AU720778B2 - Synthetic mammalian alpha-N-acetylglucosaminidase and genetic sequences encoding same - Google Patents

Synthetic mammalian alpha-N-acetylglucosaminidase and genetic sequences encoding same Download PDF

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AU720778B2
AU720778B2 AU76124/96A AU7612496A AU720778B2 AU 720778 B2 AU720778 B2 AU 720778B2 AU 76124/96 A AU76124/96 A AU 76124/96A AU 7612496 A AU7612496 A AU 7612496A AU 720778 B2 AU720778 B2 AU 720778B2
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acetylglucosaminidase
recombinant
seq
sequence
nucleic acid
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Donald Stewart Anson
Lianne Blanch
John Joseph Hopwood
Hamish Steele Scott
Birgit Weber
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Womens and Childrens Hospital Adelaide
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Womens and Childrens Hospital Adelaide
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Description

WO 97/19177 PCT/AU96/00747 SYNTHETIC MAMMALIAN a-N-ACETYLGLUCOSAMINIDASE AND GENETIC SEQUENCES ENCODING SAME FIELD OF THE INVENTION The present invention relates generally to mammalian a-N-acetylglucosaminidase and to genetic sequences encoding same and to the use of these in the investigation, diagnosis and treatment of subjects suspected of or suffering from a-N-acetylglucosaminidase deficiency.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
BACKGROUND TO THE INVENTION The increasing sophistication of recombinant DNA technology is greatly facilitating the efficacy of many commercially important industries including areas of medical and pharmaceutical research and development. The ability to purify native proteins and subsequently clone genetic sequences encoding these proteins is an important first step in the development of a range of therapeutic and diagnostic procedures. However, practitioners have faced many difficulties in purifying target molecules to an extent sufficient to determine amino acid sequences to permit the development of oligonucleotide probes to assist in the cloning of genetic sequences encoding the target molecules.
WO 97/19177 PCT/AU96/00747 -2- Such difficulties have been particularly faced in the research and development of lysosomal enzymes. An important lysosomal enzyme is a-N-acetylglucosaminidase (EC 2.1.50).
This enzyme acts as a exoglycosidase in lysosomes to hydrolyse the terminal a-Nacetylglucosamine residues present at the non-reducing terminus of fragments of heparan sulphate and heparin (Hopwood, 1989). A deficiency in this lysosomal hydrolase is responsible for the pathogenesis of Sanfilippo B (Mucopolysaccharidosis type IIB [MPS- IIIB]) syndrome (von-Figura and Kresse, 1972; O'Brien, 1972). This is an autosomal recessive disorder of glycosaminoglycan catabolism leading to storage and excretion of excessive amounts ofheparan sulphate and a variety of clinical phenotypes, but classically presenting with progressive mental retardation in conjunction with skeletal deformities (McKusick and Neufeld, 1983).
There is a need, therefore, to purify a-N-acetylglucosaminidase and to clone genetic sequences encoding same to permit development of a range of therapeutic and diagnostic procedures to assist in the diagnosis and treatment of disease conditions arising from a-Nacetylglucosaminidase deficiency.
SUMMARY OF THE INVENTION One aspect of the invention provides an isolated nucleic acid molecule comprising a sequence ofnucleotides which encodes or is complementary to a sequence which encodes a mammalian a-N-acetylglucosaminidase or fragment or derivative thereof.
A second aspect of the invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which is capable of hybridising under at least low stringency conditions to a nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3 or a complementary strand or a homologue, analogue or derivative thereof.
Another aspect of the invention is directed an isolated nucleic acid molecule which is at least 40% identical to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID WO 97/19177 PCT/AU96/00747 -3- NO:3 or to a complementary strand thereof or a homologue, analogue or derivative thereof.
A further aspect of the present invention provides a nucleic acid molecule comprising a sequence ofnucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolysing the terminal a-N-acetylglucosamine residues present at the nonreducing terminus of fragments ofheparan sulphate and heparin residues and wherein said nucleotide sequence is capable of hybridising under low stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 1.
A further aspect of the invention is directed to a genetic construct comprising a sense molecule, for the expression or over-expression of a-N-acetylglucosaminidase in prokaryotic or eukaryotic cells.
A further aspect of the present invention is directed to synthetic a-N-acetylglucosaminidase or like molecule.
A further aspect of the invention contemplates antibodies to a-N-acetylglucosaminidase and preferably synthetic a-N-acetylglucosaminidase or a like molecule.
In still yet another aspect of the present invention there is contemplated a method of diagnosing a mutation or other abberations in the a-N-acetylglucosaminidase gene in a human or animal patient.
Another aspect contemplates a method of treating patients suffering from a-Nacetylglucosaminidase deficiency, such as in MPS-IIIB, said method comprising administering to said patient an effective amount of a-N-acetylglucosaminidase or active like form thereof.
Another aspect of the present invention is directed to a pharmaceutical composition comprising a recombinant mammalian a-N-acetylglucosaminidase or an active fragment WO 97/19177 PCT/AU96/00747 -4or derivative thereof and one or more pharmaceutically acceptable carriers and/or diluents.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a photographic representation of a-N-acetylglucosaminidase purified from human placenta following SDS/PAGE. Lane 1: M, standards (kDa); Lanes 2 and 3 purified a-N-acetylglucosaminidase from human placenta. Lane 4 and 5, bovine serum albumin.
Figure 2 is a photographic representation of an SDS/polyacrylamide gel showing the molecular weights of recombinant a-N-acetylglucosaminidase polypeptides produced in CHO cells before and after PNGase F digestion. The 50 mM NaCI and 75 mM NaCl fractions are indicated. Molecular weights of a-N-acetylglucosaminidase polypeptides are indicated on the left of the figure. Molecular weights of marker proteins are indicated on the right hand side of the figure (lane Single and three letter abbreviations of conventional amino acid residues as used herein are defined in Table 1.
Suitable amino acid substitutions referred to herein are defined in Table 2.
Codes for non-conventional amino acid residues as used herein are defined in Table 3.
WO 97/19177 WO 97/91 77PCT/AU96/00747 TABLE 1 Amino Acid Three-letter One-letter Abbreviation Symbol It Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cystemne Cys C Glutamine Gin Q Glutamic acid Glu E Glycine Gly G Histidmne His H Isoleucine le I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanie Phe F Proline Pro P Serine Ser S Threonmne Thr T Tryptophan Trp W Tyrosine Tyr Y Valine, Val V Any residue Xaa X WO 97/19177 WO 9719177PCT/AU96/00747 -6- TABLE 2 Suitable residues for amino acid substitutions Origfinal Residue Ala Arg Asn Asp Cys Gin Glu Gly His lie Leu Lys Met Phe Ser Thr Trp Tyr Val Exepl=Subtiuins Ser Lys Gin; H-is Glu Ser Asn Asp Pro Asn; Gin Leu; Val le; Val Arg; Gin; Gin Leu; Ile Met; Leu; Tyr Thr Ser Tyr Trp; Phe lie; Leu WO 97/19177 WO 9719177PCT/AU96/00747 -7- TABLE 3 Non-conventional Code Non-conventional Code aino acid ammno acid rz-ammnobutyric acid a-amimo-rL-methylbutyrate aminocyclopropanecarboxylate aminoisobutyric acid aminonorbornylcarboxylate cyclohexylalanine cyclopentylalanime D-alanine D-arginine D-aspartic acid D-cystemne D-glutammne D-glutamic acid D-histidmne D-isoleucmne D-leucine D-lysine D-methionine D-ornithmne D-phenylalanine D-prolmne D-serine D-threonine D-arytophan Abu Mgabu Cpro Aib Norb Chexa Cpen Dal Darg Dasp Dcys Dgln Dglu.
Dhis Dile Dleu Dlys Dmet Dorn Dphe Dpro Dser Dthr Dtrp L-N-methylalanine L-N-methylarginme L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid L-N-methylhistidmne L-N-methylisolleucine L-N-methylleucine L-N-methyllysine L-N-methylmethionine L-N-methylnorleucine L-N-methylnorvaline L-N-methylornithine L-N-methylphenylalanine L-N-methylproline L-N-methylserine L-N-methylthreonine L-N-methyltrytophan L-N-methyltyrosine L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine L-norvaline Nmala, Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmhis Nmile Nmleu Nmlys Nnimet Nmnle Nmnva Nmorn Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe Nva WO 97/19177 WO 9719177PCT/AU96/00747 -8- D-tyrosmne D-valine D-a-methylalanine D-iz-methylarginine D-a-methylasparagmne D-z-methylaspartate D-cc-methylcysteine D-a-methylglutamine D-iz-methylhistidine D-a-methylisoleucmne D-a-methylleucine D-a-methyllysmne D-a-methylmethionine D-rz-methylornithine D-a-methylphenylalanine D-a-methylproline D-a-methylserine D-ni-methylthreonine D-a-methylarytophan D-a-methyltyrosine D-a-methylvaline D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine Dtyr Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmhis Dmile Dnileu Dinlys Dnimet Dmorn Dmphe Dmpro Dmser Dmthr Dmtrp Dmty Dmval Dnmala Dnmarg Dnmn Dnmasp Dnmcys a-methyl-aminoisobutyrate u-methyl-y-aminobutyrate a-methylcyclohexylalanine a-inethylcylcopentylalanine a-methyl-iz-napthylalanine a-methylpenicfllamine N-(4-axninobutyl)glycine N-(2-.aiinoethyl)glycine N-(3-aminopropyl)glycine N-amino-a-methylbutyrate rt-napthylalanine N-benzylglycine N-(2-carbamylethyl)glycine N-(carbamylmethyl)glycine N-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycine N-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycine N-cylcododecylglycine N-cyclooctylglycine N-cyclopropylglycine N-cycloundecylglycine N-(2 ,2-diphenylethyl) glycine N-(3 ,3-diphenyipropyl) glycine Maib Mgabu Mchexa Mcpen Manap Mpen Nglu Naeg Norn Nmaabu Anap Nphe Ngln Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Ncund Nbhm Nbhe WO 97/19177 WO 9719177PCT/AU96/00747 -9- D-N-methylglutamine D-N-methylglutmmte D-N-methylhistidine D-N-methylisoleucine D-N-methylleucmne D-N-methyllysine N-methylcyclohexylalanine D-N-methylornithine N-methylglycine N-methylaminoisobutyrate N-(l1-methylpropyl)glycine N-(2-methylpropyl)glycine D-N-methyltryptophan D-N-methyltyrosine D-N-rnethylvaline y-aminobutyric acid L-t-butylglycine L-ethylglycine L-homophenylalanine L-a-methylarginine L-a-methylaspartate L-rz-methylcystemne L-a-methylglutamine L-ra-methylhistidine L-a-methylisoleucine Dnmgln Dnglu Dnimhis Dnmile Dnmleu Dnmlys Nmchexa Dnmorn Nala Nniaib Nile Nleu Dnmtrp Dnmtyr Dnval Gabu Thug Etg Hphe Marg Masp Mcys Mgln Mhis Mile N-(3-guanidinopropyl) glycine N-(l1-hydroxyethyl)glycmne N-(hydroxyethyl))glycine N (imidazolylethy1)) glyc-ine N-(3-indolylyethyl) glycine N-methyl-y-aminobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-methylphenylalanine D-N-methylproline D-N-methylserine D-N-methylthreonine N-(l1-methylethyl)glycine N-methyla-napthylaianine N-methylpenicillaine N-(p-hydroxyphenyl)glycine N-(thiomethyl)glycine penicillamine L-a-methylalanine, L-az-methylasparagine L-a-methy-t-butylglycine L-methylethylglycine L-rt-methylglutainate L-rz-nethylhomo phenylalanine N-(2-methylthioethyl) glycine L-rt-methyllysine Narg Nthr Nser Nis Nhtrp Nmgabu Dnnimet Nmcpen Dnmphe Dnmpro Dninser Dnmthr NvaI Nmanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mlys L-az-methylleucine Mleu WO 97/19177 WO 9719177PCT/AU96/00747 L-a-methylmethionine L-iz-methylnorvalime L-a-methylphenylaianine L-a-methylserine L-et-methylarytophan L-a-methylvaline ,2-diphenylethyl) carbamylmethyl)glycine 1-carboxy-l1-(2,2-diphenylethylaniino)cyclopropane Mmet Mnva Mphe Mser Mtrp Mval Nnbhm Nmbc L-a-methyinorleucine L-a-methylornidhine L-cc-methylproline L-a-methylthreonine L-ix-methyltyrosine L-N-methylhomo phenylalanie ,3-diphenylpropyl) carbaniylmethyl)glycine Mnle Morn Mpro Mthr Mtyr Nmhphe Nnbhe WO 97/19177 PCT/AU96/00747 11 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes, or are complementary to a sequence which encodes, a mammalian a-N-acetylglucosaminidase or fragment or derivative thereof or its like molecule.
Preferably, the mammal is a human, livestock animal, companion animal, wild animal or laboratory test animal rabbit, rat, mouse or guinea pig). Most preferably, the mammal is a human. Conveniently, the a-N-acetylglucosaminidase is isolatable from the liver, kidney or placenta. However, the present invention extends to all mammalian a-Nacetylglucosaminidase enzymes and from any anatomical or cellular source and/or any biological fluid source, such as but not limited to plasma, serum, cell extract or lymph fluid.
Although a preferred embodiment of the present invention contemplates the use of human a-N-acetylglucosaminidase or genomic or recombinant cDNA) genetic sequences encoding same in the investigation, diagnosis and/or treatment of human subjects (i.e.
homologous system), one skilled in the art will appreciate that the enzyme or genetic sequences encoding same from a non-human animal may also be useful. Such a heterologous system is encompassed by the present invention.
The term "nucleic acid molecule" as used herein shall be taken to refer to any RNA or DNA (eg. cDNA) molecule, whether single-stranded or double-stranded or in a linear or covalently-closed form. The nucleic acid molecule may also be DNA corresponding to the entire genomic gene or a substantial portion thereof or a fragment or derivative thereof.
The nucleic acid molecule of the present invention may constitute solely the nucleotide sequence encoding a-N-acetylglucosaminidase or a a-N-acetylglucosaminidase-like molecule or may be part of a larger nucleic acid molecule. Accordingly, the present invention extends to the isolated genomic a-N-acetylglucosaminidase gene. The non- WO 97/19177 PCT/AU96/00747 -12translated sequences in a larger nucleic acid molecule may include vector, transcriptional and/or translational regulatory sequences, promoter, terminator, enhancer, replication or signal sequences or non-coding regions (eg intron sequences) of an isolated genomic gene.
