Biosynthesis of truncated O-glycans in the T cell line Jurkat. Localization of O-glycan initiation

Glycoproteins from the human T leukemia cells Jurkat were found to bind to the GalNAc alpha 1----Ser/Thr-specific lectin from Salvia sclarea seeds. The analysis of the O-linked saccharides of immunopurified leukosialin, the major [3H]glucosamine-labeled glycoprotein in Jurkat cell lysate, revealed the presence of mainly GalNAc alpha 1----Ser/Thr with only minor amounts (approximately 17%) of more complex O-glycans. A comparison between Jurkat and K562 cell glycosyltransferase involved in the biosynthesis of O-linked carbohydrates showed that a markedly lower activity of UDP-Gal:GalNAc alpha 1----Ser/Thr beta 1----3galactosyltransferase is apparently responsible for the presence of truncated O-glycans in the Jurkat cell line. The O-glycosylation defect makes Jurkat cells an ideal model to study the initiation of O-linked saccharides. Pulse-chase experiments with [35S] methionine showed that the addition of GalNAc to leukosialin is responsible for the decreased mobility of the mature glycoprotein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, no biosynthetic intermediates between the O-glycan-free precursor and the fully O-glycosylated form could be detected either with an anti-leukosialin antiserum or with the GalNAc-specific lectin. Lowering the chase temperature to 15 degrees C completely inhibited the transfer of GalNAc to the peptide core indicating that O-glycan initiation takes place in the first Golgi elements and not in transitional vesicles between endoplasmic reticulum and Golgi. In addition, treatment of the cells with monensin did not inhibit GalNAc transfer to leukosialin apoprotein. These results indicate that the initiation of O-glycosylation in Jurkat cells starts in the cis-Golgi stacks.     

Coexpression of cancer-associated carbohydrate antigens,Tn and sialyl Tn

The expression of cancer-associated antigens, Tn and sialyl Tn, was examined using monoclonal antibodies, MLS 128 and MLS 102, recognizing these two antigens, respectively. A cell lysate from a human carcinoma cell line, LS 180 cells, was analysed by Western blotting using these two antibodies. Three glycoprotein bands were discernible with each antibody, of which two, corresponding to 250 and 210 kDa, were reactive with both the antibodies. LS 180 cells were metabolically labelled with³H-glucosamine and then the lysate from these cells was applied to two immunoaffinity columns. Sixty-five per cent of the Tn antigenic glycoproteins, based on radioactivity, bound to the MLS 102 affinity column. On the other hand, 45% of the sialyl Tn antigenic glycoproteins bound to the MLS 128 affinity column. These results indicate that some Tn and sialyl Tn antigens were expressed on the same polypeptide chains.The presence of non-sialylated GalNAc residues on the polypeptide chain with many Sia-GalNAc residues appears to be due to the incapability of three consecutive moieties of GalNAc-Ser/Thr to accept sialic acid.Abbreviations PSMF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - GalNAc N-acetylgalactosamine - Sia sialic acid     

A new murine IgG1 anti-Tn monoclonal antibody with in vivo anti-tumor activity

The Tn antigen (GalNAc α-O-Ser/Thr) is heterogeneously synthesized by a variety of tumors and contains an epitope defined by lectins and antibodies as a cluster of αGalNAc carbohydrates synthesized within a peptide sequence, which is rich in serine and/or threonine. The Tn antigen has been utilized as a target in vaccine experiments and also used as a biomarker for prognosis of different cancer forms. In this paper, we present a new monoclonal antibody, GOD3-2C4, with the clear hallmarks of an anti-Tn antibody. It was generated through somatic cell hybridization after immunization of a mouse with a tumor cell line and a Tn carrying mucin. The antibody recognizes synthetic Tn antigen and binds breast, colon, lung, ovarian and pancreas cancer. The GOD3-2C4 antibody has antibody-dependent cellular cytotoxicity activity against Jurkat cells in vitro, and for the first time, it can be shown that an anti-Tn antibody has a significant in vivo effect on a human cancer cell line grown as a xenograft in severe combined immunodeficiency mice.     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Elucidation of an essential structure recognized by an anti-GalNAc alpha-Ser(Thr) monoclonal antibody (MLS 128).

