variation of (l), (n) or (in) Preferably, a purified and isolated nucleic acid molecule of the invention comprises
(0 a nucleic acid sequence comprising the sequence of SEQ ID NO 1, 13, 21, 43, 56, or 65 wherein T can also be U, (ii) nucleic acid sequences complementary to (l), preferably complementary to the full nucleic acid sequence of SEQ ID NO 1, 13, 21, 43, 56, or 65, (in) a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid of (I) or (n) and preferably having at least 18 nucleotides, or (iv) a nucleic acid molecule differing from any of the nucleic acids of (I) to (in) in codon sequences due to the degeneracy of the genetic code The invention also contemplates a nucleic acid molecule comprising a sequence encoding a truncation of a KLK-L protein, an analog, or a homolog of a KLK-L Protein or a truncation thereof (KLK-
L Proteins and truncations, analogs and homologs of KLK-L Proteins are also collectively referred to herein as "KLK-L Related Proteins")
The nucleic acid molecules of the invention may be inserted into an appropriate expression vector, l e a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence Accordingly, recombinant expression vectors adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements linked to the nucleic acid molecule
The recombinant expression vector can be used to prepare transformed host cells expressing KLK- L Related Proteins Therefore, the invention further provides host cells containing a recombinant molecule of the invention The invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a recombinant molecule comprising a nucleic acid molecule of the invention, in particular one which encodes an analog of a KLK-L Protein, or a truncation of a KLK-L Protein The invention further provides a method for preparing KLK-L Related Proteins utilizing the purified and isolated nucleic acid molecules of the invention In an embodiment a method for preparing a KLK-L Related Protein is provided comprising (a) transferring a recombinant expression vector of the invention into a host cell, (b) selecting transformed host cells from untransformed host cells, (c) culturing a selected transformed host cell under conditions which allow expression of the KLK-L Related Protein, and (d) isolating the KLK-L Related Protein
The invention further broadly contemplates an isolated KLK-L Protein comprising an amino acid sequence as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
The KLK-L Related Proteins of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins This may be accomplished, for example, by the synthesis of N-terrrunal or C-terminal fusion proteins
The invention further contemplates antibodies having specificity against an epitope of a KLK-L Related Protein of the invention Antibodies may be labeled with a detectable substance and used to detect proteins of the invention in tissues and cells
The invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the invention and/or to proteins of the invention Therefore, the invention also relates to a probe comprising a nucleic acid sequence of the invention, or a nucleic acid sequence encoding a protein of the invention, or a part thereof The probe may be labeled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleic acid molecule of the invention including nucleic acid molecules coding for a protein which displays one or more of the properties of a protein of the invention
The invention still further provides a method for identifying a substance which binds to a protein of the invention comprising reacting the protein with at least one substance which potentially can bind with the protein, under conditions which permit the formation of complexes between the substance and protein and detecting binding Binding may be detected by assaying for complexes, for free substance, or for non- complexed protein The invention also contemplates methods for identifying substances that bind to other intracellular proteins that interact with a KLK-L Related Protein Methods can also be utilized which identify compounds which bind to KLK-L gene regulatory sequences (e g promoter sequences)
Still further the invention provides a method for evaluating a compound for its ability to modulate the biological activity of a KLK-L Related Protein of the invention For example a substance which inhibits or enhances the interaction of the protein and a substance which binds to the protein may be evaluated In an embodiment, the method comprises providing a known concentration of a KLK-L Related Protein, with a substance which binds to the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and removing and/or detecting complexes Compounds which modulate the biological activity ot a protein ot the invention may also be identified using the methods of the invention by comparing the pattern and level of expression of the protein of the invention in tissues and cells, in the presence, and in the absence of the compounds
The proteins of the invention and substances and compounds identified using the methods of the invention, and peptides of the invention may be used to modulate the biological activity of a KLK-L
Related Protein of the invention, and they may be used in the treatment of conditions such as cancer (e g breast, testicular, and prostate cancer) Accordingly, the substances and compounds may be formulated into compositions for administration to individuals suffering from cancer
Therefore, the present invention also relates to a composition comprising one or more of a protein of the invention, a peptide of the invention, or a substance or compound identified using the methods of the invention, and a pharmaceutically acceptable carrier, excφient or diluent A method for treating or preventing cancer is also provided comprising administering to a patient m need thereof, a KLK-L Related
Protein of the invention, or a composition of the invention
Other objects, features and advantages of the present invention will become apparent from the following detailed description It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in relation to the drawings in which
Figure 1 shows an approximate 300 Kb of contiguous genomic sequence around chromosome 19ql3 3 - ql3 4 represented by 8 contigs, each one shown with its length in Kb The contig numbers refer to those reported in the Lawrence Livermore National Laboratory website Note the localization of the seven known genes (PSA, KLK2, Zyme, NES1, HSCCE, neuropsin and TLSP) (see abbreviations for full names of these genes) All genes are represented with arrows denoting the direction of transcription The gene with no homology to human kailikreins is termed UG (unknown gene) The five new kallikrein-hke genes (KLK-L 1 to KLK-L5) were numbered from the most centromeπc to the most telomeπc Numbers just below or just above the arrows indicate appropriate Kb lengths in each contig Gene lengths and distances between genes are rounded to the nearest 6 5 kb The site of the gap is marked with an asterisk Figure 2 shows a contiguous genomic sequence around chromosome 19ql3 3- ql3 4 Genes are represented by horizontal arrows denoting the direction of the coding sequence Distances between genes are in base pairs
Figure 3 shows tissue expression of the prostase/KLK-Ll gene as determined by RT-PCR Actin and PSA are control genes Interpretations are presented in Table 9 Figure 4 shows the sequence of PCR product obtained with cDNA from female breast tissue using prostase/KLK-Ll primers Primer sequences are underlined The sequence is identical to the sequence obtained from prostatic tissue
Figure 5 is a blot showing the results of experiments for hormonal regulation of the prostase/KLK- LI gene in the BT-474 breast carcinoma cell lines DHT = dihydrotestosterone Steroids were added at 10 M final concentrations Actin (not regulated by steroid hormones), pS2 (up-regulated by estrogens) and PSA (up-regulated by androgens and progestins), are control genes Prostase/KLK-Ll is up-regulated by androgens and progestins Figure 6 is a schematic diagram showing comparison of the genomic structure of PSA, KLKl,
KLK2, zyme, neuropsin and prostase/KLK-Ll genes Exons are shown by open boxes and introns by the connecting lines Arrow head shows the start codons and the vertical arrow represents stop codons Letters above boxes indicate relative positions of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases The intron phase refers to the location of the intron within the codon, I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons Numbers inside boxes indicate exon lengths in base pairs
Figure 7 shows the genomic organization and partial genomic sequence of the KLK-L2 gene Intronic sequences are not shown except for the splice junctions. Introns are shown with lower case letters and exons with capital letters. The start and stop codons are encircled and the exon -intron junctions are boxed The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are inside triangles Putative polyadenylation signal is underlined
Figure 8 shows an approximate 300 Kb region of almost contiguous genomic sequence around chromosome 19ql3.3- ql3.4. Genes are represented by horizontal arrows denoting the direction of the coding sequence Distances between genes are mentioned in base pairs
Figure 9 shows the alignment of the deduced amino acid sequence of KLK-L2 with members of the kal krein multi-gene family. Genes are (from top to bottom) • Prostase/KLK-Ll, enamel matrix serine proteinase 1 (EMSPl) (GenBank accession # NP_004908), KLK-L2, zyme (GenBank accession # Q92876), neuropsin (GenBank accession # BAA28673), trypsin-hke serine protease (TLSP) (GenBank accession # B AA33404), PSA (GenBank accession # P07288), KLK2 (GenBank accession # P20151),
KLKl (GenBank accession # NP_002248), and trypsinogen ( GenBank accession # P07477) (See SEQ.ID NOs 68-77) Dashes represent gaps to bring the sequences to better alignment The residues of the catalytic triad are represented by (•---•) and the 29 invariant serine protease residues by (I or •---•) Conserved areas around the catalytic triad are boxed The predicted cleavage sites are indicated by ( ) The dotted area represents the kalhkrein loop sequence The trypsin like cleavage pattern is indicated by (©)
Figure 10(A) shows a dendrogram of the predicted phylogenetic tree for some kal krein genes
Neighbor-joining/UPGMA method was used to align KLK-L2 with other members of the kalhkrein gene family Gene names and accession numbers are listed in Figure 9 The tree grouped the classical kailikreins
(KLKl, KLK2, and PSA) together and aligned the KLK-L2 gene in one group with EMSP, prostase, and TLSP (B) Plot of hydrophobicity and hydrophilicity of KLK-L2
Figure 11 is a blot showing tissue expression of KLK-L2 gene as determined by RT-PCR Actin and PSA are control genes Interpretations are presented in Table 12
Figure 12 is a blot showing hormonal regulation of the KLK-L2 gene in BT-474 breast carcinoma cell lines DHT = dihydrotestosterone Steioids were at 108 M final concentrations Actin (not regulated by steioid hormones), pS2 (up-regulated by estrogens) and PSA (upregulated by androgens and progestins), are control genes KLK-L2 is upregulated by estrogens and progestins
Figure 13 are blots of EtBr-stained agarose gels Total RNA was extracted from normal, benign, and cancer tissues and used to generate cDNA PCR was performed on cDNA
Figure 14 shows an approximate 300 Kb region of almost contiguous genomic sequence around chromosome 19q I3 3- ql3 4 Genes are represented by horizontal arrows denoting the direction of the coding sequence Gene lengths and distances between genes are rounded to the nearest 05 kb The site of the gap is marked with an asterisk Telomeπc to TLSP there are likely another three kallikrein-hke genes Figure 15 shows the genomic organization and partial genomic sequence of the KLK-L3 gene
Intronic sequences are not shown except for the splice junctions Introns are shown with lower case letters and exons with capital letters For the full sequence, see SEQ ID NO 21 The start and stop codons are encircled and the exon -intron junctions are boxed The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are inside triangles Putative polyadenylation signal is underlined
Figure 16 is a plot of hydrophobicity and hydrophilicity, comparing the pattern of the KLK-L3 with that of the zyme gene Note the hydrophobic region around the first twenty amino acids, likely representing the signal peptide
Figure 17 is an alignment of the deduced amino acid sequence of KLK-L3 with members of the kalhkrein multi-gene family Genes are (from top to bottom and in brackets is the GenBank accession #) PSA (P07288), KLK2 (P20151), KLKl (NP002248), trypsinogen (P07477), KLK-L3 (AF135026), trypsin- like serine protease (TLSP) (BAA33404), neuropsin (BAA28673), zyme (Q92876), human stratum corneum chymotryptic enzyme (HSCCE) (AAD49718), and prostase/KLK-Ll (AAD21581) (See SEQ ID NOs 78 to 84) Dashes represent gaps to bring the sequences to better alignment The residues of the catalytic triad are bold and in italics, and the 29 invariant serine protease residues are denoted by ( ♦ )
Cysteine residues are marked by (•) Conserved areas around the catalytic triad are highlited in black The arrow heads ( "*■ ) represent the potential cleavage sites The dotted area represents the kalhkrein loop sequence
Figure 18 is a dendrogram of the predicted phylogenetic tree for some serine proteases and kal krein genes Neighbor-joimng/UPGMA method was used to align KLK-L3 with other members of the kalhkrein gene family Gene names and accession numbers are listed in Figure 17 The tree grouped the classical kailikreins (KLKl , KLK2, and PSA) together and aligned the KLK-L3 gene in one group with TLSP, neuropsin, and NES 1 genes KLK-L4 (SEQ ID NO 43) lies further telomeπc to TLSP (21)
Figure 19 is a blot showing tissue expression of the KLK-L3 gene as determined by RT-PCR Actin and PSA are control genes
Figure 20 shows hormonal regulation of the KLK-L3 gene in the BT-474 breast carcinoma cell line DHT = dihydrotestosterone Steroids were at 10 8 M final concentrations Actin (not regulated by steroid hormones), pS2 (up-regulated by estrogens) and PSA (upregulated by androgens and progestins), aie control genes KLK L3 is upregulated by progestins, estrogens and androgens, in that order
Figure 21 is a schematic diagram showing the comparison of the genomic structure of PSA, KLK2, neuropsin, NES1, and KLK-L3 genes Exons are shown by black boxes and introns by the connecting lines Arrowheads show the start codon, and arrows show the stop codon Letters above boxes indicate relative positions of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases The intron phase refers to the location of the intron within the codon, I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons Numbers inside boxes indicate exon lengths base pairs Figure 22 shows a comparative genomic structure of the ESTs (Table 16), the clone from The German Genome Project, and the long form of KLK-L4 Exons are represented by solid bars and introns by the connecting lines Exon numbers on top of solid bars refer to GenBank submission #AF135024 The EST IDs represent GenBank accession numbers Asterisks represent the positions of stop codons Horizontal arrows indicate the direction of the PCR primers (described in Table 15) and arrowheads their position along the exons Vertical dotted lines show alignment of identical fragments Figure 23 shows tissue expression of the KLK-L4 gene as determined by RT-PCR Actin and PSA are control genes KLK-L4 is highly expressed in breast, prostate, salivary gland and testis
Figure 24 in the Upper Panel is a Diagram showing the comparative genomic structure of the long KLK-L4 form and the short KLK-L4 variant Exons are represented by boxes and introns by the connecting lines Exon numbers refer to SEQ ID NO 43 and GenBank Accession No AF135024 The black region indicates the extra fragment (214 bp) that is found in the long, but not in the short form of the gene The positions of the stop codons of the two forms are marked with asterisks Frame shifting occurs as a result of utilization of an alternative splice site, and a stop codon is generated at the beginning of exon 4 in the short form The Lower Panel shows PCR products of the amplification of the KLK-L4 gene using L4-R1 and L4-X1 primers (Figure 22 and Table 15) Note the predominant long form and a minor band representing the short form of KLK-L4 mRNA (M), Markers with sizes in bp shown on the left Tissues used (1), salivary gland, (2), mammary gland, (3), prostate, (4), testis, (5), uterus, (6), breast cancer tissue, (7), negative control
Figure 25 shows the genomic organization and partial genomic sequence of the KLK-L4 gene Intronic sequences are not shown except for the splice junction areas Introns are shown with lower case letters and exons with capital letters For full sequence, see SEQ ID NO 43 or GenBank Accession
#AF135024 The start and stop codons are encircled and the exon -intron junctions are underlined The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are boxed The putative polyadenylation signal is underlined
Figure 26 is a plot of hydrophobicity and hydrophilicity of the KLK-L4 protein, as compared with the glandular kal krein gene 2 (KLK2) Note the hydrophobic region at the amino terminus, suggesting presence of a signal peptide
Figure 27 shows an alignment of the deduced am o acid sequence of KLK-L4 with members of the kalhkrein multi-gene family Genes are (from top to bottom, and in brackets are the GenBank accession #) KLK-L 1/prostdse (AAD21581). enamel matrix serine proteinase 1 (EMSP) (NP_004908), KLK-L2 (AF135028), PSA (P07288), KLK2 (P20151), KLKl (NP_002248), trypsinogen (P07477), zyme (Q92876), KLK-L4 (AF135024), trypsin-hke serine protease (TLSP) (BAA33404), KLK-L3 (AF135026),neuropsιn (BAA28673), and the normal epithelial cell-specific 1 gene (NES1) (043240) (See SEQ ID NOs 78-88) Dashes represent gaps to bring the sequences to better alignment The residues of the catalytic triad are typed in bold, and conserved motifs around them are highlighted in grey The 29 invariant serine protease residues are denoted by (•), and the cysteine residues by (♦ ) The predicted cleavage sites are indicated by (*) The dotted area represents the kalhkrein loop sequence The trypsin-hke cleavage pattern of KLK-L4 with the D residue, is indicated by (©) Figure 28 shows an approximate 300 Kb region of almost contiguous genomic sequence around chromosome 19ql3 3- ql3 4 Genes are represented by horizontal arrows denoting the direction of the coding sequence Their lengths are shown on top of each arrow Distances between genes are mentioned in base pairs below the arrows The distance between KLKl and PSA is not accurately known For gene names, see under abbreviations Figure 29 shows is a dendrogram of the predicted phylogenetic tree for some kalhkre and serine protease genes The neighbor-joining/UPGMA method was used to align KLK-L4 with other serine proteases and members of the kalhkrein gene family The tree grouped the classical kailikreins (KLKl, KLK2, and PSA) together and aligned the KLK-L4 gene in one group with zyme, NES1, neuropsin, KLK- L3, and TLSP Other serine proteases were aligned in different groups, as shown Figure 30 is a blot showing the hormonal regulation of the KLK-L4 gene in the BT-474 breast carcinoma cell line DHT = dihydrotestosterone Steroids were added at 108 M final concentrations Actin (not regulated by steroid hormones), pS2 (up-regulated by estrogens) and PSA (upregulated by androgens and progestins) are control genes KLK-L4 is up-regulated by androgens and progestins and to a lesser extent by estrogens H2O was used to check for PCR specificity in all PCR reactions For more details, see text
Figure 31 is a schematic diagram showing the comparison of the genomic structure of PSA, KLK2, neuropsin, NES 1, and KLK-L4 genes Exons are shown by black boxes and introns by the connecting lines The arrowhead shows the start codons and the arrow the stop codons Letters above boxes indicate the relative positions of the amino acids of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases The intron phase refers to the location of the intron within the codon, I, the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons Numbers inside boxes indicate exon lengths in base pairs The question mark indicates the possibility of more untranslated bases
Figure 32 is a diagram showing the comparative genomic structure of the three splice forms of KLK-L5, the classical kalhkrein form, related protein- 1, and related proteιn-2 Exons are represented by solid bars and introns by the connecting lines Exon numbers refer to SEQ ID NO 56 and GenBank
Accession #AF135025 Start codons are represented by the inverted arrowhead (T) and stop codons are represented by asterisks (*) Primer locations are represented by vertical arrowheads (A) and their directions by horizontal arrows For primer sequences and codes see Table 17 and SEQ ID NOs 61-64, and 9-12
Figure 33 shows the genomic organization and partial genomic sequence of the KLK-L5 gene
Intronic sequences are not shown except for short sequences around the splice junctions Introns are shown with lower case letters and exons with capital letters For full sequence, see SEQ ID NO 56 The start and stop codons are encircled and the exon -intron junctions are underlined The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are boxed
Putative polyadenylation signal is underlined The extra intron of the related protein- 1 form is represented by non-bold capital letters between brackets When this intron is spliced, the frame continues with codon AAC (asparagine, N, instead of lysine, K) until it encounters the stop codon TAA (encircled)
Figure 34 is a schematic diagram showing the comparison of the genomic structure of PSA, KLK2, neuropsin, NES1, KLK-L4 and KLK-L5 genes Exons are shown by solid bars and introns by the connecting lines Arrowhead marks the site of the start codon, and the arrow represents the stop codon Letters above boxes indicate relative positions of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases The intron phase refers to the location of the intron with the codon, I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons Numbers inside boxes indicate exon lengths in base pairs Question marks indicate that exact length is not accurately known
Figure 35 is a plot of hydrophobicity and hydrophilicity of KLK-L5 protein compared to prostate specific antigen (PSA) The hydrophobic N-terminus may harbor a signal and activation peptide
Figure 36 shows an alignment of the deduced ammo acid sequence of KLK-L5 with members of the kalhkrein multigene family (See SEQ ID NOs 78-81, 83, 84) Dashes represent gaps to bring the sequences to better alignment The residues of the catalytic triad are represented by bold letters, and the 29 invariant serine protease residues are marked with (•) The cysteine residues are marked by (♦ ) Conserved areas are highlighted in grey The predicted cleavage sites in signal peptide are indicated by (4)
The dotted area represents the kalhkrein loop sequence A vertical arrow marks the trypsin like cleavage site
Figure 37 is a dendrogram of the predicted phylogenetic tree for some serine proteases and other kalhkrein proteins Neighbor-joining/UPGMA method was used to align KLK-L5 with other serine proteases and members of the kalhkrein gene family The tree grouped the classical kailikreins (hKl, hK2, and PSA) together and aligned the KLK-L5 protein in one group with NES 1 and neuropsin Other serine proteases were aligned in different groups
Figure 38 shows tissue expression of the KLK-L5 gene as determined by RT-PCR The upper band (905 base pairs, bp) is the classical form (see Figure 32, the middle (776 bp) the related protein- 1 , and the lower band (644 bp) the related proteιn-2 For splice variant discussion see text The primers used were L5-F2 and L5-R2, as shown in Table 17
Figure 39 shows hormonal regulation of the KLK-L5 gene in the LnCaP prostatic carcinoma cell line, BT-474 and T-47D breast carcinoma cell lines Steroids were at 108 M final concentration Actin (not regulated by steroid hoi mones) was used as a control gene Note detection ot three isotorms only in LNCaP
Figure 40 shows the expression of the KLK-L5 gene in breast cancer ( 1-17) and normal ( 18) tissues Note complete absence of expression in 12 cancer tissues. For isotorms see also Figure 38 Figure 41 shows the full structure of a KLK-L6 nucleic acid molecule,
Figure 42 is a plot of hydrophobicity and hydrophilicity of KLK-L6 protein compared to prostate specific antigen (PSA).