Reference herein to a "gene" is to be taken in its broadest context and includes: a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e.
introns, and untranslated sequences); (ii) mRNA or cDNA corresponding to the coding regions exons) optionally comprising or 3'-untranslated sequences of the gene; or (iii) synthetic, amplified DNA fragments or other recombinant nucleic acid molecules produced in vitro and comprising all or a part of the coding region and/or or 3'-untranslated sequences of the gene.
The term "gene" is also used to describe synthetic or fusion molecules encoding all or part of a functional product. A functional product is one which comprises a sequence of nucleotides or is complementary to a sequence of nucleotides which encodes a functional polypeptide, in particular a polypeptide having the catalytic activity of a-Nacetylglucosaminidase or a homologue, analogue or derivative thereof.
For the present purpose, "homologues" of a nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.
"Analogues" of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including WO 97/19177 PCT/AU96/00747 -13radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.
"Derivatives" of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence similarity to said sequence or a part thereof. Generally, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues. Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed. Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.
Preferably, a homologue, analogue or derivative of an a-N-acetylglucosaminidase gene according to any embodiments described herein, comprises a sequence of nucleotides of at least 10 contiguous nucleotides derived from SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand thereof, wherein the sequence of said homologue, analogue or derivative is at least 40% identical to SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand thereof or wherein said homologue, analogue or derivative is capable of hybridising to said sequence under at least low stringency hybridisation conditions.
For the purposes of nomenclature, the nucleotide sequence set for in SEQ ID NO: 1 relates to the cDNA encoding the human a-N-acetylglucosaminidase enzyme.
The nucleotide sequence set forth in SEQ ID NO:3 relates to the genomic gene equivalent WO 97/19177 PCT/AU96/00747 -14of the cDNA encoding the human liver a-N-acetylglucosaminidase enzyme. Those skilled in the art will be aware that the specific exon sequences described in SEQ ID NO:3 correspond to the coding regions of the a-N-acetylglucosaminidase gene, said exon regions further comprising the entire open reading frame of the cDNA sequence set forth in SEQ ID NO:1, when aligned in a head-to-tail configuration. The intron sequences of SEQ ID NO:3, which correspond to non-coding regions of the gene which are spliced from the primary transcription product thereof, although not explicitly defined, may be readily deduced by those skilled in the art, when provided with the exon sequence data provided in the nucleotide sequence listing.
The nucleotide sequence of the present invention may correspond to the sequence of the naturally-occurring a-N-acetylglucosaminidase gene or may comprise a homologue, analogue or derivative thereof which contains single or multiple nucleotide substitutions, deletions and/or additions. All such homologues, analogue or derivatives encode a-Nacetylglucosaminidase or a-N-acetylglucosaminidase-like molecules or a homologue, analogue or derivative thereof as contemplated by the present invention. The length of the nucleotide sequence may vary from a few bases, such as in nucleic acid probes or primers, to a full length sequence.
The present invention is particularly directed to the nucleic acid in cDNA form and particularly when inserted into an expression vector. The expression vector may be replicable in a eukaryotic or prokaryotic cell and may either produce mRNA or the mRNA may be subsequently translated into a-N-acetylglucosaminidase or like molecule.
Particularly preferred eukaryotic cells include CHO cells but may be in any other suitable mammalian cells or cell lines or non-mammalian cells such as yeast or insect cells.
In an alternative embodiment, the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or are complementary to a sequence which encodes a polypeptide capable of hydrolysing the a-N-acetylglucosamine residues from the non-reducing terminus of heparan sulphate and heparin fragments and wherein said nucleotide sequence is capable of hybridising under at least low stringency conditions WO 97/19177 PCT/AU96/00747 to a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a homologue, analogue or derivative thereof.
A second aspect of the invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which is capable of hybridising under at least low stringency conditions to a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand or a homologue, analogue or derivative thereof.
Preferably, hybridisation is possible under at least medium stringent conditions. More preferably, hybridisation is possible under high stringent conditions.
For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et al (1989) or Ausubel et al (1987) which are herein incorporated by reference.
A low stringency is defined herein as being a hybridisation and/or wash carried out in 4-6X SSC/0.1-0.5% w/v SDS at 37-45"C for 2-3 hours. A medium stringency hybridisation and/or wash is carried out in 1-4X SSC/0.25-0.5% w/v SDS at 45"C for 2-3 hours and a high stringency hybridisation and/or wash is carried out 0.1-IX SSC/0.1% w/v SDS at 60"C for 1-3 hours.
Alternative conditions of stringency may be employed to those specifically recited herein.
Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash. Those skilled in the art will be aware that the conditions for hybridisation and/or wash may vary depending upon the nature of the hybridisation membrane or the type of hybridisation probe used. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of parameters affecting hybridisation between nucleic acid molecules, reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.
WO 97/19177 PCT/AU96/00747 -16- Those skilled in the art will be aware that the nucleotide sequences set forth in SEQ ID NO:1 and SEQ ID NO:3 may be used to isolate the corresponding genes from other human tissues or alternatively, from the tissues or cells of other species, without undue experimentation. Means for the isolated of such related sequences will also be known to those skilled in the art, for example nucleic acid hybridisation, polymerase chain reaction, antibody screening of expression libraries, functional screening of expression libraries, or complementation of mutants, amongst others. The present invention is not to be limited by the source from which the specific gene sequences described herein have been isolated or by the means used to isolate said sequences.
In one embodiment, a related genetic sequence comprising genomic DNA, or mRNA, or cDNA is contacted with a hybridisation effective amount of a genetic sequence which encodes a-N-acetylglucosaminidase, or its complementary nucleotide sequence or a homologue, analogue, derivative or functional part thereof, and then said hybridisation is detected using a suitable detection means.
The related genetic sequence may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the related genetic sequence originates from an animal species or a human. More preferably, the related genetic sequence originates from a human.
Preferably, the genetic sequence which encodes a-N-acetylglucosaminidase (i.e probe or latter genetic sequence) comprises a sequence of nucleotides of at least 10 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 50 nucleotides and even still more preferably at least 100 nucleotides derived from the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3 or a complementary sequence or a homologue, analogue or derivative thereof.
Preferably, the detection means is a reporter molecule capable of giving an identifiable signal a radioisotope such as 32 P or S or a biotinylated molecule) covalently attached to the a-N-acetylglucosaminidase probe.
WO 97/19177 PCT/AU96/00747 -17- In an alternative embodiment, the detection means is a polymerase chain reaction.
According to this embodiment, two opposing non-complementary nucleic acid "primer molecules" of at least 10 nucleotides in length, more preferably at least 20 nucleotides in length, derived from the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3 may be contacted with a nucleic acid "template molecule" and specific nucleic acid molecule copies of the template molecule amplified in a polymerase chain reaction.
The opposing primer molecules are selected such that they are each capable of hybridising to complementary strands of the same template molecule, wherein DNA polymerase-dependant DNA synthesis occurring from a first opposing primer molecule will be in a direction toward the second opposing primer molecule.
Accordingly, both primers hybridise to said template molecule such that, in the presence of a DNA polymerase enzyme, a cofactor and appropriate substrate, DNA synthesis occurs in the 5' to 3' direction from each primer molecule towards the position on the DNA where the other primer molecule is hybridised, thereby amplifying the intervening
DNA.
Those skilled in the art are aware of the technical requirements of the polymerase chain reaction and are capable of any modifications which may be made to the reaction conditions. For example, of the polymerase chain reaction may be used in any suitable format, such as amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others, to isolate a related a-N-acetylglucosaminidase gene sequence or identify a mutation in an a-N-acetylglucosaminidase genetic sequence. Such variations of the polymerase chain reaction are discussed in detail by McPherson et al (1991), which is incorporated herein by reference. The present invention encompasses all such variations, the only requirement being that the final product of the reaction is an isolated nucleic acid molecule which is capable of encoding a-N-acetylglucosaminidase or a homologue, WO 97/19177 PCT/AU96/00747 -18analogue or derivative thereof.
In a preferred embodiment, the first primer molecule is preferably derived from the sense strand of a gene which encodes a-N-acetylglucosaminidase, in particular from the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a homologue, derivative or analogue thereof and the second primer molecule is preferably derived from the antisense strand of said gene.
Those skilled in the art will be aware that it is not essential to the performance of the invention that the primer molecules be derived from the same gene.
According to this embodiment, the nucleic acid primer molecule may further consist of a combination of any of the nucleotides adenine, cytidine, guanine, thymidine, or inosine, or functional analogues or derivatives thereof, capable of being incorporated into a polynucleotide molecule provided that it is capable of hybridising under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NO: 1 or SEQ ID NO:3 or a homologue, analogue or derivative thereof.
The nucleic acid primer molecules may further be each contained in an aqueous pool comprising other nucleic acid primer molecules. More preferably, the nucleic acid primer molecule is in a substantially pure form.
The nucleic acid template molecule may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the related genetic sequence originates from a cell, tissue, or organ derived from an animal species or a human. More preferably, the related genetic sequence originates from a cell, tissue, or organ derived from a human.
Accordingly, a third aspect of the present invention extends to an isolated nucleic acid molecule which is at least 40% identical to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3 or to a complementary strand thereof or a homologue, analogue WO 97/19177 PCT/AU96/00747 -19or derivative thereof.
Preferably, the percentage identity to SEQ ID NO: 1 or SEQ ID NO:3 is at least about still more preferably at least about 65 yet still more preferably at least about 80% and even still more preferably at least about 85-95%.
In an even more preferred embodiment, the present invention provides an isolated nucleic acid molecule which is at least 40% identical to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or to a complementary strand thereof or a homologue, analogue or derivative thereof and is capable of hybridising under at least low stringency conditions to a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3.
In a particularly preferred embodiment, the isolated nucleic acid molecule described herein is further capable of encoding a sequence of amino acids which is capable of carrying out the enzyme reaction catalysed by a a-N-acetylglucosaminidase enzyme.
The isolated nucleic acid molecule of the present invention is also useful for developing a genetic construct comprising a sense molecule, for the expression or over-expression of a-N-acetylglucosaminidase in prokaryotic or eukaryotic cells. Particularly preferred eukaryotic cells include CHO cells but may be in any other suitable mammalian cells or cell lines or non-mammalian cells such as yeast or insect cells.
The term "sense molecule" as used herein shall be taken to refer to an isolated nucleic acid molecule of the invention as described herein, which is provided in a format suitable for its expression to produce a recombinant polypeptide, when said sense molecule is introduced into a host cell.
In a particularly preferred embodiment, a sense molecule which encodes the a-Nacetylglucosaminidase comprises a sequence of nucleotides set forth in SEQ ID NO: 1 or SEQ ID NO:3 or a complementary strand, homologue, analogue or derivative thereof.
WO 97/19177 PCT/AU96/00747 In a most particularly preferred embodiment, the sense molecule of the invention comprises the sequence of nucleotides set forth in SEQ ID NO: 1 or a complementary strand, homologue, analogue or derivative thereof.
Those skilled in the art will be aware that expression of a sense molecule may require the nucleic acid molecule of the invention to be placed in operable connection with a promoter sequence to produce a "sense construct". The choice of promoter for the present purpose may vary depending upon the level of expression of the sense molecule required and/or the tissue-specificity or developmental-specificity of expression of the sense molecule which is required. The sense construct may further comprise a terminator sequence and be introduced into a suitable host cell where it is capable of being expressed to produce a recombinant polypeptide gene product.
In the context of the present invention, a sense molecule which corresponds to a genetic sequence or isolated nucleic acid molecule which encodes a-N-acetylglucosaminidase polypeptide or a homologue, analogue or derivative thereof, placed operably under the control of a suitable promoter sequence, is introduced into a cell using any suitable method for the transformation of said cell and said genetic sequence or isolated nucleic acid molecule is expressed therein to produce said polypeptide.
The present invention clearly extends to genetic constructs designed to facilitate expression of any nucleic acid molecule described herein.
A genetic construct of the present invention comprises the foregoing sense molecule, placed operably under the control of a promoter sequence capable of regulating the expression of the said nucleic acid molecule in a prokaryotic or eukaryotic cell, preferably a mammalian cell such as a CHO cell, a yeast cell, insect cell or bacterial cell.
The said genetic construct optionally comprises, in addition to a promoter and sense molecule, a terminator sequence.
The term "terminator" refers to a DNA sequence at the end of a transcriptional unit WO 97/19177 PCT/AU96/00747 -21 which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.
Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. A promoter is usually, but not necessarily, positioned upstream or of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of said sense molecule in a cell.
Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the sense molecule and/or to alter the spatial expression and/or temporal expression of said sense molecule. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense molecule, thereby conferring copper inducibility on the expression of said molecule.
Placing a sense molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is WO 97/19177 PCT/AU96/00747 -22generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, the genes from which it is derived.
Again, as is known in the art, some variation in this distance can also occur.
Examples of promoters suitable for use in genetic constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in animal, human, yeast, insect or bacterial cells. The promoter may regulate the expression of the said molecule constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or plant pathogens, or metal ions, amongst others. Preferably, the promoter is capable of regulating expression of a sense molecule in a cell derived from an animal species or human.
In a particularly preferred embodiment, the promoter is derived from the genomic gene encoding a-N-acetylglucosaminidase, preferably the human a-N-acetylglucosaminidase gene. In a more preferred embodiment, however, the promoter is derived from the nucleotide sequence set forth in SEQ ID NO:3 or is at least capable of hybridising to nucleotide residues 1 to 989 of SEQ ID NO:3 or at least 20 contiguous nucleotides derived therefrom.
In an even more particularly preferred embodiment, the promoter is the CMV promoter sequence or a promoter sequence derived therefrom.
An alternative embodiment of the invention is directed to a genetic construct comprising a promoter or functional derivative, part fragment, homologue, or analogue thereof, WO 97/19177 PCT/AU96/00747 -23derived from the a-N-acetylglucosaminidase genomic gene defined by SEQ ID NO: 3.
Preferably, said genetic construct further comprises the a-N-acetylglucosaminidase sequence defined by SEQ ID NO: 1 placed in operably connection with said promoter.
A further aspect of the present invention is directed to synthetic a-N-acetylglucosaminidase or like molecule.
The term "synthetic" as used herein shall be taken to include both recombinant and chemically-synthesised molecules produced by the sequential addition of amino acid residues or groups of amino acid residues in defined order.
