WO1995015175A1 - Galectine-8 et proteines analogues a la galectine-8 et molecules d'adn les codant - Google Patents

Galectine-8 et proteines analogues a la galectine-8 et molecules d'adn les codant Download PDF

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WO1995015175A1
WO1995015175A1 PCT/US1994/013679 US9413679W WO9515175A1 WO 1995015175 A1 WO1995015175 A1 WO 1995015175A1 US 9413679 W US9413679 W US 9413679W WO 9515175 A1 WO9515175 A1 WO 9515175A1
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galectin
protein
dna molecule
sequence
dna
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PCT/US1994/013679
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Yahiel Zick
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Yeda Research And Development Co., Ltd. At The Weizmann Institute Of Science
Rycus, Avigail
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Priority to EP95903624A priority Critical patent/EP0812202A4/fr
Priority to US08/647,960 priority patent/US5908761A/en
Publication of WO1995015175A1 publication Critical patent/WO1995015175A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4726—Lectins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is generally in the field of mammalian S-type lectin proteins, now designated galectins, which are thiol-dependent and specifically bind ⁇ -galactoside residues.
  • the present invention relates to a new S- type mammalian lectin, termed hereinafter as "galectin-8", and to galectin-8-li e proteins, to DNA molecules coding therefor and to antibodies raised against said proteins.
  • the invention further relates to pharmaceutical compositions comprising said proteins for the purpose of cell growth regulation in general, and more particularly for inhibition of cell proliferation and for treatment of tumors.
  • Lectins are involved in a wide variety of cellular functions, many of which are related to their only common feature, the ability to bind carbohydrates specifically and reversibly, and to agglutinate cells [reviewed in (1)].
  • Animal lectins are classified as C-lectins, which are Ca 2* -dependent and are structurally related to the asialoglycoprotein receptor, and galectins, previously known as S-type lectins, which are thiol- dependent and specifically bind ⁇ -galactoside residues.
  • C-lectins which are Ca 2* -dependent and are structurally related to the asialoglycoprotein receptor
  • galectins previously known as S-type lectins, which are thiol- dependent and specifically bind ⁇ -galactoside residues.
  • S-type lectins previously known as S-type lectins
  • Galectins require fulfillment of two criteria: affinity for ⁇ -galactosides and significant sequence similarity in the carbohydrate recognition domain (CRD) (4), the relevant amino acids residues of which have been determined by X-ray crystallography (5) .
  • galectin-1 and -2 The function of galectin-1 and -2 is not yet fully understood, although there is evidence that they might be involved in regulation of cell growth (8); cell adhesion (7); cell transformation (9); and embryogenesis (10). Larger galectins (galectin-3) (previously known as CBP-35, Mac-2, RL-29) do exist ((11) and references therein). These are monomeric 29-35 kDa mosaic proteins, composed of an N-terminal half made of tandem repeats characteristic of the collagen gene superfamily, and a C-terminal half homologous to galectins-1 and -2 (11).
  • Galectin-3 also binds laminin, and is implicated as component of growth regulatory systems; mediator of cell-cell and cell-matrix interactions; modulator of immune response; marker of neoplastic transformation, and indicator for metastatic potential of melanoma cells.
  • Galectin-4 was cloned from rat intestine (12), and an homologous protein was cloned from nematode (13).
  • Galectins may functionally substitute each other.
  • the absence of any major phenotypic abnormalities in mice carrying a null mutation in the gene encoding galectin-1 suggests that other protein(s), presumably galectin-3, are capable of functionally substituting for galectin-1, at least at early stages of embryogenesis.
  • Galectin-8 has the characteristic properties of other galectins (2,3), and it is structurally related (34% identity) to rat galectin-4 (12).
  • a novel protein of 35 Kd which has the characteristic properties of galectins was cloned from rat liver cDNA expression library.
  • This protein was originally called by us RL-30 protein.
  • the nomenclature of S-type lectins has recently been changed to galectins (2). Since names for galectins 1-7 were already assigned (3), this new protein has now been named galectin-8, but it is to be understood that this is the same protein formerly called by us RL-30.
  • the present invention provides a biologically active S-type lectin named galectin-8 and galectin- 8-like proteins and fragments thereof selected from:
  • the present invention relates to an isolated DNA sequence encoding galectin-8 or a galectin-8-like protein.
  • the isolated DNA sequence of the invention is one that encodes a polypeptide product of prokaryotic or eukaryotic host expression, said product having all or part of the primary structural conformation of galectin-8 or of a galectin-8-like protein and having the biological activity of galectin-8.
  • the above DNA sequence of the invention may be any one of the group consisting of:
  • the above DNA sequence of the invention is one selected from:
  • a DNA molecule having a coding nucleotide sequence which is homologous to the DNA molecule of (i), (ii) or (iii), which is derived from a mammal other than rats and which encodes a polypeptide having a similar biological activity to that encoded by the sequences of (i), (ii) or (iii);
  • a fragment of the coding sequence of (i)-(iv) which encodes a polypeptide which essentially retains the biological properties of the polypeptide encoded by the unfragmented DNA molecule;
  • a DNA molecule comprising the coding DNA sequence of a fragment of (i)-(v) and additional DNA sequences in the 3' and 5' ends.