Figure 43 shows an alignment of the deduced amino acid sequence of KLK-L6 with members of the kallikrem multigene family. (See SEQ. ID. NOs. 78-81, 83, 84). Dashes represent gaps to bring the sequences to better alignment
Figure 44 is a dendrogram of the predicted phylogenetic tree for some serine proteases and other kalhkrein proteins. Neighbor-joining UPGMA method was used to align KLK-L6 with other serine proteases and members of the kalhkrein gene family. DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See for example, Sambrook, Fπtsch, & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M..J. Gait ed. 1984); Nucleic Acid Hybridization B.D. Hames & S.J. Higgins eds. (1985); Transcription and Translation B.D. Hames & S.J. Higgins eds (1984); Animal Cell Culture R.I. Freshney, ed. (1986), Immobilized Cells and enzymes IRL Press, (1986); and B. Perbal, A Practical Guide to Molecular Cloning (1984) 1. Nucleic Acid Molecules of the Invention As hereinbefore mentioned, the invention provides an isolated nucleic acid molecule having a sequence encoding a KLK-L Protein. The term "isolated" refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical reactants, or other chemicals when chemically synthesized. An "isolated" nucleic acid may also be free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) from which the nucleic acid is derived The term "nucleic acid" is intended to include DNA and
RNA and can be either double stranded or single stranded In an embodiment, a nucleic acid molecule encodes a KLK-L Protein comprising an amino acid sequence as shown in SEQ.ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, preferably a nucleic acid molecule comprising a nucleic acid sequence as shown in SEQ.ID.NO. 1, 13, 21, 43, 56, or 65. The invention includes nucleic acid sequences complementary to a nucleic acid encoding a KLK-L
Protein comprising an amino acid sequence as shown in SEQ.ID.NO. 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, preferably the nucleic acid sequences complementary to a full nucleic acid sequence shown in SEQ.ID NO. 1 , 13, 21, 43, 56, or 65 The invention includes nucleic acid molecules having substantial sequence identity or homology to nucleic acid sequences of the invention or encoding proteins having substantial identity or similarity to the amino acid sequence shown in in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67 Preferably, the nucleic acids have substantial sequence identity for example at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% nucleic acid identity, more preferably 90% nucleic acid identity, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity "Identity" as known in the art and used herein, is a relationship between two or more amino acid sequences or two or more nucleic acid sequences, as determined by comparing the sequences It also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences Identity and similarity are well known terms to skilled artisans and they can be calculated by conventional methods (for example see Computational Molecular Biology, Lesk, A M ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M and Griffin, H G eds , Humana Press, New Jersey, 1994, Sequence Analysis m Molecular Biology, von He je, G Acadmeic Press, 1987, and Sequence Analysis Primer, Gnbskov, M and Devereux, J eds M Stockton Press, New York, 1991, Caπllo, H and Lipman, D , SIAM J Applied Math 48 1073, 1988) Methods which are designed to give the largest match between the sequences are generally preferred Methods to determine identity and similarity are codified in publicly available computer programs including the GCG program package (Devereux J et al , Nucleic Acids Research 12(1) 387, 1984), BLASTP, BLASTN, and FASTA (Atschul, S F et al J Molec Biol 215 403-410, 1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S et al NCBI NLM NIH Bethesda. Md 20894, Altschul, S et al J Mol Biol 215 403-410, 1990)
Isolated nucleic acid molecules encoding a KLK-L Protein, and having a sequence which differs from a nucleic acid sequence of the invention due to degeneracy in the genetic code are also within the scope of the invention Such nucleic acids encode functionally equivalent proteins (e g , a KLK-L Protein) but differ in sequence from the sequence of a KLK-L Protein due to degeneracy in the genetic code As one example, DNA sequence polymorphisms within the nucleotide sequence of a KLK-L Protein may result in silent mutations which do not affect the ammo acid sequence Variations in one or more nucleotides may exist among individuals within a population due to natural allelic variation Any and all such nucleic acid variations are within the scope of the invention DNA sequence polymorphisms may also occur which lead to changes in the amino acid sequence of a KLK-L Protein These amino acid polymorphisms are also within the scope of the present invention
Another aspect of the invention provides a nucleic acid molecule which hybridizes under stringent conditions, preferably high stringency conditions to a nucleic acid molecule which comprises a sequence which encodes a KLK-L Protein having an amino acid sequence shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67 Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1989), 6 3 1-6 3 6 For example, 6 0 x sodium chloride/sodium citrate (SSC) at about 45°C, tollowed by a wash of 2 0 x SSC at 50°C may be employed The stringency may be selected based on the conditions used in the wash step By way of example, the salt concentration in the wash step can be selected from a high stringency of about 0 2 x SSC at 50°C In addition, the temperature in the wash step can be at high stringency conditions, at about 65°C It will be appreciated that the invention includes nucleic acid molecules encoding a KLK-L
Related Protein including truncations of a KLK-L Protein, and analogs of a KLK-L Protein as described herein It will further be appreciated that variant forms of the nucleic acid molecules of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention (See for example, splice vaπants of KLK-L5, SEQ ID NO 58, 59, and 60 ) An isolated nucleic acid molecule of the invention which comprises DNA can be isolated by preparing a labelled nucleic acid probe based on all or part of a nucleic acid sequence of the invention The labeled nucleic acid probe is used to screen an appropriate DNA library (e g a cDNA or genomic DNA library) For example, a cDNA library can be used to isolate a cDNA encoding a KLK-L Related Protein by screening the library with the labeled probe using standard techniques Alternatively, a genomic DNA library can be similarly screened to isolate a genomic clone encompassing a gene encoding a KLK-L Related Protein Nucleic acids isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques
An isolated nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid encoding a KLK-L Related Protein using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA It is possible to design synthetic oligonucleotide primers from the nucleotide sequence of the invention for use in PCR A nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques The nucleic acid so amplified can be cloned into an appropriate vector and characteπzed by DNA sequence analysis cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al , Biochemistry,
18, 5294-5299 (1979) cDNA is then synthesized from the mRNA using reverse transcπptase (for example, Moloney MLV reverse transcπptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcπptase available from Seikagaku America, Inc , St Petersburg, FL)
An isolated nucleic acid molecule of the invention which is RNA can be isolated by cloning a cDNA encoding a KLK-L Related Protein into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a KLK-L Related Protein For example, a cDNA can be cloned downstream of a bacteriophage promoter, (e g a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by conventional techniques Nucleic acid molecules of the invention may be chemically synthesized using standard techniques Methods of chemically synthesizing polydeoxynucleotides are known, including but not limited to solid- phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e g , Itakura et al U S Patent No 4,598,049, Caruthers et al U S Patent No 4,458,066, and ltakura U S Patent Nos 4,401,796 and 4,373,071) Determination ot whether a pai ticular nucleic acid molecule encodes a KLK-L Related Protein can be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the expressed protein in the methods described herein A cDNA encoding a KLK-L Related Protein can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein
The initiation codon and untranslated sequences of a KLK-L Related Protein may be determined using computer software designed for the purpose, such as PC/Gene (IntelhGenetics Inc , Calif ) The intron-exon structure and the transcription regulatory sequences of a gene encoding a KLK-L Related Protein may be confirmed by using a nucleic acid molecule of the invention encoding a KLK-L Related Protein to probe a genomic DNA clone library Regulatory elements can be identified using standard techniques The function of the elements can be confirmed by using these elements to express a reporter gene such as the lacZ gene which is operatively linked to the elements These constructs may be introduced into cultured cells using conventional procedures or into non-human transgenic animal models In addition to identifying regulatory elements in DNA, such constructs may also be used to identify nuclear proteins interacting with the elements, using techniques known in the art
In a particular embodiment of the invention, the nucleic acid molecules isolated using the methods described herein are mutant klk-l gene alleles The mutant alleles may be isolated from individuals either known or proposed to have a genotype which contributes to the symptoms of for example, cancer (e g breast, testicular, brain, colon, and prostate cancer) Mutant alleles and mutant allele products may be used in therapeutic and diagnostic methods described herein For example, a cDNA of a mutant klk-l gene may be isolated using PCR as descπbed herein, and the DNA sequence of the mutant allele may be compared to the normal allele to ascertain the mutatιon(s) responsible for the loss or alteration of function of the mutant gene product A genomic library can also be constructed using DNA from an individual suspected of or known to carry a mutant allele, or a cDNA library can be constructed using RNA from tissue known, or suspected to express the mutant allele A nucleic acid encoding a normal klk-l gene or any suitable fragment thereof, may then be labeled and used as a probe to identify the corresponding mutant allele in such libraries Clones containing mutant sequences can be purified and subjected to sequence analysis In addition, an expression library can be constructed using cDNA from RNA isolated from a tissue of an individual known or suspected to express a mutant klk-l allele Gene products made by the putatively mutant tissue may be expressed and screened, for example using antibodies specific for a KLK-L Related Protein as described herein Library clones identified using the antibodies can be purified and subjected to sequence analysis
The sequence of a nucleic acid molecule of the invention, or a fragment of the molecule, may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule An antisense nucleic acid molecule may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art 2. Proteins of the Invention An amino acid sequence ot a KLK L Protein comprises a sequence as shown in Tables 1 to 5 or SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
In addition to proteins comprising an amino acid sequence as shown in Tables 1 to 5 or SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, the proteins of the present invention include truncations of a KLK-L Protein, analogs of a KLK-L Protein, and proteins having sequence identity or similarity to a KLK-L Protein, and truncations thereof as described herein (I e included in KLK-L Related Proteins) Truncated proteins may comprise peptides of between 3 and 70 amino acid residues, ranging in size from a tπpeptide to a 70 mer polypeptide
The truncated proteins may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end The truncated proteins may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end The proteins of the invention may also include analogs of a KLK-L Protein, and/or truncations thereof as described herein, which may include, but are not limited to a KLK-L Protein, containing one or more amino acid substitutions, insertions, and/or deletions Amino acid substitutions may be of a conserved or non-conserved nature Conserved amino acid substitutions involve replacing one or more amino acids of a KLK-L Protein amino acid sequence with ammo acids of similar charge, size, and/or hydrophobicity characteristics When only conserved substitutions are made the resulting analog is preferably functionally equivalent to a KLK-L Protein Non-conserved substitutions involve replacing one or more am o acids of the KLK-L Protein amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics
One or more amino acid insertions may be introduced into a KLK-L Protein Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length
Deletions may consist of the removal of one or more amino acids, or discrete portions from a KLK-L Protein sequence The deleted amino acids may or may not be contiguous The lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 20 to 40 amino acids The proteins of the invention include proteins with sequence identity or similarity to a KLK-L
Protein and/or truncations thereof as described herein Such KLK-L Proteins include proteins whose amino acid sequences are comprised of the amino acid sequences of KLK-L Protein regions from other species that hybridize under selected hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain a KLK-L Protein These proteins will generally have the same regions which are characteristic of a KLK-L Protein Preferably a protein will have substantial sequence identity for example, about 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% identity, preferably 90% identity, more preferably at least 95%, 96%, 97%, 98%, or 99% identity, and most preferably 98% identity with an amino acid sequence shown in Tables 1 to 5 or SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
A percent amino acid sequence homology, similarity or identity is calculated as the percentage ot aligned amino acids that match the reference sequence using known methods as described herein
The invention also contemplates isoforms of the proteins of the invention An isoform contains the same number and kinds of amino acids as a protein of the invention, but the isoform has a different molecular structure Isoforms contemplated by the present invention preferably have the same properties as a protein of the invention as described herein
The present invention also includes KLK-L Related Proteins conjugated with a selected protein, or a marker protein (see below) to produce fusion proteins Additionally, immunogenic portions of a KLK- L Protein and a KLK-L Protein Related Protein are within the scope of the invention
A KLK-L Related Protein of the invention may be prepared using recombinant DNA methods Accordingly, the nucleic acid molecules of the present invention having a sequence which encodes a KLK- L Related Protein of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e g replication defective retroviruses, adenoviruses and adeno- associated viruses), so long as the vector is compatible with the host cell used
The invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology Methods m Enzymology 185, Academic Press, San Diego, CA (1990) Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art The necessary regulatory sequences may be supplied by the native KLK-L Protein and/or its flanking regions The invention further provides a recombinant expression vector comprising a DNA nucleic acid molecule of the invention cloned into the expression vector in an antisense orientation That is, the DNA molecule is linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to the nucleic acid sequence of a protein of the invention or a fragment thereof Regulatory sequences linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance a viral promoter and/or enhancer, or regulatory sequences can be chosen which direct tissue or cell type specific expression of antisense RNA
The recombinant expression vectors of the invention may also contain a marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention Examples of marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG The markers can be introduced on a separate vector from the nucleic acid of interest The lecombinant expiession vectors may also contain genes which encode a fusion moiety which provides increased expression ot the recombinant protein, increased solubility of the recombinant protein, and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification For example, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein Typical fusion expression vectors include pGEX (Amrad Corp , Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein
The recombinant expression vectors may be introduced into host cells to produce a transformant host cell "Transformant host cells" include host cells which have been transformed or transfected with a recombinant expression vector of the invention The terms "transformed with", "transfected with", "transformation" and "transfection" encompass the introduction of a nucleic acid (e g a vector) into a cell by one of many standard techniques Prokaryotic cells can be transformed with a nucleic acid by, for example, electroporation or calcium-chloride mediated transformation A nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, pofectin, electroporation or microinjection Suitable methods for transforming and transfecting host cells can be found in Sambrook et al (Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells For example, the proteins of the invention may be expressed in bacterial cells such as E coli, insect cells (using baculovirus), yeast cells, or mammalian cells Other suitable host cells can be found in Goeddel, Gene Expression Technology Methods in Enzymology 185, Academic Press, San Diego, CA (1991)
A host cell may also be chosen which modulates the expression of an inserted nucleic acid sequence, or modifies (e g glycosylation or phosphorylation) and processes (e g cleaves) the protein in a desired fashion Host systems or cell lines may be selected which have specific and characteristic mechanisms for post-translational processing and modification of proteins For example, eukaryotic host cells including CHO, VERO, BHK, HeLA, COS, MDCK, 293, 3T3, and WI38 may be used For long-term high-yield stable expression of the protein, cell lines and host systems which stably express the gene product may be engineered
Host cells and in particular cell lines produced using the methods descπbed herein may be particularly useful in screening and evaluating compounds that modulate the activity of a KLK-L Related Protein
The proteins of the invention may also be expressed in non-human transgenic animals including but not limited to mice, rats, rabbits, guinea pigs, micro-pigs, goats, sheep, pigs, non-human primates (e g baboons, monkeys, and chimpanzees) [see Hammer et al (Nature 315 680-683, 1985), Palmiter et al
(Science 222 809-814, 1983), Brinster et al (Proc Natl Acad Sci USA 82 44384442, 1985), Palmiter and
Brinster (Cell 41 343-345, 1985) and U S Patent No 4,736,866)] Procedures known in the art may be used to intioduce a nucleic acid molecule ot the invention encoding a KLK-L Related Protein into animals to produce the founder lines of transgenic animals. Such procedures include pronuclear microinjection, retrovirus mediated gene transfer into germ lines, gene targeting in embryonic stem cells, electroporation of embryos, and sperm-mediated gene transfer The present invention contemplates a transgenic animal that carries the KLK-L gene in all their cells, and animals which carry the transgene in some but not all their cells. The transgene may be integrated as a single transgene or in concatamers The transgene may be selectively introduced into and activated in specific cell types (See for example, Lasko et al, 1992 Proc. Natl. Acad. Sci. USA 89: 6236). The transgene may be integrated into the chromosomal site of the endogenous gene by gene targeting. The transgene may be selectively introduced into a particular cell type inactivating the endogenous gene in that cell type (See Gu et al Science 265: 103-106).
The expression of a recombinant KLK-L Related Protein in a transgenic animal may be assayed using standard techniques. Initial screening may be conducted by Southern Blot analysis, or PCR methods to analyze whether the transgene has been integrated. The level of mRNA expression in the tissues of transgenic animals may also be assessed using techniques including Northern blot analysis of tissue samples, in situ hybridization, and RT-PCR. Tissue may also be evaluated lmmunocytochemically using antibodies against KLK-L Protein.
Proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merπfield, 1964, J. Am. Chem. Assoc. 85:2149- 2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed E Wansch, Vol. 15 I and II, Thieme, Stuttgart).
N-terminal or C-terminal fusion proteins comprising a KLK-L Related Protein of the invention conjugated with other molecules, such as proteins, may be prepared by fusing, through recombinant techniques, the N-terminal or C-termmal of a KLK-L Related Protein, and the sequence of a selected protein or marker protein with a desired biological function. The resultant fusion proteins contain KLK-L
Protein fused to the selected protein or marker protein as described herein. Examples of proteins which may be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc 3. Antibodies KLK-L Related Proteins of the invention can be used to prepare antibodies specific for the proteins. Antibodies can be prepared which bind a distinct epitope in an unconserved region of the protein An unconserved region of the protein is one that does not have substantial sequence homology to other proteins. A region from a conserved region such as a well-characteπzed domain can also be used to prepare an antibody to a conserved region of a KLK-L Related Protein. Antibodies having specificity for a KLK-L Related Protein may also be raised from fusion proteins created by expressing fusion proteins in bacteria as described herein
The invention can employ intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g. a Fab, (Fab)2 fragment, or Fab expression library fragments and epitope-binding fragments theieof), an antibody heavy chain, and antibody light chain, a genetically engineered single chain Fv molecule (Ladner et al, U S Pat No 4,946,778), or a chimenc antibody, for example, an antibody which contains the binding specificity ot a muπne antibody, but in which the remaining portions are of human origin Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art
4. Applications of the Nucleic Acid Molecules, KLK-L Related Proteins, and Antibodies of the
Invention
The nucleic acid molecules, KLK-L Related Proteins, and antibodies of the invention may be used in the prognostic and diagnostic evaluation of cancer (e g breast, testicular, and prostate cancer) or other conditions, and the identification of subjects with a predisposition to cancer (Section 4 1 1 and 4 1 2) Methods for detecting nucleic acid molecules and KLK-L Related Proteins of the invention, can be used to monitor conditions including cancer, by detecting KLK-L Related Proteins and nucleic acid molecules encoding KLK-L Related Proteins It would also be apparent to one skilled in the art that the methods described herein may be used to study the developmental expression of KLK-L Related Proteins and, accordingly, will provide further insight into the role of KLK-L Related Proteins The applications of the present invention also include methods for the identification of compounds that modulate the biological activity of KLK-L or KLK-L Related Proteins (Section 4 2) The compounds, antibodies etc may be used for the treatment of cancer (Section 4 3) 4.1 Diagnostic Methods A variety of methods can be employed for the diagnostic and prognostic evaluation of conditions including cancer (e g breast, testicular, and prostate cancer), and the identification of subjects with a predisposition to such conditions Such methods may, for example, utilize nucleic acid molecules of the invention, and fragments thereof, and antibodies directed against KLK-L Related Proteins, including peptide fragments In particular, the nucleic acids and antibodies may be used, for example, for (1) the detection of the presence of KLK-L mutations, or the detection of either over- or under-expression of KLK-L mRNA relative to a non-disorder state or the qualitative or quantitative detection of alternatively spliced forms of KLK-L transcripts which may correlate with certain conditions or susceptibility toward such conditions, and (2) the detection of either an over- or an under-abundance of KLK-L Related Proteins relative to a non- disorder state or the presence of a modified (e g , less than full length) KLK-L Protein which correlates with a disorder state, or a progression toward a disorder state
The methods descπbed herein may be performed by utilizing pre-packaged diagnostic kits comprising at least one specific KLK-L nucleic acid or antibody described herein, which may be conveniently used, e g , in clinical settings, to screen and diagnose patients and to screen and identify those individuals exhibiting a predisposition to developing a disorder Nucleic acid-based detection techniques are described, below, in Section 4 1 1 Peptide detection techniques are described, below, in Section 4 1 2 The samples that may be analyzed using the methods of the invention include those which are known or suspected to express KLK-L or contain KLK-L Related Proteins The samples may be derived from a patient or a cell culture, and include but are not limited to biological fluids, tissue extracts, treshly harvested cells, and lysates ot cells which have been incubated in cell cultures
Oligonucleotides or longer fragments derived trom any of the nucleic acid molecules of the invention may be used as targets in a microarray The microarray can be used to simultaneously monitor the expression levels of large numbers of genes and to identify genetic variants, mutations, and polymorphisms The information from the microarray may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents
The preparation, use, and analysis of microarrays are well known to a person skilled in the art (See, for example, Brennan, T M et al (1995) U S Pat No 5,474,796, Schena, et al (1996) Proc Natl Acad Sci 93 10614-10619, Baldeschweiler et al (1995), PCT Application W095/251116, Shalon, D et al (1 995) PCT application WO95/35505, Heller, R A et al (1997) Proc Natl Acad Sci 94 2150-2155, and Heller, M J et al (1997) U S Pat No 5,605,662 ) 4.1.1 Methods for Detecting Nucleic Acid Molecules of the Invention The nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleic acid sequences of the invention in samples Suitable probes include nucleic acid molecules based on nucleic acid sequences encoding at least 5 sequential amino acids from regions of a KLK-L Protein, preferably they comprise 15 to 30 nucleotides A nucleotide probe may be labeled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32P, 3H, 14C or the like Other detectable substances which may be used include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and luminescent compounds An appropnate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al,
1989, Molecular Cloning, A Laboratory Manual (2nd ed ) The nucleic acid probes may be used to detect genes, preferably in human cells, that encode KLK-L Related Proteins The nucleotide probes may also be useful in the diagnosis of cancer, in monitoring the progression of cancer, or monitoring a therapeutic treatment The probe may be used in hybridization techniques to detect genes that encode KLK-L Related
Proteins The technique generally involves contacting and incubating nucleic acids (e g recombinant DNA molecules, cloned genes) obtained from a sample from a patient or other cellular source with a probe of the present invention under conditions favorable for the specific annealing of the probes to complementary sequences in the nucleic acids After incubation, the non-annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected
The detection of nucleic acid molecules of the invention may involve the amplification of specific gene sequences using an amplification method such as PCR, followed by the analysis of the amplified molecules using techniques known to those skilled in the art Suitable primers can be routinely designed by one ot skill in the art
Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities involving klk I structure, including point mutations, insertions, deletions, and chromosomal rearrangements For example, direct sequencing, single stranded conformational polymorphism analyses, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical mismatch cleavage, and oligonucleotide hybridization may be utilized
Genotyping techniques known to one skilled in the art can be used to type polymorphisms that are in close proximity to the mutations in a klk-l gene The polymorphisms may be used to identify individuals in families that are likely to carry mutations If a polymorphism exhibits linkage disequalibπum with mutations in a klk-l gene, it can also be used to screen for individuals in the general population likely to carry mutations Polymorphisms which may be used include restriction fragment length polymorphisms (RFLPs), single-base polymorphisms, and simple sequence repeat polymorphisms (SSLPs)
A probe of the invention may be used to directly identify RFLPs A probe or primer of the invention can additionally be used to isolate genomic clones such as YACs, BACs, PACs, cosmids, phage or plasmids The DNA in the clones can be screened for SSLPs using hybridization or sequencing procedures
Hybridization and amplification techniques described herein may be used to assay qualitative and quantitative aspects of klk-l expression For example, RNA may be isolated from a cell type or tissue known to express klk-l and tested utilizing the hybridization (e g standard Northern analyses) or PCR techniques referred to herein The techniques may be used to detect differences in transcript size which may be due to normal or abnormal alternative splicing The techniques may be used to detect quantitative differences between levels of full length and/or alternatively splice transcripts detected in normal individuals relative to those individuals exhibiting cancer symptoms or other disease conditions
The primers and probes may be used in the above descπbed methods in situ l e directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections
4.1.2 Methods for Detecting KLK-L Related Proteins
Antibodies specifically reactive with a KLK-L Related Protein, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect KLK-L Related Proteins in various samples (e g biological materials) They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of KLK-L Related Proteins expression, or abnormalities in the structure, and/or temporal, tissue, cellular, or subcellular location of a KLK-L Related Protein Antibodies may also be used to screen potentially therapeutic compounds in vitro to determine their effects on cancer, and other conditions In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies The antibodies of the invention may also be used in vitro to determine the level of KLK-L expression in cells genetically engineered to produce a KLK-L Related Protein
The antibodies may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of a KLK-L Related Protein and the antibodies Examples of such assays are radioimmunoassays, enzyme immunoassays (e g ELISA), lmmunofluorescence, immunoprecipitation, latex agglutination, hemagglutmation, and histochemi il tests The antibodies may be used to detect and quantity KLK-L Related Proteins in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states
In particular, the antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect a KLK-L Related Protein, to localize it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression
Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a KLK-L Related Protein Generally, an antibody of the invention may be labeled with a detectable substance and a KLK-L Related Protein may be localised m tissues and cells based upon the presence of the detectable substance Examples of detectable substances include, but are not limited to, the following radioisotopes (e g , 3 H, 14C, 35S, 125I, 131I), fluorescent labels (e g , FITC, rhodamine, lanthanide phosphors), luminescent labels such as lurrunol, enzymatic labels (e g , horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylchohnesterase), biotinyl groups (which can be detected by marked avidin e g , streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or caloπmetπc methods), predetermined polypeptide epitopes recognized by a secondary reporter (e g , leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steπc hindrance Antibodies may also be coupled to electron dense substances, such as ferπtin or colloidal gold, which are readily visualised by electron microscopy The antibody or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies etc For example, the carrier or support may be nitrocellulose, or glass, polyacrylamides, gabbros, and magnetite The support material may have any possible configuration including spherical (e g bead), cylindrical (e g inside surface of a test tube or well, or the external surface of a rod), or flat (e g sheet, test strip) Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against KLK-L Related Protein By way of example, if the antibody having specificity against a KLK-L Related Protein is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labeled with a detectable substance as described herein
Where a radioactive label is used as a detectable substance, a KLK-L Related Protein may be localized by radioautography The results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains 4.2 Methods for Identifying or Evaluating Substances/Compounds
The methods described herein are designed to identify substances that modulate the biological activity of a KLK-L Related Protein including substances that bind to KLK-L Related Proteins, or bind to other proteins that interact with a KLK-L Related Protein, to compounds that interfere with, or enhance the interaction of a KLK-L Related Protein and substances that bind to the KLK-L Related Protein or other proteins that interact with a KLK-L Related Protein Methods are also utilized that identify compounds that bind to KLK L regulatory sequences The substances and compounds identified using the methods ot the invention include but aie not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members ot random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e g polyclonal, monoclonal, humanized, anti-idiotypic, chimenc, single chain antibodies, fragments, (e g Fab, F(ab)2, and Fab expression library fragments, and epitope-binding fragments thereof)], and small organic or inorganic molecules The substance or compound may be an endogenous physiological compound or it may be a natural or synthetic compound
Substances which modulate a KLK-L Related Protein can be identified based on their ability to bind to a KLK-L Related Protein Therefore, the invention also provides methods for identifying substances which bind to a KLK-L Related Protein Substances identified using the methods of the invention may be isolated, cloned and sequenced using conventional techniques A substance that associates with a polypeptide of the invention may be an agonist or antagonist of the biological or immunological activity of a polypeptide of the invention The term "agonist' , refers to a molecule that increases the amount of, or prolongs the duration of, the activity of the polypeptide The term "antagonist" refers to a molecule which decreases the biological or immunological activity of the polypeptide Agonists and antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules that associate with a polypeptide of the invention
Substances which can bind with a KLK-L Related Protein may be identified by reacting a KLK-L Related Protein with a test substance which potentially binds to a KLK-L Related Protein, under conditions which permit the formation of substance-KLK-L Related Protein complexes and removing and/or detecting the complexes The complexes can be detected by assaying for substance-KLK-L Related Protein complexes, for free substance, or for non-complexed KLK-L Related Protein Conditions which permit the formation of substance-KLK-L Related Protein complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein
The substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof To facilitate the assay of the components, antibody against KLK-L Related Protein or the substance, or labeled KLK-L Related Protein, or a labeled substance may be utilized The antibodies, proteins, or substances may be labeled with a detectable substance as described above
A KLK-L Related Protein, or the substance used in the method of the invention may be insolubilized For example, a KLK-L Related Protein, or substance may be bound to a suitable carrier such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, lon- exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc The insolubilized protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical ethods, toi example, cyanogen bromide coupling
The invention also contemplates a method tor evaluating a compound for its ability to modulate the biological activity of a KLK-L Related Protein ot the invention, by assaying for an agonist or antagonist (1 e enhancer or inhibitor) of the binding of a KLK-L Related Protein with a substance which binds with a KLK-L Related Protein The basic method for evaluating if a compound is an agonist or antagonist of the binding of a KLK-L Related Protein and a substance that binds to the protein, is to prepare a reaction mixture containing the KLK-L Related Protein and the substance under conditions which permit the formation of substance-KLK-L Related Protein complexes, in the presence of a test compound The test compound may be initially added to the mixture, or may be added subsequent to the addition of the KLK-L Related Protein and substance Control reaction mixtures without the test compound or with a placebo are also prepared The formation of complexes is detected and the formation of complexes in the control reaction but not in the reaction mixture indicates that the test compound interferes with the interaction of the KLK-L Related Protein and substance The reactions may be carried out in the liquid phase or the KLK- L Related Protein, substance, or test compound may be immobilized as descπbed herein The ability of a compound to modulate the biological activity of a KLK-L Related Protein of the invention may be tested by determining the biological effects on cells
It will be understood that the agonists and antagonists l e inhibitors and enhancers that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or allosteπc sites
The invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of KLK-L Related Protein with a substance which is capable of binding to the KLK-L Related Protein Thus, the invention may be used to assay for a compound that competes for the same binding site of a KLK-L Related Protein The invention also contemplates methods for identifying compounds that bind to proteins that interact with a KLK-L Related Protein Protein-protein interactions may be identified using conventional methods such as co-immunoprecipitation, crosshnking and co-purification through gradients or chromatographic columns Methods may also be employed that result in the simultaneous identification of genes which encode proteins interacting with a KLK-L Related Protein These methods include probing expression libraries with labeled KLK-L Related Protein
Two-hybrid systems may also be used to detect protein interactions in vivo Generally, plasmids are constructed that encode two hybrid proteins A first hybrid protein consists of the DNA-binding domain of a transcription activator protein fused to a KLK-L Related Protein, and the second hybrid protein consists of the transcription activator protein's activator domain fused to an unknown protein encoded by a cDNA which has been recombined into the plasmid as part of a cDNA library The plasmids are transformed into a strain of yeast (e g S cerevisiae) that contains a reporter gene (e g lacZ, luciferase, alkaline phosphatase, horseradish peroxidase) whose regulatory region contains the transcription activator's binding site The hybrid proteins alone cannot activate the transcription of the reporter gene However, rntei action ot the two hybrid proteins reconstitutes the functional activator protein and results in expression ot the reporter gene, which is detected by an assay for the reporter gene product
It will be appreciated that fusion proteins may be used in the above-described methods In particular, KLK-L Related Proteins fused to a glutathione S-transferase may be used in the methods The reagents suitable for applying the methods of the invention to evaluate compounds that modulate a KLK-L Related Protein may be packaged into convenient kits providing the necessary materials packaged into suitable containers The kits may also include suitable supports useful in performing the methods of the invention 4.3 Compositions and Treatments The proteins of the invention, substances or compounds identified by the methods descπbed herein, antibodies, and antisense nucleic acid molecules of the invention may be used for modulating the biological activity of a KLK-L Related Protein, and they may be used in the treatment of conditions such as cancer (e g prostate, testicular, brain, uterine, thymus, ovarian, colon, ovarian, or breast cancer) Accordingly, the substances, antibodies, peptides, and compounds may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo By "biologically compatible form suitable for administration in vivo" is meant a form of the active substance to be administered in which any toxic effects are outweighed by the therapeutic effects The active substances may be administered to living organisms including humans, and animals Administration of a therapeutically active amount of a pharmaceutical composition of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result For example, a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual Dosage regima may be adjusted to provide the optimum therapeutic response For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation
The active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc ), oral administration, inhalation, transdermal application, or rectal administration Depending on the route of administration, the active substance may be coated in a material to protect the substance from the action of enzymes, acids and other natural conditions that may inactivate the substance
The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa , USA 1985) On this basis, the compositions include, albeit not exclusively, solutions of the active substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and lso-osmotic with the physiological fluids Based upon their homology to genes encoding kalhkrein, nucleic acid molecules ot the invention may be also useful in the treatment ot conditions such as hypertension, cardiac hypertrophy, arthritis, inflammatory disorders, neurological disorders, and blood clotting disorders
Vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids, may be used to deliver nucleic acid molecules to a targeted organ, tissue, or cell population Methods well known to those skilled in the art may be used to construct recombinant vectors which will express antisense nucleic acid molecules of the invention (See, for example, the techniques described in Sambrook et al (supra) and Ausubel et al (supra))
The nucleic acid molecules comprising full length cDNA sequences and/or their regulatory elements enable a skilled artisan to use sequences encoding a protein of the invention as an investigative tool in sense (Youssoufian H and H F Lodish 1993 Mol Cell Biol 13 98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem 60 631-652) regulation of gene function Such technology is well known in the art, and sense or antisense o gomers, or larger fragments, can be designed from various locations along the coding or control regions Genes encoding a protein of the invention can be turned off by transfectmg a cell or tissue with vectors which express high levels of a desired KLK-L-encoding fragment Such constructs can inundate cells with untranslatable sense or antisense sequences Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases Modifications of gene expression can be obtained by designing antisense molecules, DNA, RNA or PNA, to the regulatory regions of a gene encoding a protein of the invention, le, the promoters, enhancers, and introns Preferably, oligonucleotides are derived from the transcription initiation site, eg, between -10 and +10 regions of the leader sequence The antisense molecules may also be designed so that they block translation of mRNA by preventing the transcπpt from binding to ribosomes Inhibition may also be achieved using ' triple helix" base-pairing methodology Triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules Therapeutic advances using triplex DNA were reviewed by Gee J E et al (In Huber B E and B I Carr (1994) Molecular and Immunologic Approaches, Futura Publishing Co, Mt Kisco N Y )
Ribozymes are enzymatic RNA molecules that catalyze the specific cleavage of RNA Ribozymes act by sequence-specific hybridization of the πbozyme molecule to complementary target RNA, followed by endonucleolytic cleavage The invention therefore contemplates engineered hammerhead motif πbozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a protein of the invention
Specific πbozyme cleavage sites within any potential RNA target may initially be identified by scanning the target molecule for πbozyme cleavage sites which include the following sequences, GUA,
GUU and GUC Once the sites are identified, short RNA sequences of between 15 and 20 πbonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable The suitability of candidate targets may also be detei mined by testing accessibility to hybridization with complementary oligonucleotides using πbonuclease piotection assays
Methods tor introducing vectors into cells or tissues include those methods discussed herein and which are suitable tor m vivo, m vitro and ex vivo therapy For ex vivo therapy, vectors may be introduced into stem cells obtained from a patient and clonally propagated for autologous transplant into the same patient (See U S Pat Nos 5,399,493 and 5,437,994) Delivery by transfection and by liposome are well known in the art
The nucleic acid molecules disclosed herein may also be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including but not limited to such properties as the triplet genetic code and specific base pair interactions
The invention also provides methods for studying the function of a polypeptide of the invention Cells, tissues, and non-human animals lacking in expression or partially lacking in expression of a nucleic acid molecule or gene of the invention may be developed using recombinant expression vectors of the invention having specific deletion or insertion mutations in the gene A recombinant expression vector may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a deficient cell, tissue, or animal
Null alleles may be generated in cells, such as embryonic stem cells by deletion mutation A recombinant gene may also be engineered to contain an insertion mutation that inactivates the gene Such a construct may then be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, electroporation, injection etc Cells lacking an intact gene may then be identified, for example by Southern blotting, Northern Blotting, or by assaying for expression of the encoded polypeptide using the methods described herein Such cells may then be fused to embryonic stem cells to generate transgenic non-human animals deficient in a polypeptide of the invention Germlme transmission of the mutation may be achieved, for example, by aggregating the embryonic stem cells with early stage embryos, such as 8 cell embryos, in vitro, transferring the resulting blastocysts into recipient females and, generating germhne transmission of the resulting aggregation chimeras Such a mutant animal may be used to define specific cell populations, developmental patterns and in vivo processes, normally dependent on gene expression The invention thus provides a transgenic non-human mammal all of whose germ cells and somatic cells contain a recombinant expression vector that inactivates or alters a gene encoding a KLK-L Related
Protein In an embodiment the invention provides a transgenic non-human mammal all of whose germ cells and somatic cells contain a recombinant expression vector that inactivates or alters a gene encoding a KLK- L Related Protein resulting in a KLK-L Related Protein associated pathology Further the invention provides a transgenic non-human mammal which doe not express a KLK-L Related Protein of the invention In an embodiment, the invention provides a transgenic non-human mammal which doe not express a KLK-L Related Protein of the invention resulting in a KLK-L Related Protein associated pathology A KLK-L Related Protein pathology refers to a phenotype observed for a KLK-L Related Protein homozygous mutant A transgenic non-human animal includes but is not limited to mouse, rat, rabbit, sheep, hamster, dog, guinea pig, micro-pig, pig, cat, goat, and non-human primates, preferably mouse
The invention also provides a transgenic non-human animal assay system which provides a model system for testing for an agent that reduces or inhibits a pathology associated with an KLK-L Related Protein, preferably a KLK-L Related Protein associated pathology, comprising
(a) administering the agent to a transgenic non-human animal of the invention, and
(b) determining whether said agent reduces or inhibits the pathology (e g KLK-L Related Protein associated pathology) in the transgenic non-human animal relative to a transgenic non-human animal of step (a) which has not been administered the agent The agent may be useful in the treatment and prophylaxis of conditions such as cancer as discussed herein The agents may also be incorporated in a pharmaceutical composition as described herein The activity of the proteins, substances, compounds, antibodies, nucleic acid molecules, agents, and compositions of the invention may be confirmed in animal experimental model systems Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED50 ( the dose therapeutically effective in 50% of the population) or LD50 (the dose lethal to 50% of the population) statistics The therapeutic index is the dose ratio of therapeutic to toxic effects and it can be expressed as the ED50/LD50 ratio Pharmaceutical compositions which exhibit large therapeutic indices are preferred
The following non-limiting examples are illustrative of the present invention Examples
Example 1 MATERIALS AND METHODS Identification of positive PAC and BAC genomic clones from a human genomic DNA library
The sequence of PSA, KLKl, KLK2, NES1 and Zyme genes is already known Polymerase chain reaction (PCR)-based amplification protocols have been developed which allowed generation of PCR products specific for each one of these genes Using these PCR products as probes, labeled with 32P, a human genomic DNA PAC library and a human genomic DNA BAC library was screened for the purpose of identifying positive clones of approximately 100-150 Kb long The general strategies for these experiments have been published elsewhere (14) The genomic libraries were spotted in duplicate on nylon membranes and positive clones were further confirmed by Southern blot analysis as described (14)
DNA sequences on chromosome 19
The Lawrence Livermore National Laboratory participates in the sequencing of the human genome project and focuses on sequencing chromosome 19 Large sequencing information on this chromosome is available at the website of the Lawrence Livermore National Laboratory (http //www- bio llnl gov/genome/gemnome html)
Approximately 300 Kb of genomic sequences were obtained from that website, encompassing a region on chromosome 19q 13 3 - 13 4, where the known kalhkrein genes are localized This 300 Kb of sequence is represented by 8 contigs of variable lengths By using a number of different computer programs, an almost contiguous sequence of the region was established as shown diagramatically in Figure 1 and Figure 28 Some of the contigs were reversed as shown in Figure 1 in order to reconstruct the area on both strands of DNA
By using the published sequences of PSA, KLK2, NESl and Zyme and the computer software BLAST 2, using alignment strategies, the relative positions of these genes on the contiguous map were identified (Figure 28) These known genes served as hallmarks for further studies An EcoRI restriction map of the area is also available at the website of the Lawrence Livermore National Laboratory Using this restriction map and the computer program WebCutter (http //www firstmarket com/cutter/cut2 html), a restriction study analysis of the available sequence was performed to further confirm the assignment and relative positions of these contigs along chromosome 19 The obtained configuration and the relative location of the known genes are presented in Figure 1 Gene prediction analysis
For exon prediction analysis of the whole genomic area, a number of different computer programs were used All the programs were initially tested using known genomic sequences of the PSA, Zyme, and NES 1 genes The more reliable computer programs, GeneBuilder (gene prediction), GeneBuilder (exon prediction), Grail 2 and GENEID-3 were selected for further use Protein homology searching
Putative exons of the new genes were first translated to the corresponding amino acid sequences BLAST homology searching for the proteins encoded by the exons of the putative new genes were performed using the BLASTP program and the Genbank databases RESULTS Relative position of PSA, KLK2, Zyme and NESl on Chromosome 19
Screening of the human BAC library identified two clones which were positive for the Zyme gene (clones BAC 288H1 and BAC 76F7) These BACs were further analyzed by PCR and primers specific for PSA, NESl, KLKl and KLK2 These analyses indicated that both BACs were positive for Zyme, PSA and
KLK2 and negative for KLKl and NESl genes
Screening of the human PAC genomic library identified a PAC clone which was positive for NESl (clone PAC 34B 1) Further PCR analysis indicated that this PAC clone was positive for NESl and KLKl genes and negative for PSA, KLK2 and Zyme Combination of this information with the EcoRI restriction map of the region allowed establishment of the relative positions of these four genes PSA is the most centromeric, followed by KLK2, Zyme and NES 1 Further alignment of the known sequences of these genes with the 300 Kb contig enabled precise localization of all four genes and determination of the direction of transcription, as shown by the arrows in Figure 1 The KLKl gene sequence was not identified on any of these contigs and appears to be further telomeric to NES 1 (since it is co-localized on the same PAC as NESl)
Identification of new genes
A set of rules was used to consider the presence of a new gene in the genomic area of interest as follows 1 Clusters of at least 3 exons should be tound
2 Only exons with high prediction score ("good" or "excellent" quality, as indicated by the searching progiams) were considered for the construction ot the putative new genes
3 Exons predicted were reliable only if they were identified by at least two different exon prediction programs
By using this strategy, eleven putative new genes were identified of which three were found on subsequent homology analysis to be known genes not previously mapped 1 e the human stratum corneum chymotrypsin enzyme (HSCCE), human neuropsin, and trypsin-hke serine protease (TLSP) Their relative location is shown in Figure 1 The five genes all have variable homologies with known human or animal kalhkrein proteins and or other known serine proteases (depicted as KLK-Ll, KLK-L2, KLK-L3, KLK-L4 and KLK-L5 in Figure 1 and KLK-Ll to KLK-L6 in Figure 28)
In Tables 1 to 5, the preliminary exon structure and partial protein sequence for each one of the newly identified genes is shown In Table 6, some proteins are presented which appear, on preliminary analysis, to be homologous to the proteins encoded by the putative new genes SEQ ID NOs 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, and 67 show ammo acid sequences of KLK-L 1-KLK-L6, and SEQ ID NOs 1, 13, 21, 43, 56, and 65 show nucleic acid sequences of the genes encoding KLK-Ll- KLK-L6 DISCUSSION
Prediction of protein-coding genes in newly sequenced DNA becomes very important after the establishment of large genome sequencing projects This problem is complicated due to the exon-intron structure of the eukaryotic genes which interrupts the coding sequence in many unequal parts In order to predict the protein-codmg exons and overall gene structure, a number of computer programs were developed All these programs are based on the combination of potential functional signals with the global statistical properties of known protein-coding regions (15) However, the most powerful approach for gene structure prediction is to combine information about potential functional signals (splice sites, translation start or stop signal etc ) together with the statistical properties of coding sequences (coding potential) along with information about homologies between the predicted protein and known protein families (16)
In mouse and rat, kailikreins are encoded by large multigene families and these genes tend to cluster in groups with a distance as small as 3 3 - 7 0 Kb (3) A strong conservation of gene order between human chromosome 19ql3 1 - ql3 4 and 17 loci in a 20-cM proximal part of mouse chromosome 7, including the kalhkrein locus, has been documented (17)
In humans, only a few kalhkrein genes were identified In fact, only KLKl, KLK2 and KLK3 (PSA) are considered to represent the human kalhkrein gene family (9) The work described herein provides strong evidence that a large number of kallikrein-hke genes are clustered within a 300Kb region around chromosome 19q l3 2 - ql3 4 The three established human kailikreins (KLKl, KLK2, KLK3), Zyme and NESl, as well as the stratum corneum chymotrypticn enzyme, neuropsin, and TLSP (trypsin-like serine protease) and another five new genes , KLK-Ll to KLK-L5, may constitute a large gene family This will bring the total number of kalhkrein or kallikrein-hke genes in this region of chromosome 19 to thirteen
The human stratum corneum chymotryptic enzyme (19), neuropsin (20) and trypsin-hke serine protease (TLSP) (21) are three previously characterized genes which have many structural similarities with the kailikreins and other members of the serine protease family However, they have not been mapped in the past Their precise mapping in the region ot the kalhkrein gene family indicates that these three genes, along with the ones that were newly identified, or are already known, constitute a family that likely originated by duplication of an ancestral gene The relative localization of all these genes is depicted in Figure 1
Kalhkrem genes are a subfamily of serine proteases, traditionally characterized by their ability to liberate lysyl-bradykinin (kalhdin) from kininogen (18) More recently, however, a new, structural concept has emerged to describe kailikreins From accumulated sequence data, it is now clear that the mouse has many genes with high homology to kalhkrein coding sequences (19-20) Richard and co-workers have contributed to the concept of a " kal krem multigene family" to refer to these genes (21-22) This definition is not based much on specific enzymatic function of the gene product, but more on its sequence homology and their close linkage on mouse chromosome 7 In humans, only KLKl meets the functional definition of a kalhkrein KLK2 has trypsin-hke enzymatic activity and KLK3 (PSA) has very weak chymotrypsin-Iike enzymatic activity These activities of KLK2 and KLK3 are not known to liberate biologically active peptides from precursors Based on the newer definition, members of the kalhkrem family include, not only the gene for the kalhkrein enzyme, but also genes encoding other homologous proteases, including the enzyme that processes the precursors of the nerve growth factor and epidermal growth factor (8) Therefore, it is important to note the clear distinction between the enzyme kalhkrein and a kal krein or a kallikrein-hke gene
In carrying out the study only exons were considered which were predicted with "good" or "excellent" quality and only exons were considered which were predicted by at least two different programs Moreover, the presence of a putative gene was only considered when at least three exons clustered coordmately m that region Additional evidence that these new genes are indeed homologous to the known kailikreins and other serine proteases comes from comparison of the intron phases As published previously (14), trypsinogen, PSA and NESl have 5 coding exons of which the first has intron phase I (the intron occurs after the first nucleotide of the codon), the second has intron phase II (the intron occurs after the second nucleotide and the codon), the third has intron phase I and the fourth has intron phase 0 (the intron occurs between codons) The fifth exon contains the stop codon The intron phases of the predicted new kallikrein-hke genes follow these rules and are shown in the respective tables Further support comes from the identification in the new genes, of the conserved ammo acids of the catalytic domain of the serine proteases, as presented in Tables 1 - 5