In one embodiment, the invention relates to recombinant a-N-acetylglucosaminidase or like molecule encoded by or expressed from the nucleic acid molecules as hereinbefore described.
In another embodiment, the synthetic a-N-acetylglucosaminidase or like molecule comprises a sequence of amino acids which is at least 40% identical to the amino acid sequence set forth in any one of SEQ ID Nos:2, 4, 5 or 6.
More preferably, the percentage identity is at least 60% and still more preferably at least or 85-90%.
A particularly preferred embodiment of the present invention provides a synthetic a-Nacetylglucosaminidase as hereinbefore defined which comprises a sequence of amino acids substantially as set forth in any one of SEQ ID Nos:2, 4, 5 or 6 or a homologue, analogue or derivative thereof.
For the purposes of nomenclature, the amino acid sequence set forth in SEQ ID NO:2 comprises the full-length translation product of the human a-N-acetylglucosaminidase gene hereinafter referred to as the "a-N-acetylglucosaminidase polypeptide" or "SEQ ID WO 97/19177 PCT/AU96/00747 -24- NO:2") produced by expression of either the cDNA sequence defined by SEQ ID NO: 1 or the genomic gene defined by SEQ ID NO:3. The a-N-acetylglucosaminidase polypeptide comprises at least seven potentially-glycosylated Asn residues, at positions 261, 272, 435, 503, 513, 526 and 532. Furthermore, the amino acid sequence of the a-Nacetylglucosaminidase polypeptide may comprise a signal peptide of approximately 23 amino acid residues in length, with a probable site for signal peptide peptidase cleavage occurring between Gly2 and Asp 24 The amino acid sequences set forth in SEQ ID Nos:4-6 relate to N-terminal and internal CNBr) amino acid sequences derived from human a-N-acetylglucosaminidase, purified as described in Example 1. As described in Example 2, the purified form of the enzyme comprises two polypeptides having approximate molecular weights of 82 and 77 kDa. The sequence set forth in SEQ ID NO:4 relates to the N-terminal sequence of the 82 kDa polypeptide, while SEQ ID NO:5 relates to the N-terminal sequence of the 77 kDa polypeptide. Furthermore, SEQ ID NO:4 comprises amino acids residues 24-43 of SEQ ID NO:2, while SEQ ID NO:5 comprises amino acid residues 59-76 of SEQ ID NO:2.
The amino acid sequence defined by SEQ ID NO:6 relates to the CNBr-cleaved peptide of purified human a-N-acetylglucosaminidase. This amino acid sequence aligns with amino acid residues 540-554 of the a-N-acetylglucosaminidase polypeptide (SEQ ID NO:2).
In the present context, "homologues" of a polypeptide refer to those polypeptides, enzymes or proteins which have a similar a-N-acetylglucosaminidase enzyme activity, notwithstanding any amino acid substitutions, additions or deletions thereto. A homologue may be isolated or derived from the same or another animal species.
Furthermore, the amino acids of a homologous polypeptide may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, charge or antigenicity, and so on.
WO 97/19177 PCT/AU96/00747 "Analogues" encompass a-N-acetylglucosaminidase polypeptides and peptide derivatives thereof notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein.
The term "derivative" in relation to the polypeptides of the invention refer to mutants, parts or fragments of a functional molecule. Derivatives include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention.
Additionally, derivatives of a polypeptide may comprise fragments or parts of an amino acid sequence disclosed herein and are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject polypeptides. Procedures for derivatizing peptides are well-known in the art.
Accordingly, this aspect of the present invention is directed to any proteinaceous molecule comprising an amino acid sequence corresponding to the full length mammalian a-Nacetylglucosaminidase enzyme or to a like molecule. The like molecule, therefore, comprises parts, derivatives and/or portions of the a-N-acetylglucosaminidase enzyme whether functional or not.
Preferably, the mammal is human but may be of non-human origin as contemplated above.
The synthetic or recombinant a-N-acetylglucosaminidase of the present invention may comprise an amino acid sequence corresponding to the naturally occurring amino acid sequence or may contain single or multiple amino acid substitutions, deletions and/or additions. The length of the amino acid sequence may range from a few residues to a full length molecule.
Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from WO 97/19177 PCT/AU96/00747 -26about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.
Amino acid insertional derivatives of a-N-acetylglucosaminidase of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with the following Table 2: The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield synthesis) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently elsewhere described such as Sambrook et al, 1989 Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratories, Cold Spring Harbor, NY.
The derivatives or like molecules include single or multiple substitutions, deletions and/or additions of any component(s) naturally or artificially associated with the a-Nacetylglucosaminidase enzyme such as carbohydrate, lipid and/or other proteinaceous moieties. For example, the present invention extends to glycosylated and non-glycosylated forms of the molecule. All such molecules are encompassed by the expression "mutants", "derivatives", "fragments", "portions" and "like" molecules. These molecules may be active or non-active and may contain specific regions, such as a catalytic region.
Particularly, preferred derivative molecules include those with altered glycosylation WO 97/19177 PCT/AU96/00747 -27patterns relative to the naturally occurring molecule. Even more particularly, the recombinant molecule is more highly glycosylated than the naturally occurring molecule.
Such highly glycosylated derivatives may have improved take-up properties and enhanced half-lives.
As indicated in the Examples, the molecular weight of purified human a-Nacetylglucosaminidase 82kDa and 77kDa) and recombinant mammalian a-Nacetylglucosaminidase produced in CHO cells 89 kDa and 79 kDa) are greater than the deduced molecular weight of the a-N-acetylglucosaminidase polypeptide set forth in SEQ ID No:2 70 kDa), suggesting that the purified and recombinant polypeptide are post-translationally modified. The data presented in Example 8 indicate further that the recombinant a-N-acetylglucosaminidase enzyme produced in CHO cells, at least, is glycosylated and that the difference in molecular weight determined for the recombinant polypeptides and the polypeptide of SEQ ID No: 2 is due almost entirely to glycosylation of the recombinant polypeptide by CHO cells. As shown in Example 9, the glycosylated recombinant a-N-acetylglucosaminidase polypeptide exhibits enzymatic activity.
The present invention also extends to synthetic a-N-acetylglucosaminidase or like molecules when fused to other proteinaceous molecules. The latter may include another enzyme, reporter molecule, purification site or an amino acid sequence which facilitates transport of the molecule out of a cell, such as a signal sequence.
The present invention extends further to post-translational modifications to the a-Nacetylglucosaminidase enzyme. The modifications may be made to the naturally occurring enzyme or following synthesis by recombinant techniques. The modifications may be at the structural level or at, for example, the electrochemical level such as modifying net charge or structural conformation of the enzyme.
Such modification may be important to facilitate entry or penetration of the enzyme into selected tissues such as cartilage or blood brain barriers or to increase circulation half-life.
WO 97/19177 PCT/AU96/00747 -28- Analogues of a-N-acetylglucosaminidase contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the enzyme.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5'-phosphate followed by reduction with NaBH 4 The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2chloromercuric-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with Nbromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with WO 97/19177 PCT/AU96/00747 -29tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. Non-naturally occurring amino acids contemplated by the present invention are incorporated herein, as Table 3.
Crosslinkers can be used, for example, to stabilise 3D conformations, using homobifunctional crosslinkers such as the bifunctional imido esters having (CH 2 n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, the enzyme could be conformationally constrained by, for example, incorporation of C, and N.-methylamino acids, introduction of double bonds between C. and C, atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
Electrochemical modifications of a-N-acetylglucosaminidase include interaction with polylysine or polyethylene glycol or other agent which effects an overall change to the net charge of the enzyme.
Advantageously, the recombinant a-N-acetylglucosaminidase is a biologically pure preparation meaning that it has undergone some purification away for other proteins and/or non-proteinaceous material. The purity of the preparation may be represented as at least WO 97/19177 PCT/AU96/00747 of the enzyme, preferably at least 60%, more preferably at least 75%, even more preferably at least 85% and still more preferably at least 95% relative to non-a-Nacetylglucosaminidase material as determined by weight, activity, amino acid homology or similarity, antibody reactivity or other convenient means.
Particularly preferred methods for the preparation and purification of recombinant a-Nacetylglucosaminidase are provided in Examples 7 and 8.
Those skilled in the art will be aware of the means of purifying a synthetic or recombinant a-N-acetylglucosaminidase from several sources without undue experimentation and for expressing the degree of purity of such a purified preparation of the enzyme.
The present invention further contemplates antibodies to a-N-acetylglucosaminidase and preferably synthetic a-N-acetylglucosaminidase or like molecule. The antibodies may be polyclonal or monoclonal, naturally occurring or synthetic (including recombinant, fragment or fusion forms). Such antibodies will be useful in developing immunoassays for a-N-acetylglucosaminidase and for identifying additional genetic sequences which are capable of expressing a-N-acetylglucosaminidase polypeptides or homologues, analogues or derivatives thereof.
Both polyclonal and monoclonal antibodies are obtainable by immunisation with an appropriate synthetic or recombinant gene product, or epitope, or peptide fragment of a gene product, in particular a a-N-acetylglucosaminidase polypeptide or a homologue, analogue or derivative thereof.
Alternatively, fragments of antibodies may be used, such as Fab fragments. The present invention extends further to encompass recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies.
A further aspect of the present invention contemplates a method of screening for mutations WO 97/19177 PCT/AU96/00747 -31or other abberations in the a-N-acetylglucosaminidase gene in a human or animal patient.
Such a method may be accomplished in a number of ways including isolating a source of DNA to be tested or mRNA therefrom and hybridising thereto a nucleic acid molecule as hereinbefore described. Generally, the nucleic acid is probe or primer size and polymerase chain reaction is a convenient means by which to analyse the RNA or DNA. Other suitable assays include the ligation chain reaction and the strand displacement amplification methods. The a-N-acetylglucosaminidase sequence can also be determined and compared to the naturally occurring sequence. Such methods may be useful in adults and children and may be adapted for a pre-natal test. The DNA to be tested includes a genomic sample carrying the a-N-acetylglucosaminidase gene, a cDNA clone and/or amplification product.
In accordance with this aspect of the present invention there is provided a method for screening for abberations in the a-N-acetylglucosaminidase gene including the absence of such a gene or a portion or a substantial portion thereof comprising isolating a sample of DNA or mRNA corresponding to a region of said DNA and contacting same with an oligonucleotide probe capable of hybridising to one or more complementary sequences within the a-N-acetylglucosaminidase gene and then detecting the hybridisation, the extent of hybridisation or the absence of hybridisation.
Alternatively, the probe is a primer and capable of directing amplification of one or more regions of said a-N-acetylglucosaminidase gene and the amplification products and/or profile of amplification products is compared to an individual carrying the full gene or to a reference date base.
Conveniently, the amplification products are sequenced to determine the presence or absence of the full gene.
The present invention extends to the use of any and all DNA-based or nucleic acid-based hybridisation and/or polymerase chain reaction formats as described herein, for the diagnosis of a disorder involving the a-N-acetylglucosaminidase gene in a human or animal patient.
WO 97/19177 PCT/AU96/00747 -32- The present invention further extends to a method of treating patients suffering from a-Nacetylglucosaminidase deficiency, such as in MPS-IIIB, said method comprising administering to said patient an effective amount of a-N-acetylglucosaminidase or active like form thereof.
Preferably, the a-N-acetylglucosaminidase is in recombinant form. Such a method is referred to as "enzyme therapy". Alternatively, gene therapy can be employed including introducing an active gene a nucleic acid molecule as hereinbefore described) or to parts of the gene or other sequences which facilitate expression of a naturally occurring a- N-acetylglucosaminidase gene.
Administration of a-N-acetylglucosaminidase for enzyme therapy may be by oral, intravenous, suppository, intraperitoneal, intramuscular, intranasal, intradermal or subcutaneous administration or by infusion or implantation. The a-Nacetylglucosaminidase is preferably as hereinbefore described including active mutants or derivatives thereof and glycosylation variants thereof. Administration may also be by way of gene therapy including expression of the gene by inclusion of the gene in viral vectors which are introduced into the animal human) host to be treated. Alternatively, the gene may be expressed in a bacterial host which is then introduced and becomes part of the bacterial flora in the animal to be tested.
Still yet another aspect of the present invention is directed to a pharmaceutical composition comprising synthetic recombinant) a-N-acetylglucosaminidase or like molecule, including active derivatives and fragments thereof, alone or in combination with other active molecules. Such other molecules may act synergistically with the enzyme or facilitates its entry to a target cell. The composition will also contain one or more pharmaceutically acceptable carriers and/or diluents. The composition may alternatively comprise a genetic component useful in gene therapy.
The active ingredients of the pharmaceutical composition comprising the synthetic or recombinant a-N-acetylglucosaminidase or mutants or fragments or derivatives thereof are WO 97/19177 PCT/AU96/00747 -33contemplated to exhibit excellent activity in treating patients with a deficiency in the enzyme when administered in an amount which depends on the particular case. The variation depends, for example, on the patient and the a-N-acetylglucosaminidase used.
For example, from about 0.5 ug to about 20 mg of enzyme per animal body or, depending on the animal and other factors, per kilogram of body weight may be administered. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. Accordingly, alternative dosages in the order of 1.0 J/g to 15 mg, 2.0 I/g to mg or 10I/g to 5mg may be administered in a single or as part of multiple doses. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules). Depending on the route of administration, the active ingredients which comprise a synthetic recombinant) a- N-acetylglucosaminidase or fragments, derivatives or mutants thereof may be required to be coated in a material to protect same from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, the low lipophilicity of a- N-acetylglucosaminidase will allow it to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer the vaccine by other than parenteral administration, the enzyme will be coated by, or administered with, a material to prevent its inactivation. For example, the enzyme may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.
Conveniently, the adjuvant is Freund's Complete or Incomplete Adjuvant. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.
The active compound may also be administered in dispersions prepared in glycerol, liquid WO 97/19177 PCT/AU96/00747 -34polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
WO 97/19177 PCT/AU96/00747 When the a-N-acetylglucosaminidase of the present invention is suitably protected as described above, the composition may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in the vaccine compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared, so that an oral dosage unit form contains between about 0.5 ug and 20 mg of active compound.
The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release reparations and formulations.
As used herein "pharmaceutically acceptable carriers and/or diluents" include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, WO 97/19177 PCT/AU96/00747 -36isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The present invention further relates to the use of a-N-acetylglucosaminidase or active fragment, mutant or derivative thereof in the manufacture of a medicament for the treatment of patients suffering from a deficiency in the naturally occurring enzyme (e.g.