  • the present invention relates to a recombinant DNA molecule comprising a coding sequence according to any of (i)-(iii) and (i)-(iv) above or a fragment thereof according to (v) or (vi) above.
  • the present invention also provides a recombinant expression vector comprising any one of the above-mentioned DNA molecules of the invention.
  • a recombinant expression vector may be one capable of being expressed in prokaryotic or eukaryotic hosts, the vector containing, in addition to any one of the above galectin-8 or galectin-8-like protein encoding sequences, various other sequences such as, for example, those sequences that are known to be important for expression of the desired sequence and the maintenance and propagation of the vector in the host cell. Construction of such recombinant expression vectors is by way of x22any of the known procedures.
  • the present invention further provides a method for preparing galectin-8 or a galectin-8-like protein or a biologically active fragment thereof, comprising culturing a suitable host cell containing the above recombinant vector of the invention under conditions promoting expression.
  • the protein of the invention may be prepared, as noted above, by expression of a recombinant vector comprising a DNA sequence encoding the protein, or it may be isolated and purified from various mammalian tissues using standard procedures for protein extraction and purification. In such purification procedures there may be employed yet another aspect of the present invention, namely, antibodies which are immunoreactive with native or recombinant galectin-8 or with a galectin-8-like protein.
  • Such antibodies may be applied in standard affinity chromatography methods to provide for the final purification steps of the galectin from various tissues.
  • the preparation of the antibodies is by standard procedures using native or recombinant galectin-8 or a fragment thereof or a galectin-8- like protein or a fragment thereof as antigen or immunogen to stimulate antibody production in suitable animals .
  • Both polyclonal and monoclonal antibodies to galectin-8 are encompassed by the invention. These antibodies can be prepared by standard procedures well-known in the art.
  • the anti-galectin-8 antibodies of the invention may also be employed in an assay method for the detection of overexpression of galectin-8 in mammalian tissue, said method comprising applying an effective amount of the antibodies to a tissue or body fluid sample obtained from a mammal and determining the extent of antibody binding to the sample.
  • assay standard procedures may be employed, such as, for example, ELISA assay procedures .
  • the present invention also provides pharmaceutical compositions comprising as active ingredient an effective amount of galectin-8 or of a mammalian galectin-8-like protein and a suitable diluent or carrier, in particular compositions for cell growth regulation, more specifically for the inhibition of cell proliferation, for example for the treatment of cancer.
  • the diluents or carriers may be any of those substances well known in the art for the preparation of pharmaceutical compositions, and likewise the compositions may be prepared by standard procedures. Actual dosages and modes of administration of the above compositions are to be determined by skilled professionals.
  • Fig. 1 depicts the cDNA sequence of galectin-8 and deduced protein sequence.
  • the cDNA sequence of 1247 base pairs (bp) contains an open reading frame from 121-1069 bp, which encodes for a protein of 316 amino acids.
  • Fig. 2 shows that galectin-8 encodes for a galectin with two homologous carbohydrate-binding regions.
  • a schematic structure of galectin-8 is presented (top) .
  • Each box represents a putative carbohydrate-binding domain, linked by a 32 amino acid long peptide. Shown are invariant amino acids preserved in most galectins analyzed so far.
  • the Arg residue indispensable for sugar binding, located at the C-terminal carbohydrate recognition domain (CRD), and its corresponding lie residue, localized to the N-terminal CRD, are shown in bold. Amino acid sequences of different galectins are presented for comparison (bottom) .
  • human galectin-1 (Galec-1); human galectin-2 (Galec-2); the carbohydrate binding domain (amino acids 128-263) of rat galectin-3 (Galec-3); N-terminal (Galec-4-Nt) and C-terminal (Galec-4-Ct) halves of galectin-4; N-terminal (CE-Nt) and C- terminal (CE-Ct) halves of a 32-kDa ⁇ -galactoside-binding protein from Caenorhabdit ⁇ s elegans; N-terminal (Galec-8-Nt) and C- terminal (Galec-8-Ct) halves of galectin-8. Residues with shared identity are boxed. Residues with shared similarity are shaded.
  • Fig. 3 shows Northern blot analysis of RNA from rat tissues probed with galectin-8 cDNA. Top, 30 ⁇ g of total RNA from the indicated tissues was electrophoresed, blotted, and probed with labeled galectin-8 PCR product as described in "Experimental Procedures". The migration of the 18S and 28S rRNA are marked. Bottom, the same blot was stripped and reblotted with cDNA encoding for GAPDH.
  • Fig. 4 shows immunoprecipita ion of zn-v ⁇ tro translation product of galectin-8 by lp-lec8 antibodies.
  • Five ml of the total 35 S-labeled galectin-8 (total), 5 ⁇ l of the fraction not precipitated by the antibodies (Sup), and 50 ⁇ l of the immunoprecipitated fraction (IP) were subjected to 12% SDS-PAGE and autoradiography.
  • Fig. 1 shows immunoprecipita ion of zn-v ⁇ tro translation product of galectin-8 by lp-lec8 antibodies.
  • FIG. 5 shows binding of tag-free recombinant galectin-8 (r- galectin-8) to lactosyl-Sepharose.