In order to test the accuracy of the computer programs, known genomic areas containing the PSA,
Zyme and KLK2 genes were tested Two of these programs (Grail 2 and GeneBuilder) were able to detect about 95% of the tested known genes Matches with expressed sequence tag sequences (EST) can also be employed for gene structure prediction in the GeneBuilder program and this can significantly improve the power of the program especially at high stringency (e g >95% homology)
In mouse, ten of the kalhkrein genes appear to be pseudogenes (9) Example 2 PROSTASE/KLK-L1 in prostate and breast tissues
The fine mapping of the prostase/KLK-Ll gene and its chromosomal localization in relation to a number of other homologous genes also mapping to the same region are described. In addition, extensive tissue expression studies were carried out that demonstrate that, in addition to prostate (which shows the highest expression), that prostase/KLK-Ll is also expressed in female breasts, testis, adrenals, uterus, colon, thyroid, brain, spinal cord and salivary glands Furthermore, the gene is up-regulated by androgens and progestins in the breast carcinoma cell line BT-474 Materials and Methods DNA sequences on chromosome 19
Large DNA sequencing data for chromosome 19 is available at the web site of the Lawrence Livermore National Laboratory (LLNL) (http://www-bio.llnl. gov/genome /genome.html). Approximately 300 Kb of genomic sequence was obtained from that web site, encompassing a region on chromosome 19ql3.3 - 13.4, where the known kalhkrein genes are localized This sequence is represented by 9 contigs of variable lengths. By using the sequences of PSA, KLK2, NESl and protease M and the alignment program BLAST 2 (37), the relative positions of these genes on the contiguous map were located. Gene prediction analysis
For exon prediction analysis of the whole genomic area, a number of different computer programs were used Originally all these programs were tested using the known genomic sequences of the PSA, protease M and NESl genes The most reliable computer programs GeneBuilder (gene predιctιon)[http://125. itba.mi.cnr.it/~webgene/genebuilder.html ] GeneBuilder (exon prediction) [http://125.itba.mi.cnr.it/~webgene/genebuilder.htm]] , Grail 2 [httpV/compbio. ornl.gov ], and GENEID-3 [httpV/apolo. imim.es/geneid htmll] were selected for further use Protein homology searching Putative exons of the newly identified gene were first translated to the corresponding amino acid sequences BLAST homology searching for the proteins encoded by the exons were performed using the BLASTP program and the GenBank databases (37) Searching expressed sequence tags (ESTs)
Sequence homology searching was performed using the BLASTN alogπthm (37) on the National Center for Biotechnology Information web server (http //www ncbi nlm nih.gov/BLAST/) against the human EST database (dbEST) Clones with > 95% homology were obtained from the I.M.A G.E (38) consortium through Research Genetics Inc, Huntsville, AL and from The Institute for Genomic Research (TIGR) (http://WWW.TIGR.ORG/ tdb/tdb.html) (Table 7). Clones were propagated, purified and then sequenced from both directions with an automated sequencer, using insert-flanking vector primers Breast cancer cell line and stimulation experiments
The breast cancer cell line BT-474 was purchased from the American Type Culture Collection (ATCC), Rockville, MD. BT-474 cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic tlasks, to near contluency The cells were then ahquoted into 24-well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 108 M Cells stimulated with 100% ethanol were included as controls The cells were cultured for 24 hours, then harvested for mRNA extraction Reverse transcriptase polymerase chain reaction
Total RNA was extracted from the breast cancer cells using Tπzol reagent (Gibco BRL) following the manufacturer's instructions RNA concentration was determined spectrophotometπcally 2 μg of total RNA was reverse transcribed into first strand cDNA using the Superscript™ preamphfication system (Gibco BRL) The final volume was 20 μl Based on the combined information obtained from the predicted genomic structure of the new gene and the EST sequences, two gene-specific primers were designed (Table 8), PCR was carried out in a reaction mixture containing 1 μl of cDNA, 10 mM Tπs-HCl (pH 8 3), 50 mM KCl, 1 5 mM MgCl2, 200 μM dNTPs (deoxynucleoside triphosphates), 150 ng of primers and 2 5 units of Amp Taq Gold DNA polymerase (Roche Molecular Systems, Branchburg, NJ, USA) on a Perk -Elmer 9600 thermal cycler The cycling conditions were 94°C for 9 minutes to activate the Taq Gold DNA polymerase, followed by 43 cycles of 94°C for 30 s, 63°C for 1 minute and a final extension at 63°C for 10 min Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining All primers for RT-PCR spanned at least 2 exons to avoid contamination by genomic DNA
Tissue expression of KLK-Ll
Total RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as descπbed above for the tissue culture experiments and used for PCR reactions with the primers described in Table 8 (SEQ ID Nos 5-12) Tissue cDNAs were amplified at various dilutions Cloning and sequencing of the PCR products
To verify the identity of the PCR products, they were cloned into the pCR 2 1-TOPO vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions The inserts were sequenced from both directions using vector-specific primers, by an automated DNA sequencer Results Identification of the prostase/KLK-Ll gene
The exon prediction strategy of the 300Kb DNA sequences around chromosome 19ql3 3 - ql3 4 identified a novel gene with a structure reminiscent of a serine protease The major features of this gene were its homology, at the amino acid and DNA level, with other human kalhkrein genes, the conservation of the catalytic triad (histidine, aspartic acid, and serine), the number of exons and the complete conservation of the intron phases EST sequence homology search
EST sequence homology search of the putative exons obtained from the gene prediction programs (as described above) against the human EST database (dbEST) revealed five expressed sequence tags (ESTs) with >95 % identity to the putative exons ot the gene (Table 7) Positive clones were obtained and the inserts were sequenced trom both directions Alignment was used to compare between the EST sequences and the exons predicted by the computer programs, and final selection ot the exon-intron splice sites was made according to the EST sequences Furthermore, many of the ESTs were overlapping, further ensuring the accuracy of the data
The coding sequence of the klk-L2 gene is shown in SEQ ID NO 1 and GenBAnk Accession # AF135023 The exons of the gene are as follows exon 1 (939-999), exon 2 (2263-2425), exon 3 (2847- 3097), exon 4 (3181-3317), and exon 5 (4588-4740) The amino acid sequence of KLK-L2 proteins are shown in SEQ ID Nos 2 and 3 Mapping and chromosomal localization of prostase /KLK-Ll gene
Alignment of the prostase/ KLK-Ll sequence and the sequences of other known kalhkrem genes withm the 300 Kb area of the contigs constructed at the Lawrence Livermore National Laboratory enabled precise localization of all genes and to determine the direction of transcription, as shown in Figure 2 The distance between PSA and KLK2 genes was calculated to be 12,508 bp The prostase/KLK-Ll gene is 26,229 bp more telomeric to KLK2 and transcribes in the opposite direction The zyme gene is about 51 Kb more telomeric to the prostase gene and transcribes in the same direction The human stratum corneum chymotryptic enzyme gene, the neuropsin gene and the NES 1 gene are all further telomeric to zyme and all transcribe in the same direction as zyme Tissue expression of the prostase/KLK-Ll gene The tissues that express the prostase/KLK-Ll gene were assessed by RT-PCR The experiments were performed at various dilutions of the cDNAs to obtain some information about the relative levels of expression RT-PCR for actin was used as a positive control and RT-PCR for the PSA cDNA was used as another positive control with tissue restricted specificity Positive ESTs for prostase/KLK-Ll were used as controls for the PCR procedure The PSA gene was found to be highly expressed in the prostate, as expected, and to a lower extent in mammary and salivary glands as also expected from recent literature reports (24, 25) Very low expression of PSA in the thyroid gland, trachea and testis was also found, a finding that accords with recent RT-PCR data by others (26)
The tissue expression of prostase/KLK-Ll is summarized in Table 9 and Figure 3 This protease is primarily expressed in the prostate, testis, adrenals, uterus, thyroid, colon, central nervous system and mammary tissues, and, at much lower levels in other tissues The specificity of the RT-PCR procedure was verified for prostase/KLK-Ll by cloning the PCR products from mammary, testicular and prostate tissues and sequencing them One example with mammary tissue is shown in Figure 4 All cloned PCR products were identical in sequence to the cDNA sequence reported for the prostase/KLK-Ll Hormonal regulation of the prostase/KLK-Ll gene The steroid hormone receptor-positive breast carcinoma cell line BT-474 was used as a model system to evaluate whether prostase/KLK-Ll expression is under steroid hormone regulation As shown in Figure 5, the controls worked as expected I e , actin positivity without hormonal regulation in all cDNAs, only estrogen up-regulation of the pS2 gene and up-regulation of the PSA gene by androgens and piogestms Prostase/KLK-Ll is up-regulated primarily by androgens and progestins, similarly to PSA This up-regulation was dose-dependent and it was evident at steroid hormone levels >10 l0 M DISCUSSION
The KLK3 gene encodes for PSA, a protein that currently represents the best tumor marker available (24) Since in rodents there are so many kalhkrein genes, the restriction of this family to only 3 genes in humans was somewhat surprising More recently, new candidate kalhkrein genes in humans have been discovered, including NESl (13) and zyme/protease M neurosin (10-12) The known kailikreins and the newly discovered kalhkrein-hke genes share the following similarities (a) they encode serine proteases (b) they have five coding exons (c) they share significant DNA and protein homologies with each other (d) they map in the same locus on chromosome 19ql3 3-ql3 4, a region that is structurally similar to an area on mouse chromosome 7, where all the mouse kalhkrein genes are localized (e) they appear to be regulated by steroid hormones Prostase/KLK-Ll is a member of the same family since these common characteristics are also shared by the newly discovered gene
The exact localization of the KLK-Ll gene and its position in relation to other genes in the area (Figure 2) was determined Prostase/KLK-Ll lies between KLK2 and zyme
Irwin et al (27) have proposed that the serine protease genes can be classified into five different groups according to intron position The established kailikreins ( KLKl, KLK2, and PSA), trypsinogen and chymotrypsinogen belong to a group that has (1) an intron just downstream from the codon for the active site histidine residue, (2) a second intron downstream from the exon containing the codon for the active site aspartic acid residue, and (3) a third intron just upstream from the exon containing the codon for the active site serine residue As seen in Figure 6, the genomic organization of prostase/KLK-Ll gene is very similar to this group of genes The lengths of the coding parts of exons 1-5 are 61,163, 263, 137 and 153 bp, respectively, which are close or identical to the lengths of the exons of the kalhkrem genes and also, similar or identical to those of other newly discovered genes in the same chromosomal region like the NES1(14), zyme/protease M neurosin (10-12) and neuoropsin (28) genes
The sensitive RT-PCR protocol reveals that the KLK-Ll enzyme is expressed m prostatic tissue and it is also expressed in significant amounts in other tissues, including testis, female mammary gland, adrenals, uterus, thyroid, colon, brain, lung and salivary glands (Figure 3 and Table 9) The specificity of the RT-PCR primers was verified by sequencing the obtained PCR products, with one example shown in Figure 4 (SEQ ID NO 4) Tissue culture studies with the breast carcinoma cell line BT-474 further confirm not only the ability of these cells to produce prostase/KLK-Ll but also its hormonal regulation (Figure 5) An interesting theme is now developing involving the group of homologous genes on chromosome 19ql3 3(PSA, KLK2, prostase, zyme, and NESl) The combined data suggest that all of them are expressed in prostate and breast tissues, and all of them are hormonally regulated All these genes may be part of a cascade pathway that plays a role in cell proliferation, differentiation or apoptosis by regulating (positively or negatively) growth factors or their receptors or cytokines, through proteolysis (30) Also interesting is the linkage of locus 19ql3 to solid tumors and ghomas (31) which raises the possibility that some of the genes in the region may be disrupted by rearrangements The KLK-1L gene encodes tor a serine protease that shows homology with other membeis ot the kal krein gene family and maps to the same chromosomal location. Many structural features of the kailikreins aie conserved in prostase/KLK-Ll . The precise mapping of this gene between the two known genes KLK2 and zyme is presented It is further demonstrated that prostase/KLK-Ll is expressed in many tissues, in addition to the prostate, including the female breast. This gene is also herein referred to as 'prostase'. It has been further demonstrated, using breast carcinoma cell lines, that prostase/KLK-Ll can be produced by these cells and that its expression is significantly up-regulated by androgens and progestins Based on information for other homologous genes in the area ( PSA, zyme, and NESl), prostase/KLK-Ll may be involved in the pathogenesis and/or progression of prostate, breast and possibly other cancers Example 3
IDENTIFICATION OF THE KLK-L2 GENE Materials and Methods DNA sequence on chromosome 19
Sequencing data of approximately 300Kb of nucleotides on chromosome 19ql3.3-ql3 4 was obtained from the web site of the Lawrence Livermore National Laboratory (LLNL) (http://www- bio.llnl.gov/genome/genome.html). This sequence was in the form of 9 contigs of different lengths. A restriction analysis study of the available sequences was performed using the "WebCutter" computer program (httpV/www.firstmarket. com/cutter/cut2.html) and with the aid of the EcoRI restriction map of this area (also available from the LLNL web site) an almost contiguous stretch of genomic sequences was constructed. The relative positions of the known kalhkrein genes: PSA (GenBank accession # X14810 ), KLK2 (GenBank accession # M18157), and zyme (GenBank accession # U60801) was determined using the alignment program BLAST 2 (37) New Gene Identification
A number of computer programs were used to predict the presence of putative new genes in the genomic area of interest. These programs were initially tested using the known genomic sequences of the
PSA, protease M and NESl genes. The most reliable computer programs GeneBuilder (gene prediction) (http://125.itba.mi.cnr.it ~webgene/genebuilder.html) GeneBuilder (exon prediction)
(http://125.itba mi.cnr.it/~webgene/genebuilder.html). Grail 2 (http://compbιo ornl.gov) and GENEID-3 (http.Z/apolo imim.es/geneid html) were selected for further use Expressed sequence tag (EST) searching
The predicted exons of the putative new gene were subjected to homology search using the BLASTN algorithm (37) on the National Center for Biotechnology Information web server (http://www ncbi.nlm.nih.gov/BLAST/) against the human EST database (dbEST). Clones with > 95% homology were obtained from the I.M.A.G.E. consortium (38) through Research Genetics Inc, Huntsville, AL (Table 10) The clones were propagated, purified and sequenced from both directions with an automated sequencer, using insert-flanking vector primers Rapid amplification of cDNA ends (5' RACE) According to the EST sequence data and the predicted sti uctuie of the gene, two gene-specitic primers were designed (Rl & R2) (Table 11) Two iounds ot RACE reactions (nested PCR) were performed with 5μl Marathon Ready™ cDNA of human testis (Clontech, Palo Alto, CA, USA) as a template The reaction mix and PCR conditions were conducted according to the manufacturer's recommendations In brief, denaturation was done for 5 min at 94°C followed by 94° C for 5 sec followed by 72°C for 2 min for 5 cycles, then 94°C for 5 sec followed by 70° C for 2 min for 5 cycles then 94°C for 5 sec followed by 65°C for 2 mm for 30 cycles for the first reaction and 25 cycles for the nested PCR reaction Tissue expression Total RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as described below for the tissue culture experiments and used for PCR reactions with the primers described in Table 11 (SEQ ID Nos 9-12, 15-20) Tissue cDNAs were amplified at various dilutions Breast cancer cell line and hormonal stimulation experiments The breast cancer cell line BT-474 was purchased from the American Type Culture Collection
(ATCC), Rockville, MD Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency The cells were then a quoted into 24-well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 10 s M Cells stimulated with 100% ethanol were included as controls The cells were cultured for 24 hours, then harvested for mRNA extraction Reverse transcriptase polymerase chain reaction Total RNA was extracted from the breast cancer cells using Tπzol reagent (Gibco BRL) following the manufacturer's instructions RNA concentration was determined spectrophotometπcally 2 μg of total RNA was reverse-transcribed into first strand cDNA using the Superscript™ preamplification system (Gibco BRL) The final volume was 20 μl Based on the combined information obtained from the predicted genomic structure of the new gene and the EST sequences, two gene-specific primers were designed (Table 1 1) and PCR was carried out in a reaction mixture containing 1 μl of cDNA, 10 mM Tπs-HCl (pH 8 3),
50 mM KCl, 1 5 mM MgCl2, 200 μM dNTPs (deoxynucleoside triphosphates), 150 ng of primers and 2 5 units of AmpliTaq Gold DNA polymerase (Roche Molecular Systems, Branchburg, NJ, USA) on a Perkin- Elmer 9600 thermal cycler The cycling conditions were 94°C for 9 minutes to activate the Taq Gold DNA polymerase, followed by 43 cycles of 94°C for 30 s, 63°C for 1 minute and a final extension at 63°C for 10 mm Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining All primers for RT-PCR spanned at least 2 exons to avoid contamination by genomic DNA To verify the identity ot the PCR products, they were cloned into the pCR 2 1-TOPO vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions The inserts were sequenced from both directions using vector-specific primers, with an automated DNA sequencer Structure analysis Multiple alignment was performed using the Clustal X software package available at ftp://ftp ebi.ac. uk/pub/software/dos/clustalw/clustalx/ (clustalxl .64b.msw exe) and the multiple alignment program available from the Baylor College of Medicine (BCM), Houston, TX, USA (kiwi.imgen.bcm.tmc edu:8808/search-launcher/launcher/html). Phylogenetic studies were performed using the Phy p software package available at: http://evolutιon genetics, washington.edu phyhp/getme.html. Distance matrix analysis was performed using the "Neighbor-Joining/UPGMA" program and parsimony analysis was done using the "Protpars" program. Hydrophobicity study was performed using the BCM search launcher programs (http://dot.imgen.bcm.tmc. edu:9331/seq-search/struc-predιct.html). Signal peptide was predicted using the "SignalP" server (http://www.cbs.dtu.dk/services/ signal). Protein structure analysis was performed by "SAPS" (structural analysis of protein sequence) program (http://dot.imgen.bcm.tmc. edu:9331/seq- search/struc-predict.html) RESULTS
Computer analysis of the genomic sequence predicted a putative new gene consisting of four exons. This gene was detected by all programs used and all exons had high prediction scores. EST sequence homology search of the putative exons against the human EST database (dbEST) revealed nine expressed sequence tag (EST) clones from different tissues with >95 % identity to the putative exons of the gene (Table 10). Positive clones were obtained and the inserts were sequenced from both directions The "Blast 2 sequences" program was used to compare the EST sequences with the predicted exons, and final selection of the exon-intron splice sites was done according to the EST sequences. The presence of many areas of overlap between the various EST sequences allowed further verification of the structure of the new gene. The coding sequence of the gene is shown in SEQ. ID. NO. 13 and GenBank Accession
#AF135028. The 3' end of the gene was verified by the presence of poly A stretches that are not present in the genomic sequence at the end of two of the sequenced ESTs One of the sequenced ESTs revealed the presence of an additional exon at the 5 'end The nucleotide sequence of this exon matches exactly with the genomic sequence. To further identify the 5' end of the gene, 5' RACE was performed but no additional sequence could be obtained However, as is the case with other kailikreins, the presence of further upstream untranslated exon(s) could not be excluded. The amino acid sequence of KLK-L2 is shown in SEQ
ID. No. 14.
Mapping and chromosomal localization of the KLK-L2 gene
Alignment of KLK-L2 gene and the sequences of other known kalhkrein genes within the 300 Kb area of interest enabled precise localization of all genes and determination of the direction of transcription, as shown by the arrows in Figure 8 The PSA gene was found to be the most centromeric, separated by
12,508 base pairs (bp) from KLK2, and both genes are transcribed in the same direction (centromere to telomere) The prostase/KLK-Ll gene is 26,229 bp more telomeric and transcribes in the opposite direction, followed by KLK-L2 The distance between KLK-Ll and KLK-L2 is about 35 Kilobases (Kb) The zyme gene is 5,981 bp more telomeric and the latter 3 genes are all transcribed in the same direction (Figure 8)
Structural characterization of the KLK-L2 gene and its protein product The KLK-L2 gene, as presented in Figure 7, is formed of 5 coding exons and 4 intervening introns, spanning an area of 9,349 bp of genomic sequence on chromosome 19ql3 3-ql3 4 The lengths of the exons are 73, 262, 257,134, and 156 bp, respectively The intron/exon splice sites (mGT AGm) and their flanking sequences are closely related to the consensus splicing sites (-mGTAAGT CAGm-) (32) The presumptive protein coding region of the KLK-L2 gene is formed of 879 bp nucleotide sequence encoding a deduced 293-amιno acid polypeptide with a predicted molecular weight of 32 KDa There are two potential translation initiation codons (ATG) at positions 1 and 25 of the predicted first exon (numbers refer to SEQ ID NO . 13 and GenBank Accession #AF135028) It is assumed that the first ATG will be the initiation codon, since (1) the flanking sequence of that codon (GCGGCCATGG) matches closely with the Kozak consensus sequence for initiation of translation (GCC A/G CCATGG) (33) and is exactly the same as that of the homologous zyme gene At this initiation codon, the putative signal sequence at the N- terminus is similar to other trypsin-hke serine proteases (prostase and EMSP) (Figure 9) The cDNA ends with a 328 bp of 3' untranslated region containing a conserved poly adenylation signal (AATAAA) located 11 bp up-stream of the poly A tail ( at a position exactly the same as that of the zyme poly A taιl)(l 1)
A hydrophobicity study of the KLK-L2 gene shows a hydrophobic region in the N-terminal region of the protein (Figure 10), suggesting that a presumed signal peptide is present By computer analysis, a 29-amino acid signal peptide is predicted with a cleavage site at the carboxyl end of Ala29 For better characterization of the predicted structural motif of the KLK-L2 protein, it was aligned with other members of the kalhkrein multi-gene family, (Figure 9), and the predicted signal peptide cleavage site was found to match with the predicted signal cleavage sites of zyme (11), KLK1(1), KLK2 (8), and KLK-Ll Also, sequence alignment supports, by analogy, the presence of a cleavage site at the carboxyl end of Ser66, which is the exact site predicted for cleavage of the activation peptide of all the other kailikreins aligned in Figure 9 Interestingly, the starting amino acid sequence of the mature protein (I I N G (S) D C ) is conserved in the prostase and enamel matrix serine proteinase 1 (EMSP) genes Thus, like other kailikreins, KLK-L2 is likely also synthesized as a preproenzyme that contains an N-terminal signal peptide (prezymogen) followed by an activation peptide and the enzymatic domain
The presence of aspartate (D) in position 239 suggests that KLK-L2 will possess a trypsin-like cleavage pattern like most of the other kailikreins (e g , KLKl , KLK2, TLSP, neuropsin, zyme, prostase, and EMSP) but different from PSA which has a serine (S) residue in the corresponding position, and is known to have a chymotrypsin like activity (Figure 9) The dotted region in Figure 9 indicates an 11-amιno acid loop characteristic of the classical kailikreins (PSA, KLKl, and KLK2) but not found in KLK-L2 or other members of the kallikrein-hke gene family (11) Homology with the kalhkrein multi-gene family
The mature 227-amιno acid sequence of the predicted protein was aligned against the GenBank database and the known kailikreins using the "BLASTP" and "BLAST 2 sequence" programs KLK-L2 is found to have 54% amino acid sequence identity and 68% similarity with the enamel matrix serine proteinase 1 (EMSPl ) gene, 50% identity with both trypsin like serine protease (TLSP) and neuropsin genes and 47%, 46%, and 42% identity with trypsinogen, zyme, and PSA genes, respectively The multiple alignment study shows that the typical catalytic triad of serine proteases is conserved in the KLK-L2 gene (H108, D153, and S245) and, as the case with all other kailikreins, a well conserved peptide motif is found around the amino acid residues of the catalytic triad [I e , histidine (WLLTAA/7C), seπne(GDSGGP), and aspartate(-DLMLI) ] (10, 11)
Twelve cysteine residues are present in the putative mature KLK-L2 protein, ten of them are conserved in all the serine proteases that are aligned in Figure 9, and would be expected to form disulphide bridges The other two cystemes (C178 and C279 ) are not found in PSA, KLKl, KLK2 or trypsinogen, however, they are found in similar positions in prostase, EMSPl, zyme, neuropsin, and TLSP genes and are expected to form an additional disulphide bond Twenty nine "invariant" amino acids surrounding the active site of serine proteases have been described (39) Of these, twenty-six are conserved in KLK-L2 One of the non-conserved amino acids (Ser210 instead of Pro) is also found in prostase and EMSPl genes, the second (Leu103 instead of Val) is also found in TLSP gene, and the third (Val174 instead of Leu) is also not conserved in prostase or EMSPl genes According to protein evolution studies, each of these am o acid changes represents a conserved evolutionary substitution to a protein of the same group (39) Evolution of the KLK-L2 gene To predict the phylogenetic relatedness of the KLK-L2 gene with other seπne proteases, the amino acid sequences of the kalhkrein genes were aligned together using the "Clustal X" multiple alignment program and a distance matrix tree was predicted using the Neighbor-joinmg/UPGMA method (Figure 10) Phylogenetic analysis separated the classical kailikreins (KLKl, KLK2, and PSA) and grouped the KLK-L2 with KLK-Ll, EMSPl, and TLSP (40, 41) Tissue expression of the KLK-L2 gene
As shown in Table 12 and Figure 11, the KLK-L2 gene is primarily expressed in the brain, mammary gland, and testis but lower levels of expression are found in many other tissues In order to verify the RT-PCR specificity, the PCR products were cloned and sequenced Hormonal regulation of the KLK-L2 gene A steroid hormone receptor positive breast cancer cell line (BT-474) was used as a model to verify whether the KLK-L2 gene is under steroid hormone regulation PSA was used as a control known to be upregulated by androgens and progestins and pS2 as an estrogen upregulated control The results indicate that KLK-L2 is up-regulated by estrogens and progestins (Figure 12) Expression of KLK-L2 in Ovarian Tissues KLK-L2 is up-regulated (overexpressed) in ovarian tumors (Figure 13)
Discussion
With the aid of computer programs for gene prediction and the available EST database, a new gene, named KLK-L2 (for kallikrem like gene 2) was identified The 3' end of the gene was verified by the presence of "poly A" stretches in the sequenced ESTs which were not found in the genomic sequence, and the start of translation was identified by the presence of a start codon in a well conserved consensus Kozak sequence
As is the case with other kailikreins, the KLK-L2 gene is composed of 5 coding exons and 4 intervening introns and, except for the second coding exon, the exon lengths are comparable to those of other members of the kalhkrein gene family (Figure 6) The exon-intron splice junctions were identified by comparing the genomic sequence with the EST sequence and were further confirmed by the conservation of the consensus splice sequence (-mGT AGm-) (32), and the fully conserved intron phases, as shown in Figure 6. Furthermore, the position of the catalytic triad residues in relation to the different exons is also conserved (Figure 6). As is the case with most other kailikreins, except PSA and HSCCE, KLK-L2 is more functionally related to trypsin than to chymotrypsin (34). The wide range of tissue expression of KLK-L2 should not be surprising since, by using the more sensitive RT-PCR technique instead of Northern blot analysis, many kalhkre genes were found to be expressed in a wide variety of tissues including salivary gland, kidney, pancreas, brain, and tissues of the reproductive system (uterus, mammary gland, ovary, and testis) (34). KLK-L2 is highly expressed in the brain. Another kalhkrein, neuropsin, was also found to be highly expressed in the brain and has been shown to have important roles in neural plasticity in mice (35). Also, the zyme gene is highly expressed in the brain and appears to have amyloidogenic potential (11). Taken together, these data point to a possible role of KLK-L2 in the central nervous system.