MPS-IIIB).
The present invention is further described with reference to the following non-limiting Examples.
EXAMPLE 1 Purification of a-N-Acetylglucosaminidase a-N-acetylglucosaminidase was purified according to the method described in Weber et al. (1996). Enzyme was purified to homogeneity from human placenta. Evidence of purity is shown following SDS/PAGE which is represented in Figure 1. All samples were reduced with dithiothreitol prior to electrophoresis.
EXAMPLE 2 Characterisation of a-N-Acetylglucosaminidase Results presented in Figure 1 show two polypeptides of about 82kDa and 77 kDa molecular weight, which correspond to a-N-acetylglucosaminidase polypeptides purified from human placenta according to Example 1.
EXAMPLE 3 Amino Acid Sequence Determination WO 97/19177 PCT/AU96/00747 -37- The N-terminal amino acid sequences the 77 kDA and 82 kDa a-N-acetylglucosaminidase polypeptides, in addition to the amino acid sequence of an internal CNBr cleavage product of these peptides, were determined using the methods of Weber et al. (1996).
The amino acid sequences are shown in Table 4.
WO 97/19177 PCT/AU9600747 -38- TABLE 4 N-Terminal amino acid sequences (SEQ ID NO:4 and SEQ ID No:5) and CNBr peptide sequence (SEQ ID No:6) determined from purified human a-N-Acetylglucosaminidase polypeptide 82 kDa DEAREAAAVRALVARLLGPG polypeptide 77 kDa KPGLDTYSLGGGGAAX'
VR
CNBr peptide WRLLLTSAPSLX' TX'P X' no residue could be identified for this position, indicating that this residue could be phosphorylated or glycosylated.
EXAMPLE 4 Cloning of a-N-Acetylglucosaminidase cDNA Oligonucleotide probes were prepared based on the partial amino acid sequences obtained for the purified a-N-acetylglucosaminidase polypeptides (Example The probes were subsequently used to screen a human peripheral blood leukocyte cDNA library. An approximately 2.6 kbp cDNA clone was isolated encoding most of the sequence of human a-N-acetylglucosaminidase (SEQ ID NO:1).
The remaining a-N-acetylglucosaminidase coding sequence was obtained from the nucleotide sequence of the corresponding genomic gene (SEQ ID NO:3), isolated by hybridisation to a human chromosome 17 library (Weber et.al. 1996).
WO 97/19177 PCT/AU96/00747 -39- The complete open reading frame is 2232 nucleotides long and encodes a 743 (plus stop codon) amino acid protein. The predicted molecular mass of the longest mature protein (minus the 23 amino acid N-terminal signal peptide) is about 79,622 daltons.
The amino acid sequence of a-N-acetylglucosaminidase is shown in SEQ ID NO:2. The deduced molecular weight of the desired amino acid sequence of a-Nacetylglucosaminidase is approximately 70kDa. The probable site of signal peptide peptidase cleavage is between amino acids 23 and 24. There are seven potential Nglycosylation sites in the sequence.
The nucleotide sequence of the corresponding a-N-acetylglucosaminidase genomic gene (SEQ ID No:3) comprises 10380 bp including 889 bp of 5' upstream sequence corresponding to at least at part of the a-N-acetylglucosaminidase promoter sequence, in addition to the nucleotide sequences ofintrons I, II, II, IV, V, in addition to 1326 bp of 3'untranslated sequence.
EXAMPLE Construction of an expression vector comprising the a-N-Acetylglucosaminidase cDNA sequence The cDNA insert of X clone pbl 33, containing bases 107 to 2575 of the a-Nacetylglucosaminidase cDNA was excised with EcoRI and subcloned into pBluescript II SK-(Stratagene). A 178 bp XmaI fragment (bases 1 to 178 of the a-Nacetylglucosaminidase cDNA) from cosmid sub-clone 6.3, containing the start codon, was cloned into the pBluescript subclone to produce a full-length cDNA sequence in addition to 101 bp of 5' non-translated sequence as well as 245 bp of 3' non-translated region including the polyadenylation-site, the polyA-tail and linkerDNA. The full length cDNA was directionally cloned into the pCDNA3 expressionvector (Invitrogen) via the EcoRI and BamHI sites.
EXAMPLE 6 WO 97/19177 PCT/AU96/00747 Expression of recombinant a-N-acetylglucosaminidase Chinese Hamster Ovary (CHO) cells were transfected with expressionvector using the DOTAP transfection reagent (Boehringer Mannheim) according to the manufacturers instructions. Cells were grown in Ham's F12 medium, 10% fetal calf serum, penicillin and streptomycin sulfate at 100 ig/ml each. Cells were grown in nonselective medium for 48 h and then incubated in medium containing 750 /g/ml G418 sulfate (Geniticin) until resistant colonies emerged.
Single cell clones were grown up and 26 of them were tested for expression of recombinant a-N-acetylglucosaminidase with a fluorogenic a-N-acetylglucosaminidase substrate.'(i.e.
N-acetylglucosamine a-linked to 4-methylumbelliferone) EXAMPLE 7 Large scale a-N-acetylglucosaminidase production 2 g of Cytodex 2 microcarrier beads were swollen in 250 ml of PBS for 3 h at 37"C with three changes of PBS and then autoclaved for 15 min at 120"C (wet cycle). The beads were then rinsed with sterile growth medium (Coons/DMEM, 10% v/v fetal calf serum, penicillin and streptomycin sulfate at 100 ig/ml each and 0.1% w/v Pluronic F68) and transferred into a Techne stirrer culture flask. The microcarrier beads were inoculated with seven confluent 175 flasks of the cell clone showing the highest expression of recombinant a-N-acetylglucosaminidase. Growth medium was added up to 200 ml and the culture incubated with a stirrer speed of 20 rpm to achieve an even distribution of cells on the beads. The cells were allowed to attach to the beads for 16 h at low speed then medium was added up to 500 ml and the stirrer speed increased to 30 rpm. After a growth phase of 48 to 72 h with daily aerating to allow gas exchange the beads were completely covered with cells and the medium was exchanged for production medium (Coons/DMEM, no fetal calf serum, penicillin and streptomycin sulfate at 100 jig/ml each, 0.1% w/v Pluronic F68 and 5 mM NH 4 C1). The glucose concentration was monitored daily and the medium replaced, when glucose fell below 5 mM every 203 days. The harvested medium contained WO 97/19177 PCT/AU96/00747 -41approximately 2 mg a-N-acetylglucosaminidase protein per dm 3 of production medium.
EXAMPLE 8 Purification of recombinant a-N-acetylglucosaminidase Production medium was dialysed against 50 mM NaAc pH 5.5 and loaded onto a heparinagarose column equilibrated in the same buffer. After washing with NaAc buffer and mM NaCI the column was eluted with 75 mM NaCI in NaAc buffer. The eluate was dialysed against 20 mM Tris/HCI pH 7.5, loaded onto a DEAE Scphacel column, washed with 25 mM NaCI in 20 mM Tris/HCl and then eluted with 50 and 75 mM NaCl in 20 mM Tris/HCI respectively.
SDS-PAGE of the two eluates showed two polypeptide bands associated with enzyme activity with apparent molecular weights of 79 and 89 kDa. The smaller a-Nacetylglucosaminidase was eluted predominantly in the 50 mM NaCI fraction whereas the 89 kDa a-N-acetylglucosaminidase polypeptide was enriched in the 75 mM NaCl fraction (Fig. 2).
The difference in apparent molecular weight of the recombinant a-Nacetylglucosaminidase polypeptides is due to the presence of additional carbohydrate side chains, since a digest with PNGase F, which cleaves offN-glycosylation groups, reduced both the 79 kDa and 89 kDa polypeptides to the polypeptide band having an apparent molecular weight of about 70 kDa (Fig. which corresponds to the approximate molecular weight deduced from primary amino acid sequence data (SEQ ID No:2).
EXAMPLE 9 Characteristics of recombinant a-N-acetylglucosaminidase No differences were observed between the enzyme activities of the 79 and 89 kDa recombinant a-N-acetylglucosaminidase polypeptides produced in CHO cells according to Example 7 and 8. With the fluorogenic N-acetylglucosamine a-linked to 4- WO 97/19177 PCT/AU96/00747 -42methylumbelliferone (4-MU) substrate, the enzyme has a pH-optimum of 4.6 with a kM of 5.34 mM and a of 3.97 x 106 pmol/min/mg. Towards a 3 H-labelled disaccharide substrate it should a pH-optimum of 4.1 with a kM of 0.0166 mM and a V. of 4.48 x 104 pmol/min/mg.
EXAMPLE Mutational analysis of Sanfilippo B patients Genomic DNA is isolated from cultivated skin fibroblasts of patients by extraction with Phenol/Chloroform and used to amplify the eight exons and adjacent intronic sequences individually by PCR Primer sequences used in the amplification reaction are readily determined from the nucleotide sequences of the a-N-acetylglucosaminidase cDNA and genomic clones, set forth in SEQ ID No:l or SEQ ID No:3. Amplification conditions are also readily determined without undue experimentation. Procedures for the design of PCR primers and amplification conditions are described in detail, for example, by McPherson et al. (1991).
Differences in the primary sequence can be identified by separating the PCR products on a polyacrylamide gel under non-denaturing conditions (SSCP gels). Base changes, insertions and deletions will lead to a different band pattern compared with the wildtype in most of the cases, which can be visualised either by autoradiography of the gel after labelling the DNA during the PCR or by staining unlabelled DNA in the gel with silver.
PCR products which show a different band pattern are sequences to identify the change.
Other patient samples can be tested for mutations and polymorphism that were found by hybridisation with wildtype- and mutation-specific oligonucleotides (ASO).
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or WO 97/19177 PCT/AU96/00747 43 more of said steps or features.
WO 97/19177 PCT/AU96/00747 -44-
REFERENCES:
1. Ausubel, F. Brent, Kingston, RE, Moore, Seidman, Smith, and Struhl, K. (1987). In: Current Protocols in Molecular Biology. Wiley Interscience (ISBN 047150338).
2. Hopwood JJ (1989) In: "Heparin: Chemical and Biological Properties, Clinical Applications" (Lane DW and Lindahl U, eds.), 190-229, Edward Arnold, London.
3. McKusick V and Neufeld E (1983) In: "The Metabolic Basis oflnherited Disease" (Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL and Brown MS, eds), Ed., 751-771, McGraw-Hill, New York.
4. McPherson, Quirke, P. and Taylor, (1991) In: PCR A Practical Approach.
Oxford University Press, Oxford. (ISBN 0-19-96322L-X).
O'Brien JS, (1972) Proc. Natl. Acad Sci. USA 69: 1720-1722.