  • Tag-free r-galectin-8 was expressed in pLysS as described under "Experimental Procedures". After centrifugation, 30 ml of the soluble bacterial proteins were purified over 5 ml of lactosyl-Sepharose. r-galectin-8 was eluted with 100 mM lactose in buffer-I, and 1 ml fractions were collected. Ten ⁇ l of the total and effluent fractions and 50 ⁇ l from each elution fraction were resolved by 12% SDS-PAGE, transferred to nitrocellulose and Western immunoblotted with lp- lec ⁇ antibodies.
  • Fig. 6 shows binding of rat hepatic galectin-8 to lactosyl- Sepharose.
  • Five g of rat liver were homogenized in buffer-I as described under "Experimental Procedures” and cytosolic extracts (25 ml) were applied over 5 ml of lactosyl-Sepharose. After extensive washing the bound proteins were eluted with 100 mM lactose in buffer-I.
  • One ml fractions (numbered 1-10) were collected and frozen for a period of 16 h at -20°C.
  • Eluted fractions (N° 3-5) were toughed, centrifuged for 15 min at 12000Xg and the pellets were resuspended in 50 ml sample buffer (34).
  • Fig. 7 shows inhibition by recombinant galectin-8 of serum- induced 3 H-thymidine incorporation into DNA.
  • 3 H-Thymidine incorporation into DNA was examined as follows: Confluent CHO cells, grown in 24-well trays (Costar), were starved for 48 hours in serum-free medium. Medium containing 10% fetal calf serum was added to the cells in the presence of rgalectin-8 at the indicated concentrations, and the cells were incubated for 14.5 hours at 37°C.
  • the medium was then washed, and the cells were incubated for 2 hours at 37°C in 1 ml of serum-free medium containing 1% BSA, 20mM Hepes (pH 7.5) and 0.5 ⁇ Ci/ml [ 3 H]- thymidine.
  • the solution was removed, cells were washed 3 times in ice-cold PBS and incubated for 30 min at 4°C in 0.5 ml ice-cold 7.5% trichloroacetic acid.
  • the pellets were washed twice with 98% ice-cold ethanol, dissolved in 0.6 ml of 0.1 M NaOH, and counted using scintillation cocktail containing xylene and Lumax (6:4 ratio, respectively).
  • Fig. 8 shows chromatography of galectin-8 over a FPLC column.
  • Approximately 100 ⁇ g protein was loaded onto Superdex-200 HR (Pharmacia) FPLC column equilibrated with buffer A (PBS, 4 mM ⁇ -mercaptoethanol, 2mM EDTA) , and run for 60 min at 0.5 ml/min.
  • O.D. was measured at 215 nm, and the column profile was obtained by running separately standard marks.
  • Galectin-8 is a novel, widely expressed protein of 35 kDa which has the characteristic properties of galectins (S-type mammalian lectins) .
  • galectin-8 is indeed a novel galectin: i. its deduced amino acid sequence contains two domains with conserved motifs that are implicated in the carbohydrate binding of galectins; ii. in vitro translation products of galectin-8 cDNA or bacterially-expressed recombinant galectin-8 are biologically active and possess sugar- binding and hemagglutination activity; iii.
  • a protein of the expected size (34 kDa), that binds to lactosyl-Sepharose and reacts with galectin-8-specific antibodies is present in rat liver and comprises ⁇ 0.025% of the total Triton-soluble hepatic proteins.
  • galectin-8 is structurally related (34% identity) to galectin-4, a soluble rat galectin with two carbohydrate- binding domains in the same polypeptide chain, joined by a link peptide. Nonetheless, several important features distinguish these two galectins: i. Northern blot analysis revealed that unlike galectin-4 that is confined to the intestine and stomach, galectin-8 is expressed in liver, kidney, cardiac muscle, lung, and brain; ii. unlike galectin-4, but similar to galectins-1 and -2, galectin-8 contains 4 Cys residues; iii. the link peptide of galectin-8 is unique and bears no similarity to any known protein; iv.
  • galectin-8 contains a unique WG-E-I motif instead of the consensus WG-E-R/K motif implicated as playing an essential role in sugar-binding of all galectins. Together with galectin-4, galectin-8 therefore represents a subfamily of galectins consisting of a tandem repeat of structurally different CRDs within a single polypeptide chain.
  • galectin-8-like protein refers to a protein derived from any mammal, including humans, which protein presents homology to galectin-8 as defined in the present invention and has the biological properties of galectin-8.
  • Galectin-8 was cloned when a ⁇ -ZAP rat liver cDNA library was screened with affinity-purified antibodies directed against a 14-amino acid peptide located at the C-terminal end of the insulin-receptor substrate 1 (IRS-1) (14). Since galectin-8 bears no sequence similarity either to IRS-1, or to the peptide used as immunogen, it was suspected that the reactivity towards IRS-1 antibodies could be due to a false positive reaction. This conclusion is supported by the fact that the anti-peptide antibodies used for screening, failed to react with purified recombinant galectin-8 either by means of immunoprecipitation, or immunoblotting.
  • CRDs carbohydrate- binding regions
  • This unique architecture is shared so far only by two galectins: rat galectin-4 (12) and its C. elegans homologue (13).
  • galectin-8 exists as a monomer (Fig.8).
  • Hepatic galectin-8 (Fig. 6) has a similar mobility on SDS-PAGE as its recombinant counterpart (Fig. 5). This suggests, though not proves, that hepatic galectin-8 is neither heavily glycosylated, nor it is subjected to extensive post-translational modifications (e. g. phosphorylation) .
