A monoclonal antibody directed to Tn antigen

A murine monoclonal antibody, MLS 128, that was assigned to an anti-Tn antibody has been established by immunizing mice with human colonic cancer cells (LS 180). MLS 128 bound to mucin glycopeptides from LS 180 cells and their asialo forms to the same extent as well as to ovine submaxillary mucin (OSM) and asialo OSM. Special non-sialylated GalNAc residue(s) attached to a certain peptide region in the antigens seems to be involved in the binding since N-acetylgalactosaminidase treatment of the antigen abolished the binding and pronase digestion diminished the binding markedly.     

Expression of the Tn antigen on T-lymphoid cell line Jurkat.

Expression of the Tn antigen on a T-lymphoid cell line, Jurkat, was investigated using an anti-Tn monoclonal antibody, MLS 128. Immunoprecipitation or immunoaffinity chromatography of a lysate of Jurkat cells led to the isolation of a 120 kDa glycoprotein carrying the Tn antigen. This glycoprotein and leukosialin (CD43) were indistinguishable on SDS-PAGE and as to immunoreactivity with MLS 128. Leukosialin from an erythroid cell line, K562, exhibited no reactivity with MLS 128 despite that this leukosialin has several GalNAc alpha-Ser(Thr) structures. Pulse-chase experiments with the Jurkat leukosialin showed that newly synthesized leukosialin acquired the antigenecity after a lag of about 30 min, whereas incorporation of GalNAc into the leukosialin occurred earlier. These results indicate that the Tn antigen is expressed on leukosialin and that its epitopic structure is more complex than GalNAc alpha-Ser(Thr).     

Enzymatic large-scale synthesis of MUC6-Tn glycoconjugates for antitumor vaccination

In cancer, mucins are aberrantly O-glycosylated, and consequently, they express tumor-associated antigens such as the Tn determinant (alpha-GalNAc-O-Ser/Thr). As compared with normal tissues, they also exhibit a different pattern of expression. In particular, MUC6, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. Recently, we have shown that the MCF7 breast cancer cell line expresses MUC6-Tn glycoproteins in vivo. Cancer-associated mucins show antigenic differences from normal mucins, and as such, they may be used as potential targets for immunotherapy. To develop anticancer vaccines based on the Tn antigen, we prepared several MUC6-Tn glycoconjugates. To this end, we performed the GalNAc enzymatic transfer to two recombinant MUC6 proteins expressed in Escherichia coli, using UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which catalyze in vivo the Tn antigen synthesis. We used either a mixture of ppGalNAc-Ts from MCF7 breast cancer cell extracts or a recombinant ppGalNAc-T1. In both cases, we achieved the synthesis of MUC6-Tn glycoconjugates at a semi-preparative scale (mg amounts). These glycoproteins displayed a high level of Tn antigens, although the overall density depends on both enzyme source and protein acceptor. These MUC6-Tn glycoconjugates were recognized by two anti-Tn monoclonal antibodies that are specific to human cancer cells. Moreover, the MUC6-Tn glycoconjugate glycosylated using MCF7 extracts as the ppGalNAc-T source was able to induce immunoglobulin G (IgG) antibodies that recognized a human tumor cell line. In conclusion, the large-scaled production of MUC6 with tumor-relevant glycoforms holds considerable promise for developing effective anticancer vaccines, and further studies of their immunological properties are warranted.     

黏蛋白型O-聚糖：结构、功能及与肿瘤的相关性

黏蛋白是细胞表面的或分泌的、具有高度O-糖基化修饰的糖蛋白。黏蛋白型O-聚糖是由多肽：N-乙酰氨基半乳糖转移酶（ppGalNAc-T）家族催化起始合成的,在肿瘤中常常伴随着黏蛋白型O-聚糖结构和数量上的改变,形成肿瘤特异聚糖结构（cancer-associated glycans）,如肿瘤Tn和T抗原等。肿瘤特异聚糖使肿瘤细胞的抗原性和黏附能力发生改变,促进肿瘤细胞的恶性增生与转移。而这些肿瘤特异聚糖结构,也为肿瘤的诊断与抗肿瘤药物或疫苗开发提供了理论基础。     

A monoclonal antibody that recognizes a cluster of a disaccharide, NeuAc alpha(2----6)GalNAc, in mucin-type glycoproteins

