A molecular basis for functional peptide mimicry of a carbohydrate antigen

Peptides may substitute for carbohydrate antigens in carbohydrate-specific immunological reactions. Using the recognition properties of an anti-Lewis Y (LeY) antibody, BR55-2, as a model system, we establish a molecular perspective for peptide mimicry by comparing the three-dimensional basis of BR55-2 binding to LeY with the binding of the same antibody to peptides. The peptides compete with LeY, as demonstrated by enzyme-linked immunosorbent assay and Biacore analysis. The computer program LUDI was used to epitope map the antibody-combining site, correlating peptide reactivity patterns. This approach identified amino acids interacting with the same BR55-2 functional residue groups that recognize the Fucalpha(1-3) moiety of LeY. Molecular modeling indicates that the peptides adopt an extended turn conformation within the BR55-2 combining site, serving to overlap the peptides with the LeY spatial position. Peptide binding is associated with only minor changes in BR55-2, relative to the BR55-2-LeY complex. Anti-peptide serum distinguishes the Fucalpha(1-3) from the Fucalpha(1-4) linkage, therefore differentiating difucosylated neolactoseries antigens. These results further confirm that peptides and carbohydrates can bind to the same antibody-binding site and that peptides can structurally and functionally mimic salient features of carbohydrate epitopes.     

A Lewis(y) epitope mimicking peptide induces anti-Lewis(y) immune responses in rabbits and mice.

Lewis(y) carbohydrate antigens are abundant on the surface of many carcinomas. Mab B3 directed against this carbohydrate antigen has been used to make an immunotoxin that is very cytotoxic to cancer cells expressing the Lewis(y) antigen. Mab B3 was also used to screen a phage-displayed peptide library and identified a peptide mimicking the Lewis(y) epitope. In this report, we demonstrate that the Lewis(y) epitope-mimicking peptide induces anti-Lewis(y)immune responses in both rabbits and mice. In addition, Lewis(y) antigens induce anti-peptide immune responses. These results indicate that carbohydrate-mimicking peptides provide a novel strategy to elicit immune responses for tumor-associated carbohydrates.     

Structural convergence of antibody binding of carbohydrate determinants in Lewis Y tumor antigens

Antibodies targeting human epithelial carcinomas bearing Lewis Y (Le(y)) carbohydrate antigens provide a striking illustration of convergent immune recognition. We report a 1.9A resolution crystal structure of the Fab of a humanized antibody (hu3S193) in complex with the Le(y) tetrasaccharide, Fuc(alpha 1-->2)Gal(beta 1-->4)[Fuc(alpha 1-->3)]GlcNAc. Comparisons of the hu3S193 and BR96 antibodies bound to Le(y) tumor antigens revealed extremely similar mechanisms for recognition of the carbohydrate epitopes. Solvent plays a critical role in hu3S193 antibody binding to the Le(y) carbohydrate epitope. Specificity for Le(y) is maintained because a conserved pocket accepts an N-acetyl group of the core Gal(beta 1-->4)GlcNAc disaccharide. Closely related blood-group determinants (Le(a) and Le(b)) cannot enter the specificity pocket, making the Le(y) antibodies promising candidates for immunotherapy of epithelial cancer.     

Structural variation and immune recognition of the P1.2 subtype meningococcal antigen

Neisseria meningitidis is a globally important cause of bacterial meningitis and septicemia. No comprehensive antimeningococcal vaccine is available, largely as a consequence of the high sequence diversity of those surface proteins that could function as components of a vaccine. One such component is the protein PorA, a major surface porin of this Gram-negative organism that has been used in a number of experimental and licensed vaccines. Here we describe a series of experiments designed to investigate the consequences for antibody recognition of sequence diversity within a PorA antigen. The binding of a 14-residue peptide, corresponding to the P1.2 subtype antigen, to the MN16C13F4 monoclonal antibody was sensitive to mutation of five out of the six residues within the epitope sequence. The crystal structure of the antibody Fab fragment, determined in complex with the peptide antigen, shows a remarkably hydrophobic binding site and interactions between the antigen and antibody are dominated by apolar residues. Nine intrachain hydrogen bonds are formed within the antigen which maintain the beta-hairpin conformation of the peptide. These hydrogen bonds involve residues that are highly conserved amongst different P1.2 sequence variants, suggesting that some positions may be conserved for structural reasons in these highly polymorphic regions. The sensitivity of antibody recognition of the antigen towards mutation provides a structural explanation for the widespread sequence variation seen in different PorA sequences in this region. Single point mutations are sufficient to remove binding capability, providing a rationale for the manner in which different meningococcal PorA escape variants arise.     