  • galectin-8 contains two putative CRDs, potential differences in sugar-binding between the domains is predicted from a critical difference in their sequence [WG-E-I vs.
  • galectin-8 lacks a classical signal sequence or a transmembrane segment. Indeed, galectin-8 was isolated from the cytosolic fraction of rat liver.
  • galectin-8 seems to be developmentally regulated. Very low levels of expression were noted in whole embryos, while high levels of expression were noted in adult tissues. In that respect galectin-8 might resemble other galectins that were implicated as regulators of cell growth and embryogenesis (8-10).
  • Antibodies - Antisera to insulin receptor substrate 1 were raised in rabbits according to standard procedures, by injection of a peptide CYASINFQKQPEDRQ corresponding to the carboxy-terminal 14 amino acids of rat liver IRS-1 (and an additional Cys residue at the N-terminal site) .
  • Antibodies were affinity-purified from the serum by adsorption onto a column of peptide coupled to Affi-gel 10, elution with 100 mM HC1 glycine pH 2.7, and immediate neutralization.
  • Anti glutathione-S- transferase (GST) antisera was a kind gift from Y. Yarden (Weizmann Institute) .
  • RNA extraction was carried out as described (16). Total RNA (30 ⁇ g) was electrophoresed, the gel was blotted onto nitrocellulose, and the blot was probed with labeled PCR product which was obtained by the following procedure. Two primers, 5 '-CCCGACAATCCCCTATGTCAGTACC-3 and 5'- GCATGGCCAGGCCTGACAACA-3 ' , were used to amplify the entire cDNA coding sequence of galectin-8, using the cloned cDNA in Bluescript as a template. The PCR products were labeled with [ - 32 P]-ATP by random priming with DECAprime II DNA labeling kit (Ambion, Austin, TX) .
  • a sense primer 5 '-GGGGGGCATATGTTGTCCTTAAGCAAT-3 ' and an antisense primer 5 '-GGGGGGGGATCCGCCATTTTGTATTTCCAG-3 ' were used to amplify the entire coding sequence of galectin-8, using the cloned cDNA in Bluescript as a template.
  • the PCR products were digested by Nde I and BamH I, gel-purified, and ligated into a pET-3a expression plasmid (Novagen) in the pLysS bacterial host. Sequencing of both expression plasmids was carried out to ensure proper, in-frame, ligation of the inserts.
  • a bacterial pellet was isolated by centrifugation, resuspended in 30 ml of buffer I (phosphate buffered saline containing 4 mM ⁇ -mercaptoethanol, 2 mM EDTA, 10 ⁇ g/ml soybean trypsin inhibitor, 2 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride, pH 7-5), and lysed by sonication. Debris were removed by centrifugation at 38,000 xg at 4 °C for 45 min. , and 30 ml of the soluble extract were passed over 5 ml of lactosyl-Sepharose.
  • buffer I phosphate buffered saline containing 4 mM ⁇ -mercaptoethanol, 2 mM EDTA, 10 ⁇ g/ml soybean trypsin inhibitor, 2 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride, pH 7-5
  • the blotted proteins were incubated with lp-lec8 antibodies at 4°C for 16 h and then were extensively washed. To detect antibody binding, a horseradish peroxidase-conjugated Protein A ECL kit (Amersham) was used according to the manufacturer's instructions.
  • galectin-8 denatures upon freezing, the frozen fractions were toughed, and centrifuged at 12, 000 xg, for 15 min. at 4°C to precipitate, and thus concentrate, galectin-8.
  • Supernatants and pellets were resuspended in sample buffer, resolved by 12% SDS-PAGE, transferred to nitrocellulose and Western immunoblotted with lp- lec8 antibodies.
  • the amount of galectin-8 in rat liver was estimated using 100,000xg supernatants that were prepared in buffer I in the presence of 1% Triton-X-100.
  • the isolated clone contained an open reading frame (ORF) (nucleotides 112-1068) with a potential initiation ATG codon at position 121.
  • This ORF coded for 316 amino acids, which form a protein of about 35 kDa.
  • the putative coding sequence was followed by a signal for translation termination (TAG) and 176 nucleotides of 3'- untranslated region. Search of the GenBank for similar nucleotide sequences revealed that this sequence is unique. This sequence, depicted in Fig. 1, has been submitted to the Gen BankTM/EMBL Data Bank with accession number U09824.
  • galectin-8 resembles a 32-kDa ⁇ -galactoside-binding protein from Caenorhabditis elegans (13) (CE-galectin) , and rat galectin-4 (galectin-4) (12), that also contain two CRDs connected by a link peptide (Fig. 2).
  • galectin-8 is 50% and 45% homologous to galectin-4 and CE-galectin., respectively.
  • galectin-8 shares 34% and 31% identity, respectively, with the above proteins. No homology with any known protein was found in the region of the link peptide.
  • galectin-8 Like other galectins, galectin-8 lacks classical signal sequence or transmembrane segment, but it contains three potential N-linked glycosylation (Asn-X-Ser/Thr) sites. Analysis of its predicted secondary structure (not shown), revealed that the N-and C- terminal domains of galectin-8 share a great degree of structural homology, as expected from their primary structure. Both domains are predicted to form several ⁇ -sheets, a structural feature of other galectins (5). The cDNA clone encoding galectin-8 may be used as a probe to isolate and characterize the full length genomic sequence encoding this protein in various mammals, for example, humans and rats, using standard procedures.