The structure of an epitopic carbohydrate recognized by a monoclonal antibody, MLS 102, was determined. A disaccharide, NeuAc alpha (2----6)GalNAc, the major prosthetic group of ovine submaxillary mucin (OSM) and related synthetic glycosides, NeuAc alpha(2----6)GalNAc alpha----Ser, NeuAc alpha(2----6)GalNAc beta----Ser, and NeuAc alpha (2----6)GalNAc beta----propyl, reacted with MLS 102 to similar extents, but the reaction was considerably weaker compared to that of OSM. This difference in reactivity could be ascribed to the occurrence of a cluster of the disaccharide on OSM. Purification of MLS 102-reactive antigens from a Triton X-100 extract of LS 180 cells by means of immunoaffinity chromatography gave mucin fractions (cMLS 102 antigen) with an OSM-like domain. Correlation between the content of the disaccharide, NeuAc alpha(2----6)GalNAc, in mucins and their reactivity with MLS 102 was observed.     

Carbohydrate recognition factors of a Talpha (Galbeta1-->3GalNAcalpha1-->Ser/Thr) and Tn (GalNAcalpha1-->Ser/Thr) specific lectin isolated from the seeds of Artocarpus lakoocha

Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha fruit, is a galactose-binding lectin and a potent mitogen of T and B cells. Knowledge obtained from previous studies on the affinity of ALA was limited to molecular and submolecular levels of Galbeta1-->3GalNAc (T) and its derivatives. In the present study, the carbohydrate specificity of ALA was characterized at the macromolecular level according to the mammalian Gal/GalNAc structural units and corresponding glycoconjugates by an enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results indicate that ALA binds specifically to tumor-associated carbohydrate antigens GalNAcalpha1-->Ser/Thr (Tn) and Galbeta1-->3 GalNAcalpha1-->Ser/Thr (Talpha). It barely cross-reacts with other common glycotopes on glycoproteins, including ABH blood group antigens, Galbeta1-->3/4GlcNAc (I/II) determinants, T/Tn covered by sialic acids, and N-linked plasma glycoproteins. Dense clustering structure of Tn/Talpha-containing glycoproteins tested resulted in 2.4 x 10(5)-6.7 x 10(5)-fold higher affinities to ALA than the respective GalNAc and Gal monomer. According to our results, the overall affinity of ALA for glycans can be ranked respectively: polyvalent Tn/Talpha glycotopes > monomeric Talpha and simple clustered Tn > monomeric Tn > GalNAc > Gal; while other glycotopes: Galalpha1-->3/4Gal (B/E), Galbeta1-->3/4GlcNAc (I/II), GalNAcalpha1-->3Gal/GalNAc (A/F), and GalNAcbeta1-->3/4Gal (P/S) were inactive. The strong specificity of ALA for Tn/Talpha cluster suggests the importance of glycotope polyvalency during carbohydrate-receptor interactions and emphasizes its value as an anti-Tn/T lectin for analysis of glycoconjugate mixtures or transformed carbohydrates.     

Binding characteristics of an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (sialyl Tn) monoclonal antibody (MLS 132).

To determine the epitopic structure for an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (anti-sialyl Tn) monoclonal antibody, MLS 132, ovine submaxillary mucin (OSM) was digested with the combination of trypsin and thermolysin and the digest fractionated by immunoaffinity column chromatography and HPLC. From tryptic digest, a major glycopeptide designated as T3 was obtained as an immunoaffinity column-bound fraction. On solid-phase radioimmunoassay, it was found that T3 exhibited strong immunoreactivity with MLS 132. On treatment with thermolysin, T3 was converted into about 50 fragments, as found on fractionation by HPLC. Several of them were strongly immunoreactive and had the same amino acid sequence, i.e. Phe-Ser*-Gly-Glu-Thr*-Ser*-Thr*-Thr*-Val-Ile-Ser*-Gly-Thr*-Asn-Val, where asterisks denote the sites of attachment of carbohydrate. Of these, one was fully sialylated, the others having one Ser or Thr with unsialylated GalNAc attached. Results of analyses of the carbohydrate attached in these glycopeptides led us to postulate that a cluster composed of four sialyl Tn antigens is the essential epitopic structure for MLS 132.     