It was initially thought that each kalhkrein enzyme has one specific physiological substrate However, the increasing number of substrates, which purified proteins can cleave in vitro, has led to the suggestion that they may perform a variety of functions in different tissues or physiological circumstances Serine proteases encode protein cleaving enzymes that are involved in digestion, tissue remodeling, blood clotting etc., and many of the kalhkrein genes are synthesized as precursor proteins that must be activated by cleavage of the propeptide. The predicted trypsin-hke cleavage specificity of KLK-L2 makes it a candidate activator of other kailikreins or it may be involved in a "cascade" of enzymatic reactions similar to those found in fibπnolysis and blood clotting (36)
In conclusion, a new member of the human kalhkrein gene family, KLK-L2 was characterized This gene is hormonally regulated and it is mostly expressed in the brain, mammary gland and testis. KLK- L2 may be useful as a tumor marker Example 4
Materials and methods Strategy for new gene discovery
Sequencing data of approximately 300 kb, around chromosome 19ql3 3-ql3.4, was obtained from the web site of the Lawrence Livermore National Laboratory (LLNL) (http://www-bio.llnl.gov/ genome/ genome, html). Different computer programs were used for putative new gene prediction, as previously described. RT-PCR for KLK-L3 cDNA
Total RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as desu ibed below and used tor PCR amplification A pinner set (L3 FI and L3-R1) was used to identity the presence of the gene in tissues, and the reverse primer (L -R1) was used with another primer (L3-F2) to amplify and clone the full cDNA of the gene These primer sequences are shown in Table 13 (SEQ ID Nos 9-12, 24-26) Tissue cDNAs were amplified at various dilutions Reverse transcriptase polymerase chain reaction.
2 μg of total RNA was reverse-transcribed into first strand cDNA using the Superscript™ preamplification system (Gibco BRL, Gaithersburg, MD) The final volume was 20 μl Based on the combined information obtained from the predicted genomic structure of the new gene and the EST sequence, two gene-specific primers (L3-F1 and L3-R1) were designed (Table 13, SEQ ID Nos 9-12, 24- 26) and PCR was carried out in a reaction mixture containing 1 μl of cDNA, 10 mM Tπs-HCl (pH 8 3), 50 mM KCl, 1 5 mM MgCl2, 200 μM dNTPs (deoxynucleoside triphosphates), 150 ng of primers and 2 5 units of AmphTaq Gold DNA polymerase (Roche Molecular Systems, Branchburg, NJ, USA) on a Perkin- Elmer 9600 thermal cycler The cycling conditions were 94°C for 9 minutes, followed by 43 cycles of 94°C for 30 s, 63°C for 1 minute, and a final extension at 63°C for 10 minutes Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining All primers for RT- PCR spanned at least 2 exons to avoid contamination by genomic DNA Breast cancer cell line and hormonal stimulation experiments
The breast cancer cell line BT-474 was purchased from the American Type Culture Collection (ATCC), Rockville, MD Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency The cells were then a quoted into 24-well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 108 M Cells stimulated with 100% ethanol were included as controls
The cells were cultured for 24 hours, then harvested for total RNA extraction by the Tπzol method (Gibco- BRL) cDNA was prepared and amplified as described above Control genes (PSA, pS2, and actin) were amplified as previously described herein Cloning and sequencing of the PCR products. To verify the identity of the PCR products, they were cloned into the pCR 2 1-TOPO vector
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer s instructions The inserts were sequenced from both directions using vector-specific primers, with an automated DNA sequencer Identification of positive PAC and BAC genomic clones from human genomic DNA libraries
The PCR product generated with primer set Z1S and Zl AS (Table 14, SEQ ID NOS 27-42), was purified and then labeled with 32P by the random primer method (Sambrook, supra) and used as a probe to screen a human genomic DNA BAC library, spotted in duplicate on nylon membranes, for identification of positive clones The filters were hybridized in 15% formamide, 500 mM Na2HP04, 7% SDS, 1% BSA
(w/v) at 65°C overnight, then washed sequentially with 2X SSC, IX SSC, 02X SSC, containing 0 1% SDS at 65°C, and then exposed to X-ray film as descnbed (Sambrook, supia) Positive clones were obtained, plated on selective LB medium, and then a single colony was transferred into LB broth for overnight cultures. A PAC clone positive for NESl was identified by a similar methodology as described elsewhere ( 14) PAC and BAC libraries were constructed by de Jong and associates (42). Purification of BAC and PAC DNA was done by a rapid alkaline lysis miniprep method, which is a modification of the standard Qiagen-Tip method. Positive clones were further confirmed by Southern blot analysis as described (Sambrook, supra) 5' Rapid amplification of cDNA ends (5' RACE)
According to the EST sequences and the computer-predicted structure of the KLK-L3 gene, two gene specific primers were designed. Two rounds of RACE reactions (nested PCR) were performed with 5μl Marathon Ready ™ cDNA of human testis (Clontech) as a template. The reaction mix and PCR conditions were selected according to the manufacturer's recommendations. Positive bands were gel- puπfied using Qiagen Gel Purification kits according to manufacturer's recommendations Gene-specific amplification of other genes from genomic DNA According to the published sequence of prostatic specific antigen (PSA), human renal kalhkrein
(KLKl), human glandular kallikrem (KLK2), normal epithelial cell-specific lgene (NESl), KLK-Ll, KLK- L2 and zyme genes, gene-specific primers were designed for each of these genes (Table 14) and developed polymerase chain reaction (PCR)-based amplification protocols which allowed us to generate specific PCR products with genomic DNA as a template The PCR reactions were carried out as described above but by using an annealing/extension temperature of 65°C. Structure analysis studies.
Multiple alignment was performed using the clustal X software package available af ftp.//ftp.ebι.ac.uk/pub/software/dos/clustalw/clustalx/(clustalx 1.64b.msw.exe) and the multiple alignment program available from the Baylor College of Medicine (BCM) search launcher (kιwι.ιmgen.bcm.tmc.edu-8808/search-launcher/launcher/html). Phylogenetic studies were performed using the Phyhp software package available from: (http://evolutιon.genetιcs washington.edu/phylip/getme.html) Distance matrix analysis was performed using the "Neighbor-Joining UPGMA" program and parsimony analysis was done using the "Prompters" program. Hydrophobicity study was performed using the BCM search launcher programs (http://dot.imgen.bcm.tmc edu-9331/seq-search/struc-predιct.html). Signal peptide was predicted using the SignalP WWW server (http://www cbs dtu.dk/services/ signal). Protein structure analysis was performed by SAPS (structural analysis of protein sequence) program (http.Z/dot.imgen bcm tmc.edu'9331/seq-search struc-predιct.html) Results:
Construction of a contiguous map of the human kalhkrein locus on chromosome 19ql3.3-ql3.4 Sequence information around the human chromosome 19ql3.3-ql3.4 locus (the proposed kal krein locus) is available at the Lawrence Livermore National Laboratory web site. Sequences of approximately 300 kb in length were obtained. These sequences were in the form of contigs of different lengths. A restriction analysis study of the contigs was performed using various computer programs With the aid ot the EcoRI lestπction map ot this area which is also available at the LLNL web site, the relative positions of these contigs was defined in relation to each other Some contigs were overlapping, enabling construction of a contiguous segment, however, three gaps were present <BLAST> analysis of these segments against the GenBank database (37) enabled the precise location of two classical kailikreins, namely PSA and KLK2 to be defined Other newly discovered serine proteases were localized which are homologous with the kalhkrein genes, namely protease M/zyme/neurosin ( 10, 11, 12), human stratum corneum chymotryptic enzyme ( HSSCE) (55), neuropsin (28), normal epithelial cell-specific 1 gene ( NESl) (13), trypsin-like serine protease ( TLSP) (GenBank accession # AF164623), KLK-Ll (SEQ ID NO 1) and KLK-L2 (SEQ ID NO 13) The gaps in the 300 kb genomic sequence were partially filled as follows
(a) The margins of the first gap were found to contain the 5' and 3' ends of the KLK2 gene, this gap was filled with the genomic structure of the KLK2 gene (GenBank Accession # M18157)
(b) The margins of the third gap (gaps are numbered from centromere to telomere) were found to have the 3' and 5' ends of the zyme gene mRNA sequence, thus, a radiolabeled probe specific for the zyme gene was used to screen a human BAC library and two positive clones were obtained Restriction analysis was performed, followed by Southern blotting and a fragment containing the zyme gene was obtained and sequenced, thus filling this gap
(c) The second gap (between KLK-Ll and KLK-L2 genes) still exists and the EcoRI restriction map of this area was used to approximately define its length (Figure 14) Further support for the relative locations of these genes was obtained by performing PCR reactions with gene-specific primers to screen genomic DNA clones The most centromeric group of genes (PSA, KLK2, KLK-Ll, KLK-L2 and zyme ) were found to be clustered in one genomic BAC clone, and the next group (HSCCE, neuropsιn,KLK-L3 and NES l) were found to be clustered together in another clone, as expected from the data of Figure 14 Cloning of the KLK-L3 gene
A putative new gene, formed of three exons, was predicted by computer analysis of the genomic sequence The predicted exons were subjected to sequence homology search against the human EST database (dbEST) and revealed an EST clone (GenBank accession # AA583908) which exhibited 99% homology with the putative gene This EST was obtained, purified and sequenced and the sequence was aligned by BLAST software (37) against the genomic area that contains the putative gene An additional exon, downstream of the predicted structure, was identified The 3' end of the gene was verified by (a) The presence of the serine residue (S) of the catalytic triad in a well-conserved region This highly conserved motif (GDSGGP) always occurs at the beginning of the last exon in all known kailikreins (b) The presence of a stop codon that is in frame with the predicted amino acid sequence (c) The presence of a 19-poly A stretch at the end of the EST that was not found in the genomic sequence
To verify the accuracy of the cDNA sequence of the gene, PCR reactions were performed using gene-specific primers for the first and last exons of the predicted structure of the gene (L3-F2 and L3-R1) with cDNA isolated from different human tissues as putative templates A positive band of the expected size was isolated from testis cDNA and fully sequenced Its sequence was aligned by BLAST against the genomic sequence to unequivocally define the exon/intron boundaries For further characterization of the 5' end of the gene, 5 'RACE reaction was performed using Marathon Ready cDNA from testis as a template This allowed identification of an additional exon that contains the start codon and 5' untranslated region The full sequence of the gene is shown in SEQ ID NO 21 (GenBank Accession # AF135026) and the amino acid sequences of KLK-L3 proteins are shown in SEQ ID Nos 22 and 23 Structural characterization of the KLK-L3 gene:
As shown in Figure 15, the KLK-L3 gene is formed of 5 coding exons and 4 intervening introns, although, as with other kailikreins, the presence of further upstream untranslated exon(s) could not be ruled out (14, 28) All of the exon /intron splice sites conform to the consensus sequence for eukaryotic splice sites (32) The gene further follows strictly the common structural features of the human kalhkrein multigene family, as described below
The predicted protein-coding region of the gene is formed of 753 bp, encoding a deduced ammo acid polypeptide with a predicted molecular weight of 27 5 kDa A potential translation initiation codon is found at position 28 of the predicted first exon (numbers of nucleotides refer to SEQ ID NO 21 or GenBAnk Accession # API 35026 This codon does not match well with the consensus Kozak sequence (33), however, it has a puπne at position (-3) which occurs in 97% of vertebrate mRNAs (43), and it is almost identical to the sequence of the zyme gene flanking the start codon It should also be noted that most kailikreins do not have the consensus G nucleotide in position (4-4) Nucleotides 6803-6808 (AGTAAA) closely resemble a consensus polyadenylation signal (44) and are followed by a stretch of 19 poly A nucleotides not found in genomic DNA, after a space of 14 nucleotides No other potential polyadenylation signals were discemable in the 3' untranslated region, suggesting that the above motif is indeed the polyadenylation signal The same polyadenylation signal motif was predicted for the KLKl and KLK2 genes Although the KLK-L3 protein sequence is unique, comparative analysis revealed that it is highly homologous to other members of the kalhkrein multigene family KLK-L3 shows 40% protein identity with the TLSP gene product and 38% and 33% identity with the KLK-L2 and KLKl proteins, respectively Hydrophobicity analysis revealed that the amino-terminal region is quite hydrophobic (Figure 16), consistent with the possibility that this region may harbor a signal sequence, analogous to other serine proteases Computer analysis of the aminoacid sequence of KLK-L3 predicted a cleavage site between amino acids 19 and 20 (GWA-DT) Sequence alignment (Figure 17) also revealed a potential cleavage site (Arg ), at a site homologous to other serine proteases (lysine (K) or arginme (R) is present in most cases) Several evenly distributed hydrophobic regions throughout the KLK-L3 polypeptide are consistent with a globular protein, similar to other kailikreins and serine proteases The dotted region in Figure 17 indicates an 11 -amino acid loop characteristic of the classical kailikreins (PSA, KLKl, and KLK2) but not found in KLK-L3 or other members of the kalhkrein multi-gene family (11, 41)
Twenty nine "invariant" amino acids surrounding the active site of serine proteases have been described Of these, twenty-six are conserved in KLK-L3 One of the unconserved amino acids (Ser168 mstead ot Pro) is also found in pi stase, KLK-L2 and enamel matrix serine pioteinase (EMSPl) genes The second (Leu instead ot Val) is also found in TLSP and KLK-L2 genes, and the third is Ala26 instead of Gly According to protein evolution studies, each of these changed amino acids represents a conserved evolutionary change to a protein of the same group (45) Twelve cysteine residues are present in the putative mature KLK-L3 protein, ten of them are conserved in all the serine proteases that are aligned in Figure 17, and would be expected to form disulphide bridges The other two (C136 and C238) are not found in PSA, KLKl, KLK2 or trypsinogen, however, they are found in similar positions in prostase, HSCCE, zyme neuropsin, and TLSP genes and are expected to form an additional disulphide bond
To predict the phylogenetic relatedness of the KLK-L3 gene with other serine proteases, the amino acid sequences of the kalhkrein genes were aligned together using the "Clustal X" multiple alignment program and a distance matrix tree was predicted using the Neighbor-joimng UPGMA method (Figure 18) Phylogenetic analysis separated the classical kailikreins (KLKl, KLK2, and PSA) and grouped KLK-L3 with TLSP, neuropsin, zyme, HSCCE and prostase/KLK-Ll, consistent with previously published studies (11, 41) Tissue expression and hormonal regulation of the KLK-L3 gene
As shown in Figure 19, the KLK-L3 gene is primarily expressed in thymus, testis, spinal cord, cerebellum, trachea, mammary gland, prostate, brain, salivary gland, ovary and skin (the latter two tissues are not shown in the figure) Lower levels of expression are seen m fetal brain, stomach, lung, thyroid, placenta, liver, small intestine, and bone marrow No expression was seen in uterus, heart, fetal liver, adrenal gland, colon, spleen, skeletal muscle, pancreas, and kidney In order to verify the RT-PCR specificity, representative PCR products were cloned and sequenced Figure 20 shows that KLK-L3 gene is regulated by steroid hormones in the human breast cancer cell line BT-474 DISCUSSION
A human kalhkrein gene locus has been defined, and the first detailed map describing the relative positions of the kailikreins and other kallikrein-hke genes has been constructed (Figure 14) This map is consistent with previous reports on the localization of the classical kailikreins and the approximate mapping of some new kailikreins by radiation hybrid and FISH techniques (9, 14, 67) It should be noted, however, that the lengths of certain segments of this map (as depicted in Figure 14) are dependent on the EcoRI restriction map of the area and are measured in terms of approximate kb units Also, the measure of intervals between genes may change slightly in the future, since some kailikreins may have extra 5'exon(s) that have not as yet been identified Kailikreins with verified 5 '-untranslated exons include NESl (14), zyme, and neuropsin (35) This map is also directional, it indicates that PSA and KLK2 genes are transcribed in the same direction (centromere to telomere) and that the rest of the kalhkrein-hke genes are transcribed in the reverse direction (Figure 14) An early report indicated that KLKl is located approximately 31 kb centromeric to PSA (9) The map described extends only 24 kb centromeric to PSA, and for this reason, KLKl was not precisely localized Thus, the exact location of the KLKl gene is still to be defined from linear chromosome 19 sequencing data The possibility still exists that this locus is extended further, and that other kallikrein-hke genes may be located upstream ot PSA or downstream from TLSP
Traditionally, kailikreins are characterized by their ability to liberate lysyl-bradykinin (kal din) tiom kininogen (2) In humans, only KLKl meets this "functional" definition KLK2 and KLK3 are assigned to the same family based on the strong structural similarities of the genes and proteins and the close localization of these genes on the same chromosomal region (20) More recently, a new structural concept has emerged to describe kailikreins Richards and co-workers introduced the concept of a "kalhkrein multigene family" in mice, to refer to these genes (20, 21) This definition is not based much on the specific enzymatic function of the gene product, but more on its sequence homology and its close linkage on mouse chromosome 7 Irwin et al (27) proposed that the serine protease genes can be classified into five different groups according to intron position as discussed above The results indicate the presence of some more common structural features that are found in all kailikreins (including the newly identified KLK-L3 gene) (1) All genes are formed of 5 coding exons and 4 intervening introns (with the possibility that some genes may have extra 5' untranslated exon(s) (24, 31, 35) (Figure 21) (2) The exon lengths are usually comparable (3) The intron phases are always conserved ( I-II-I-O) (see Figure 21 for description of intron phases) (4) These genes are clustered in the same chromosomal region, apparently without any intervening non kallikrein-hke genes (Figure 14) Thus, all the recently identified serine proteases that are present m this region ( zyme, HSCCE, neuropsin, NESl, prostase/KLK-Ll, KLK-L2 and TLSP), together with the newly identified kallikrein-hke gene (KLK-L3), could be considered members of the expanded human kalhkrein multigene family
The chromosomal band 19ql3 is nonrandomly rearranged in a variety of human solid tumors including ovarian cancers (46), and the currently available data indicate that the kalhkrein gene locus is related to many malignancies At least three kalhkrein genes (PSA, zyme and NESl) are down regulated in breast cancer (10, 13, 47, 48), and NESl appears to be a novel tumor suppressor gene (29) Furthermore, PSA exhibits potent antiangiogenic activity (49) It is possible that some of these kailikreins are involved in a cascade pathway, similar to the coagulation or apoptotic process, whereby pro-forms of proteolytic enzymes are activated and then act upon downstream substrates Such activity was found for the KLK2 gene product which acts upon and activates pro PSA (50, 51)
The expanded human kalhkrein gene family has similar number of members as the rodent family of genes Some new compelling data have raised the possibility that at least some of these genes behave as tumor suppressors (29), as negative regulators of cell growth (52), as antiangiogenic (49) and apoptotic (53) molecules The paramount diagnostic value of some members is also well-established (24, 54) For these reasons, it is important to check all members of this family of genes as potential diagnostic or prognostic markers or as candidate therapeutic targets The newly identified KLK-L3 gene is expressed in many tissues, including skin, thymus, central nervous system, breast, prostate, and testis The wide range of tissue expression of KLK-L3 should not be surprising since, by using the more sensitive RT-PCR technique, many kalhkrein genes were found to be expressed in a wide variety of tissues For example, PSA, KLK2, prostase/KLK-Ll, and KLK-L2 are now known to be expiessed in breast and many other tissues (30, 54)
Like many other kailikreins, KLK-L3 is regulated by steroid hormones but in a more complex fashion than PSA and KLK2 which are up-regulated by androgens and progestins (71). In the breast carcinoma cell line studied, KLK-L3 appears to be up-regulated by progestins > estrogens > androgens (Figure 20)
Example 5 Materials and Methods DNA sequence on chromosome 19 and prediction of new genes
Sequencing data of approximately 300Kb of nucleotides, around chromosome 19ql3.3-ql3.4, was obtained from the web site of the Lawrence Livermore National Laboratory (LLNL) (http://www http://www-bio.llnl.gov/genome/genome.html) and an almost contiguous stretch of genomic sequences was constructed. A number of computer programs were used to predict the presence of putative new genes in this genomic area.
Expressed sequence tag (EST) searching The predicted exons of the putative new gene were subjected to homology search using the
BLASTN algorithm (37) on the National Center for Biotechnology Information web server (http://www ncbi.nlm nih gov/BLAST/) against the human EST database (dbEST). Clones with > 95% homology were obtained from the I.M.A.G.E. consortium (38) through Research Genetics Inc, Huntsville, AL. The clones were propagated, purified and sequenced from both directions with an automated sequencer, using lnsert- flanking vector primers
Rapid amplification of cDNA ends (3' RACE)
According to the EST sequence data and the predicted structure of the gene, two gene-specific primers were designed and two rounds of RACE reactions (nested PCR) were performed with 5μl Marathon Ready™ cDNA of human testis (Clontech, Palo Alto, CA, USA) as a template. The reaction mix and PCR conditions used were according to the manufacturer's recommendations.
Tissue expression
Total RNA isolated from 26 different human tissues was purchased from Clontech. cDNA was prepared as described below, and used for PCR reactions with different sets of primers (Table 15, SEQ.IDNOs 46-55, 9-12) Tissue cDΝAs were amplified at various dilutions Breast cancer cell line and hormonal stimulation experiments
The breast cancer cell line BT-474 was purchased from the American Type Culture Collection (ATCC), Rockville, MD. Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency. The cells were then a quoted into 24- well tissue culture plates and cultured to 50% confluency. 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 10"8 M Cells stimulated with 100% ethanol were included as controls The cells were cultured tot 24 houis, then harvested for mRNA extraction Reverse transcriptase polymerase chain reaction
Total RNA was extracted fiom the breast cancer tissues and cell lines using Tπzol™ reagent (Gibco BRL) following the manufacturer's instructions RNA concentration was determined spectrophotometπcally. 2 μg of total RNA was reverse-transcribed into first strand cDNA using the Superscript™ preamphfication system (Gibco BRL). The final volume was 20 μl. Based on the combined information obtained from the predicted genomic structure of the new gene and the EST sequences, two gene-specific primers were designed (L4-F1 and L4-R1, see Table 15, SEQ.IDNOs. 46 and 47) and PCR was carried out in a reaction mixture containing 1 μl of cDΝA, 10 mM Tπs-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM dΝTPs (deoxynucleoside triphosphates), 150 ng of primers and 2.5 units of AmpliTaq Gold DΝA polymerase (Roche Molecular Systems, Branchburg, ΝJ, USA) on a Perkin-Elmer 9600 thermal cycler. The cycling conditions were 94°C for 9 minutes to activate the Taq Gold DΝA polymerase, followed by 43 cycles of 94°C for 30 s, 63°C for 1 minute and a final extension at 63°C for 10 min Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. All primers for RT-PCR spanned at least 2 exons to avoid contamination by genomic DΝA.
To verify the identity of the PCR products, they were cloned into the pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The inserts were sequenced from both directions using vector-specific primers, with an automated DΝA sequencer Normal and malignant breast tissues
Normal breast tissues were obtained from women undergoing reduction mammoplasties. Breast tumor tissues were obtained from female patients at participating hospitals of the Ontario Provincial Steroid Hormone Receptor Program The normal and tumor tissues were immediately frozen in liquid nitrogen after surgical resection and stored in this manner until extracted The tissues were pulverized with a hammer at dry ice temperature and RNA was extracted as described above, using Tπzol reagent.
Structure analysis
Multiple alignment was performed using the Clustal X software package available af ftp://ftp.ebi.ac.uk/pub/software/dos/clustalw/ clustalx/fclustalxl 64b. msw exe] and the multiple alignment program available from the Baylor College of Medicine (BCM), Houston, TX, USA [kiwumgen.bcm.tmc edu.8808/search-launcher/launcher/html] Phylogenetic studies were performed using the Phylip software package available at: http://evolutιon. genetics washington.edu/phyhp/getme.html. Distance matrix analysis was performed using the "Neighbor- Joining/UPGMA" program and parsimony analysis was done using the "Protpars" program Hydrophobicity study was performed using the BCM search launcher programs [http.//dot. imgen.bcm.tmc.edu 9331/seq-search/struc-predιct.html]. Signal peptide was predicted using the "SignalP" server [http://www.cbs. dtu.dk/services/ signal]. Protein structure analysis was performed by the "SAPS" (structural analysis of protein sequence) program [http://dot.imgen.bcm.tmc.edu- 9331/seq- search struc-predict.html]. Results Cloning of the KLK-L4 gene
Computer analysis ot the genomic sequence around chromosome 19 q 13 3-q 13 4 predicted a putative new gene formed of at least 3 exons To experimentally verify the existence of this gene, the putative exons were subjected to sequence homology search against the human expressed sequence tag (EST) database (dbEST), and four EST clones with > 97% homology were identified (Table 16) All ESTs were cloned from testicular tissue These clones were obtained and inserts were sequenced from both directions Sequences were then compared with the computer-predicted structure and final selection of the intron/exon splice sites was made according to the EST sequences.