  • galectin-8 may be used to generate, by standard procedures, fragments containing only a portion of the full-length galectin-8 sequence, where each fragment essentially retains at least one of the biological activities of galectin-8. These fragments are termed 'biologically active fragments'.
  • galectin-8 sequence may also be used to generate analogs of galectin-8 (herein termed "galectin-8-like proteins") or fragments thereof, such analogs having at least one amino acid residue added, deleted or replaced by another in comparison to the native galectin-8 sequence, and such analogs essentially retaining the biological activity of their non-modified progenitor molecules.
  • galectin-8 contains a unique link peptide region, antibodies against this region are not expected to cross-react with other galectins.
  • a peptide corresponding to positions 168- 182 in the link peptide of galectin-8 (and an additional Cys residue at the N-terminal site) of the sequence CQISKETIQKSGKLHL was synthesized, purified, and polyclonal antibodies against it were raised in rabbits by standard procedures.
  • the antibodies (denoted lp-lec8) were affinity-purified over a column of immobilized peptide. Ip-lec8 antibodies reacted specifically with galectin-8 both by means of immunoprecipitation (IP) and immunoblotting (IB).
  • Galectin-8 J-n-vitro-translated galectin-8 is biologically active. Galectin-8 cDNA was transcribed and translated in vitro using a TNT (Promega) kit. An 35 S-labeled product of the expected size (34 kDa) was synthesized (Fig. 4). This in vit o-translated product was indeed galectin-8 since it could be immunoprecipitated with lp-lec8 antibodies described in Example 2 (Fig. 4).
  • galectin-8 As predicted by its primary amino acid sequence, in vitro-translated galectin-8 exhibited the key feature of galectins, namely, capacity to bind to a column of lactosyl- Sepharose in the presence of reducing agents, and to be eluted with 0.1 M lactose (not shown).
  • galectin-8 it was expressed in bacteria as a GST-fusion protein.
  • GST-galectin-8 remained bound to glutathione-Sepharose beads, and could be eluted with glutathione (not shown) .
  • GST-galectin-8 retained its sugar- binding capacity and could be purified by binding to lactosyl- Sepharose and elution with 0.1 M lactose (not shown).
  • 3 mg GST-galectin-8 could be purified in such a way from 1 liter of bacterial extracts.
  • GST-galectin-8 also maintained hemagglutination activity. Half and maximal activities were obtained with 0.1 and 1 ⁇ g/ml of GST-galectin-8, respectively.
  • a tag-free rgalectin-8 was expressed employing a pET-3a expression plasmid (Novagen) in the pLysS bacterial host.
  • galectin-4 that precipitates and cannot be extracted with buffers that preserve its lectin activity ( 12)
  • rgalectin-8 could be readily extracted from bacteria in a soluble form, rgalectin-8 was not subjected to major proteolytic cleavage, as it migrated at the expected size of 34 kDa.
  • rgalectin-8 retained its sugar-binding activity and 1.2 mg protein/liter bacteria were obtained following its purification over lactosyl-Sepharose column (Fig.
  • IPTG IP-galectin-8 or rgalectin-8
  • approximately 5 mg protein are loaded onto a column of antibodies covalently linked to Affi-Gel 15 beads (Pharmacia) .
  • the bound proteins are then eluted with HCl/glycine buffer (pH 2.8) and immediatly neutralized.
  • Endogenous galectin-8 is present in rat liver
  • a cytosolic (100,000xg supernatant) liver extract was prepared, applied to a column of lactosyl-Sepharose, and proteins retained specifically by the column were eluted with 0.1M lactose.
  • Advantage was taken of the fact that hepatic galectin-8 denatures and precipitates upon freezing. Fractions, eluted from the lactosyl-Sepharose column, were therefore frozen at -20°C, thawed, and centrifuged to precipitate, and thus concentrate, the hepatic galectin-8.
  • Galectin-8 is widely expressed. Tissue distribution and cellular localization of galectin-8.
  • Identifying tissues where galectin-8 is highly expressed provides important clues related to its possible function and involvement in development. More important, determining whether galectin-8, like other galectins, is externalized, is of fundamental importance in attempts to assess its mode of action. Three different approaches may be used to gain a detailed tissue distribution of galectin-8. i. Northern blot analysis of rat tissues; ii. to ascertain that the level of mRNA indeed reflects the level of expression of galectin-8, the abundance of galectin- 8 in various tissues may be determined by Western blot analysis using anti-rgalectin-8 antibodies.
  • galectin-8 is prone to proteolysis, freshly isolated tissues are directly homogenized in 4M guanidinium-HCl to inactivate all proteases .
  • the amount of galectin-8 in the tissue under study is determined following SDS-PAGE, Western blotting, and probing with anti-rgalectin-8 antibodies. iii.
  • tissues of interest e. g. liver and brain
  • in situ hybridization of brain slices indicated that galectin-8 is specifically expressed in the hypocampus, cerebellum, and brain stem, with little expression in the cortex (not shown) .
  • Table I Tissue Distribution of galectin-8 mRNA according to Northern Blot Analysis.
  • RNA from the indicated rat tissues was electrophoresed, blotted, and probed as described in legend to Fig. 3.