The human repertoire of antibody specificities against Thomsen-Friedenreich and Tn-carcinoma-associated antigens as defined by human monoclonal antibodies

Summary Human monoclonal antibodies specific for tumour-associated Thomsen-Friedenreich (TF) [Gal(1–3)GalNAc()-O-] and Tn [GalNAc()-O-] glycoproteins were prepared using peripheral blood lymphocytes from healthy blood donors. The B lymphocytes were either directly transformed with Epstein-Barr virus (EBV) or transformed after an in vitro stimulation period with synthetic glycoproteins. The EBV-transformed lymphocytes were subsequently fused with a mouse-human heteromyeloma to secure antibody production and stability. IgM antibodies exhibiting different patterns of specificity for synthetic TF and Tn antigens were obtained, including antibodies specific for the and forms of different Gal(1–3)GalNAc-O- and GalNAc-O- conjugates and antibodies agglutinating neuraminidase-treated erythrocytes. Several of the human monoclonal antibodies showed an increased binding to cultured carcinoma cells as compared to melanoma cells. This straightforward approach for the production of human monoclonal antibodies demonstrates the possibility of investigating the reactivity pattern of tumour-binding antibodies from peripheral blood lymphocytes. The binding patterns of these monoclonal antibodies show that healthy donors carry different fine specificities against synthetic TF/Tn antigens and that these antibodies react with different tumour cells.     

Comparison of antigen constructs and carrier molecules for augmenting the immunogenicity of the monosaccharide epithelial cancer antigen Tn

We have demonstrated previously that the optimal method for inducing an antibody response against defined cancer antigens is covalent conjugation of the antigen to keyhole limpet hemocyanin (KLH) and use of the potent saponin adjuvant QS-21. Single molecules of glycolipids (tetrasaccharides, pentasaccharides, or hexasaccharides) and MUC1 peptides (containing between one and five MUC1 tandem repeats) conjugated to KLH have proven sufficient for antibody recognition and vaccine construction. However, cancer specificity of monoclonal antibodies against the monosaccharide Tn and disaccharide sTn comes largely from recognition of clusters (c) of these molecules on the cell surface. Tn consists of a monosaccharide (GalNAc) O-linked to serine or threonine on epithelial cancer mucins which are uniquely rich in serines and threonines. We test here several Tn constructs: Tn monosaccharide, Tn(c) prepared on a triple threonine backbone, and Tn prepared on a partially or fully glycosylated MUC1 backbone. We determine that Tn(c) is more effective than Tn, and conjugation to KLH is more effective than conjugation to BSA or polystyrene beads for inducing ELISA reactivity against Tn, and FACS reactivity against Tn-positive tumor cells. Surprisingly, MUC1 glycosylated with Tn at three or five sites per 20 amino acid MUC1 tandem repeat and conjugated to KLH, induced the strongest antibody response against Tn and tumor cells expressing Tn, and had the additional advantage of inducing antibodies against MUC1.     

Construction and expression of anti-Tn-antigen-specific single-chain antibody genes from hybridoma producing MLS128 monoclonal antibody

Anti-Tn-antigen monoclonal antibody MLS128 has affinity for three consecutive Tn-antigens (Tn3) more than Tn2. The major aim of this study was to isolate genes encoding MLS128 variable domains to produce a large quantity of recombinant MLS128 antibodies, in turn, allowing the conduct of studies on precise interactions between Tn3- or Tn2-epitopes and MLS128. This study describes cloning of the variable region genes of MLS128, construction of the variable region genes in single-chain variable fragments (scFv) and two scFvs conjugated with human IgG(1) hinge and Fc regions (scFv-Fc) types, and their respective expression in bacterial and mammalian cell. MLS128 scFv protein with the expected specificity and affinity was successfully prepared from inclusion bodies accumulating in Escherichia coli. Construction, expression and purification of two types of MLS128-scFv-Fc proteins with differing linker lengths in Chinese hamster ovary cells demonstrated that the purified scFv-Fc proteins had binding activity specific to the glycoprotein-expressing Tn-antigen clusters. These results revealed that VL and VH genes cloned from the hybridoma represent those of MLS128 and that recombinant antibodies produced from these genes should provide sufficient amounts of binding domains for use in 3D structural studies such as NMR and X-ray analysis.     