6. Sambrook, Fritsch, and Maniatis, T. (1989) In:"Molecular Cloning" a laboratory manual, Cold Spring Harbour.
7. Von Figura, K, and Kresse H, (1972) Biochem Biophys. Res. Commun, 48: 262-269 8. Weber B, Scott H, Blanch L, Clements P, Morris CP, Anson D, Hopwood J, (1996) Nature Genetics (submitted) WO 97/19177 PCT/AU96/00747 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: WOMEN'S AND CHILDREN'S HOSPITAL (US ONLY:HOPWOOD JJ, WEBER B SCOTT HS, BLANCH LC, ANSON D) (ii) TITLE OF INVENTION: SYNTHETIC MAMMALIAN a-N-ACETYLGLUCOSAMINIDASE AND GENETIC SEQUENCES ENCODING SAME (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: AU PN6748/95 FILING DATE: 23-NOV-1995 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER:PCT INTERNATIONAL FILING DATE: 22-NOV-1996 (viii) ATTORNEY/AGENT INFORMATION: NAME: HUGHES, DR E JOHN L REFERENCE/DOCKET NUMBER: MRO/EJH/JMC (ix) TELECOMMUNICATION INFORMATION: TELEPHONE:+61 3 9254 2777 TELEFAX: +61 3 9254 2770 WO 97/19177 PCT/AU96/00747 -46- INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2575 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens TISSUE TYPE: Peripheral Blood CELL TYPE: Leukocyte (ix) FEATURE: NAME/KEY: CDS LOCATION: 102..2333 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CCCGGGCTTA GCCTTCGGGT CCACGTGGCC GGAGGCCGGC AGCTGATTGG ACGCGGGCCG CCCCACCCCC TGGCCGTCGC GGGACCCGCA GGACTGAGAC C ATG GAG GCG GTG Met Glu Ala Val GCG GTG GCC GCG GCG GTG GGG GTC CTT CTC CTG GCC GGG GCC GGG Ala Val Ala Ala Ala Val Gly Val Leu Leu Leu Ala Gly Ala Gly GCG GCA GGC GAC Ala Ala Gly Asp GAG GCC CGG GAG GCG GCG GCC Glu Ala Arg Glu Ala Ala Ala GTG CGG GCG Val Arg Ala CTC GTG Leu Val GCC CGG CTG CTG GGG CCA GGC Ala Arg Leu Leu Gly Pro Gly CCC GCG GCC GAC Pro Ala Ala Asp 45 TTC TCC GTG TCG GTG Phe Ser Val Ser Val 257 GAG CGC GCT CTG GCT GCC AAG CCG GGC TTG GAC ACC TAC AGC CTG GGC Glu Arg Ala Leu Ala Ala Lys Pro Gly Leu Asp Thr Tyr Ser Leu Gly GGC GGC Gly Gly GGC GCG GCG CGC Gly Ala Ala Arg CGG GTG CGC GGC TCC ACG GGC GTG GCG Arg Val Arg Gly Ser Thr Gly Val Ala GCC GCG GGG CTG CAC CGC TAC CTG CGC GAC TTC TGT GGC Ala Ala Gly Leu His Arg Tyr Leu Arg Asp Phe Cys Gly TGC CAC Cys His 100 WO 97/19177 WO 9719177PCT/AU96/00747 -47 GTG GCC TGG TCC GGC TOT Val Ala Trp Ser Gly Ser 105 CAG CTG CGC Gin Leu Arg CTG CCG CGG CCA CTG Leu Pro Ai-g Pro Leu 110 CCA GC Pro Ala 115 GTG CCG GGG Vai Pro Gly CAG AAT GTG Gin Asn Val 135 CTG ACC GAG GCC ACG CCC AAC AGG TAC Leu Thr Glu Ala Thr Pro Asn Arg Tyr 125 CGC TAT TAC Arg Tyr Tyr 130 TGC ACG CAA AGC TAC TCC TTC GTG TGG TGG GAC TGG GCC Cys Thr Gin Ser Tyr Ser Phe Val Ti-p Ti-p Asp Ti-p Ala 140 145 CGC TGG Arg Ti-p 150 GAG CGA GAG ATA GAC TGG ATG GCG CTG AAT GGC ATC AAC CTG Giu Arg Giu Ile Asp Ti-p Met Ala Leu Asn Gly Ile Asn Leu CTG GCC TGG AGC GGC CAG GAG GCC ATC TGG CAG CGG GTG TAO CTG Leu Ala Ti-p Ser Gly Gin Giu Ala Ile Ti-p Gin Arg Val Tyr Leu 170 175 180 641 GCO TTG GGC CTG Ala Leu Gly Leu CAG GCA GAG ATC AAT GAG TTC TTT ACT Gin Ala Giu Ile Asn Giu Phe Phe Thi- 190 GCC TTC CTG Ala Phe Leu CCC C-TG CCC Pro Leu Pro 215 TGG GGG CGA ATG Ti-p Gly Arg Met AAC CTG CAC ACC Asn Leu His Thr TGG GAT GGC Ti-p Asp Gly 210 CAG CAC CG Gi1n His Arg CCC TCC TGG CAC Pro Ser Ti-p His ANG CAG CTT TAC Lys Gin Leu Tyr GTC CTG Val Leu 230 GAC CAG ATG CGC Asp Gin Met Arg TTC GGC ATG ACC CCA GTG CTG OCT GCA Phe Gly Met Thi- Pro Val Leu Pro Ala 240 GCG GGG CAT OTT Ala Gly His Val GAG OCT GTC ACC Giu Ala Val Th- GTG TTC CCT CAG Val Phe Pro Gin 881 AAT GTC ACG AAG Aen Val Thi- Lys 000 AGT TOO 000 CAC TTT AAC TOT Gly Sei- Ti-p Gly His Phe Asn Cys 270 TOO TAO TCC Ser Tyr Ser 275 ATC ATO GG Ile Ile Gly 290 TGO TOO TTC OTT OTO OCT COG GAA GAO CCC ATA TTC CCC Cys Ser Phe Leu Leu Ala Pro Giu Asp Pro Ile Phe Pro 977 1025 AGO OTO TTC OTO OGA GAG CTG ATO AAA GAG TTT 000 ACA GAO CAC ATC Ser Leu Phe Leu Arg Oiu Leu Ile Lys Giu Phe Giy Thi- Asp His Ile 295 300 305 WO 97/19177 PCT/AU96/00747 -48- TAT GGG Tyr Gly 310 GCC GAC ACT TTC AAT GAG ATG CAG CCA CCT TCC TCA GAG CCC Ala Asp Thr Phe Ann Giu Met Gin Pro Pro Ser Ser Glu Pro 1073 TCC TAC CTT 0CC Ser Tyr Leu Ala 325 GTG GAT ACT GAG Val Asp Thr Glu GCA GCC Ala Ala 330 ACC ACT GCC GTC TAT GAG GCC ATG ACT OCA Thr Thr Ala Val Tyr Glu Ala Met Thr Ala 335 340 1121 1169 GTG TOG CTG CTC CAA GGC TGG CTC TTC Val Trp Leu Leu Gin Gly Trp Leu Phe 350 CAG CAC Gin His 355 CAG CCG CAG Gln Pro Gin GTG CCC CGT Val Pro Arg 375 TGG GGG CCC GCC Trp Gly Pro Ala ATC AGO GCT GTG Ile Arg Ala Val CTG OGA GCT Leu Gly Ala 370 GAG AGC CAG Glu Ser Gin 1217 1265 GGC CGC CTC CTG Gly Arg Leu Leu CTG GAC CTG Leu Asp Leu TTT GCT Phe Ala 385 CCT GTG Pro Val 390 TAT ACC CGC ACT Tyr Thr Arg Thr TCC TTC CAG GGC CAG CCC TTC ATC TOG Ser Phe Gin Gly Gin Pro Phe Ile Trp 400 1313 ATG CTG CAC AAC TTT GGG GGA AAC CAT GOT CTT TTT GOA Met Leu His Asn Phe Gly Gly Asn His Gly Leu Phe Gly GCC CTA Ala Leu 420 1361 GAG GCT GTG AAC GGA GGC CCA GAA GCT Glu Ala Val Ann Gly Gly Pro Giu Ala 425 GCC COC CTC TTC CCC AAC TCC Ala Arg Leu Phe Pro Ann Ser 430 435 1409 ACC ATG GTA GGC ACG GOC Thr Met Val GTG GTC TAT Val Val Tyr 455 CCA GAT TTG Pro Asp Leu 470 GTC TCC CAC Val Ser His 485 GTG TAC AAC Val Tyr Asn Gly Thr Gly 440 ATG 0CC CCC GAG GOC ATC AGC Met Ala Pro Giu Gly Ile Ser 445 CAG AAC GAA Gin Ann Glu 450 GAC CCA OTO Asp Pro Val 1457 TCC CTC ATG GCT GAG CTG GOC TO CGA Ser Leu Met Ala Glu Leu Gly Trp Arg 460 1505 GCA GCC TGG Ala Ala Trp ACC AGC TTT GCC Thr Ser Phe Ala CGO CGG TAT GGG Arg Arg Tyr Gly 1553 CCG GAC GCA GGG GCA GCG TGO Pro Asp Aia Gly Ala Ala Trp 490 TGC TCC GGG GAG GCC TGC AGG Cys Ser Gly Giu Ala Cys Arg 505 510 CTA CTG CTC Leu Leu Leu COO AGT Arg Ser 500 1601 GOC CAC AAT Gly His Asn CGT AGC CCG Arg Ser Pro 515 1649 WO 97/19177 WO 9719177PCT/AU96/00747 -49 CTG GTC AGG CGG CCG TCC CTA CAG ATG AAT ACC AGC ATC TGG TAC AAC 1697 Leu Vai Arg CGA TCT GAT Arg Ser Asp 535 A±-g Pro Ser Leu Gin Met An 525 Thr Ser Ile Trp Tyr An 530 GTG TTT GAG GCC TGG CGG Val Phe Giu Ala Trp Arg 540 CTG CTG CTC ACA TCT GCT CCC Leu Leu Leu Thr Ser Ala Pro 545 1745 TCC CTG Ser Leu 550 GCC ACC AGC Aia Thr Ser CCC CCC TTC CGC TAC GAC CTG Pro Ala Phe Arg Tyr Asp Leu 555 560 CTG GAC CTC ACT Leu Asp Leu Thr 1793 CAG GCA GTG CAG Gin Ala Val Gin CTG GTC AGC TTG Leu Val Ser Leu TAT GAG GAG Tyr Giu Giu GCA AGA Ala Arg 580 GGA GGC Gly Gly 595 1841i AGC GCC TAC CTG Ser Ala Tyr Leu AACG GAG CTG CCC TCC CTG TTG AGG GCT Lys Glu Leu Ala Ser Leu Leu Arg Ala 590 1889 GTC CTG GCC Vai Leu Ala GAC AGC CC Asp Ser Arg 615 GAG CTG CTG Giu Leu Leu CCG GCA CTG GAC GAG GTG CTG GCT AGT Pro Ala Leu Asp Giu Val Leu Ala Ser 605 610 1937 1985 TTC TTG CTG GCC Phe Leu Leu Gly TGG CTA GAG CAG Trp Leu Giu Gin CGA GCA GCG Arg Ala Ala GCA GTC AGT GAG GCC GAG Ala Val Ser Glu Ala Glu 630 CAT TTC TAC GAG CAG AAC AGC CGC TAC Asp Phe Tyr Giu Gin Asn Ser Arg Tyr 640 2033 CAG CTG ACC TTG Gin Leu Thr Leu 645 AAG CAG CTG GCG Lys Gin Leu Ala TGG CCC CCA CAA GGC AAC Trp Gly Pro Ciu Gly An 650 CTG GAC TAT CC Leu Asp Tyr Ala 2081 2129 TTG GTG GCC AAC Leu Val Ala An TAC ACC CCT CC Tyr Thr Pro Ax-g TGG CGC Trp Arg 675 CTT TTC CTG GAG GCG CTG Leu Phe Leu Glu Ala Leu 680 CAA CAG CAC CAG TTT GAC Gin Gin His Gin Phe Asp 695 CIT CTC AGC AAG CAG AG Val Leu Ser Lys Gin AZ-g 710 GTT GAC ACT GTG GCC CAC GCC Vai Asp Ser Val Ala Gin Cly 685 AAA AAT CTC TIC CAA CTG GAG Lys Ann Vai Phe Gin Leu Glu 700 705 ATC CCT TTC Ile Pro Phe 690 CAG CCC TTC Cln Ala Phe 2177 2225 TAC CCC AGC Tyr Pro Ser 715 CAG CCC CGA CCA GAC ACT GTG Gin Pro Arg Giy Asp Thr Val 720 2273 WO 97/19177 WO 9719177PCT/AU96/00747 50 GAC CTG GCC AAG AAG ATC TTC CTC AAA TAT TAC CCC GGC TGG GTG GCC Asp Lou Ala Lys Lys Ile Phe Lou Lys Tyr Tyr Pro Gly Try, Val Ala 725 730 735 740 GGC TCT TGG TGATAGATTC GCCACCACTG GGCCTTGTTT TCCGCTAATT Gly Ser Trp CCAGGGCAGA TTCCAGGGCC CAGAGCTGGA CAGACATCAC AGGATAACCC AGGCCTGGGA GGAGGCCCCA CGGCCTGCTG GTGGGGTCTG ACCTGGGGGG ATTGGAGGGA AATGACCTGC CCTCCACCAC CACCCAAAGT GTGGGATTAA AGTACTGTTT TCTTTCCACT TAAAAAAAAA AAAAAAGTCG AGCGGCCGCG AATTC INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 743 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 2321.
2370 2430 2490 2550 2575 (ix) FEATURE:
NAME/KEY:
LOCATION:
(ix) FEATURE:
NAME/KEY:
LOCATION:
(ix) FEATURE: NAMvE/KEY:
LOCATION:
(ix) FEATURE:
NAME/KEY:
LOCATION:
(ix) FEATURE:
NAME/KEY:
LOCATION:
(ix) FEATURE: Potentially-glycosylated Asn site, 261 Potentially-glycosylated Asn site, 272 Potentially-glycosylated Asn site, 435 Potentially-glycosylated Asn site, 503 Potentially-glycosylated Asn site, 513 WO 97/19177 WO 9719177PCT/AU96/00747 -51 NAME/KEY: Potentially-glycosylated Asn site, LOCATION: 526 (ix) FEATURE: NAME/KEY: Potentially-glycosylated Asn site, LOCATION: 532 Gly Val Leu Arg Glu Ala Giy Pro Ala Lys Pro Gly Val Arg Vai Arg Tyr Leu Gin Leu Arg Giu Ala Thr 125 Ser Tyr Ser 140 Asp Trp Met 155 Gin Giu Ala Ala Giu Ile Arg Met Gly 205 His Ile Lys 220 Leu Ala Ala Val Asp Phe Asp Thr Gly Ser so Asp Phe Pro Arg Asn Arg Val Trp Gin His Arg Val Leu Asp 230 Gin Met Arg Ser Phe 235 Giy Met Thr W O 97/19177 PCT/AU96/00747 52 Val Leu Pro Ala Phe Ala Gly His Val Pro Giu Ala Val Thr Arg Val 245 250 255 Phe Pro Gin Val Asn Val Thr Lys Met Gly Ser Trp Gly His Phe Asn 260 265 270 Cys Ser Tyr Ser Cys Ser Phe Leu Leu Ala Pro Glu Asp Pro Ile Phe 275 280 285 Pro Ile Ile Gly Ser Leu Phe Leu Arg Giu Leu Ile Lys Giu Phe Gly 290 295 300 Thi- Asp His Ile Tyr Gly Ala Asp Thr Phe Asn Giu Met Gin Pro Pro 305 310 315 320 Ser Ser Giu Pro Ser Tyr Leu Ala Ala Ala Thr Thr Ala Val Tyr Glu 325 330 335 Ala Met Thr Ala Val Asp Thr Giu Ala Val Trp Leu Leu Gin Gly Ti-p 340 345 350 Leu Phe Gin His Gin Pro Gin Phe Ti-p Gly Pro Ala Gin Ile Arg Ala 355 360 365 Val Leu Gly Ala Val Pro Arg Gly Arg Leu Leu Val Leu Asp Leu Phe 370 375 380 Ala Glu Ser Gin Pro Val Tyr Thi- Arg Thr Ala Ser Phe Gin Gly Gin 385 390 395 400 Pro Phe Ile Ti-p Cys Met Leu His Asn Phe Gly Gly Asn His Gly Leu 405 410 415 Phe Gly Ala Leu Glu Ala Vai Asn Gly Gly Pro Giu Ala Ala Arg Leu 420 425 430 Phe Pro Asn Ser Thi- Met Val Gly Thi- Gly Met Ala Pro Giu Gly Ile 435 440 445 Ser Gin Asn Glu Val Val Tyr Ser Leu Met Ala Giu Leu Gly Ti-p Arg 450 455 460 Lys Asp Pro Val Pro Asp Leu Ala Ala Ti-p Val Thr Ser Phe Ala Ala 465 470 475 480 Arg Arg Tyr Gly Val Ser His Pro Asp Ala Giy Ala Ala Ti-p Arg Leu 485 490 495 Leu Leu Arg Ser Val Tyr Asn Cys Ser Giy Giu Ala Cys Ax-g Gly His 500 505 510 Asn Arg Ser Pro Leu Val Arg Arg Pro Ser Leu Gin Met Asn Thr Ser 515 520 525 WO 97/19177 PCT/AU96/00747 53 Ile Trp Tyr Ann Arg Ser Asp Val Phe Giu Ala Trp Arg Leu Leu Leu 530 535 540 Thr Ser Ala Pro Ser Leu Ala Thr Ser Pro Ala Phe Arg Tyr Asp Leu 545 550 555 560 Leu Asp Leu Thr Arg Gin Ala Val Gin Giu Leu Val Ser Leu Tyr Tyr 565 570 575 Giu Glu Ala Arg Ser Ala Tyr Leu Ser Lys Giu Leu Ala Ser Leu Leu 580 585 590 Arg Ala Gly Gly Val Leu Ala Tyr Giu Leu Leu Pro Ala Leu Asp Glu 595 600 605 Val Leu Ala Ser Asp Ser Arg Phe Leu Leu Gly Ser Trp Leu Giu Gin 610 615 620 Ala Az-g Ala Ala Ala Val Ser Glu Ala Giu Ala Asp Phe Tyr Giu Gin 625 630 635 640 Ann Ser Arg Tyr Gin Leu Thr Leu Trp Gly Pro Giu Gly Ann Ile Leu 645 650 655 Asp Tyr Ala Ann Lys Gin Leu Ala Gly Leu Val Ala Ann Tyr Tyr Thr 660 665 670 Pro A±-g Trp Arg Leu Phe Leu Giu Ala Leu Val Asp Ser Val Ala Gin 675 680 685 Gly Ile Pro Phe Gin Gin His Gin Phe Asp Lys Asn Val Phe Gin Leu 690 695 700 Giu Gin Ala Phe Val Leu Ser Lys Gin Arg Tyr Pro Ser Gin Pro Arg 705 710 715 720 Gly Asp Thr Val Asp Leu Ala Lys Lys Ile Phe Leu Lys Tyr Tyr Pro 725 730 735 Gly Trp Val Ala Gly Ser Trp 740 INFORMATION FOR SEQ ID NO:3: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 10380 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 97/191 77 PCTIAU96/00747 54- (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (viii) POSITION IN GENOME: cHRomosoME/SEGMENT: Chromosome 17 (ix) FEATURE: NAME/KEY: exon 1 LOCATION: 990. .1372 (ix) FEATURE: NAME/KEY: exon 2 LOCATION: 2115. .2262 (ix) FEATURE: NAME/KEY: exon 3 LOCATION: 3056. .3202 (ix) FEATURE: NAME/KEY: exon 4 LOCATION: 3387. .3472 (ix) FEATURE: NAME/KEY: exon LOCATION: 5667. .5923 (ix) FEATURE: NAME/KEY: exon 6 LOCATION: 7745. .8955 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATAATGAGCA GTGAGGACGA TCAGAGGTCA CCTTCCTGTC TTGGTTTTGG CAGGTTTTGA CCAGTTTCTT TGCTGCATTC TGTTTTATCA GCGGGGTCTT GTGACCTTTT ATCTTGTGCT GACCTCCTGT CTCATCCTGT GACGAAGGCC TAACCTCCTG GGAATTCAGC CCAGCAGGTC TCTGCCTCAT TTTACCCAGC CCCTGTTCAA GATGGAGTCG CTCTGGTTGG AAACTTCTGA CAAAATGACA GCTCCTGTTA TGTTGCTGCT GCTGCCGCCA. ATGGACAGCC TTTAACGTGC CCGCCAGCCC TGCTCCACCG CCGGCCTGGG CTCACATGGC CCC.ATCCCTC CTCGAACCTC CTAGCCTGTT AGTTACTCAA ATCTGCAAGC TCTCTGCCTT CTCAGGGCCT TCAATAAATG CATTTCTTCT GTCTGGAAGG CTCTTCCTTT CCCTCTTCTA GCCAATTCCT ATTCATCCCT GAGTTTCAGA TTAAAAGTCA CTTCCTTTGG AAACCTTACT TCGCTACTTC GCTACTTACT GCACTACTTC GCAGCATCAC AACTATGATG GAAATCCTTA CTTACGTTAA ATATCTGGTT 120 180 240 300 360 420 480 540 600 WO 97/19177 WO 9719177PCT/AU96/00747 55 TCTAGGTCAC CTCCCTGACG GGGACGGTAG GGACCGTCTT TAGCTATGGC AGTGCCTGAT ACAAAATAAA CTCCAAATGT
TGGAAGTTAT
GTCCCCTTCG
CCCCAAGGGA
CCTTCGGGTC
GCCGTCGCGG
GGGTCCTTCT
CCGTGCGGGC
CGGTGGAGCG
GCGCGGCGCG
GCTACCTGCG
CGCGGCCACT
CCGAAGCTTC
ATCGGGAGGC
GTGTGGCCTT
GAAAGAAGAC
TTTGTGGTGC
GAACCTGCGG
CCTGCCACAC
TGTGGGGGCA
GTGGCATGAA
TGCAGGGGAC
TGGGTCCCAG
GGGATGGGGG
GACACTGCCC
TGGTGGGACT
TTGCGTGTGA AAGCGCGTTT GCCCTGGAGC CGGGGTCACA GTATCCTGGT ACCCGGAAGC CACGTGGCCG GAGCCGGCAG GACCCGCAGG ACTGAGACCA CCTGGCCGGG GCCGGGGGCG GCTCGTGGCC CGGCTGCTGG CGCTCTGGCT GCCAAGCCGG CGTGCGGGTG CGCGGCTCCA CGACTTCTGT GGCTGCCACG GCCAGCCGTG CCGGGGGAGC CCCGCGTCCG CCCGAGGCGC TGAGCGGGGA GCGCTGGCCG GAGCCAGCCA CTCTGCCTTT GCCTACCGTG CAGTGTTATT ACAATTGGTG ATGAGTGAAT ACTGAGGAAG GACGCCTCCA TATGGAGTGA TGTGTTCACA GGGATTCCCC GTTCCAGGAA AGTGGAATAT GCCACCCAAA GAGTGCCTCA GAAGCCCAGC TGTGCAGCAG AAGGGCCGAG
TACCCGAAGG
CGCTCCCCAC
CGCGACTCCT
CTGATTGGAC
TGGAGGCGGT
CGGCAGGCGA
GGCCAGGCCC
GCTTGGACAC
CGGGCGTGGC
TGGCCTGGTC
TGACCGAGGC
TTACCCCCTC
GAAGGCCCAG
CAGAGCCTCG
GTGAGGATTT
TTTCTTGCCT
TCCCCCACCC
CAGCTGTCCT
AACACCGTGC
TACCCGCCAG
CCCGG3TACCT CTCGTTCATC AGTAGGGAAG GTATTTATTA GATGGTTGGA CGCTCTGTGA GGGCCAGCGG CGCGTGCGGT CACGAGACGC
GGCCCTGAGC
GCGGGCCGCC
GGCGGTGGCC
CGAGGCCCG4G
CGCGGCCGAC
CTACAGCCTG
GGCCGCCGCG
CGGCTCTCAG
CACGCCCAAC
CCGGAGCCGC
CTGCGCCGCC
GCTGGCCCAC
GCACGATGAT
CCGGGCTTAG
CCACCCCCTG
GCGGCGGTGG
GAGGCGGCGG
TTCTCCGTGT
GGCGGCGGCG
GGGCTGCACC
CTGCGCCTGC
AGGTACCGCC
TGCCACCCAA
TCCAGCAGCT
CTGAAAAACG
GGGCATAGAA
660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 TCCTCCCCCA CCTTCTCTTT TACAGGCCTG TGTTCCAGCG CCCCTGCCCA TCTGTTAGAC AGAGGAGGGG CTCTGGCAGT GCTAGAGGGC CCTGGGAGAG GGTCTCAGCT CCACCTGGGG TTTGGAGCCC CTCCCCTCTC CTCTAGGTGG
ATTTGTTCCA
GCAGGTACCG
GGGCCCGCTG
GGGCCGTGGA CCCTCCAGGG TGGGATGCGC CTATTACCAG AATGTGTGCA CGCAAAGCTA GGAGCGAGAG ATAGACTGGA TGGCGCTGAA
CCCTGCTCAT
CTCCTTCGTG
TGGCATCAAC
I
WO 97/19177 WO 9719177PCT/AU96/00747
CTGGCACTGG
TTCCCCACCC
GGCGCAGTGT
ACTGAGGCTT
TAGGCGGGCG
CGTCTCTACT
AAAAAAAAAA
CCCTATGCAG
TCCCTGGAAG
TTGCCCTGCC
GGGGAAAGCC
CACCTACTCA
TTGTCTCAGT
ATGAATGAAT
CCTGGCCTTG
GGCCTGGGGG
CATCAAGCAG
AGATGGTCAT
GGGGGGCTGC
CACCACCCTT
TGACCCCAGT
CCGCTCACCC
AAAAACTTAA
GGCCGAGTTG
AAACCCTGTC
CCCATCTACT
CCTGGAGCGG
TCCTCTATGG
CTCTCTCTAG
CCGGCCGGGC
GATCAGGAGT
AAAATACAAA
AAAAAAACTG
CCAATCACCT
CTTCTGTGCT
CTTCCATCTG
CCTTTGTGCC
CAGGTCTCTT
AGCCCTAGCA
GAATGAAGAT
GGCCTGACCC
56 CCAGGAQCC ATCTGGCAGC CGGGAGCCAC CGTAGGTGTT AAGTGCTTTC AGCGTGCACA GCGGTGGCTC ACGCCTGTCA TCAGGAGATC GAGACCATCC
GAAATAGCAA
AGGCTTCCAG
GGTCCCTTGG
GCAATGGCTG
GCACTCTTGC
CCATCTCCTC
TCCTCTGGGC
CCCAGCACAA
GAATATATTT
AGGCAGAGAT
CCTGGGCAAC
TTTGAGGAGT
ATCCAACTCA
CTCCAGGCTC
TCCATGAAGC
GGGTGCGTGC
TTCACCCGCC
GTGGCTTGGG
TCCCAGCACT
TGGCCAACAT
AGAGCGAGAC
GGGGCTCCTT
TGGGCAGCTC
TGCTTAAGCT
CTTCTAAGGC
ACCCATGCGA CAAAGGCAAC TGTGGGCTCC TTGATGGCAG AGAAGCAATG AGTGAATGGT CTATGTGTGG GCCCTTCTTC
CCACTGTCCC
CCCCAGCATG
CCTCCTAAAA
TCGGGAGGCC
TGTGAAACCC
TCTGTCTAAA
CCCCCATCTC
TAGATCTGCC
CTTCACACAG
CTTCCTGTTG
ACAGTGAACT
CGTTCGGATT
TGTTGAATGA
CTCAGGTGTA
CTGCCTTCCT
CCTCCTGGCA
AATCGGTGAT
TAACTGAGGC
TGGCCATGCT
TCCTTCGGCA
AGGTGAGGTT
AACATGACTT
CACTTTGGGA
CAACATGGTG
CGCCTGTAAT
AGACGTTGCA
2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 CGAATGGGCA ACCTGCACAC CTTTACCTGC AGGTAALAAGG GGGCCCAGGA AGGGTGGTAT GTGTATCCTG GGAGATGAGG CCTTCTGTTC CTCCAGCACC GCTGCCTGCA TTCGCGGGGC CCTCCACTTA GCTCAGAGAG GCTCTGQGCC GGGCGCAGTG GGCGGATCAC CTGAGGTCAG TCTACTAAAA ATATAAAAAT TAGGAGGCTG AGACAGGAGA
CAATGAGTTC
CTGGGATGGC
ATGGAAAAGG
TAGGCCGGCC
GCCTTCTCAT
GGGTCCTGGA
ATGTTCCCGA
GGAATTTTAT
GCTCACGCCT
TTTACTGGTC
CCCCTGCCCC
GAAGGGGCAG
CCAGGGCTCT
AGGACAGCAG
CCAGATGCGC
GGCTGTCACC
TCCCTTCTAG
GTAATCCCAG
GAGTTCGAGA CCAGCCTGGC TAGCTGGGCA TGGTGGCACG ATTGCTTAAA CCTGGGAGGC GTGAGTCAAG ATCACGCCAT TGCACTCCAG CCTGGGTGAC GAGCGAAACT CTGTCTCAAA
I