Rational design and synthesis of peptide ligands for an anti-carbohydrate antibody and their immunochemical characterization

Molecular mimics of carbohydrates present an alternative source of compounds to target pathways involving protein-carbohydrate interactions. Certain peptides act as molecular mimics of carbohydrates in binding to anti-carbohydrate antibodies. A series of potential peptide ligands for the anti-carbohydrate antibody SYA/J6, directed against Shigella flexneri Y, was designed by molecular modeling based on a crystal structure of the antibody complex with a carbohydrate-mimetic peptide. These octapeptides were synthesized using solid-phase peptide synthesis, and their recognition by the antibody was investigated. The results shed light on the nature of peptide-carbohydrate mimicry.     

The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide

The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.     

Characterization of monoclonal antibodies specific for the Lewis a human blood group determinant

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.     

Monosaccharides modulate HCV E2 protein-derived peptide biological properties

A hepatitis C virus E(2) protein-derived sequence was selected for studying the effect of N-glycosylation on the peptide chain's conformational structure. The results suggested that the (534)TDVF(537) motif contained in peptide 33402 ((529)WGENDTDVFVLNNTRY(544)) had a type III beta-turn, relevant in antigen recognition of polyclonal antibodies, binding to human cells, and binding to HLA DRB1 *0401 molecules. N-Glycopeptides derived from this sequence contained monosaccharides in Asn(532). N-Glycopeptides presented differences in peptide chain structure compared to non-glycosylated peptides. Peptide 33402 specifically bound to human cells, specificity becoming lost when it was N-glycosylated. N-Glycosylation decreased antigen recognition of mouse polyclonal sera against this sequence. N-Glycopeptide binding to HLA DRB1 *0401 molecules was similar to that presented by non-glycosylated peptide, indicating that N-glycosylation did not affect binding to HLA DRB1 *0401 molecules. N-Glycosylation induced changes at structural and functional level which could be relevant for modulating human cell binding properties and antibody recognition.     

Peptide mimicking sialyl-Lewis(a) with anti-inflammatory activity

Peptides mimicking carbohydrate structure sialyl-Lewis a (SA-Le(a)) have been selected from a diverse dodecapeptide library using monoclonal antibody (MAb) NS19-9. Families of peptides with a consensus sequence consisting of three to nine amino acids and peptides that do not show a conserved core amino acid region were identified. Peptide DLWDWVVGKPAG was selected based on the consensus sequence DXXDXXVG shared with other peptides and strong binding in Western blot. Peptide competes with antibody binding to its native carbohydrate antigen, SA-Le(a), at 50% inhibitory concentration (IC(50)), 700 microM, implying that it represents a structural mimic of the carbohydrate epitope recognized by MAb. Statistically significant reduction of neutrophil recruitment into the intraperitoneal cavity was observed upon administration of this peptide in a murine acute inflammation model in vivo. Results suggest that the peptide mimic of SA-Le(a) carbohydrate might bind to E-selectin and block its interaction with another ligand, sialyl-Lewis X (SA-LeX), expressed on neutrophils.     

Y and blood group B type 2 glycolipid antigens accumulate in a human gastric carcinoma cell line as detected by monoclonal antibody. Isolation and characterization by mass spectrometry and NMR spectroscopy