Surface plasmon resonance and NMR analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen clusters

Tn-antigens are tumour-associated carbohydrate antigens that are involved in metastatic processes and are associated with a poor prognosis. MLS128 monoclonal antibody recognizes the structures of two or three consecutive Tn-antigens (Tn2 or Tn3). Since MLS128 treatment inhibits colon and breast cancer cell growth [Morita, N., Yajima, Y., Asanuma, H., Nakada, H., and Fujita-Yamaguchi, Y. (2009) Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Biosci. Trends 3, 32-37.], understanding the interaction between MLS128 and Tn-clusters may allow us to the development of novel cancer therapeutics. Although MLS128 was previously reported to have specificity for Tn3 rather than Tn2, similar levels of Tn2/Tn3 binding were unexpectedly observed at 37°C. Thus, thermodynamic analyses were performed via surface plasmon resonance (SPR) using synthetic Tn2- and Tn3-peptides at 10, 15, 20, 25 and 30°C. SPR results revealed that MLS128's association constants for both antigens were highly temperature dependent. Below 25°C MLS128's association constant for Tn3-peptide was clearly higher than that for Tn2-peptide. At 30°C, however, the association constant for Tn2-peptide was higher than that for Tn3-peptide. This reversal of affinity is due to the sharp increase in K(d) for Tn3. These results were confirmed by NMR, which directly measured MLS128-Tn binding in solution. This study suggested that thermodynamic control plays a critical role in the interaction between MLS128/Tn2 and MLS128/Tn3.     

Solid-phase synthesis of a pentavalent GalNAc-containing glycopeptide (Tn antigen) representing the nephropathy-associated IgA hinge region

Incomplete or aberrant glycosylation leading to Tn antigen (GalNAcα1-Ser/Thr) expression on human glycoproteins is strongly associated with human pathological conditions, including tumors, certain autoimmune diseases, such as the idiopathic IgA nephropathy, and may modulate immune homeostasis. In addition, the Tn antigen is highly expressed by certain pathogens and plays a role in host-pathogen interactions. To enable experimental approaches to study interactions of the Tn antigen with the immune system and analyze anti-Tn antibody responses in infection or disorders, we generated a Tn-expressing resource that can be used for high-throughput screening. In consideration of IgA nephropathy in which the hinge region is incompletely glycosylated, we used this hinge sequence that encodes five potential glycosylation sites as the ideal template for the synthesis of a Tn antigen-expressing glycopeptide. Inclusion of an N-terminal biotin in the peptide enabled binding to streptavidin-coated ELISA plates as monitored using Helix pomatia agglutinin or anti-Tn monoclonal antibody. We also found that the biotinylated IgA-Tn peptide is a functional acceptor for β1-3-galactosylation using recombinant T-synthase (β1-3-galactosyltransferase). Besides its immunochemical functionality as a possible diagnostic tool for IgA nephropathy, the peptide is an excellent substrate for glycan elongation and represents a novel template applicable for glycan-antigen-associated diseases.     

Structural basis for recognition of breast and colon cancer epitopes Tn antigen and Forssman disaccharide by Helix pomatia lectin

Helix pomatia agglutinin (HPA) is a lectin that has been used extensively in histopathology, since its binding to tissue sections from breast and colon cancers is correlated with the worst prognosis for the patients. The lectin recognizes alpha-d-N-acetylgalactosamine (alphaGalNAc) containing epitopes which are only present in cancer cell lines having a high likelihood to undergo metastasis, such as the HT29 cancer colon cell line. Several breast cancer cell lines have also been shown to be labeled, although IGROV1, an ovarian cancer cell line, is not. Inhibition studies, using GalNAc monosaccharides, are reported here, showing that the labeling is dependent upon the presence of carbohydrate epitopes. The crystal structures of the lectin complexed with two GalNAc containing epitopes associated with cancer, the Tn (alphaGalNAc-Ser) and Forssman (alphaGalNAc1-3GalNAc) antigens, show the lectin's specificity for GalNAc is due to a particular network of hydrogen bonds. A histidine residue makes hydrophobic contact with the aglycon, rationalizing the preference for GalNAc bearing an additional sugar or amino acid in the alpha position. These structures provide the molecular basis for the use of HPA in metastasis research.     

Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy

Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.     