  • the intensity of the signal corresponding to the galectin-8 probe was determined by densitometry and is presented as percentage of the strongest signal (normalized to GAPDH) which was obtained in lung (100%).
  • galectin-8 in different rat tissues was examined by Northern blots (Fig. 3). A single mRNA transcript of ⁇ 3 kb hybridized with galectin-8 PCR product probe. Unlike galectin-4, which is confined to intestine and stomach (12) , galectin-8 mRNA is highly expressed in lung, and to a lower extent in liver, kidney, spleen, hind-limb, and cardiac muscle (Fig. 3, Table 1). Lower levels of expression were detected in brain and almost no expression was found in whole rat embryos.
  • galectin-8nt has any sugar-binding activity, and whether galectin-8ct might function independently of its N-terminal half
  • galectin-8nt and galectin-8ct are amplified by PCR and proper restriction sites are introduced. Expression of each domain either as a GST-fusion protein or as tag-free domain are carried out as described above (Example 4).
  • tag-free galectin-8ct the Met residue placed within the MCS of pET-3d is utilized as the start-site. Purification of galectin-8nt and galectin-8ct is carried out as described above (Example 4) .
  • the cDNA coding for galectin-8 was introduced into four different eukaryotic high expression plasmids: pcDNA I Amp (Invitrogene) ; pREP8 (Invitrogene) ; pBPV-II, and pMAMneo
  • MMTV-LTR promoter is of particular use if constitutive overexpression of galectin-8 induces growth arrest or prevents adhesion of the transfected cells. Sequencing of the vector/insert boundaries is carried out, to ensure proper integration of the insert. a. Transient expression of galectin-8- Northern blot analysis of RNA and Western immunoblotting with lp-lec8 antibodies, has indicated that COS-7 cells express low levels of endogenous galectin-8. These cells are therefore appropriate targets to study transient expression of galectin-8. COS-7 cells are plated in DMEM/10% FCS at 2x 10 6 cells /10 cm plate, 24 h before transfection.
  • Stable expression of galectin-8- The above expression plasmids are used for stable transfection of galectin-8 DNA into Chinese Hamster Ovary (CHO) cells that have relatively low amount of endogenous galectin-8. Stable transfectants are identified by their ability to accumulate galectin-8 in the cytosol, or to secrete galectin-8 into the medium. Conditioned-medium is collected, concentrated by Amicon Centricon-10 micro concentrator, and lyophilized. Cytosolic extracts are prepared by boiling in "sample buffer" and the presence of galectin-8 is detected by immunoblotting with galectin-8 antibodies. Cells expressing the highest concentration of galectin-8 are further propagated.
  • rgalectin-8 the biological activity of rgalectin-8 is compared with that of its individually-expressed domains. i. Hemagglutination activity of rgalectin-8, rgalectin-8nt and rgalectin-8ct is assayed as previously described (17). Rabbit erythrocytes are trypsin-treated and fixed with glutaraldehyde.
  • hemagglutination activity of serial dilutions of rgalectin-8 is compared with those of rgalectin-8nt and rgalectin-8ct. If rgalectin-8, like galectin-1, is capable of forming homodimers, and if both CRDs of galectin-8 are capable of sugar binding, then rgalectin-8 is expected to express hemagglutination activity.
  • galectin-8 has reduced or no sugar-binding activity, and if rgalectin-8 fails to dimerize, then rgalectin-8, having a single functional CRD at the C-terminal domain, might fail to express hemagglutination activity.
  • galectin-8 as having a function different from cross-linking glycoconjugates.
  • Carbohydrate-binding specificity of whole galectin-8 and its individually-expressed domains is compared to previously determined specificity of other galectins, including galectin-4. To avoid possible alterations in the native structure of galectin-8 (e.g.
  • rgalectin-8 due to carboxymethylation and iodination 5 ⁇ g of purified rgalectin-8 (or individual domains) are incubated with 100 ⁇ l of lactosyl-Sepharose; conditions that result in quantitative binding of rgalectin-8. Binding specificity may be determined by the capacity of various saccharides (e.g. thiodigalactose, thiodiglucose) to inhibit binding of rgalectin-8 (or individual domains), when compared with lactose. If galectin- 8nt expresses, as predicted, altered or markedly reduced carbohydrate-binding activity, binding activity may be restored by site-directed mutagenesis, where the Ile-90 residue is mutated to Arg.
  • various saccharides e.g. thiodigalactose, thiodiglucose
  • Site-directed mutagenesis is carried out using "Altered Sites II in vitro mutagenesis systems" (Promega) according to the manufacturer's manual.
  • Ile-90 is mutated to Arg to determine how such substitution affects hemagglutination activity and sugar binding specificity of rgalectin-8nt and whole galectin-8.
  • Arg-253, located within the WG-E-R motif at the C-terminal CRD may be mutated to lie, and the effect of this mutation on the biological activity of galectin-8 is assessed. If Arg-253->Ile mutation markedly reduces or abolishes the in vitro biological activity of galectin-8, then the biological consequences of overexpression of this negative- dominant mutant will be compared with cells that overexpress the native form of galectin-8.
  • alkylation of rgalectin-8 may be carried out with iodoacetamide or with N- ethyl-maleimide.
  • the modified product is then subjected to rechromatography over lactosyl-Sepharose column and is eluted with water.