As shown in Figure 22, three ESTs match almost perfectly with the predicted 3 exons (exons 3, 4, 5) of the gene and one EST matches perfectly with predicted exons 3 and 5. However, each of the ESTs extends further upstream with different exonic patterns, suggesting the presence of different splice variants Attempts to translate these clone sequences demonstrated the presence, in some ESTs, of interrupting stop codons in all three possible reading frames. A homology search of the three common exons against the GenBank database revealed a cDNA sequence from the German Human Genome Project. This clone has an identical exon 2 as the long form of KLK-L4 gene [this form will be described below] but has an extended exon 3 that ends with a stop codon (Figure 22). This clone was isolated from uterine tissue and is translated by software into a truncated protein product of 196 amino acids which is followed by a 3' untranslated region [GenBank accession # AL050220]
Screening of cDNAs from 26 different tissues by RT-PCR, using gene-specific primers for exons 3 and 5 [L4-F1 and L4-R1] (Table 15 & Figure 22) revealed that this gene is expressed in many tissues Four tissues that show the highest level of expression [salivary gland, mammary gland, prostate, and testis] (Figure 23) and uterus [the EST clone AL050220 was isolated from this tissue] were selected for identification of the full structure of the gene. Different PCR reactions were performed using one reverse primer (L4-R1) together with each of the forward primers located in upstream exons that were found in the different EST clones [primers L4-B, L4-D, L4-E] (Table 15 & Figure 22) The PCR reactions were performed under different experimental conditions, using the EST clones as positive controls, and the PCR products were sequenced None of these forms were found in any of the tissues, except in testis where all three forms were found
By RT-PCR of the KLK-L4 gene using primers L4-R1 and L4-F1, it was found that the gene is expressed in a wide variety of tissues (Figure 23). In order to obtain the structural forms that exist in these tissues, a homology study was performed Aligning the predicted polypeptide of the KLK-L4 gene with all other kailikreins and kallikrein-hke genes, suggested, by homology, that at least two more exons should be present upstream of the predicted three exons The genomic fragment upstream of the third exon was subjected to further computer analysis for gene prediction, and exon 2 was identified based on a) a consensus exon/intron splice site b) preservation intron phase II after this exon, in agreement to intron phases of all other known kailikreins c) presence of the histidine residue of the catalytic triad (H76) surrounded by a well-conserved peptide motif [see below] just before the end of this exon d) comparable exon length to other kalhkrein genes A potential first exon was also predicted from the upstream genomic sequence, based on the preserved intron phase (phase I), and the existence ot an in-frame start codon that is located at a comparable distance [in relation to other kailikreins] from the end of this exon To verity this predicted structure, a PCR reaction was performed using one reverse primer (L4-R1) together with another forward primer that is located in the predicted first exon (primer L4-X1) (Table 15 & Figure 22) Two main PCR bands were obtained from the tissues examined, the expected 819 bp band (predominant) and an additional minor band of about 650 bp (Figure 24) Cloning and sequencing of these two bands revealed that the gene exists in two main forms in these tissues, the long form [SEQ ID No 43 or GenBank Accession No AF135024] and another form [referred to as the short KLK-L4 variant] that utilizes an upstream alternative splice donor site, located inside exon 3, thus creating an mRNA product that that is 214 bp shorter This alternative splice site causes frame-shifting of the coding region that will generate a predicted stop codon at the beginning of exon 4, giving rise to a truncated protein product that does not contain the serine residue of the catalytic triad (Figures 24 and 25)
Aligning the long KLK-L4 form with the ESTs (Figure 22) demonstrated that all ESTs utilize a different splice donor site located 80 bp downstream from the end of exon 3 These additional 80 bp contain an in-frame stop codon at nucleotide position 5505 which will lead to the formation of a shorter polypeptide product They also utilize an alternative polyadenylation signal located at position 8706 [numbers refer to SEQ ID No 43 or or GenBank Accession No AF135024] The clone from the German Genome Project utilizes another splice donor site that is located further downstream, mside tron 3, and ends up with a poly A tail without having a fourth or fifth exon The same stop codon (position 5505) will be in-frame, and therefore, a truncated protein product is predicted to be formed (Figure 22)
In order to obtain the 3 'end of the gene, a 3 'RACE reaction was performed, and an additional 375 bp fragment of 3' untranslated region, downstream from PCR primer L4-R1, was obtained This fragment was further confirmed to be present in all tissues tested, by performing a PCR reaction using primers L4-F1 and L4-R3 (Table 15 & Figure 22) This fragment ends with a putative polyadenylation signal variant (TATAAA)
Structural characterization of the KLK-L4 gene and its protein product
The long form of the KLK-L4 gene is presented in Figure 25 (SEQ ID NO 43) KLK-L4 is formed of five coding exons and four intervening introns, spanning an area of 8,905 bp of genomic sequence on chromosome 19ql3 3-ql3 4 The lengths of the coding regions are 52, 187, 269,137 and 189 bp, respectively The predicted protein coding region of the gene is formed of 831 bp, encoding a deduced
277-amino acid protein with a predicted molecular mass of 30 6 kDa (Figure 25) The intron/exon splice sites (mGT AGm, where m is any base) and their flanking sequences are agreement with the consensus splice site sequence A potential translation initiation codon is present at position 45 of the predicted first exon [numbers refer to SEQ ID No 43] The cDNA extends at least 382 bp further downstream from the stop codon and a putative polyadenylation signal (TATAAA) is present at the end of this region (Figure 25)
Hydrophobicity analysis revealed that the amino-terminal region is quite hydrophobic (Figure 26), consistent with the possibility that this region may harbor a signal sequence, analogous to other serine proteases Figure 26 also shows the presence of several evenly distributed hydrophobic regions throughout the KLK-L4 polypeptide, which are consistent with a globular protein, similar to other serine proteases ( 13) Computer analysis of the amino acid sequence of KLK-L4 predicted a cleavage site between amino acids 20 and 21 (GVS-QE) Sequence homology with other serine proteases (Figure 27) predicted another potential cleavage site (Lys25) in close proximity Most other kailikreins are activated by cleavage after arginine or lysine Thus, the protein product is very likely to be a secreted protein The dotted region in Figure 27 indicates an 11-amιno acid loop characteristic of the classical kailikreins (PSA, KLKl, and KLK2) which is not found in KLK-L4 or other members of the kalhkrein multi-gene family (11,13, 35) Amino acid sequences for KLK-L4 proteins are shown in SEQ ID NO 44 and 45 Sequence analysis of eukaryotic serine proteases indicates the presence of twenty nine invariant amino acids (39) Twenty eight of them are conserved in the KLK-L4 protein and the remaining ammo acid (Q182 instead of P) is not conserved among all other kailikreins (Figure 27) Ten cysteine residues are present in the putative mature KLK-L4 protein These are conserved in all the serine proteases that are aligned in Figure 27, and would be expected to form disulphide bridges The presence of aspartate (D) in position 239 suggests that KLK-L4 will possess a trypsin-hke cleavage pattern, similarly to most of the other kailikreins [e g , KLKl, KLK2, TLSP, neuropsin, zyme, prostase, and EMSP] but different from PSA which has a serine (S) residue in the corresponding position, and is known to have chymotrypsin like activity (Figure 27) (2,40) Mapping and chromosomal localization of the KLK-L4 gene Alignment of the KLK-L4 gene and the sequences of other known kalhkrem genes within the 300
Kb area of interest [the human kalhkrein gene family locus], enabled precise localization of all known genes and to determine the direction of transcription, as shown by the arrows in Figure 28 The PSA gene lies between KLKl and KLK2 genes and is separated by 13, 319 base pairs (bp) from KLK2, and both genes are transcribed in the same direction (centromere to telomere) All other kallikrein-hke genes are transcribed in the opposite direction KLK-L4 is 13 kb centromeric from KLK-L6 [SEQ ID NO 65], and
21 kb more telomeric to KLK-L5 [SEQ ID NO 56] Homology with the kalhkrein multi-gene family
Alignment of the amino acid sequence of the KLK-L4 protein (long form) against the GenBank database and the known kailikreins, using the BLAST algorithm (37), indicated that KLK-L4 has 51% amino acid sequence identity with the TLSP and zyme genes, 49% identity with KLK-L2 and 47% and 45% identity with PSA and KLK2 genes, respectively Multiple alignment study shows that the typical catalytic triad of serine proteases is conserved in the KLK-L4 gene (H108, D153, and S245) and, as is the case with all other kailikreins, a well conserved peptide motif is found around the ammo acid residues of the catalytic triad [l e histidine (WLLTAAHC), serine (GDSGGP), and aspartate (DLMLI)] (Figure 27) (1, 11, 13, 35) In addition, several other residues were found to be fully or partially conserved among the human kalhkre gene family, as further shown in Figure 27 To predict the phylogenetic relatedness of the KLK-L4 gene with other serine proteases, the amino acid sequences of the kalhkrein genes were aligned together using the "Clustal X" multiple alignment program and a distance matrix tree was predicted using the Neighbor- joining/UPGMA method (Figure 29) Phylogenetic analysis separated the classical kailikreins (KLKl, KLK2, and PSA) and grouped KLK-L4 with zyme, TLSP, KLK-L3, neuropsin, and NES 1 genes, consistent with previously published studies (41) and indicating that this group of genes probably arose from a common ancestral gene by duplication Tissue expression and hormonal regulation of the KLK-L4 gene
As shown in Figure 23, the KLK-L4 gene is primarily expressed in mammary gland, prostate, salivary gland and testis, but, as is the case with other kailikreins, lower levels of expression are found in many other tissues In order to verify the RT-PCR specificity, the PCR products were cloned and sequenced A steroid hormone receptor-positive breast cancer cell line (BT-474) was used as a model, to verify whether the KLK-L4 gene is under steroid hormone regulation PSA was used as a control gene, known to be up-regulated by androgens and progestins and pS2 as an estrogen up-regulated control gene in the same cell line Preliminary results indicate that KLK-L4 is up-regulated by progestins and androgens and to a lower extent by estrogens (Figure 30) Expression of KLK-L4 in breast cancer tissues and cell lines
To characterize the extent and frequency of expression of the KLK-L4 gene in breast tumors, cDNA derived from 3 normal and 19 malignant breast tissues and 3 breast cancer cell lines was used The data were interpreted by comparison of band intensities Out of the 19 tumors, KLK-L4 gene expression was undetectable in 7, lower than normal tissues in 9, comparable to the normal tissues in 1, and higher than normal tissues in 2 tumors Without hormonal stimulation, the BT-474 and T-47D cell lines had no detectable KLK-L4 mRNA, while the MCF-7 cell line was positive These preliminary results suggest that this gene is down-regulated in the majority (16/19) of breast tumors Discussion
The established kailikreins (KLKl, KLK2, and PSA), trypsinogen and chymotrypsinogen belong to a group that has (1) an intron just downstream from the codon for the active site histidine residue, (2) a second intron downstream from the exon containing the codon for the active site aspartic acid residue, and (3) a third intron just upstream from the exon containing the codon for the active site serine residue Figure 31 shows that KLK-L4 meets the above mentioned criteria, moreover, is located in close proximity to other kallikrem genes on the chromosomal locus 19ql3 3-ql3 4 (Figure 28) The preliminary findings, supporting that the KLK-L4 gene may be down-regulated in a subset of breast cancers, is not surprising There is now growing evidence that many of the kailikreins and kal krein-like genes that are clustered in the same chromosomal region (Figure 28) are related to malignancy PSA is the best marker for prostate cancer so far (24) A recent report provided evidence that PSA has antiangiogenic activity, and that this activity may be related to its function as a serine protease (49) This study suggested that other serine proteases, including new members of the kalhkrein multigene family of enzymes, should also be evaluated for potential antiangiogenic activity (49) Recent reports suggest that hK2 (encoded by the KLK2 gene) could be another useful diagnostic marker for prostate cancer (57, 58) NESl appears to be a tumor suppressor gene (29) The protease M gene was shown to be differentially expressed in primary breast and ovarian tumors ( 10), and the human stratum corneum chymotryptic enzyme has been shown to be expressed at abnormally high levels in ovarian cancer (59) Another recently identified kallikrein-hke gene, located close to KLK-L4 and tentatively named tumor- associated, differentially expressed gene- 14 (TADG14) [an alternatively spliced form of neuropsin, see Figure 28] was found to be overexpressed in about 60% of ovarian cancer tissues (59) Also, prostase/KLK-Ll, another newly discovered kallikrein-hke gene, is speculated to be linked to prostate cancer (41) Thus, extensive new literature suggests multiple connections of many kalhkrein genes to various forms of human cancer
The removal of intervening RNA sequences (introns) from the pre-messenger RNA in eukaryotic nuclei is a major step in the regulation of gene expression (60) RNA splicing provides a mechanism whereby protein isoform diversity can be generated and the expression of particular proteins with specialized functions can be restricted to certain cell or tissue types during development (60) The sequence elements in the pre-mRNA at the 5' and 3' splice sites in metazoans have very loose consensus sequence, only the first and the last two bases (GT AG) of the introns are highly conserved (Sambrook, supra) These sequences cannot be the sole determinants of splice site selection, since identical, but not ordinarily active, consensus sequences can be found within both exons and introns of many eukaryotic genes Other protein factors and sequences downstream of the splice sites are also involved
The existence of multiple splice forms is frequent among kailikreins Distinct RNA species are transcribed from the PSA gene, in addition to the major 1 6-kb transcript (61) Several distinct PSA transcripts have been descπbed by Reigman et al (7) Interestingly, one of these clones lacks the 3' untranslated region and the first 373 nucleotides of the open reading frame, and has an extended exon that contains a stop codon, a pattern that is comparable with some alternative forms of the KLK-L4 cDNA, as described here (Figure 22) Heuze et al , reported the cloning of a full-length cDNA corresponding to a 2 1 kb PSA mRNA This form results from the alternative splicing of intron 4 and lacks the serine residue that is essential for catalytic activity (61) Also, Reigman et al reported the identification of two alternatively spliced forms of the human glandular kalhkrein 2 (KLK2) gene (62) A novel transcπpt of the tissue kalhkrein gene (KLKl) was also isolated from the colon (63) Interestingly, this transcript lacks the first two exons of the tissue kalhkrein gene, but the last three exons were fully conserved, a pattern that is similar to the findings with some ESTs containing parts of the KLK-L4 gene (Figure 22) Neuropsin, a recently identified kallikrein-hke gene, was found to have two alternatively spliced forms, in addition to the major form (59, 64) Here, the cloning of the KLK-L4 gene is described and the identification of a number of alternative mRNA forms These forms may result from alternative splicing (Sambrook, supra), retained intronic segment (7), or from the utilization of an alternative transcription initiation site (63) Because the long form of KLK-L4 and the major alternative splice variant [short KLK-L4 variant] (Figure 24) have an identical 5' sequence required for translation, secretion and activation, it is possible to assume that both mRNAs encode for a secreted protein (61)
In order to investigate the relative predominance of the long KLK-L4 and related forms, cDNA from various tissues was amplified by PCR Although, in general, it is difficult to use PCR for quantitative compaπsons between mRNA species, in this experiment, [mRNAs ot comparable sizes, using one set of primers under identical conditions], such a comparison is reasonable (62) In all five normal tissues examined [breast, prostate, testis, salivary gland and uterus] the long form of KLK-L4 was the predominant, with minimal level of expression of the short form (Figure 24) The presence of alternatively spliced forms may be related to malignancy Recent literature suggests that distinct molecular forms of PSA could be expressed differently by malignant versus benign prostate epithelium (65). Aberrant PSA mRNA splicing in benign prostatic hyperplasia, as opposed to prostate cancer, has been described by Henttu et al (66). In addition, it has been recently postulated that different prostatic tissues potentially harboring occult cancer could account for the presence of various forms of PS A (65).
Example 6 Materials and Methods DNA sequence on chromosome 19
Sequencing data of approximately 300Kb of nucleotides on chromosome 19ql3.3-ql3.4 was obtained from the web site of the Lawrence Livermore National Laboratory (LLNL) (http://www- bio.llnl.gov/genome/genome.html). This sequence was in the form of 9 contigs of different lengths. Restriction enzyme analysis, long PCR strategies, followed by DNA sequencing, BAC and PAC library screening and end sequencing of selected clones, were used to construct a contiguous genomic region, representing the complete human kalhkrein gene locus. New gene identification
A number of computer programs were used to predict the presence of putative new genes within the contiguous genomic area of interest. The ability of these programs for predicting new genes was first examined by using the genomic sequences of the known kailikreins as testing parameters. The most reliable computer programs; GeneBuilder (gene prediction) (http://125.itba.mi.cnr.it/ -webgene/genebuilder.html), GeneBuilder (exon prediction) (http://125.itba.mi.cnr.it ~webgene/genebuilder.html), Grail 2
(http://compbio.ornl.gov), and GENEID-3 (http://apolo.imim.es/geneid.html) were selected for further use Expressed sequence tag (EST) searching
The predicted exons of the putative new gene were subjected to homology search using the BLASTN algorithm (37) on the National Center for Biotechnology Information web server (http://www ncbi.nlm.nih.gov/BLAST/) against the human EST database (dbEST). A clone with > 95% homology was obtained from the I.M.A G.E consortium (38) through Research Genetics Inc, Huntsville, AL This clone was propagated, purified and sequenced from both directions with an automated sequencer, using msert- flanking vector primers Rapid amplification of cDNA ends (RACE) According to the EST sequence and the predicted structure of the gene, two sets of gene-specific primers were designed for 5' and 3' RACE reactions. Two rounds of RACE reactions (nested PCR) were performed for each type of RACE with 5μl Marathon Ready™ cDNA of human testis and prostate (Clontech, Palo Alto, CA, USA) as templates. The reaction mix and PCR conditions were selected according to the manutacturei 's recommendations In brief, the initial denaturation was for 5 min at 94°C, followed by 94°C tor 5 s and 72°C tor 2 min, tor 5 cycles, then, 94°C for 5 s and 70° C for 2 min, for 5 cycles, then, 94°C tor 5 s and 65°C for 2 mm for 30 cycles for the first reaction and 25 cycles for the nested PCR reaction Tissue expression
Total RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as described below for the tissue culture experiments and used for PCR reactions After aligning all known kalhkrein genes, two primers (KLK-L5-R1 and KLK-L5-F1) (Table 17, SEQ ID NOs 61-64, 9-12, and Figure 32) were designed from areas with relatively low homology Tissue cDNAs were amplified at various dilutions Due to the high degree of homology between kailikreins, and to exclude non-specific amplification, PCR products were cloned and sequenced Normal and malignant breast tissues
Normal breast tissues were obtained from women undergoing reduction mammoplasties Breast tumor tissues were obtained from female patients at participating hospitals of the Ontario Provincial Steroid Hormone Receptor Program The normal and tumor tissues were immediately frozen in liquid nitrogen after surgical resection and stored in this manner until extracted The tissues were pulverized with a hammer under liquid nitrogen and RNA was extracted as described below, using Tπzol reagent Breast and prostate cancer cell lines and hormonal stimulation experiments
The breast cancer cell lines BT-474 and T-47D, and the LNCaP prostate cancer cell line were purchased from the American Type Culture Collection (ATCC), Rockville, MD Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovme insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency The cells were then ahquoted into 24- well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal- stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media at a final concentration of 108 M Cells stimulated with 100% ethanol were included as controls The cells were cultured for 24 hours, then harvested for mRNA extraction
Reverse transcriptase polymerase chain reaction (RT-PCR) Total RNA was extracted from the cell lines or tissues using Tπzol reagent (Gibco BRL) following the manufacturer's instructions RNA concentration was determined spectrophotometπcally 2 μg of total RNA was reverse-transcribed into first strand cDNA using the Superscript™ preamplification system (Gibco BRL) The final volume was 20 μl Based on the combined information obtained from the predicted genomic structure of the new gene and the EST sequences, two gene-specific primers were designed (KLK-L5-F1 and KLK-L5-R1) (Table 17) and PCR was carried out in a reaction mixture containing 1 μl of cDNA, 10 M Tπs-HCl (pH 8 3), 50 mM KCl, 1 5 mM MgCl2, 200 μM dNTPs (deoxynucleoside triphosphates), 150 ng of primers and 2 5 units of AmpliTaq Gold DNA polymerase (Roche Molecular Systems, Branchburg, NJ, USA) on a Perkin-Elmer 9600 thermal cycler The cycling conditions were 94°C toi 9 minutes to activate the Taq Gold DNA polymerase, followed by 43 cycles of 94°C tor 30 s, 63°C for 1 minute and a final extension step at 63°C for 10 min Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining All primers for RT-PCR spanned at least 2 exons to avoid contamination by genomic DNA To verify the identity of the PCR products, they were cloned into the pCR 2.1-TOPO vector
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions The inserts were sequenced from both directions using vector-specific primers, with an automated DNA sequencer Structure analysis
Multiple alignment was performed using the Clustal X software package available at ftp://ftp.ebi ac.uk/pub/software/dos/clustalw/clustalx (clustalxl .64b.msw.exe) and the multiple alignment program available from the Baylor College of Medicine (BCM), Houston, TX, USA (kiwi.imgen.bcm.tmc. edu:8808/search-iauncher/launcher/html). Phylogenetic studies were performed using the Phylip software package available at: http://evolutιon.genetιcs washington.edu/phyhp/getme.html. Distance matrix analysis was performed using the "Neighbor-Joining UPGMA" program and parsimony analysis was done using the "Protpars" program. Hydrophobicity study was performed using the BCM search launcher programs (http://dot.ιmgen.bcm.tmc.edu:9331/seq-search/struc-predιct.html). Signal peptide was predicted using the "SignalP" server (http://www.cbs.dtu.dk/services/ signal) Protein structure analysis was performed by "SAPS" (structural analysis of protein sequence) program (http://dot.ιmgen.bcm.tmc.edu:9331/seq- search struc-predict.html) RESULTS
Identification of the KLK-L5 gene
Computer analysis of the genomic area of interest (300Kb around chromosome 19ql3.3-ql3.4) predicted a putative gene comprised of at least three exons Screening of the human expressed sequence tag (EST) database revealed an EST clone (GenBank Accession #394679) with 99% homology with the predicted exons This clone was obtained, purified, and sequenced. The full-length sequence of the EST was compared with the genomic area containing the putative new gene and showed 100% homology with certain areas (exons), which were separated by introns This alignment indicated that the new gene was comprised of 7 exons. Sequence homology comparisons and phylogenetic analysis revealed that this new gene is structurally similar to known kailikreins and other serine proteases (see below). Since four other new kalhkrein-like genes were discovered in this area, this gene was named KLK-L5 (for kallikrein-hke gene 5) Attempts to translate the coding region in all three possible reading frames indicated that only one reading frame will produce a full-length polypeptide chain without interrupting in-frame stop codons Further support for the correctness of this reading frame was obtained by demonstrating that only this frame will preserve the three amino acid residues necessary for serine protease activity (catalytic triad) and the conserved motifs around them An m-frame methionine start codon was found in the second exon This start codon falls within a typical consensus Kozak sequence (CCACCATGG) (33) Thus, the gene will have at least one 5' untranslated exon, similarly to other kallikrein-hke genes [e.g. zyme, the normal epithelial cell- specific 1 gene (NES l) (14), and neuropsin (35)] 5' and 3' RACE reactions were performed in order to obtain the 5' and 3' ends of the gene. No more sequence was obtained by 5* RACE. However, 3' RACE enabled identification ot the 3' untranslated region of the gene. The additional sequence ends with a poiy-A stretch that does not exist in the genomic structure, thus marking the 3' end of the gene and the start of the poly-A tail. Splice variants of the KLK-L5 gene
Homology analysis of the KLK-L5 gene with other kailikreins revealed the presence of an additional 3' exon, an observation that has not been reported for any other member of the kal krem multigene family Furthermore, two different PCR bands were obtained with the 3' RACE. Sequencing of these bands revealed that this gene has at least two splice forms at its 3' end; one form in which the last exon is a single continuous fragment, and another form in which the last exon is split into two exons, with an intervening intron. In order to identify the full structure of other possible splice variants of the gene, PCR was performed using two primers (L5-F2 and L5-R2) (Table 17 and Figure 32). cDNA from 26 different tissues were used as templates and the reaction was performed under different experimental conditions (annealing temperature, MgCl2 concentration). Three distinct bands were observed in many tissues. These bands were excised, gel-purified, and sequenced. As shown in Figure 32, the KLK-L5 gene was found to have 3 molecular forms:
1 ) One form (referred to, from now on, as the "classical" form) represents a typical kallikrein-hke serine protease with five coding exons and four intervening introns (Figure 32). As is the case with some other kailikreins, a 5' untranslated exon is also present, and the possibility of further upstream untranslated exon(s) could not be excluded. Exons 1, 2 and 3 were present at the aforementioned EST. The start codon is present in the second exon (numbers refer to SEQ.ID.NO. 56 or GenBank Accession # AF135025). The stop codon is located in the sixth exon, followed by a 3' untranslated region, and a typical polyadenylation signal (AATAAA) is located 16 bp before the poly-A tail (Figure 33). This form of KLK-L5 spans a genomic length of 5,801 bp on chromosome 19ql3.3-q l3.4 The lengths of the coding regions of the exons are 37, 160, 260, 134, and 156 bp, respectively (Figures 33 and 34). The predicted protein-coding region is formed of 747 bp, encoding a deduced 248-amιno acid protein with a predicted molecular mass of 26 7 kDa. The intron/exon splice sites (GT....AG) and their flanking sequences are in agreement with the consensus splice site sequence.