Further characterization of the binding properties of two monoclonal antibodies recognizing human Tn red blood cells

The terminal alpha anomeric Ga1NAc residue is an essential sugar for the Tn glycotope, human blood group A determinant, and Forssman antigen. In a previous study [King M.J., Parson S.F., Wu A,M., Jones N., Transfusion 31: 142-149, 1991] we defined two monoclonal antibodies (MoAbs, BRIC66 and BRIC111) reacting with human Tn red blood cells. However, more advanced studies of these two MoAbs were hampered by the lack of availability of Gal/GalNAc related glycotopes. In order to use these antibodies as powerful probes to elucidate structural changes during life processes, we have characterized in detail the combining sites of these two MoAbs using enzyme-linked immunosorbent (ELISA) and inhibition assays with an extended glycan/ligand collection. From the results, it has been established that BRIC66 demonstrated multiple specificities and its reactivity towards glycotopes was defined as: Ga1NAc alpha1-->Ser/Thr (Tn) > or = Ga1NAc alpha1-->3(LFuc alpha1-->2)Gal (Ah) > Ga1NAcalpha1-->3Galbeta1-->4Glc (AL) > Ga1NAalpha1-->3Gal (A) GalNAc alpha1-->3GalNAc > Gal or Glc. Another MoAb, BRIC111, mainly bound Tn-glycophorin. The best ligand for this MoAb was Tn-containing glycopeptides (M.W. < 3.0 x 10(3) Da) from asialo ovine salivary mucin (OSM), which was approximately 70 and 58 times more active than Ga1NAc and monomeric Ga1NAc alpha1-->Ser/Thr (Tn), respectively, suggesting that the active glycotopes present in glycophorin for BRIC111 binding also exist in OSM. The N-acetyl group at carbon-2 and configuration at carbon-2 and carbon-4 of the alpha anomeric Ga1NAc are required for the binding of either MoAb. Identification of these binding properties should aid in the selection of these MoAbs and the conditions required for biological studies and clinical applications.     

Tn antigen and UDP-Gal:GalNAc alpha-R beta 1-3Galactosyltransferase expression in human breast carcinoma.

The serine/threonine O-linked carbohydrates GalNAc alpha and Gal beta 1-3GalNAc alpha, referred to as Tn and T antigens, respectively, appear to be more prevalent in some human carcinomas than in surrounding tissues. Tn/T antigens may represent incomplete synthesis of O-linked oligosaccharides, due to decreased activity of specific glycosyltransferases, or alternatively, increased glycosidases activity in tumors which may expose these internal O-linked oligosaccharide sequences. To explore these possibilities, we measured UDP-Gal:GalNAc alpha-R beta 1-3 galactosyltransferase (beta 3Gal-T) and Gal beta 1-3GalNAc alpha-R beta 1-3 galactosidase in a series of human breast tumors. In addition, glycoproteins extracted from the tumors were separated by SDS-PAGE and stained with the lectins HPA (GalNAc alpha-R reactive) and PNA (Gal beta-3GalNAc alpha-R reactive). The relative levels of HPA- to PNA-reactive glycoproteins in the carcinomas correlated inversely with beta 3Gal-T activities. The results suggest that Tn antigen expression in human breast carcinoma is due in part to low beta 3Gal-T activity, a situation similar to that observed previously in haematopoietic cells of individuals with a condition called Tn syndrome.     

Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells

CD175 or Tn antigen is a carbohydrate moiety of N-acetylgalactosamine (GalNAc)α1-O- linked to the residue of amino acid serine or threonine in a polypeptide chain. Despite the chemical simplicity of the Tn antigen, its antigenic structure is considered to be complex and the clear determinants of Tn antigenicity remain poorly understood. As a consequence, a broad variety of anti-Tn monoclonal antibodies (mAbs) have been generated. To further investigate the nature and complexity of the Tn antigen, we generated seven different anti-Tn mAbs of IgM and IgG classes raised against human Jurkat T cells, which are Tn-positive due to the low activity of T-synthase and mutation in specific chaperone Cosmc. The binding analysis of anti-Tn mAbs with the array of synthetic saccharides, glycopeptides and O-glycoproteins revealed unexpected differences in specificities of anti-Tn mAbs. IgM mAbs bound the terminal GalNAc residue of the Tn antigen irrespective of the peptide context or with low selectivity to the glycoproteins. In contrast, IgG mAbs recognized the Tn antigen in the context of a specific peptide motif. Particularly, JA3 mAb reacted to the GSPP or GSPAPP, and JA5 mAb recognized specifically the GSP motif (glycosylation sites are underlined). The major O-glycan carrier proteins CD43 and CD162 and isoforms of CD45 expressed on Jurkat cells were precipitated by anti-Tn mAbs with different affinities. In summary, our data suggest that Tn antigen-Ab binding capacity is determined by the peptide context of the Tn antigen, antigenic specificity of the Ab and class of the immunoglobulin. The newly generated anti-Tn IgG mAbs with the strong specificity to glycoprotein CD43 can be particularly interesting for the application in leukemia diagnostics and therapy.     