To determine the epitopic structure for an anti-GalNAc alpha-Ser(Thr) (anti-Tn) monoclonal antibody, MLS 128, asialo-ovine submaxillary mucin was digested with various proteases, and the digests were fractionated by immunoaffinity column chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassaying, it was found that GP-I was strongly immunoreactive, whereas GP-1-5 were poorly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity. From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was as strongly immunoreactive as GP-I. GP-II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an alpha-GalNAc residue is attached. Other anti-Tn monoclonal antibodies, NCC-LU-35 and CA 3239, showed essentially the same reactivity to these glycopeptides as MLS 128 did. The glycopeptides (GP-1-5), which exhibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Ser, GalNAc-Thr, GalNAc-Ser-(GalNAc)-Ser, and GalNAc-Thr-(GalNAc)-Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies.     

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.     

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.     

Monoclonal antibodies toward different Tn-amino acid backbones display distinct recognition patterns on human cancer cells. Implications for effective immuno-targeting of cancer

The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.     

O-glycosylation of leukosialin in K562 cells. Evidence for initiation and elongation in early Golgi compartments

The O-glycosylation of leukosialin, a major sialoglycoprotein found on leukocytes, has been studied in the human erythroleukemic cell line K562. The appearance of its O-linked chains has been followed in pulse-chase experiments with [35S]methionine by immunoprecipitation with an anti-peptide antiserum as well as with a lectin from Salvia sclarea seeds (SSA) specific for GalNAc-Ser/Thr and the peanut (Arachis hypogaea) agglutinin (PNA) which recognizes Gal beta 1----3GalNAc-Ser/Thr structures. An O-glycan-free precursor was converted into the fully O-glycosylated mature form within the 10-min labeling period and no intermediates carrying only GalNAc-Ser/Thr structures could be detected. The ionophore monensin was used in order to slow down intracellular traffic and thus O-glycan synthesis. The drug partly inhibited the transport from rough endoplasmic reticulum (RER) to the Golgi and also the cell-surface expression of leukosialin. It was found to have a marked effect on the synthesis of O-linked carbohydrate structures of leukosialin since the amount of O-glycans containing only GalNAc or NeuNAc alpha 2----6GalNAc was significantly increased after monensin treatment. Under these conditions the biosynthesis of the N-glycan on leukosialin was completely arrested in an endoglycosidase-H-sensitive step of processing, whereas the O-glycans already contained galactose and sialic acid although at a reduced level. On the other hand, the small amounts of leukosialin expressed on the cell surface of monensin-treated cells carried the same glycans as those remaining blocked inside the cell. In addition, immunocytochemical studies using SSA and PNA on untreated K562 cells suggested the absence of detectable amounts of GalNAc-Ser/Thr-bearing glycoproteins in the RER as well as in the Golgi. In contrast Gal beta 1----3GalNAc structures could be detected on intracellular membranes which were tentatively identified as the cis-Golgi. Together these results lead us to the following conclusions: N-glycan transfer occurs in the RER before the initiation of O-glycans which takes place at the entrance of the protein into the Golgi; further elongation of O-glycans with galactose and sialic acid follows very rapidly, probably before the final processing of N-glycans to complex-type structures.     

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.     

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.     

Short synthetic glycopeptides successfully induce antibody responses to carcinoma-associated Tn antigen.

Glycopeptides containing a tumor-associated carbohydrate antigen (mono-, tri- or hexa-Tn antigen) as a B-cell epitope and a CD4+ T-cell epitope (PV: poliovirus or TT: tetanus toxin) were prepared for immunological studies. Several Tn antigen residues [FmocSer/Thr (alpha-GalNAc)-OH] were successively incorporated into the peptide sequence with unprotected carbohydrate groups. The tri- and hexa-Tn glycopeptides were recognized by MLS128, a Tn-specific monoclonal antibody. The position of the tri-Tn motif in the peptide sequence and the peptide backbone itself do not alter its antigenicity. As demonstrated by both ELISA and FACS analysis, the glycopeptides induced high titers of anti-Tn antibodies in mice, in the absence of a carrier molecule. In addition, the generated antibodies recognized the native Tn antigen on cancer cells. The antibody response obtained with a D-(Tn3)-PV glycopeptide containing three alpha-GalNAc-D-serine residues is similar that obtained with the Tn6-PV glycopeptide. These results demonstrate that short synthetic glycopeptides are able to induce anticancer antibody responses.     