WO 97/19177 WO 9719177PCT/AU96/00747 -57- GTTTGATTTC TGACTCTGCT ACTAATTAGC CAAACAAACA AGCTCTGGAC GTAGGCCTGG TGTGTGACTT CGGGCAGATG GGGATCTCTA CCCACTTCGC GCACAGAGTG CTAGCAAATG TTGTCCCTCC TGTTCCTCTG TTCAAGGCTC ACCTGAGACC TCCTTTGCAC CTQCTAATGT GCCTCCCTGC CCGCCTCTTT AATAAACTTG GTCCAGCTGT GGCAAGAAAG GTGCCCAAGG AATGTGGGGA CAAGGACAGG GCAGGAAGGG CTCAGGGCAG TGGCTAATGC TTGTAATCCT AGGAGTTAAA GACCAGCCTG ATTAGCCAGG CGTGGTGGCG GAATCTCTTG AAGCCAGGGG AGCCTGGGTG ACAGAGTGAG TAAACACCTC ATGTTCTCAC GAAGGGGAAC ATCACACACC TTGGGACAGA TACTTAATGC GCAAACCACC ATGGCACATG ACATGACTGC TCTGTGCCTC AGTTTCCTTA TGTAGGGTTT GTAATTATCT CTCGATCTAT GCAGCCCTTG GGAGTGGCAG CAGGGGTGCT TGCTTCCCAG CCATCCTCTC ACATGTGGTT TCCCCTCCTT CAGGAAGCCT TGCTAGTGCC CTGGCTCCCA TACTCTCGTA GGACTTAATG GCCCCCATCA CCAGGTGGCA GGAAACTCAC CTGAGGCTGC CAGAACTGGC TGTGCTGGGT GCTTACCCCT GATAGGAGAG ATATGTTGGC AATATATGTG GGTTCCGCTC TCCTCTGCCG AGCCCAGCCT TGAAAAATGA GTGTTGCTTG AGCGTTTTGG GAGGCTGAGG CGTATGGATC GCCAACATGG CGAAACCCCA TCTCTACTAA GGCTCCTGTA ATCCCAGCTA CTCGGTAGGC CCAGAGACTG CAGTGAGCCG AGATCACACC ACTCCGTCTC AAAAAAAAAA AAAAAAAAAG TCATAGTGGG AGTTGAACAA TGAGAACAAC GGGGCCTTTC GCGGTGTGGG GGTCAAGGGG ATGCGGGGCT GAAAACCTAG ATGATGGGTT TATACCTATG CAACAAACCT GCATGTTCTG
CTTGTAAAAT
CTGTGACTTT
CCAGTGTCCC
GGGAAAAGTC
CCGCATGACC
CATGCCAGTG
TCATTCATTC
CCTGGGAGGC
TGAAGGATAC
GGAGAGAGGG
GACGGACGCT
ACCTGCGGTC
AAGTACAAAA
TGAGGCATGA
ACTTCACTCC
GAAAGAAAAT
ATGGACACAG
AGGAGTAGCA
GATGGGTGCA
CACAGAACTG
3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460
AACTGAAAGT
CCCAGCACTT
GGCTAACACA
CGGGCACCTG
AGGCAGAGCT
AGACTCTGCC
ATAATTAAAA AAAAAAAAAA TGGGAGGCCG AGACGGGCGG GTGAAACTCA GTCTCTACTA TAGTCCCAGC TACTAGGGAG TGCAGTGAGC TGAGAATGCG TCAAAAAAAA AAAAAAAAAG
AAGCTGGGTG
ATCACAAGGT
AAAATACAAA
GCTGAGGCAG
CCACTGCACT
AAAGAAAAAG
CGGTGGCCCA CACCTGTAAT CAGCAGATCG AGACCATCCT AAATTAGCCG GGTGTGGTGG GAGAATGGCA TGAACCTGGG CCAGCCTGGG GGACAGAGTG GAGCGTTGCT TGTTTCAGGC WO 97/19177 W097/9177PCT/AU96/00747
CACAGGAAGG
AGGACTGTAG
TGTAGAGAAG
ATATCTGAGC
AGTTGGGGCC
ATATTCCCCA
CACATCTATG
CTTGCCGCAG
GGTGGGAGAG
CAGAGGGACT
TGTACACATG
ACAACCTGGG
TCCTTCCCAG
GGAGAGATAG
GCAGAGAGCA
TTGGCAAGGC
TTTTGCTCCC
ACTTTAACTG
TCATCGGGAG
G-GGCCGACAC
CCACCACTGC
CCCCCCAGAC
TGAAAGTTTT
TAGCCTGTAC
TGTTGAACAC
CACTAGGGTG
TTCCTACTCC
CCTCTTCCTG
TTTCAATGAG
CGTCTATGAG
CCTCAAAAAG
GGAATAATGC CTCGCCATAA CGTTGTCTCA GTGAATCCCA TCACACCTCG CGCTCCTATT TGTCTCGTTT GCTTTTCCTG -58-
TCAGAGAAGG
AAAGCCATAG
TATGGTGAAC
TTCCCTCAGG
TGCTCCTTCC
CGAGAGCTGA
ATGCAGCCAC
GCCATGACTG
AAGGGAGTAG
CACACAGTAC
CTGTGGTTGA
TCCTGGCCGT
TAAACTGGGA
CTAGCTATGA
CAGGAGGTGA
GCCAGGGCTC
TGACTTAGTG
TGAGCAGTAC
TGTGAGATGG
TTTTTTGAGA
CACTGCAACC
GGGACTATAG
GCTCTGTCAC
CTGAGTTCAA
CGCCTGGCCC AGAGCAAAGG CCACTAAGAG
CTGCGTGGTT
TAAGTGCGCA
GAGTGGAGCA
AGGAAGGGAA
ACCAGCAGCA
AAGACCTGAA
AGGCTGGAGC
GATTCTCCTG
CAATTTTTGT
ACGATCTCAG
TCCTGAGTAG
AGTAGAGACG
TTCGCCCACT
TGGCCAGGGA AGGAGAAGAA AGGCAAGAGA AACCAGGAGC ACTATGGCGG CTTCCATGAA
GCTGAAGTAG
TCCTGCTACA
GAGGGCGCCC
CAGATCAGCT
GCAGCAGCAG
GGAGAAGCTA
ACAGTGGCAC
CCTCAGCCTC
TTGTTTTGAG
TTCACTGCAA
CTGGGATTAC
GGGTTTCGCT
TCAGCCTCCC
GCACTGCAGG
AAACTGTGAA
AAGTCAGGAA
CAGCCTTTCT
CAGCCCGAGC
CATTTTTTTT
AATCTCATCT
CCGAGTAGCT
ATAGAGTCTC
CCTCTGCTTC
TCAATGTCAC
TTCTGGCTCC
TCAAAGAGTT
CTTCCTCAGA
CAGGTACAGT
CAGATGTCAG
TTTATAGTTT
GAGGTGAGCT
GTGACTTATG
CTACCTCATA
ACAAGGATTT
GTGGATGTGC
CTTCCTGCCA
GAGAGGCGTC
TGCTCCAAGT
CAAATTCTCA
CAGATTTCAC
TTCGTCTCCT
GCACCCGCCA
CCAGGCTGGA
GCGATTCTCC
CGGCTAATTT
AAACTCCTGA
GTGAGCCACC
GAAGATGGGC
GGAAGACCCC
TGGCACAGAC
GCCCTCCTAC
GCCTGGGTGG
TAGGGGTAGG
ACCAAGCACG
CTGGAAGCCA
ACTCATGACC
GGTAGAATAA
TGTTTCATCT
CTAAAGGCAC
CTTAAAGGAG
TGTGTTGGCC
GTGACCCAAA
GGCCCTACCC
TCTGTTGCTG
AGGTTCAAGC
CCACGCCCGG
GTGCAGTGGC
TGCCTCAGCC
TTGTATTTTT
CCTCAAATGA
TTGCCTGGCC
5520 5580 5640 5700 57.60 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 AGGCGCCCCC CAACCACACT ATGTAGGTCA AGCTGGTTTC AAAGTGCTGG GATTACAGGT WO 97/19177 WO 97/91 77PCT/AU96/00747 -59- A-AACAGGTTT CACCATGGTG GCCAGACTGG TCTCAAACTC AATTTTTGTA TTTTTAGTAG CTGACCTCAG GTGAACTGCC CACCTCAGCC TCCCAAAGTA CTGGTATTAC
CACTGCGACT
AGTGCAGTGG
GGCCTTGATT
CACAATCTCA
TTGTTTTTGA GACAGAATCT GCTCACTGCA ACTTCTGCCT GTGCCTCTAC CTCCCGAGTA GCCGG3GATTA CAGGCACCTG TTGTATTTTT AGTATAGACA GCTCAAGTGA TCCACCTGCT ACGCCCATTC AGAAACCTCC AAGCTGCTGA ACCCTGTCCT GCTGGTGGGG GTCATGGGAA AACATTCCCT GGGCCCTCTG ACAGTGGATA CTGAGGCTGT TTCTGGGGGC CCGCCCAGAT GTTCTGGACC TGTTTGCTGA CAGCCCTTCA TCTGGTGCAT CTAGAGGCTG TGAACGGAGG GGCACGGGCA TGGCCCCCGA GAGCTGGGCT GGCGAAAGGA GCCCGGCGGT ATGGGGTCTC AGTGTGTACA ACTGCTCCGG CGGCCGTCCC TACAGATGAA TGGCGGCTGC TGCTCACATC CTGCTGGACC TCACTCGGCA AGAAGCGCCT ACCTGAGCAA TATGAGCTGC TGCCGGCACT AGCTGGCTAG AGCAGGCCCG
GGGTTTCCCC
TCAGCCCCTC
ATGTTTTAAG
GGAGTTTTCA
GCCATGACCT
TTTCATCACT
GTGGCTGCTC
CAGGGCTGTG
GAGCCAGCCT
GCTGCACAAC
CCCAGAAGCT
GGGCATCAGC
CCCAGTGCCA
CCACCCGGAC
GGAGGCCTGC
TACCAGCATC
TGCTCCCTCC
GGCAGTGCAG
GGAGCTGGCC
GGACGAGGTG
AGCAGCGGCA
ACATTGGCCA
AGAGTACTGG
GAGCCCTCTG
GAGGGACGCG
GGGATAGACA
CCTCTTCTCT
CAAGGCTGGC
CTGGGAGCTG
GTGTATACCC
TTTGGGGGAA
GCCCGCCTCT
CAGAACGAAG
GATTTGGCAG
GCAGGGGCAG
TACTCTGTCG
CATGGGTTCA
CCATTACGCT
GGCTGGTCTG
GATTATAGGT
GGTAACTCTC
TATGTGCCAC
GTCGTCTGTA
GTTCCCCCTA
TCTTCCAGCA
TGCCCCGTGG
GCACTGCCTC
ACCATGGTCT
TCCCCAACTC
TGGTCTATTC
CCTGGGTGAC
CGTGGAGGCT
AGGCGTGATC
CCCAGACTGG
AGTGATTCTT
AGGCTAATTT
GAACTCCTGG
GTGGGCCACC
ATGTTCACCC
AGAGCGTCCC
GAGTGGGGTG
CCTCCTGTCC
CCAGCCGCAG
CCGCCTCCTG
CTTCCAGGGC
TTTTGGAGCC
CACCATGGTA
CCTCATGGCT
CAGCTTTGCC
ACTGCTCCGG
GCTGGTCAGG
GTTTGAGGCC
CCGCTACGAC
TGAGGAGGCA
CGTCCTGGCC
CTTGCTGGGC
TTTCTACGAG
71.40 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 AGG~GGCCACA ATCGTAGCCC TGGTACAACC GATCTGATGT CTGGCCACCA GCCCCGCCTT GAGCTGGTCA GCTTGTACTA TCCCTGTTGA GGGCTGGAGG CTGGCTAGTG ACAGCCGCTT GTCAGTGAGG CCGAGGCCGA CAGAACAGCC GCTACCAGCT GACCTTGTGG GGGCCAGAAG GCAACATCCT GGACTATGCC WO 97/19177 WO 9719177PCT/AU96/00747 60 TACTACACCC AACAAGCAGC TGGCGGGGTT GGTGGCCAAC GAGGCGCTGG TTGACAGTGT GGCCCAGGGC CTCGCTGGCG GCTTTTCCTG
AATGTCTTCC
CGAGGAGACA
GCCGGCTCTT
GATTCCAGGG
CACGGCCTGC
ACCACCCAAA
GGGTCTGTCA
TGGCCCTGGG
TGTTTGTTTG
GGCTC-ACTGC
AGCTGGGACT
TCTTGCTCTG
CCTCCCGGGT
GTGCCATCAC
CCAGGCTGGT
TGGGATTACA
CCCTGACCTT
TTGTCCTTTG
TATTTGGAGG
TCAGTTCCAA
CATGAGACCA
ACTGGCTTCA
GACAGTGACC
AAAAAATTTT
AGTGGCAGGA
AACTGGAGCA
CTGTGGACCT
GGTGATAGAT
CCCAGAGCTG
TGGTGGGGTC
GTGTGGGATT
AAPATGAGAAG
GTGGGACCTG
TGACGGAGCC
AACCTCCGCC
ATAGGCATGC
TCGCCCAGGT
TCAAGCAATT
GCCTGGCTAA
CTTTAACTCC
GGAGTGAGCC
TGAACGTAGA
CTGGCATGCC
ATTTTCCCCA
TGTAATCAAC
CCCCCTCCTT
AGTTGGAGTT
CACGAAACAC
TTTCTTTCTT
TCTGGCTCCG
GGCCTTCGTT
GGCCAAGAAG
TCGCCACCAC
GACAGACATC
TGACCTGGGG
AAAGTACTGT
GTCACTGCTG
TTCTCCCATC
TTGGTCTGTT
TCCTGGGTTC
ACCACCACAC
TGGAGTTTAG
CTCCTGCCTC
TTTTTGTATT
TGAACTCAAG
ACCGTGCCCG
ATGCCCTTCT
ACAGCAACAG
CACACACCAA
CAGAGACAGC
CCCAACGGTT
CCCATGACCC
AGGGAAACCC
ATCCCTTTCC AACAGCACCA CTCAGCAAGC AGAGGTACCC ATCTTCCTCA AATATTACCC TGGGCCTTGT TTTCCGCTAA ACAGGATAAC CCAGGCCTGG GGATTGGAGG GAAATGACCT TTTCTTTCCA CTTAAACTGA CCACGCTTGG GAGGACTCAG CCTTGCCTCA CGTCCCTGTT
GTTTGACAAA
CAGCCAGCCG
CGGCTGGGTG
TTCCAGGGCA
GAGGAGGCCC
GCCCTCCACC
TGAGTCCCCT
GGCTATAGCA
TTTGTTTGTT
GCACAGTCTC
CCTCCCCGGT
CAAGATGGAG
GCAACCTCTG
GCCCAGGCTT
AAGCAATTCT
CAGGCTAATT
TGGCACCATA
AGTCTACCAG
TTTCATAGAG
TGATCCACCT
GCCATGTCTC
GTTGCAGGAA
TCACCAACAC
GCAGCAGACA
ATCAGATCCC
ACAAGTCCTG
CCTTCCCCTC
TTATTATGTT
GAGTACAATG
TGTGCCTCAG
TTTTTTTTTC
TTGGTTTACT
8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 GGAGTTAGGA CTACGGGCCT ATAAGGTTTC ACCATGTTGG GCCTCGGCCT TCCAAAGTGC TCTTTTTAAC ACTAATGTTA AACCTCTTTT CAAACCATGT AGAAGACTTC TGTGACCAAA TCAGCTGGGT GTCCTCCAAT ACAGGOTTAG GGTGCAGATC ATCCCTGGAA CTTCTGACTA TTTGGAGTCA ACTCATTTGC TATTGCTTTA TTACAGAGGA CTGTCATCCA GAATGACTGC TGGCATGATC TCGGCTCACT TTTTGAGACA GGGTCTCACT TCACCCAGGC TGGAGTGCAG WO 97/19177 PCT/AU96/00747 -61- ACAGCCTCCA TCCCCCCCAA ACCCCACGCC TCAGCGCCCC ACCCCGCAAG TGGCTGGGAC 10380 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: Modified-site LOCATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Asp Glu Ala Arg Glu Ala Ala Ala Val Arg Ala Leu Val Ala Arg Leu 1 5 10 Leu Gly Pro Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: WO 97/19177 PCT/AU96/00747 -62- ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: Modified-site, glycosylated or phosphorylated, wherein Xaa may be any amino acid residue, preferably Arg.
LOCATION: 16 (xi) SEQUENCE DESCRIPTION: SEQ ID Lys Pro Gly Leu Asp Thr Tyr Ser Leu Gly Gly Gly Gly Ala Ala Xaa Val Arg INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: Modified-site, glycosylated or phosphorylated, wherein Xaa may be any amino acid residue, preferably Ala LOCATION: 12 (ix) FEATURE: NAME/KEY: Modified-site, glycosylated or phosphorylated, wherein Xaa may be any amino acid residue, preferably Ser LOCATION: 14 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Trp Arg Leu Leu Leu Thr Ser Ala Pro Ser Leu Xaa Thr Xaa Pro 1 5 10

Claims (68)

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a mammalian a-N-acetylglucosaminidase and being substantially as set forth in SEQ ID NO: 1 or SEQ ID NO:3, a nucleotide sequence which is complementary to SEQ ID NO:1 or SEQ ID NO:3, or a nucleotide sequence having at least 80% similarity to any one of the aforementioned sequences.