A monoclonal antibody (mAb), BR55-2, was generated from mice immunized with MCF-7 human breast carcinoma cells. This mAb specifically detected glycolipids with the Y determinant Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)-beta 1----3Gal beta 1----4Glc beta 1----1 Cer and the Y-related B-active difucosylated determinant Gal alpha 1----3Gal(2----1 alpha Fuc) beta 1----4GlcNAc(3----1 alpha Fuc) beta 1----3Gal beta 1----4Glc beta 1----1 Cer, but was not reactive with related monofucosylated glycolipids of type 2 chain (X-antigen, blood group H), type 1 chain (Lea antigen, blood group H and B) or with difucosylated type 2 and type 1 chain structures (A blood group antigen or blood group B and Leb, respectively). A series of glycolipids with Y and blood group B type 2 determinants were detected in human gastric adenocarcinoma cell line KATO III with mAb BR55-2 and with a previously characterized anti-blood group B mAb PA83-52 (Hansson, G. C., Karlsson, K.-A., Larson, G., McKibbin, J. M., Blaszczyk, M., Herlyn, M., Steplewski, Z., and Koprowski, H. (1983) J. Biol. Chem. 258, 4091-4097). The isolated antigens were structurally characterized by mass spectrometry of permethylated and permethylated-reduced derivatives and by proton NMR spectroscopy. In a chromatogram binding assay, mAb BR55-2 and mAb PA83-52 detected minor components with slower mobility than the Y-6 and blood group B-7-type 2 structures. The detection of a B type 2 determinant is the first chemical evidence for the presence of an autologous difucosyl blood group B type 2 antigen in human adenocarcinoma cells.     

Molecular Dynamics Simulations of the TSSPSAD Peptide Antigen in Free and Bound with CAMPATH-1H Fab Antibody States: The Importance of the β-Turn Conformation

Humanized CAMPATH-1H antibody has been found to have biological applications through the recognition of the CD52 antigen. A peptide mimotope of the CD52 antigen with the sequence T₁SSPSAD₇ has been co-crystallized with the CAMPATH-1H antibody. A plethora of hydrogen bond interactions were found to mediate antigen recognition. An important feature of peptide’s bound conformation was the type I β-turn found in the S₃PSA₆ peptide’s fragment. Paradoxically, this fact has been underestimated from other researchers. In order to further investigate the importance of this structural feature and its significance in antibody/antigen binding we have performed molecular dynamics simulations in explicit water of the T₁SSPSAD₇ peptide in both antibody free and bound states. We have found that the turn structure has been perfectly retained in the bound state but it was eliminated in the free state. This fact implies that the turn structure of the peptide is unstable in aqueous environment and it is induced upon antibody binding. Analysis of the trajectories revealed also several other important features of the antibody/antigen binding mode.     

Effect of glycosylation on MUC1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions

MUC1 mucin is a large transmembrane glycoprotein, of which the extracellular domain is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is underglycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above) as well as the normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc), and TF (Gal beta1-3 GalNAc) carbohydrates. In the present study, NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRP core epitope region to the recognition and binding of a monoclonal antibody, Mab B27.29, raised against the intact tumor-associated MUC1 mucin. Four peptides were studied: a MUC1 16mer peptide of the sequence Gly1-Val2-Thr3-Ser4-Ala5-Pro6-Asp7-Thr8-Arg9-Pro10-Ala11-Pro12-Gly13-Ser14-Thr15-Ala16, two singly Tn-glycosylated versions of this peptide at either Thr3 or Ser4, and a doubly Tn-glycosylated version at both Thr3 and Ser4. The results of these studies showed that the B27.29 MUC1 B-cell epitope maps to two separate parts of the glycopeptide, the core peptide epitope spanning the PDTRP sequence and a second (carbohydrate) epitope comprised of the Tn moieties attached at Thr3 and Ser4. The implications of these results are discussed within the framework of developing a glycosylated second-generation MUC1 glycopeptide vaccine.     

Defining the Recognition Elements of Lewis Y-Reactive Antibodies

Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2) response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ) B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY) to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1–4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens.     

A monoclonal antibody against human colonic adenoma recognizes difucosylated Type-2-blood-group chains

A monoclonal antibody C14/1/46/10 showing preferential binding to membranes of human colorectal carcinomas over normal colon mucosae was obtained by immunization of mice with extra-nuclear membranes of a human colonic adenoma. Binding and inhibition of binding assays using blood cells or glycoproteins with known blood-group activities indicated that the antibody recognizes a carbohydrate antigen co-existing with the blood-group-H determinant: Fucα1→2Gal. Inhibition assays with structurally defined oligosaccharides showed that the antigenic determinant involves difucosylated Type-2-blood-group chains with the structure:      

Inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, HIV-1 specific antibody

The HIV envelope has evolved a dense array of immunologically "self" carbohydrates that efficiently protect the virus from antibody recognition. Nonetheless, one broadly neutralising antibody, IgG1 2G12, has been shown to recognise a cluster of oligomannose glycans on the HIV-1 surface antigen gp120. Thus the self carbohydrates of HIV are now regarded as potential targets for viral neutralisation and vaccine design. Here, we show that chemical inhibition of mammalian glycoprotein synthesis, with the plant alkaloid kifunensine, creates multiple HIV (2G12) epitopes on the surface of previously non-antigenic self proteins and cells, including HIV gp120. This formally demonstrates the structural basis for self/non-self discrimination between viral and host glycans, by a neutralising antibody. Moreover, this study provides an alternative protein engineering approach to the design of a carbohydrate vaccine for HIV-1 by chemical synthesis.     