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.     

α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway

Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcβ1-3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galβ1-3/4GlcNAcβ1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), β-galactosidase (BbgIII), and β-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.     

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.     

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).     

Carcinoma autoantigens T and Tn and their cleavage products interact with Gal/GalNAc-specific receptors on rat Kupffer cells and hepatocytes

We studied interactions of isolated Thomsen-Friedenreich (T)- and Tn-specific glycoproteins with the Gal/GalNAc-specific receptors on rat Kupffer cells and compared them to those with rat hepatocytes. Immunoreactive T and Tn are specific pancarcinoma epitopes. Electron microscopy of gold-labelled T and Tn antigens revealed their specific binding to Kupffer cells, followed by their uptake via the coated pit/vesicle pathway of receptor-mediated endocytosis. Preincubation of Kupffer cells with GalNAc and GalNAc-BSA, but not GlcNAc or GlcNAc-BSA specifically inhibited binding of the T and Tn glycoproteins. Desialylated, isologous erythrocytes (T RBC) are known to bind to the Gal/GalNAc receptors of rat Kupffer cells and hepatocytes. This attachment was specifically inhibited by T and Tn in a concentration-dependent manner: 50% T RBC-Kupffer cell contacts were inhibited at 8.5.10(-6) mM T and 8.5.10(-5) mM Tn antigen concentrations, respectively. The corresponding figures for hepatocytes were 6.10(-6) mM T and 1.2.10(-6) mM Tn antigen. Amino-terminal cleavage products of the T glycoprotein, possessing clusters terminating in non-reducing Gal/GalNAc, inhibited T RBC binding to Kupffer cells and hepatocytes usually at 10(-2) to 10(-5) mM concentrations, whereas GalNAc, galactose and galactose glycosides inhibited at millimolar concentrations. Galactose-unrelated carbohydrates were inactive at concentrations greater than or equal to 50 mM.     

Role of O-glycosylation and expression of CD43 and CD45 on the surfaces of effector T cells in human T cell leukemia virus type 1 cell-to-cell infection

We used replication-dependent retroviral vectors to identify cell surface antigens involved in the cell-to-cell transmission of human T cell leukemia virus type 1 (HTLV-1). We generated monoclonal antibodies (MAbs) against Jurkat T cells and selected several IgM MAbs that strongly inhibited HTLV-1 but not human immune deficiency virus type 1 (HIV-1) cell-to-cell infection. These MAbs recognized the so-called Tn antigen (GalNAcα1-O-Ser/Thr) that arises on Jurkat cells from a mutation in the T-synthase-specific chaperone Cosmc and the consequent loss of O-glycan elongation. Anti-Tn MAbs precipitated two major O-glycan carrier proteins, CD43 and CD45, and caused a strong aggregation of Jurkat cells. The restoration of O-glycosylation in Jurkat cells by stably transducing the wild-type Cosmc gene resulted in a 3- to 4-fold increase in the level of surface expression of CD43 and enhanced HTLV-1 transmission 10-fold in comparison to that of parental cells. The short hairpin RNA (shRNA) knockdown of CD43 or CD45 expression in Jurkat-Cosmc, HBP-ALL, and CEM T cells decreased HTLV-1 infection severalfold. The knockdown of CD45 in Jurkat cells severely reduced both HTLV-1 and HIV-1 infections, but Cosmc coexpression partially rescued infection. HTLV-1 proteins, which assembled in small patches on Jurkat cells, formed large clusters on the surface of Jurkat-Cosmc cells. These data indicate that large aggregates of HTLV-1 assemblies are more infectious than multiple clustered virions. We suggest that heavily O-glycosylated CD43 and CD45 molecules render cells less adhesive, prevent inappropriate cell-cell contacts, and favor the assembly of HTLV-1 particles into large, highly infectious structures on the surface of T cells.     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.