  • Alkylation, that stabilizes galectin-1 may preserve and stabilize rgalectin-8 activity (i.e. binding affinity to lactosyl-Sepharose), and enables increase of the half-life of rgalectin-8 and better study of its effects on cultured cells under the oxidizing environment of tissue culture medium.
  • galectin-8 The effects of galectin-8 on cell adhesion and on regulation of cellular growth are examined.
  • galectin-1 One of the well characterized effects of galectin-1 is its ability to inhibit myoblast adhesion to laminin (15).
  • galectin-8 shares a similar property the effects of overexpression of galectin-8 on cell adhesion are studied.
  • COS-7 cells are co-transfected with an expression vector for ⁇ -galactosidase (pSM ⁇ Gal) at a 1:20 ratio to the galectin-8 vector.
  • pSM ⁇ Gal expression vector for ⁇ -galactosidase
  • Cells expressing ⁇ -galactosidase are easily distinguished by a blue staining after histochemical reaction with X-gal, 36 h following transfection. Alterations in adhesion of blue cells as a function of time are monitored.
  • Control cells are cotransfected with pSM ⁇ Gal and pcDNA-IR (which contains an insert encoding for the insulin receptor) . If positive results are obtained, thio-D- glucose (TDG) is added to inhibit lectin-carbohydrate interactions and study the contribution of the carbohydrate- binding domains to this effect.
  • TDG thio-D- glucose
  • CHO cells transfected with the pMAMneo-galectin-8 plasmid (which has a dexamethasone-inducible MMTV-LTR promoter) is used. Their adhesive properties to the culture dish, before and after induction, are compared. If positive results are obtained, the effects of TDG on cell adhesion and the effects of exogenously-added rgalectin-8 on non- induced cells are determined.
  • galectin-8 Function of galectin-8 as a cytostatic factor and cell growth regulator.
  • mGBP a single-domain homologue of galectin-8, was shown to be a cell growth-regulatory molecule and a cytostatic factor that binds to a specific cell surface receptor (8).
  • rat hepatoma (Fao) cells are used as a model system.
  • Another model is mouse embryo fibroblasts (MEF), that were already shown to be subjected to the growth inhibitory action of mGBP (8). Growth inhibition induced by purified rgalectin-8 is assessed by several parameters: i.
  • the reversibility of the galectin-8 effects on these parameters may then be evaluated.
  • the relation between sugar binding and the biological activity of rgalectin-8 may be further assessed by the ability of 10 mM TDG to compete for rgalectin-8 binding.
  • Successful results lead to the second stage of the study, where it is determined whether growth inhibition is related to the growth state, as is in the case of mGBP and cytokines .
  • cells stationed in Go by serum starvation, and cells rescued from Go by serum stimulation are treated with galectin-8 for different times, and its potency to attenuate or inhibit cell growth is evaluated. Inhibition of DNA synthesis was monitored in control and rgalectin-8-treated CHO cells as described in the legend to Fig. 7.
  • rgalectin-8 inhibits serum-induced [ 3 H] thymidine incorporation in a dose-dependent manner. Half-maximal effects are obtained at 0.5 ⁇ M and maximal effects at 2 ⁇ M rgalectin-8, GST alone is without effect.
  • galectin-8 antibodies as diagnostic tools for neoplastic transformation.
  • compositions prepared by well-known standard procedures, containing anti-galectin-8 antibodies may be used to detect overexpression of this protein following neoplastic transformation in general, and in metastatic melanoma cells in particular, and accordingly, to determine whether overexpression of galectin-8 can serve as an early signal for neoplastic transformation, and/or the development of metastatic melanoma.
  • the anti-galectin-8 antibodies mat serve as a diagnostic tool for early detection of the above disease.
  • the presence of a subject's own anti-galectin-8 antibodies can also serve as such a diagnostic tool, which endogenous anti-galectin-8 antibodies may be assayed with purified galectin-8.

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Abstract

L'invention se rapporte à une nouvelle lectine de type S de mammifère, nommée galectine-8, ainsi qu'à des proteínes analogues à la galectine-8, à des fragments de celles-ci, à des molécules d'ADN les codant et à des compositions pharmaceutiques comprenant lesdites protéines. Il a été démontré que la galectine-8, une protéine de 35 kDa d'expression très répandue, est impliquée dans la régulation de la croissance cellulaire, en particulier dans l'inhibition de la prolifération cellulaire.