2. The isolated nucleic acid molecule of claim 1, wherein said nucleotide sequence is approximately 2.6 kb in length.
3. The isolated nucleic acid molecule of claim 1 or 2 wherein the mammal is a human.
4. The isolated nucleic acid molecule of any one of claims 1 to 3 comprising the nucleotide sequence substantially as set forth in SEQ ID NO:1 or complementary thereto or having at least 80% similarity thereto. se*.: o S 5. The isolated nucleic acid molecule of any one of claims 1 to 3 comprising the nucleotide sequence substantially as set forth in SEQ ID NO:3 or complementary thereto or having at least 80% similarity thereto.
6. The isolated nucleic acid molecule according to any one of claims 1 to wherein the a-N-acetylglucosaminidase encoded by said molecule comprises an amino acid sequence substantially as set forth in SEQ ID NO:2 or at least 80% similar thereto.
7. The isolated nucleic acid molecule according to any one of claims 1 to 6 wherein said molecule is carried by a vector capable of replication in a eukaryotic cell or a prokaryotic cell. 64
8. The isolated nucleic acid molecule according to claim 7 wherein the vector is an expression vector.
9. The isolated nucleic acid molecule according to claim 8 wherein the expression vector is capable of being expressed in cells derived from a eukaryote. The isolated nucleic acid molecule according to claim 9 wherein the expression vector is further capable of being expressed in cells derived from a mammal.
11. The isolated nucleic acid molecule according to claim 10 wherein the expression vector is further capable of being expressed in CHO cells.
12. A recombinant mammalian a-N-acetylglucosaminidase or enzymatically active fragment or derivative thereof comprising an amino acid sequence substantially as set forth in SEQ ID NO:2 or having at least 80% similarity thereto.
13. The recombinant mammalian a-N-acetylglucosaminidase or enzymatically active fragment or derivative thereof according to claim 12 in substantially pure form.
14. The recombinant mammalian a-N-acetylglucosaminidase or enzymatically yeast or insect cells The recombinant mammalian a-N-acetylglucosaminidase or enzymatically active fragment or derivative thereof of claim 14 when expressed in mammalian cells.
16. The recombinant mammalian a-N-acetylglucosaminidase according to claim 14 or 15, wherein the cells are capable of glycosylating said recombinant mammalian -\-acetylglucosaminidase.
17. The recombinant mammalian a-N-acetylglucosaminidase according to claim 16 wherein the cells are capable of N-glycosylating said recombinant mammalian a-N-acetylglucosaminidase.
18. The recombinant mammalian a-N-acetylglucosaminidase according to claim 17 wherein the cells are CHO cells.
19. The recombinant mammalian a-N-acetylglucosaminidase according to any one of claims 12 to 18 wherein said recombinant a-N-acetylglucosaminidase is in a glycosylated form. The recombinant mammalian a-N-acetylglucosaminidase according to claim 19 wherein the molecular weight of the glycosylated form is at least approximately 79 kDa as determined using SDS/PAGE.
21. The recombinant a-N-acetylglucosaminidase according to claim 20 wherein the molecular weight of the glycosylated form is approximately 79 kDa to 89 kDa as determined using SDS/PAGE. S 20 22. The recombinant mammalian a-N-acetylglucosaminidase according to any one of claims 12 to 21 comprising a sequence of amino acids substantially the same as a human a-N-acetylglucosaminidase. too.
23. The recombinant mammalian a-N-acetylglucosaminidase according to any one 25 of claims 12 to 22 when fused to another proteinaceous molecule. t.
24. The recombinant mammalian a-N-acetylglucosaminidase according to claim 23 wherein the other proteinaceous molecule is an enzyme, reporter molecule, purification site and/or a signal sequence. 66 A recombinant a-N-acetylglucosaminidase when produced by expression of a nucleic acid molecule according to any one of claims 7 to 11.
26. The recombinant a-N-acetylglucosaminidase according to claim 25 when glycosylated.
27. A method of diagnosing a mutation in a gene which encodes a-N-acetylglucosaminidase in a human patient, said method comprising contacting genomic DNA or RNA derived from said patient with one or more isolated DNA molecules or oligonucleotides each comprising at least 10 contiguous nucleotides derived from and specific to SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand thereof for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means.
28. The method according to claim 27 wherein the detection means is a reporter molecule covalently attached to the isolated DNA molecule or oligonucleotide. 9
29. The method according to claim 28 wherein the reporter molecule is 32p, 3 5 S or biotin. The method according to claim 27 wherein the detection means is a polymerase chain reaction format. 9*9*
31. The method according to claim 30 wherein the polymerase chain reaction 25 format is selected from the list comprising SSCP, AMD, AFLP, IRS-PCR, iPCR or RT- PCR, amongst others.
32. The method according to claim 31 wherein the polymerase chain reaction format is SSCP. The method according to any one of claims 27 to 32 wherein the isolated DNA m ule or oligonucleotide comprises at least 20 contiguous nucleotides derived 67 from and specific to SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand thereof.
34. The method according to claim 33 wherein the isolated DNA molecule or oligonucleotide comprises at least 50 contiguous nucleotides derived from and specific to SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand thereof. The method according to claim 34 wherein the isolated DNA molecule or oligonucleotide comprises at least 100 contiguous nucleotides derived from and specific to SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand thereof.
36. A method for treating a patient suffering from a-N-acetylglucosaminidase deficiency, said method comprising administering to said patient an effective amount of a recombinant mammalian a-N-acetylglucosaminidase or an enzymatically active fragment or derivative thereof according to claim 12 or 13.
37. The method according to claim 36 wherein the mammalian a-N-acetylglucosaminidase comprises a sequence of amino acids substantially the *O same as the amino acid sequence of human a-N-acetylglucosaminidase. S
38. The method according to claim 37 wherein the patient is suffering from mucopolysaccharidosis type IIIB.
39. The method according to any one of claims 36 to 38 wherein the recombinant 25 a-N-acetylglucosaminidase is produced in mammalian cells. The method according to claim 39 wherein the mammalian cells are capable of glycosylating the recombinant a-N-acetylglucosaminidase produced therein.
41. The method according to claim 40 wherein the recombinant :,ST ^cetylglucosaminidase is in a glycosylated form.
42. The method according to claim 41 wherein the glycosylated form of the recombinant a-N-acetylglucosaminidase has a molecular weight of at least approximately 79 kDa as determined using SDS/PAGE.
43. The method according to claim 42 wherein the glycosylated form of the recombinant a-N-acetylglucosaminidase has a molecular weight of approximately 79 kDa to 89 kDa as determined using SDS/PAGE.
44. A method for treating a patient suffering from a-N-acetylglucosaminidase deficiency, said method comprising administering to said patient an effective amount of a recombinant mammalian a-N-acetylglucosaminidase or an enzymatically active fragment or derivative thereof, wherein said recombinant mammalian a-N-acetylglucosaminidase is produced by expression of a nucleic acid molecule according to any one of claims 7 to 11.
45. The method according to any one of claims 36 to 44 wherein administration of 00o' the recombinant mammalian a-N-acetylglucosaminidase is by oral, intravenous, suppository, intraperitoneal, intramuscular, intranasal, intradermal or subcutaneous administration, by infusion or implantation or by enzyme replacement therapy or by gene therapy. S. 46. The method according to claim 45 wherein the method of administration is by enzyme replacement therapy. 25 47. A pharmaceutical composition comprising a recombinant mammalian a-N- "i acetylglucosaminidase or an enzymatically active fragment or derivative thereof according to claim 12 or 13 and one or more pharmaceutically acceptable carriers and/or diluents.
48. The pharmaceutical composition according to claim 47 wherein the S io binant mammalian a-N-acetylglucosaminidase comprises a sequence of amino -U acid ubstantially the same as human a-N-acetylglucosaminidase. G <t 69
49. The pharmaceutical composition according to claim 47 or 48 wherein the recombinant mammalian a-N-acetylglucosaminidase is produced in a mammalian cell.
50. The pharmaceutical composition according to claim 49 wherein the mammalian cell is a CHO cell line which is capable of glycosylating the recombinant mammalian a-N-acetylglucosaminidase.
51. The pharmaceutical composition according to any one of claims 47 to wherein the a-N-acetylglucosaminidase is glycosylated.
52. The pharmaceutical composition according to claim 51 wherein the recombinant a-N-acetylglucosaminidase has a molecular weight of at least approximately 79 kDa as determined using SDS/PAGE.
53. The pharmaceutical composition according to claim 52 wherein the 9 recombinant a-N-acetylglucosaminidase has a molecular weight of approximately 79 kDA to 89 kDa as determined using SDS/PAGE.
54. A pharmaceutical composition comprising a recombinant mammalian a-N-acetylglucosaminidase or an enzymatically active fragment or derivative thereof and one or more pharmaceutically acceptable carriers and/or diluents wherein said S recombinant mammalian a-N-acetylglucosaminidase is produced by expression of a nucleic acid molecule according to any one of claims 7 to 11. A pharmaceutical composition comprising a recombinant mammalian a-N-acetylglucosaminidase or an enzymatically active fragment or derivative thereof and one or more pharmaceutically acceptable carriers and/or diluents when used in the method according to any one of claims 36 to 46. S- 56. 7 Use of a recombinant mammalian a-N-acetylglucosaminidase or an enz atically active fragment or derivative thereof according to claim 12 or 13 in the manufacture of a medicament for the treatment of a a-N-acetylglucosaminidase deficiency in a patient.
57. The use according to claim 56 wherein the recombinant mammalian a-N-acetylglucosaminidase comprises a sequence of amino acids substantially the same as the amino acid sequence of human a-N-acetylglucosaminidase.
58. The use according to claim 56 or 57 wherein the patient is suffering from mucopolysaccharidosis type IIIB.
59. The use according to claim 58 wherein the recombinant a-N-acetylglucosaminidase is expressed in mammalian cells. The use according to claim 59 wherein the cells are CHO cells.
61. The use according to any one of claims 56 to 60 wherein the a-N-acetylglucosaminidase is glycosylated.
62. The use according to claim 61 wherein the recombinant a-N-acetylglucosaminidase has a molecular weight of at least approximately 79 kDa das determined using SDS/PAGE.
63. The use according to claim 62 wherein the recombinant a-N-acetylglucosaminidase has a molecular weight of approximately 79 kDa to 89 kDa as determined using SDS/PAGE.
64. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolyzing the terminal a-N-acetylglucosamine residues present at the non- reducing terminus of fragments of heparan sulphate and heparin and wherein said A C otide sequence is capable of hybridizing under high stringency conditions to the n leotide sequence set forth in SEQ ID NO:1. LU4 71 An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolyzing the terminal a-N-acetylglucosamine residues present at the non- reducing terminus of fragments of heparan sulphate and heparin and wherein said nucleotide sequence is capable of hybridizing under high stringency conditions to the nucleotide sequence set forth in SEQ ID NO:3.
66. A recombinant polypeptide comprising a sequence of amino acids corresponding to the amino sequence set forth in SEQ ID NO:2 or having at least similarity thereto and encoded by a nucleic acid molecule which is capable of hybridizing to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 under high stringency conditions.
67. A genetic construct comprising the nucleic acid molecule according to any one of claims 1 to 11, 64 and 65 operably connected in the sense orientation to a promoter sequence such that said genetic construct is capable of being expressed in a eukaryotic or prokaryotic cell to produce a recombinant mammalian a-N-acetylglucosaminidase or an enzymatically active fragment or derivative thereof.
68. The genetic construct according to claim 67 wherein the promoter is capable of regulating expression of the recombinant a-N-acetylglucosaminidase in a mammalian cell. O 0
69. The genetic construct according to claim 68 wherein the promoter is the CMV promoter sequence or a promoter derived therefrom. The genetic construct according to any one of claims 67 to 69 further comprising a transcription terminator sequence.
71. The genetic construct according to any one of claim 67 to 70 when used to express or over-express a-N-acetylglucosaminidase in a eukaryotic or prokaryotic cell.
72. An antibody specifically reactive with, or raised using, a recombinant a-N-acetylglucosaminidase according to any one of claims 12 to 26 or an antigenic fragment thereof.
73. The antibody according to claim 72 further defined as a polyclonal antibody molecule.
74. The antibody according to claim 72 further defined as a monoclonal antibody molecule.
75. A monoclonal antibody which specifically binds to a recombinant a-N-acetylglucosaminidase, said a-N-acetylglucosaminidase comprising the amino acid sequence set forth in SEQ ID NO:2.
76. An isolated nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO:1 or complementary to the nucleotide sequence set forth in S.SEQ ID NO:1.
77. An isolated nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO:3 or complementary to the nucleotide sequence set forth in SEQ :25 ID NO:3.
78. An isolated nucleic acid molecule according to any one of claims 1 to 11, 64, 76 and 77, substantially as described herein and with reference to any one of Examples 4 to 6.
79. A recombinant a-N-acetylglucosaminidase or fragment or derivative thereof according to any one of claims 12 to 26 and 66, substantially as described herein and with reference to any one of Examples 1 to 3 and 6 to 9.
80. A method of diagnosis according to any one of claims 27 to 35, substantially as described herein and with reference to Example
81. A method of treatment according to any one of claims 36 to 46, substantially as described herein.
82. A pharmaceutical composition according to any one of claims 47 to substantially as described herein.
83. The use of a recombinant a-N-acetylglucosaminidase or a fragment or 15 derivative thereof according to any one of claims 56 to 63, substantially as described herein.
84. A genetic construct according to any one of claims 67 to 71, substantially as described herein and with reference to any one of Examples 5 to 7.
85. An antibody according to any one of claims 72 to 75, substantially as described S* herein. Dated this 14th day of April, 2000. WOMEN'S AND CHILDREN'S HOSPITAL By its Patent Attorneys MADDERNS C A OVI/
AU76124/96A 1995-11-23 1996-11-22 Synthetic mammalian alpha-N-acetylglucosaminidase and genetic sequences encoding same Expired AU720778B2 (en)

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PCT/AU1996/000747 WO1997019177A1 (en) 1995-11-23 1996-11-22 SYNTHETIC MAMMALIAN α-N-ACETYLGLUCOSAMINIDASE AND GENETIC SEQUENCES ENCODING SAME

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