2) The second mRNA form, encoding the KLK-L5-related protein- 1, is an alternatively spliced form in which the last exon is split into two separate exons with an additional intervening intron (Figure
32). This splitting of the last exon results in the utilization of another stop codon at position 9,478, thus creating a deduced 254-amιno acid protein that is 6 ammo acids longer than the "classical" KLK-L5 form and its carboxyterminal end is different in sequence by 19 ammo acids (Figure 32) This variant has a predicted molecular mass of 27 1 kDa (for base numbering please see SEQ.ID NO 56 and GenBank Accession # AF 135025)
3) The third mRNA form, encoding for KLK-L5-related proteιn-2, is similar to the classical form except that the fourth exon is missing (Figure 32) This leads to frame shifting of the coding region, and an earlier m-frame stop codon will be encountered at position 9,180 The protein-coding region of this form consists of 336 bp, encoding toi a piedicted 1 1 1-amιno acid piotein with a molecular mass of 12 kDa This protein will lack both the serine and aspartate residues characteristic of serine proteases
Amino acid sequences for KLK-L5 proteins are shown in SEQ ID NOs 57 to 60 Structural analysis of the classical KLK-L5 gene Figure 35 shows a comparative hydrophobicity analysis of the KLK-L5 and the prostate-specific antigen (PSA) proteins The amino terminal regions of both genes are quite hydrophobic, indicating that this region of KLK-L5 is possibly harboring a signal peptide analogous to PSA Figure 35 also shows the presence of several evenly distributed hydrophobic regions throughout the KLK-L5 polypeptide, which are consistent with a globular protein, similar to other serine proteases (13) Figure 36 shows the alignment of KLK-L5 protein with another 10 members of the same gene family The dotted region in Figure 36 indicates an 11 -ammo acid loop characteristic of the classical human kailikreins (PSA, hKl and hK2) but not found in KLK-L5 protein or other members of the kalhkrein multigene family (11, 13, 35) Sequence analysis of eukaryotic serine proteases indicates the presence of twenty nine invariant amino acids (39) Twenty eight of them are conserved in the KLK-L5 polypeptide and the remaining amino acid (S156 instead of P) is not conserved among all other kailikreins (Figure 36) Twelve cysteine residues are present in the putative mature KLK-L5 protein, ten of them are conserved in all kailikreins, and the remaining two (C133, and C235 ) are present in most of the other kalhkrein-like proteins but not in the classical kailikreins and they are expected to form an additional disulphide bridge (Figure 36)
The presence of aspartate (D) at position 194 suggests that KLK-L5 will possess a trypsin-hke cleavage pattern, similarly to most of the other kailikreins (e g , hKl, hK2, TLSP, neuropsin, zyme, prostase, and EMSP) but different from PSA, which has a serine (S) residue in the corresponding position, and is known to have chymotrypsin like activity (Figure 36) (54) Homology with other members of the kalhkrein multigene family
Although the protein encoded by the KLK-L5 gene is unique, it has a high degree of homology with the other kalhkrein-like genes The KLK-L5 protein (classical form) has 48% amino acid sequence identity and 57% overall similarity with neuropsin, 46% identity with the normal epithelial cell-specific 1 gene product (NESl) and 38% identity with both PSA and hK2 proteins Multiple alignment shows that the typical catalytic triad of serine proteases is conserved in the KLK-L5 protein (H62, D108, and S200) (Figures 33 and 36) In addition, a well-conserved peptide motif is found around the amino acid residues of the catalytic triad as is the case with other serine proteases [1 e , histidine (VLTAA /C), serine
(GDSGGP), and aspartate (DLRLL)] ( 11, 12) (Figure 36) Figure 36 also shows other amino acid residues that are completely conserved between kailikreins and kalhkrein-like proteins To predict the phylogenetic relatedness of the KLK-L5 protein with other serine proteases, the amino acid sequences of the kalhkrein proteins were aligned together using the "Clustal X" multiple alignment program and a distance matrix tree was predicted using the Neighbor-joining UPGMA and Protpars parsimony methods Figure 37 shows the phylogenetic analysis which separated the classical kailikreins (hKl, hK2, and PSA) and clustered KLK-L5 with NES 1 and neuropsin proteins in a separate group away from other serine proteases, consistent with previously published studies (27, 41) and indicating that this group of genes probably arose from a common ancestral gene, by gene duplication
Tissue expression and hormonal regulation of the KLK-L5 gene
As shown in Figure 38, the KLK-L5 gene is primarily expressed in the salivary gland, stomach, uterus, trachea, prostate, thymus, lung, colon, brain, breast and thyroid gland, but, as is the case with other kailikreins, lower levels of expression are found in some other tissues (testis, pancreas, small intestine, spinal cord). In order to verify the RT-PCR specificity, the PCR products were cloned and sequenced The three splice forms of the gene were expressed in most of these tissues However, the relative abundance of each form was different among tissues (Figure 38).
In order to investigate whether the KLK-L5 gene is under steroid hormone regulation, two breast cancer cell lines (BT-474 and T-47D) and a prostate cancer cell line (LNCaP) were used as models. In LNCaP cells, the gene was up-regulated only by androgen and progestin. Only in this cell line all 3 isoforms were expressed. In BT-474 cells, KLK-L5 was found to be up-regulated, at the mRNA level, by estrogen and androgen, and to a lesser extent by the progestin The rank of potency was estrogen>androgen>progestιn However, the rank of potency for the T-47D cell line was androgen>progestιn>estrogen . In both of these cell lines, only the short isoform (related proteιn-2) was present (Figure 39). KLK-L5 is down regulated in breast cancer
Expression of the KLK-L5 gene, at the mRNA level, was compared between seventeen breast cancer tissues and one normal breast tissue, by RT-PCR. The gene is not expressed at all in 12 tumors (Figure 40) In all breast tissues (normal and malignant) the short isoform (related proteιn-2) was predominant, with the exception of one tumor, which expressed only the classical form (Figure 40, lane 8). These results should be interpreted as preliminary, since the number of tumors and normal tissues tested is relatively small. Mapping and chromosomal localization of the KLK-L5 gene The knowledge of extensive genomic sequence on chromosome 19ql3.3-ql3.4 enabled the precise localization of 14 kallikrein-hke genes and determination of the direction of transcription, as shown by the arrows in Figure 28. Only PSA and KLK2 transcribe from centromere to telomere; the rest of the genes are transcribed in the reverse direction. The KLKl gene was found to be the most centromeric, and the KLK-L6 gene the most telomeπc (KLK-L6; SEQ.ID. N0.65). KLK-L5 is 21.3 Kb centromeric to KLK- L4 (SEQ.ID.NO. 43) and 1.6 kb more telomeric to the trypsin-hke serine protease gene (TLSP) (Figure 28)
DISCUSSION
As shown in Figure 34, kailikreins are characterized by the following common structural features' (a) All genes are formed of 5 coding exons and 4 intervening introns [some genes may have extra 5( untranslated exon(s)] (14, 35) (b) The exon lengths are usually comparable (c) The intron phases are always conserved (I-II-I-0) (see legend of Figure 34 for definition of intron phases), (d) These genes are clustered in the same chromosomal region, without any intervening non-kal krein-hke genes (Figure 28) (e) The histidine residue of the catalytic triad of serine proteases is located near the end of the second coding exon; the aspartate residue in the middle of the third coding exon; and serine at the beginning of the fitth coding exon As shown in Figure 34, all these criteria apply to the newly identified KLK-L5 gene Thus, KLK-L5 should be considered a new member of the kalhkrein multigene family
Serine proteases and kailikreins are synthesized as "preproenzymes" that contain an N-terminal signal peptide (pre-zymogen), followed by a short activation peptide and the enzymatic domain (41, 54) PreproPSA has 24 additional residues that constitute the pre-region (signal peptide, 17 residues), and the propeptide (7 residues) (67) The signal peptide directs the protein to and across the endoplasmic reticulum (ER) In the ER, the signal peptide is removed and the resulting proPSA is transported to the plasma membrane, where it is secreted The hydrophobicity study (Figure 35) indicates that the amino terminal region of the KLK-L5 protein is harboring a signal peptide Also, computer analysis of the amino acid sequence of KLK-L5 predicted a cleavage site between ammo acids 17 and 18 (SQA-AT) Thus, the protein product is very likely a secreted protein
The presence of alternatively spliced forms is not a unique feature of the KLK-L5 gene, several other kailikreins are known to be expressed m various alternatively spliced forms In addition to the major 1 6-kb transcript, several RNA species are transcribed from the PSA gene (61) Furthermore, others (69, 70) have descπbed several PSA isoforms Retained introns and loss of complete exons have been reported in some of these forms In addition, Riegman et al reported the identification of two alternatively spliced forms of the human glandular kalhkrein (KLK2) gene (62) and Liu et al isolated three alternative forms of the same gene (68) A novel transcript of the tissue kalhkrein gene was isolated from the colon (63) Neuropsin, a recently identified kallkrein- ke gene, was found to have two alternatively spliced forms in addition to the major form (35, 64) Here, the cloning of the classical kalhkrem form and two unique splice forms of the KLK-L5 gene are described Because the classical form and the splice forms all have the same 5' sequence required for translation, secretion and activation as do other kailikreins, I e a 5' leader sequence, a signal peptide, and a proregion, it is reasonable to assume that all three mRNA forms should produce a secreted protein Preliminary findings identifying forms of KLK-L5 predominant in certain tissues are presented in Figures 35 and 40
The preliminary results indicate that KLK-L5 is up-regulated by steroid hormones in breast and prostate cancer cell lines (Figure 39) These results are not surprising, since many other kalhkrein genes are also regulated by steroid hormones The differences in the rank of potency of steroid hormones among different cell lines could be attributed to differences in the abundance of the steroid hormone receptors between them as described elsewhere
In conclusion, a new member of the human kalhkrein gene family, KLK-L5, has been characterized which maps to the human kalhkrein locus (chromosome 19ql3 3-ql3 4) This gene has two related splice forms in addition to the main form KLK-L5 is expressed in a variety of tissues, appears to be down-regulated in breast cancer and its expression is influenced by steroid hormones Since a few other kailikreins are already used as valuable tumour markers, KLK-L5 may also find a similar clinical application
Example 7 Using the Materials and Methods substantially as set out in Example 6, the present inventors identified another novel gene ot the kalhkrein multigen family referred to as KLK-L6 The full structure ot the KLK-L6 gene is shown in Figure 41 Exons 1, 2, 3, 4, 5, 6, and 7 are at nucleic acids 1172-1281, 2561-2695, 2781-2842, 3714-3885, 5715-5968, 6466-6602, and 7258-7520 The nucleic acid sequence ot the KLK-L6 gene is also shown in SEQ ID NO 65 and amino acid sequences for the KLK-L6 protein are shown in SEQ ID Nos 66 and 67. (See also GenBank Accession # AF161221)
Figure 42 shows a comparative hydrophobicity analysis of KLK-L6 and the prostate-specific antigen (PSA) The amino terminal regions of both genes are quite hydrophobic indicating that this region of KLK-L6 is possibly harboring a signal peptide analogous to PSA
Multiple alignment of KLK-L6 was carried out using the Clustal X software program as described herein (Figure 43)
To predict the phylogenetic relatedness of the KLK-L6 protein with other serine proteases, the amino acid sequences of the kalhkrein proteins were aligned together using the "Clustal X" multiple alignment program and a distance matrix tree was predicted using the Neighbor-joining/UPGMA and
Protpars parsimony methods Figure 44 shows the phylogenetic analysis which separated the classical kailikreins (hKl, hK2, and PSA) and placed KLK-L6 in a separate group
Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles All modifications coming within the scope of the following claims are claimed
All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety
FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION
1 Evans BAE, Yun ZX, Close JA, Tregear GW, Kitamura N, Nakamshi S, et al Structure and chromosomal localization ot the human renal kalhkrein gene Biochemistry 1988,27 3124-3129
2 Clements JA The glandular kalhkrein family of enzymes Tissue-specific and hormonal regulation Endocr Rev 1989,10 393-419
3 Evans BA, Dπnkwater CC, Richards Rl Mouse glandular kalhkrein genes Structure and partial sequence analysis of the kalhkrein gene locus J Biol Chem 1987,262 8027-8034
4 Dπnkwater CC, Evans BA, Richards Rl Kailikreins, kinins and growth factor biosynthesis Trends Biochem Sci 1988b,13 169-172 5 Ashley PL, MacDonald RJ Tissue-specific expression of kal krein-related genes in the rat
Biochemistry 1985,24 4520-5427
6 Gerald WL, Chao J, Chao L Sex dimorphism and hormonal regulation of rat tissue kal krein mRNA Biochim Biophys Acta 1986,867 16-23
7 Riegman PHJ, Vlietstra RJ, van der Korput JAGM, Romijn JC, Trapman J Characterization of the prostate-specific antigen gene A novel human kallikrein-hke gene Biochem Biophys Res
Commun 1989,159 95-102
8 Sched ch LJ, Bennetts BH, Morns B J Primary structure of a human glandular kalhkrein gene DNA 1987,6 429-437
9 Riegman PH, Vlietstra RJ, Suurmeijer L, Cleutjens CBJM, Trapman J Characterization of the human kalhkrein locus Genomics 1992,14 6-11
10 Anisowicz A, Sotiropoulou G, Stenman G, Mok SC, Sager R A novel protease homolog differentially expressed in breast and ovarian cancer Mol Med 1996,2 624-636
11 Little SP, Dixon EP, Norπs F, Buckley W, Becker GW, Johnson M, et al Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer's disease brain J Biol Chem 1997,272 25135-25142
12 Yamashiro K, Tsuruoka N, Kodama S, Tsuμmoto M, Yamamura Y, Tanaka T, et al Molecular cloning of a novel trypsin-hke serine protease (neurosin) preferentially expressed in brain Biochim Biophys Acta 1997,1350 11-14
13 Liu XL, Wazer DE, Watanabe K, Band V Identification of a novel serine protease-like gene, the expression of which is down-regulated during breast cancer progression Cancer Res
1996,56 3371-3379
14 Luo L, Herbnck J-A, Scherer SW, Beatty B, Squire J, Diamandis EP Structural characterization and mapping of the normal epithelial cell-specific 1 gene Biochem Biophys Res Commun 1998,247 580-586 15 Milanesi L, Kolchanov N, Rogozin I, Kel A, Titov I Sequence functional inference In ' Guide to human genome computing , ed M J Bishop, Academic Press, Cambridge, 1994, 249-312 16 Burset M, Guigo R Evaluation of gene structure prediction programs Genomics 1996,34 353-
367 Nadeau J, Grant P, Kosowsky M Mouse and human homology map Mouse Genome 1991 ,89 31-36 Schachter M Kailikreins (kininogenases) - a group of serine proteases with bioregulatory actions
Pharmacol Rev 1980,31 1-17 Morris BJ, Catanzaro DF, Richards Rl, Mason AJ, Shine J Kalhkrein and renin Molecular biology and biosynthesis Clin Sci 1981 ,61 351s-353s Richards Rl, Catanzaro DF, Mason AJ, Morris BJ, Baxter JD, Shine J Mouse glandular kalhkrein genes Nucleotide sequence of cloned cDNA coding for a member of the kalhkrein arginyl estero- peptidase group of serine proteases J Biol Chem 1982,257 2758-2761 Van Leeuwen BH, Evans B A, Tregear GW, Richards Rl Mouse glandular kalhkrein genes
Identification, structure and expression of the renal kalhkrein gene J Biol Chem 1986, 261 5529- 5535 Evans B A, Richards Rl Genes for the α and γ subunits of mouse nerve growth factor EMBO
J 1985, 4 133-138 Rogozin IB, Milanesi L, Kolchanov NA Gene structure prediction using information on homologous protein sequence Comput Apphc Biosci 1996,12 161-170 Diamandis, E P Prostate specific antigen-its usefulness in clinical medicine Trends Endocπnol Metab , 9 310-316, 1998 Diamandis, E P , Yu H , and Sutherland, D J Detection of prostate-specific antigen l mmunoreacti vi ty in breast tumours Breast Cancer Res Treat , 32 301-310, 1994 Ishikawa, T , Kashiwagi, H , Iwakami, Y , et al Expression of alpha-fetoprotein and prostate specific antigen genes in several tissues and detection of mRNAs in normal circulating blood by reverse trancπptase-polymerase chain reaction Jpn J Oncol , 28 723-728, 1998 Irwin, D M , Robertson, K A , and MacGilhvary, R T J Mol Biol 212 31-45, 1988 Yoshida, S , Taniguchi, M , Hirata, A , and Shiosaka, S Sequence bovine prothrombin gene J Mol Biol , 212 31-45, 1988 Goyal, J , Smith, K M , Cowan, J M , et al The role of NESl serine protease as a novel tumor suppressor Cancer Res , 58 4782-4786, 1998 Diamandis, E P , and Yu, H New biological functions of prostate specific antigen9 J Clin
Endocπnol Metab , 80 1515-1517, 1995 Reifenberger, J , reifenberger, G , Liu, L , James, C D et al Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and lp Am J Pathol ,145 1 175-90, 1994 Iida, Y (1990) Quantification analysis of 5'-splιce signal sequence in mRNA precursors
Mutations in 5 '-splice signal sequence of human 3-globιn gene and β-thalassemia J Theor Biol 145 523-533 Kozak, M (1991) An analysis of vertebrate mRNA sequences Intimations of translational contiol J Cell Biol 1 15 887-892 Clements, J ( 1997) The molecular biology ot the kailikreins and their roles in inflamation In S Farmer (ed ), The kinin system, pp 71 -97 New York Academic Press Yoshida, S , Taniguchi, M , Hirata, A , and Shiosaka, S (1998) Sequence analysis and expression of human neuropsin cDNA and gene Gene 213 9-16 Takayama, T K, Fujikawa, K , Davie, E W (1997) Characterization of the precursor of prostate specific antigen Activation by trypsin and by human glandular kal krein J Biol Chem 272 21582-21588 Altschul, S F et al , Nucleic Acids Res 25 3389-3402, 1997 Lennon, G et al, Genomics 33 151-152, 1996 Dayhoff, M O , Natl Biomed Res Found 5 (Suppl 3) 79-81, 1998 Simmer, J P , et al, J Dent Res 77 377-386, 1998 Nelson, P S et al, PNAS 96 3114-3119, 1999 Osoegawa, K et al, Genomics 52 1-8, 1999 Kozak, M , Nucleic Acid Res 15 8125-8148, 1987 Proudfoot, N J and Brownlee, C G , Nature 263 211-214, 1976 Mιyata, T et al. J Mol Evol 12 219-236, 1979 Mitelman, F , Catalog of Chromosome Aberrations in Cancer, 5th ed Wiley-Liss, New York, pp 3067-3198 Yu, H et al, Clin Cancer res 4 1489-1497, 1998 Sauter, E R , Cancer Epidemol Biomarkers Prev 5 967-970, 1996 Fortier, A H et al, J Natl Cancer Inst 91 1635-1640, 1999 Kumar, A , 1998, Cancer res 57 3111-3114, 1997 Lovgren, J Biochem Biophys Res Commun 238 549-555, 1987 Lai. L C et al. Int J Cancer 66 743-746, 1996 Balbay, M D et al, Proc Amer Assoc Cancer Res 40 225-226, 1999 Rittenhouse, H G et al Crit Rev Clin Lab Sci 35 275-368, 1998 Hansson, L et al, J Biol Chem 269 19420-19426, 1994 Stephenson, S et al J Biol Chem 27 23210 23214, 1999 Stenman, U H Clin Chem 45 753-754, 1999 Black, M H , Clin Chem 45 790-799, 1999 Underwood, L J et al, Cancer Res 59 4435-4439, 1999 Adams, M D et al, Curr Opin Cell Biol 8 331-339, 1996 Heuze, N et al, cancer Res 59 2820-2824, 1999 Riegman, P H , Mol Cell Endocπnol 76 181- 190 Chen, L M Braz J Med Biol Res 27 1829-1838, 1994 Mitsui, S et al, Eur J Biochem 260 627-634 Baffa, R , Urology, 47 795-800, 1996 Henttu, P et al, Int J Cancel 45 654-660, 1990 McCormack, RT et al, Uiology 45 729-744,1995 Liu, X F , et al, Biochem Biophys Res Commun 264833-839, 1999 Riegman, PH et al, Biochem Biophys Res Commun 155 181-188,1988 Lundwall, A and H Lilja, FEBS Lett 214317-322, 1987 Zarghami.N et al, Br J Cancer 75 579-88,1997
TABLE 1. Predicted exons of the putative gene KLK-Ll . The translated protein sequences of each exon (open reading frame) are shown.
Exon Putative c oding No. of Translated protein sequence EST Intron Stop Catalytic Exon prediction
No.1 region2 bases match3 phase4 codon5 triad6 program7
From(bp) To (bp)
2 2263 2425 163 S VSGSCSQI1NGEDCSPHSQPWQAALVMENELFCSGV + II - H A,B,D LVHPQWVLSAAHCFQ
3 2847 3109 263 NSYTIGLGLHSLEADQEPGSQMVEAS SVRHPEYNRPL + I D A,B,C,D LANDL LI LDESVSESDTIRSISIASQCPTAGNSCLVSG WGLLANGELT
4 3180 3317 137 GRMPTVLQCVNVSVVSEEVCS LYDPLYHPS FCAGG + 0 - A,B,C,D GQDQKDSCN
5 4588 4737 150 GDSGGPLICNGYLQG VSFGKAPCGQVGVPGVYTN C + - + S A,B,C FTEWIE TVQAS
1. Conventional numbering of exons in comparison to the five coding exons of PSA,as described in Ref.14.
2. Nucleotide numbers refer to the related contig
3. (+) = >95% homology with published human EST sequences.
4.Intron phase:0=the intron occurs between codons;I=the intron occurs after the first nucleotide of the codon;
II=the intron occurs after the second nucleotide of the codon. 5.(+) denotes the exon containing the stop codon.