O-glycosylation in Echinococcus granulosus: identification and characterization of the carcinoma-associated Tn antigen

In the present work we demonstrate that the cancer-associated O-glycosylated Tn antigen (GalNAc-O-Ser/Thr) is expressed by the cestode Echinococcus granulosus. This antigen was detected in both larval and adult worm extracts, with the highest specific activity observed in the adult excretion/secretion preparation. Histochemical analysis showed that Tn is preferentially expressed in the parenchyma in both parasite stages and the external part of tegument in adult worms. A similar pattern was observed for sialyl-Tn, a related O-linked antigen. Tn glycoproteins from protoscoleces were resolved by SDS-PAGE in two main components of 43 and 49 kDa. After purification, this material was reactive with lectins which bind GlcNAc/sialic acid, GalNAc, and T antigen. In a preliminary evaluation, high levels of Tn antigen were detected in serum samples from patients with hydatid cyst, suggesting that the measure of Tn in serum could be a biomarker of this disease, although extensive work is necessary in order to determine the clinical usefulness of this assay. The results reported here, the first evidence of O-glycosylation pathways in E. granulosus and the presence of Tn antigen in cestodes, suggest that the evaluation of O-glycosylated antigens might give new insights in the host-parasite relationship.     

High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins

Plant lectins have been studied as histological markers and promising antineoplastic molecules for a long time, and structural characterization of different lectins bound to specific cancer epitopes has been carried out successfully. The crystal structures of Vatairea macrocarpa (VML) seed lectin in complex with GalNAc-α-O-Ser (Tn antigen) and GalNAc have been determined at the resolution of 1.4 Å and 1.7 Å, respectively. Molecular docking analysis of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addition to the specific biological effects of VML, the structural and binding similarities between it and other lectins commonly used as histological markers (e.g., VVLB4 and SBA) strongly suggest VML as a candidate tool for cancer research.     

Catfish (Clarias batrachus) serum lectin recognizes polyvalent Tn [alpha-D-GalpNAc1-Ser/Thr], Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc1-Ser/Thr], and II [beta-D-Galp(1-->4)-beta-D-GlcpNAc1-] mammalian glycotopes

A new calcium dependent GalNAc/Gal specific lectin was isolated from the serum of Indian catfish, Clarias batrachus and designated as C. batrachus lectin (CBL). It is a disulfide-linked homodecameric lectin of 74.65kDa subunits and the oligomeric form is essential for its activity. Binding specificity of CBL was investigated by enzyme-linked lectin-sorbent assay using a series of simple sugars, polysaccharides, and glycoproteins. GalNAc was more potent inhibitor than Gal; and alpha glycosides of both were more inhibitory than their beta counterparts. CBL showed maximum affinity for human tumor-associated Tn-antigens (GalNAcalpha1-Ser/Thr) at the molecular level and was 3.5 times higher than GalNAc. CBL interacted strongly with polyvalent Tn and Talpha (Galbeta1,3GalNAcalpha1-) as well as multivalent-II (Galbeta1,4GlcNAcbeta1-) antigens containing glycoproteins and intensity of inhibition was 10(3)-10(5) times more than monovalent ones. The overall specificity of CBL lies in the order of polyvalent Tn, Talpha and II>monovalent TnMe-alphaGalNAc>monovalent Talpha> Me-betaGalNAc>Me-alphaGal>monovalent T>GalNAc>monovalent F>monovalent II>Me-betaGal>Gal.