PCT/US1994/013679 1993-12-05 1994-12-05 Galectine-8 et proteines analogues a la galectine-8 et molecules d'adn les codant WO1995015175A1 (fr)

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US08/647,960 US5908761A (en) 1993-12-05 1994-12-05 Galectin-8 and galectin-8-like proteins and DNA molecules coding therefor

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Cited By (11)

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WO1997031107A2 (fr) * 1996-02-20 1997-08-28 Coles John G Apoptose induite par des lectines de serum humain et procede de detection d'apoptose
EP0804458A1 (fr) * 1995-01-11 1997-11-05 The Trustees of Columbia University in the City of New York Developpement de sondes d'adn et de reactifs immunologiques specifiques de molecules exprimees sur une surface cellulaire et genes associes a la transformation
WO1998032851A1 (fr) * 1997-01-23 1998-07-30 Incyte Pharmaceuticals, Inc. Deux homologues humains de la galectine-5
WO2000018922A2 (fr) * 1998-10-01 2000-04-06 Incyte Genomics, Inc. Proteines humaines associees a des glucides
US6281333B1 (en) * 1996-10-09 2001-08-28 Incyte Genomics, Inc. Human galectin homolog
US6468768B1 (en) 1996-10-09 2002-10-22 Human Genome Sciences, Inc. Galectin 9 and 10SV polynucleotides
WO2003072606A2 (fr) * 2002-02-28 2003-09-04 Yeda Research And Development Company Ltd. Agents de liaison de glycoproteines cd44 et methodes d'utilisation associees
US6872522B1 (en) 1996-06-25 2005-03-29 Michael Mecklenburg Broad specificity affinity arrays: a qualitative approach to complex sample discrimination
EP1767634A1 (fr) * 2004-06-14 2007-03-28 Galpharma Co., Ltd. Nouvelle proteine de modification galectine 8 et utilisation de celle-ci
US8076087B2 (en) 2002-11-14 2011-12-13 Medical Research Fund of Tel Aviv Souraskv Medical Center Galectin sequences and compositions and methods utilizing same for treating or diagnosing arthritis and other chronic inflammatory diseases
CN111053890A (zh) * 2020-01-02 2020-04-24 中国科学院水生生物研究所 来源于鳜鱼的半乳糖凝集素-8在制备抑菌剂中的应用

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TWI411685B (zh) * 2011-01-05 2013-10-11 Univ Nat Cheng Kung 腸病毒檢測及純化方法、大量增加腸病毒之方法、及腸病毒疫苗之製造方法

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JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, issued 15 December 1993, J. HERRMANN et al., "Primary Structure of the Soluble Lactose Binding Lectin L-29 from Rat and Dog and Interaction of Its Non-collagenous Proline-, Glycine-, Tyrosine-rich Sequence with Bacterial and Tissue Collagenase", pages 26704-26711. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, No. 8, issued 15 March 1993, Y. ODA et al., "Soluble Lactose-binding Lectin from Rat Intestine With Two Different Carbohydrate-binding Domains in the Same Peptide Chain", pages 5929-5939. *
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See also references of EP0812202A4 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804458A4 (fr) * 1995-01-11 2002-08-14 Univ Columbia Developpement de sondes d'adn et de reactifs immunologiques specifiques de molecules exprimees sur une surface cellulaire et genes associes a la transformation
EP0804458A1 (fr) * 1995-01-11 1997-11-05 The Trustees of Columbia University in the City of New York Developpement de sondes d'adn et de reactifs immunologiques specifiques de molecules exprimees sur une surface cellulaire et genes associes a la transformation
US6811972B1 (en) 1995-01-11 2004-11-02 The Trustees Of Columbia University In The City Of New York Development of DNA probes and immunological reagents specific for cell surface-expressed molecules and transformation-associated genes
WO1997031107A3 (fr) * 1996-02-20 1997-11-27 John G Coles Apoptose induite par des lectines de serum humain et procede de detection d'apoptose
WO1997031107A2 (fr) * 1996-02-20 1997-08-28 Coles John G Apoptose induite par des lectines de serum humain et procede de detection d'apoptose
US7662560B2 (en) 1996-06-25 2010-02-16 Michael Mecklenburg Broad specificity affinity arrays: a qualitative approach to complex sample discrimination
US6872522B1 (en) 1996-06-25 2005-03-29 Michael Mecklenburg Broad specificity affinity arrays: a qualitative approach to complex sample discrimination
US6468768B1 (en) 1996-10-09 2002-10-22 Human Genome Sciences, Inc. Galectin 9 and 10SV polynucleotides
US6281333B1 (en) * 1996-10-09 2001-08-28 Incyte Genomics, Inc. Human galectin homolog
WO1998032851A1 (fr) * 1997-01-23 1998-07-30 Incyte Pharmaceuticals, Inc. Deux homologues humains de la galectine-5
WO2000018922A3 (fr) * 1998-10-01 2000-10-26 Incyte Genomics Inc Proteines humaines associees a des glucides
WO2000018922A2 (fr) * 1998-10-01 2000-04-06 Incyte Genomics, Inc. Proteines humaines associees a des glucides
WO2003072606A2 (fr) * 2002-02-28 2003-09-04 Yeda Research And Development Company Ltd. Agents de liaison de glycoproteines cd44 et methodes d'utilisation associees
WO2003072606A3 (fr) * 2002-02-28 2003-12-18 Yeda Res & Dev Agents de liaison de glycoproteines cd44 et methodes d'utilisation associees
US8076087B2 (en) 2002-11-14 2011-12-13 Medical Research Fund of Tel Aviv Souraskv Medical Center Galectin sequences and compositions and methods utilizing same for treating or diagnosing arthritis and other chronic inflammatory diseases
EP1767634A1 (fr) * 2004-06-14 2007-03-28 Galpharma Co., Ltd. Nouvelle proteine de modification galectine 8 et utilisation de celle-ci
EP1767634A4 (fr) * 2004-06-14 2008-10-22 Galpharma Co Ltd Nouvelle proteine de modification galectine 8 et utilisation de celle-ci
CN111053890A (zh) * 2020-01-02 2020-04-24 中国科学院水生生物研究所 来源于鳜鱼的半乳糖凝集素-8在制备抑菌剂中的应用

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