6.H=histidine,D=aspartic acid,S=serine.The aminoacids of the catalytic triad are bold and underlined. 7. A = GeneBuilder (gene analysis), B = GeneBuilder (exon analysis), C = Grail 2,
D = GENEID-3
TABLE 2. Predicted exons of the putative gene KLK-L2. The translated protein sequences of each exon (open reading frame) are shown*
Exon Putative coding sequence"1 No. of Translated protein sequence EST Intron Stop Catalytic Exon prediction No.1 From(bp) To(bp) bases match3 phase4 codon5 triad6 program7
1 15,361 15,433 73 MATARPPWMWVLCALITALLLGVT + I - - -
2 17,904 18,165 262 EHVLANNDVSCDHPSNTVPSGSNQDLGAGAGEDARSDDSSSR1IN + II H A.B.C.D GSDCDMHTQPWQAALLLRPNQLYCGAVLVHPQWLLTAAHCRK K
3 18,903 19,159 257 VFRVRLGHYSLSPVYESGQ MFQGVKSIPHPGYSHPGHSND MLI + I - D C,D KLNRRIRPTKDVRPINVSSHCPSAGTKCLVSG GTT SPQ
4 19,245 19,378 134 VHFP VLQCLN1SVLSQ RCEDAYPRQIDDTMFCAGDKAGRDSC + 0 - - B,C Q
5 24,232 24,384 153 GDSGGPVVCNGSLQGLVSWGDYPCARPNRPGVYTNLC FTKWI + - + S A,B,C QETIQANS
All footnotes same as χaβιe l
TABLE 3. Predicted exons of the putative gene K.LK-L3. The translated protein sequences of each exon (open reading frame) are shown*
* All footnotes same as Table 1
TABLE 4. Predicted exons of the putative gene KLK-L4. The translated protein sequences of each exon (open reading frame) are shown
Exon Putative coding region'' No. of Translated protein sequence EST Intron Stop Catalytic Exon prediction
No.' Fro (bp) To(bp) bases match3 phase4 codon5 triad6 program7
2 24,945 25,120 176 ESSKV NTNGTSGFLPGGYTCFPHSQPWQAALLVQGRLL + II - H C CGGVLVHPKWVLTAAHCL E
3 25,460 25,728 269 GLKVYLGKHALGRVEAGEQVREVVHSIPHPEYRRSPTHL + I D A,B,C,D NHDHDIMLLELQSPVQLTGYIQT PLSHNNRLTPGTTCRV SGWGTTTSPQ
4 26,879 27,015 137 VNYPKTLQCANIQ RSDEECRQVYPG ITDNMLCAGT E + 0 - - A,B,C,D GGKDSCE
5 28,778 28,963 189 GDSGGPLVCNRTLYGIVSWGDFPCGQPDRPGVYTRVSRY + - + S A,B,C VLWIRETIRKYETQQQK LKGPQ
* All footnotes same as Table 1
TABLE 5. Predicted exons of the putative gene K-LK--L5. The translated protein sequences of each exon (open reading frame) are shown
Exon Putative coding region No. of Translated protein sequence EST Intron Stop Catalytic Exon prediction
No.1 From(bp) To(bp) bases match3 phase4 codon5 triad6 program7
2 1588 1747 160 LSQAATPKIFNGTECGRNSQPWQVGLFEGTSLRCGGV - II - H A,B,C LIDHRWVL TAAHCSG
3 3592 3851 260 SRYWVRLGEHS SQLDWTEQIRHSGFSVTHPGYLGAS + I D A,B,C,D TSHEHDLRLLRLRLPVRVTSSVQPLPLPNDCATAGTEC HVSGWGITNHPR
4 4806 4939 134 NPFPDLLQC NLS1VSHATCHGVYPGRITSNMVCAGG + 0 - - A,B,C,D VPGQDACQ
* All footnotes same as Table 1
TABLE 6. Homology between the predicted amino acid sequences of the newly identified putative genes and protein sequences deposited in Genbank
No. Gene identity Homolgous known protein Identity%
(number of amino acids)
1 KLK-Ll • Human stratum corneum chymotryptic enzyme 44(101/227)
• Rat kallikrein 40( 96/237)
• Mouse glandular kallikrein K22 39( 94/236)
• Human glandular kallikrein 38( 93/241)
• Human prostatic specific antigen 37( 91/241)
• Human protease M 37( 87/229)
2 KLK-L2 • Human neuropsin 48(106/219)
• Human stratum corneum chymotryptic enzyme 47(103/216)
» Human protease M 45( 99/219)
» Human trypsinogen I 45(100/221)
» Rat trypsinogen 44( 98/220)
3 KLK-L3 » Human neuropsin 44(109/244)
> Rat trypsinogen 4 39( 95/241)
> Human protease M 38( 98/253)
Human glandular kallikrein 37( 94/248)
Human prostatic specific antigen 36( 89/242)
4 KLK-L4 Human protease M 52(1 18/225)
Human neuropsin 51(1 16/225)
Mouse neuropsin 51(116/226)
Human glandular kallikrein 48(1 13/234)
Human prostatic specific antigen 47(108/227)
5 KLK-L5 Human neuropsin 44( 81/184)
Rat trypsinogen I 42( 76/178)
Rat trypsinogen II 42( 75/178)
Human protease M 41( 73/178)
Table 7. Expressed sequence tags with >95% homology to exons of the prostase/KLK-Ll gene.
GenBank # Source Tissue homologous exons
AA551449 I.M.A.G.E. prostate 3,4,5
AA533140 I.M.A.G.E prostate 4,5
AA503963 I.M.A.G.E. prostate 5
AA569484 I.M.A.G.E. prostate 5
AA336074 TIGR endometπum 2,3
Table 8 Pi imers used tor reveise transci iption-polymeiase chain reaction (RT-PCR) analysis ot various genes
Gene Primer name Sequence1 Product size
(base pairs)
Prostase RS TGACCCGCTGTACCACCCCA 278
(KLK-Ll) RAS GAATTCCTTCCGCAGGATGT pS2 PS2S GGTGATCTGCGCCCTGGTCCT 328
PS2AS AGGTGTCCGGTGGAGGTGGCA
PSA PSAS TGCGCAAGTTCACCCTCA 754
PSAAS CCCTCTCCTTACTTCATCC
Actin ACTINS ACAATGAGCTGCGTGTGGCT 372
ACTIN AS TCTCCTTAATGTCACGCACGA
1. All nucleotide sequences are given in the 5'-→3' orientation.
Table 9 Tissue expression ot pi stase/KLK-Ll by RT-PCR analysis
Expression level
High medium low o Expression
Prostate Mammary gland Salivary glands Stomach
Testis Colon Lung Heart
Adrenals Spinal cord Brain Spleen
Uterus Bone marrow Placenta
Thyroid Thymus Liver
Trachea Pancreas
Cerebellum Kidney Fetal brain Fetal liver Skeletal muscle Small intestine
Table 10. EST clones with >95% homology to exons of KLK-L2
GENBANK # Tissue of Origin I.M.A.GE. ID Homologous exons
W73140 Fetal heart 344588 4,5
W73168 Fetal heart 344588 3,4,5
AA862032 Squamous cell carcinoma 1485736 4,5
AI002163 Testis 1619481 3,4,5
N80762 Fetal lung 300611 5
W68361 Fetal heart 342591 5
W68496 Fetal heart 342591 5
AA292366 Ovarian tumor 725905 1,2
AA394040 Ovarian tumor 726001 5
Table 11. Primers used for reverse transcription polymerase chain reaction (RT-PCR) analysis.
Gene Primer name Sequence1 Product size
(base pairs)
KLK-L2 KS GGATGCTTACCCGAGACAGA 342
KAS GCTGGAGΛGATGAACATTCT
pS2 PS2S GGTGATCTGCGCCCTGGTCCT 328
PS2AS AGGTGTCCGGTGGAGGTGGCA
PSA PSAS TGCGCAAGTTCACCCTCA 754
PSAAS CCCTCTCCTTACTTCATCC
Actin ACTINS ACAATGAGCTGCGTGTGGCT 372
ACTINAS TCTCCTTAATGTCACGCACGA
KLK-L2 Rl CCGAGACGGACTCTGAAAACTTTCTTCC
R2 TGAAAACTTTCTTCCTGCAGTGGGCGGC
1. All nucleotide sequence are given in the 5' 3' orientation.
Table 12 Tissue expiession ot KLK-L2 by RT-PCR analysis
sion level high Medium low No Expression
Brain Salivary gland Uterus Stomach
Mammary gland Fetal brain Lung Adrenal gland
Testis Thymus Heart Colon
Prostate Fetal liver Skeletal muscle
Thyroid Spleen
Trachea Placenta
Cerebellum Liver
Spinal cord Pancreas Small intestine Kidney Bone marrow
TABLE 13 . Primers used for reverse transcrintion polymerase chain reaction (RT-PCR) analysis. Gene Primer name Sequence1
KLK-L3 L3-F1 CATGCAGTGTCTCATCTCAG L3-F2 CATGGAGGAGGAAGGAGATG L3-R1 CTTCGGCCTCTCTTGGTCTT
PSA PSAS TGCGCAAGTTCACCCTCA PSAAS CCCTCTCCTTACTTCATCC
Actin ACTINS ACAATGAGCTGCGTGTGGCT ACTINAS TCTCCTTAATGTCACGCACGA
1. All nucleotide sequence are given in the 5' →3' orientation.
TABLE 14. Primers used for gene-specific PCR amplification of the kallikrein genes using DNA as a template.
Primer Sequence1 Coordinates GenBank Gene name name accession #
Z1S GACCCTGACATGTGACATCTA 979-999 U62801 Zyme
Z1AS GCCACTGCCTGATGGAGACTG 1422-1402
GL3-F1 AACATCAGCATCCTGGAGAA 7324-7343 AF135026 KLK-L3
LL3-R1 CTTCGGCCTCTCTTGGTCTT 8051-8060
L2-1 GGGTCAGAGCTGCAGAGAAG 11104-11123 AF135028 KLK-L2
L2-2 GGGCCTGTCGTCTGCAATGG 11522-11541
KLK-LL ATGGCCACAGCAGGAAATCC 1411-1430 AF113141 KLK-Ll GGTCACTTGTCTGCGCAGAC 1990-2019
PS CCCAACCCTGTGΓΠTTCTC 3634-3653 M33105 PSA2
PAS GGCCCTCCTCCCTCAGA 4143-4118
K1S ATCCCTCCATTCCCATCTTT 2-22 M18157 KLKl3
K1AS CACATACAATTCTCTGGTTC 324-30
K2S AGTGACACTGTCTCAGAATT 131-150 AF024605 KLK24
K2AS CCCCAATCTCACCAGTGCAC 580-561
NS GCTTCCCTACCGCTGTGCT 552-570 AF055481 NES l5
NAS CACTCTGGCAAGGGTCCTG 763-744
1. all nucleotide sequences are given in the 5'- 3' orientation
2. prostate specific antigen
3. human renal kallikrein
4. human glandular kallikrein
5. normal epithelia cell-specific 1 gene.
TABLE 15. Primers used for reverse transcription polymerase chain reaction (RT-PCR) analysis.
Gene Primer name Sequence
KLK-L4 L4-F1 AACTCTACAATGTGCCAACA
L4-R1 TTATTGTGGGCCCTTCAACC L4-R3 GGATGGTCCATTTATAGGAC
L4-A AGGCTGCCCTACTAGTGCAA L4-B ATATTGCCTAGGTGGATGTG L4-D AAGACTTCAAGGAGCCAAGC L4-E GACCCTTCACCTCCCAAAAT
L-4-X1 CTAGTGATCGCCTCCCTGAC pS2 PS2S GGTGATCTGCGCCCTGGTCCT PS2AS AGGTGTCCGGTGGAGGTGGCA
PSA PSAS TGCGCAAGTTCACCCTCA PSAAS CCCTCTCCTTACTTCATCC
Actin ACTINS ACAATGAGCTGCGTGTGGCT
ACTINAS TCTCCTTAATGTCACGCACGA
1. All nucleotide sequence are given in the 5'—»3' orientation.
TABLE 16. EST clones with >95% homology to exons of KLK-L4 GenBank # Tissue of I.M.A.GE. ID origin
AA399955 Testis 743113 AA401397
AA846771 Testis 1392889
AI002101 Testis 1619045
AI032327 Testis 1644236
TABLE 17. Primers used for reverse transcription polymerase chain reaction (RT-PCR) analysis.
Gene Primer name Sequence1
KLK-L5 KLK-L5-F1 TCAGCCAGGCAGCCACACCG
KLK-L5-R1 TTGGTGATGCCCCAGCCTGA
L5-F2 CCACACCGAAGATTTTCAAT L5-R2 GCCCCTCCTTCATTTATA
PSA PSAS TGCGCAAGTTCACCCTCA PSAAS CCCTCTCCTTACTTCATCC
Actin ACTINS ACAATGAGCTGCGTGTGGCT ACTINAS TCTCCTTAATGTCACGCACGA
1. All nucleotide sequence are given in the 5'→ 3' orientation.

Claims

We Claim:
1 An isolated KLK LI, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 nucleic acid molecule of at least 30 nucleotides which hybridizes to SEQ ID NO 1, 13, 21, 43, 56, or 65, respectively, or the complement of SEQ ID NO 1, 13, 21, 43, 56, or 65, under stringent hybridization conditions
2 An isolated nucleic acid molecule which comprises
(i) a nucleic acid sequence encoding a protein having substantial sequence identity with an amino acid sequence of a KLK-Ll, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 protein as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively, (n) a nucleic acid sequence encoding a protein comprising an amino acid sequence of a KLK-Ll, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 protein as shown m SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively, (in) nucleic acid sequences complementary to (1),
(iv) a degenerate form of a nucleic acid sequence of (l),
(v) a nucleic acid sequence capable of hybridizing under stringent conditions to a nucleic acid sequence in (l), (n) or (in), (vi) a nucleic acid sequence encoding a truncation, an analog, an allelic or species variation of a protein comprising an amino acid sequence of a KLK-Ll, KLK-L2,
KLK-L3, KLK-L4, KLK-L5, or KLK-L6 protein as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively, or (vn) a fragment, or allelic or species variation of (I), (n) or (in)
3 A purified and isolated nucleic acid molecule of the invention comprises (I) a nucleic acid sequence comprising the sequence of SEQ ID NO 1, 13, 21, 43,
56, or 65 wherein T can also be U, (n) nucleic acid sequences complementary to (I), preferably complementary to the full nucleic acid sequence of SEQ ID NO 1, 13, 21, 43, 56, or 65, (in) a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid of (I) or (n) and preferably having at least 18 nucleotides, or
(iv) a nucleic acid molecule differing from any of the nucleic acids of (I) to (in) in codon sequences due to the degeneracy of the genetic code 4 An isolated nucleic acid molecule which encodes a protein which binds an antibody of a KLK-Ll , KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 protein 5 A regulatory sequence of an isolated nucleic acid molecule as claimed in any of the preceding claims fused to a nucleic acid which encodes a heterologous protein
6 A vector comprising a nucleic acid molecule of any of the preceding claims
7 A host cell comprising a nucleic acid molecule of any of the preceding claims An isolated KLK LI piotein compnsmg an amino acid sequence ot SEQ ID NO 2 oi 3 An isolated KLK L2 protein comprising an amino acid sequence of SEQ ID NO 14 An isolated KLK L3 protein comprising an amino acid sequence of SEQ ID NO 22 or 23 An isolated KLK-L4 protein comprising an amino acid sequence of SEQ ID NO 44 or 45 An isolated KLK-L5 protein comprising an amino acid sequence of SEQ ID NO 57, 58, 59, or 60 An isolated KLK-L6 protein comprising an amino acid sequence of SEQ ID NO 66 or 67 An isolated protein having at least 65% amino acid sequence identity to an amino acid sequence of SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67 A method for preparing a protein as claimed in any of the preceding claimd comprising (a) transferring a vector as claimed in claim 6 into a host cell,
(b) selecting transformed host cells from untransformed host cells,
(d) culturing a selected transformed host cell under conditions which allow expression of the protein, and
(e) isolating the protein A protein prepared in accordance with the method of claim 15 An antibody having specificity against an epitope of a polypeptide as claimed in claim 8, 9, 10, 11, 12, or 13 An antibody as claimed in claim 17 labeled with a detectable substance and used to detect the protein in biological samples, tissues, and cells A probe comprising a sequence encoding a protein as claimed in claim 8, 9, 10, 11, 12, or 13, or a part thereof A method of diagnosing and monitoring conditions mediated by a protein as claimed in claim 8, 9, 10,
11, 12, or 13, by determining the presence of a nucleic acid molecule encoding the protein as claimed in any of the preceding claims or determining the presence of the protein A method as claimed in claim 20 wherein the condition is cancer A method for identifying a substance which associates with a protein as claimed in claim 8, 9, 10, 11,
12, or 13 comprising (a) reacting the protein with at least one substance which potentially can associate with the protein, under conditions which permit the association between the substance and protein, and (b) removing or detecting protein associated with the substance, wherein detection of associated protein and substance indicates the substance associates with the prtoein A method for evaluating a compound for its ability to modulate the biological activity of a protein as claimed in claim 8, 9, 10, 11, 12, or 13 comprising providing a known concentration of the protein with a substance which associates with the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and removing and/or detecting complexes A method for detecting a nucleic acid molecule encoding a protein comprising an amino acid sequence of SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67 in a biological sample comprising the steps of 55 -
(a) hybudizing a nucleic acid molecule ot claim 2 to nucleic acids ot the biological sample, thereby forming a hybridization complex, and
(b) detecting the hybridization complex wherein the presence of the hybridization complex correlates with the presence of a nucleic acid molecule encoding the protein in the biological sample
25 A method as claimed in claim 24 wherein nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step
26 A method for treating a condition mediated by a protein as claimed in claim 8, 9, 10, 11, 12, or 13 comprising administering an effective amount of an antibody as claimed in claim 17 or a substance or compound identified in accordance with a method claimed in claim 22 or 23
27 A method as claimed in claim 26 wherein the condition is cancer
28 A composition comprising one or more of a nucleic acid molecule or protein claimed in any of the preceding claims, or a substance or compound identified using a method as claimed in any of the preceding claims, and a pharmaceutically acceptable carrier, excipient or diluent 29 Use of one or more of a nucleic acid molecule or protein claimed in any of the preceding claims, or a substance or compound identified using a method as claimed in any of the preceding claims in the preparation of a pharmaceutical composition for treating a condition mediated by a protein as claimed in any of the preceding claims
30 A transgenic non-human mammal which doe not express a KLK-Ll, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 protein as claimed in claim 8, 9, 10, 11, 12, or 13, respectively, resulting in a
KLK-Ll, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 protein associated pathology, respectively
31 A transgenic animal assay system which provides a model system for testing for an agent that reduces or inhibits an a KLK-Ll, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 protein associated pathology comprising
(a) administering the agent to a transgenic non-human animal as claimed in claim 26, and
(b) determining whether said agent reduces or inhibits a KLK-Ll, KLK-L2, KLK-L3, KLK-L4, KLK- L5, or KLK-L6 protein associated pathology in the transgenic non-human animal relative to a transgenic non-human animal of step (a) which has not been administered the agent
EP00908878A 1999-03-11 2000-03-09 Human kallikrein-like genes Withdrawn EP1159431A2 (en)

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Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6943236B2 (en) 1997-02-25 2005-09-13 Corixa Corporation Compositions and methods for the therapy and diagnosis of prostate cancer
CA2503475A1 (en) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion or inhibition of angiogenesis and cardiovascularization
JP4495349B2 (en) 1999-01-28 2010-07-07 ジェン−プローブ・インコーポレーテッド Nucleic acid sequences for detecting cancer genetic markers in biological samples
US7034132B2 (en) 2001-06-04 2006-04-25 Anderson David W Therapeutic polypeptides, nucleic acids encoding same, and methods of use
JP2003528584A (en) * 1999-10-07 2003-09-30 シエーリング アクチエンゲゼルシャフト DNA encoding PROST07 polypeptide
JP2004512540A (en) 2000-11-01 2004-04-22 マウント・サイナイ・ホスピタル Ovarian cancer detection
EP1341804A4 (en) * 2000-11-17 2005-08-24 Nuvelo Inc Novel nucleic acids and polypeptides
AUPR402201A0 (en) * 2001-03-27 2001-04-26 Queensland University Of Technology Polynucleotides and polypeptides linked to cancer and/or benign tumours
JP2005508172A (en) * 2001-11-07 2005-03-31 ミレニアム・ファーマシューティカルズ・インコーポレイテッド Methods for identifying modulators of fractalkine receptors and methods for using modulators of fractalkine receptors
CA2497061A1 (en) * 2002-08-28 2004-03-11 Mount Sinai Hospital Methods for detecting endocrine cancer using kallikrein 13 (klk13) as a biomarker
EP1664331A2 (en) * 2003-08-30 2006-06-07 Bayer HealthCare AG Diagnostics and therapeutics for diseases associated with kallikrein 9 (klk9)
WO2005078123A1 (en) * 2004-02-18 2005-08-25 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with kallikrein 5 (klk5)
WO2013173923A1 (en) * 2012-05-25 2013-11-28 Diamedica, Inc. Formulations of human tissue kallikrein-1 for parenteral delivery and related methods
SI2854841T1 (en) 2012-06-04 2017-06-30 Diamedica Inc. Human tissue kallikrein 1 glycosylation isoforms
US20190023786A1 (en) * 2016-01-12 2019-01-24 Palleon Pharmaceuticals Inc. Use of siglec-7 or siglec-9 antibodies for the treatment of cancer
JP2020510023A (en) 2017-03-09 2020-04-02 ダイアメディカ， インコーポレイテッド Tissue kallikrein 1 dosage form
CN112480237B (en) * 2019-09-12 2023-10-17 上海津曼特生物科技有限公司 Fusion protein and preparation method and application thereof
CN114414812B (en) * 2020-12-21 2022-09-20 华中科技大学同济医学院附属同济医院 Application of biomarker combination in preparation of reagent for diagnosing fulminant myocarditis and medicine for fulminant myocarditis

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5786148A (en) * 1996-11-05 1998-07-28 Incyte Pharmaceuticals, Inc. Polynucleotides encoding a novel prostate-specific kallikrein
EP0869178A1 (en) * 1997-04-02 1998-10-07 Smithkline Beecham Corporation SAF-3, Sialoadhesin family member-3
US6146845A (en) * 1997-04-02 2000-11-14 Smithkline Beecham Corporation Polynucleotides encoding a sialoadhesin family member-2 (SAF-2)
NZ531664A (en) * 1998-09-01 2005-07-29 Genentech Inc Pro1317 polypeptides and sequences thereof with homology to the semaphorin B glycoprotein family
CA2347835A1 (en) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion or inhibition of angiogenesis and cardiovascularization

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
CHRISTINA V. OBIEZU ET AL.: "Higher human kallikrein gene 4 (KLK4) expression indicates poor prognosis on ovarian cancer patients", CLINICAL CANCER RESEARCH, vol. 7, August 2001 (2001-08-01), pages 2380 - 2386 *
CRAIG H DAY ET AL.: "Characterization of KLK4 expression and detection of KLK4-specific antibody in prostate cancer patient sera", ONCOGENE, vol. 21, 2002, pages 7114 - 7120 *
MASAZUMI MATSUMURA ET AL.: "Substrates of the prostate-specific Serine protease Prostase/KLK4 defined by positional-scanning peptide libraries", THE PROSTATE, vol. 62, 2005, pages 1 - 13 *
See also references of WO0053776A3 *
STEPHEN A MYERS ET AL.: "Kallikrein 4 (KLK4), a new member of the human kallikrein gene family is upregulated by estrogen and progesterone in the juman endometrial cancer line, KLE", THE JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, vol. 86, no. 5, 2001, pages 2323 - 2326 *
THOMAS K. TAKAYAMA ET AL.: "Characterization of hK4 (Prostase), a Prostate-specific Serine protease: Activation of the precursor of Prostate specific antigen (pro-PSA) and single-chain urokinase-type Plaminogen activator and degradation of prostatic acid phosphatase", BIOCHEMISTRY, vol. 40, 2001, pages 15341 - 15348 *
vol. 39 *
YING DONG ET AL.: "Human kallikrein 4 (KLK4) is highly expressed in serous ovarian carcinomas", CLINICAL CANCER RESEARCH, vol. 7, August 2001 (2001-08-01), pages 2363 - 2371 *
ZHIJUN XI ET AL.: "Kallikrein 4 is a predominantly nuclear protein and is overexpressed in prostate cancer", CANCER RESEARCH, vol. 64, 1 April 2004 (2004-04-01), pages 2365 - 2370 *
ZHIJUN XI ET AL.: "Kallikrein 4 is associated with paclitaxel resistance in ovarian cancer", GYNECOLOGIC ONCOLOGY, vol. 94, 2004, pages 80 - 85 *

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AU3139900A (en) 2000-09-28
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JP2002538789A (en) 2002-11-19
WO2000053747A1 (en) 2000-09-14

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