A peptide mimetic of an anti-CD4 monoclonal antibody by rational design

The development of rational methods to design 'continuous' sequence mimetics of discontinuous regions of protein sequence has, to now, been only marginally successful. This has been largely due to the difficulty of constraining the recognition elements of a mimetic structure to the relative conformational and spatial orientations present in the parent molecule. Using peptide mapping to determine 'active' antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, we have developed a peptide mimic of an anti-CD4 antibody, containing antigen contact residues from multiple CDRs. The design described is a 27-residue peptide formed by juxtaposition of residues from 5 CDR regions. It displays an affinity for the antigen (CD4) of 0.9nM, compared to 2nM for the parent antibody ST40. Nevertheless, the mimetic shows low biological activity in an anti-retroviral assay.     

High idiotypic connectivity of the VH7183-encoded antibodies directed to a murine embryonic carbohydrate antigen, Lewis Y, as ascertained by syngenic anti-idiotype monoclonal antibodies

The Lewis Y Ag is a carbohydrate Ag which is closely related to a well-known murine embryonic Ag, the stage-specific embryonic Ag-1 (SSEA-1), in its biochemical structure. It is expressed at the surface of murine embryonic cells as well as many murine cancer cells. For the analysis of idiotopes carried by the anti-Lewis Y antibodies, we generated two syngenic anti-idiotypic mAb, Id-A1 and Id-B4 (both BALB/c IgG1), which are directed to the idiotypic determinants carried by the anti-Lewis Y mAb, AH-6 (BALB/c IgM). Both Id-A1 and Id-B4 (Ab2) recognized paratope-related idiotopes carried by the AH-6 antibody (Ab1); they specifically inhibited the binding of AH-6 to the Lewis Y Ag. The high idiotypic connectivity of anti-Lewis Y antibodies was noted; the polyclonal anti-idiotype antibody, produced in the sera of BALB/c mice by immunizing AH-6 antibody, cross-reacted with several anti-Lewis Y mAb which has been established in different laboratories. Id-B4 and Id-A1 seem to represent such cross-reactive anti-idiotypic antibodies. Id-A1 recognized an idiotope carried by two out of six panel Ab1 mAb directed to the Lewis Y Ag. Id-B4 reacted with four out of the six panel antibodies, and was considered to recognize a recurrent idiotope of anti-Lewis Y antibodies which occurs more commonly than the idiotope recognized by Id-A1. All of the anti-Lewis Y antibodies which carry idiotopes that react with Id-A1 or Id-B4 were encoded by the VH genes of the VH7183 family; the most D-J proximal VH gene family in BALB/c mice, which is known to be preferentially expressed in embryonic B cells. Immunization of BALB/c mice with keyhole limpet hemocyanin-conjugated Id-B4 and/or Id-A1 induced a significant titer of anti-Lewis Y antibodies (Ab1-like Ab3) in the sera.     

Identification of a peptide mimic of the L2/HNK-1 carbohydrate epitope

The L2/HNK-1 carbohydrate is carried by many neural recognition molecules and is involved in neural cell interactions during development, regeneration in the peripheral nervous system, synaptic plasticity, and autoimmune-based neuropathies. Its key structure consists of a sulfated glucuronic acid linked to lactosaminyl residues. Because of its biological importance but limited availability, the phage display method was used to isolate a collection of peptide mimics that bind specifically to an L2/HNK-1 antibody. The phages isolated from a 15-mer peptide library by adsorption to this antibody share a consensus sequence of amino acids. The peptide mimicked several important functions of the L2/HNK-1 carbohydrate, such as binding to motor neurons in vitro, and preferential promotion of in vitro neurite outgrowth from motor axons compared with sensory neurons. A scrambled version of the peptide had no activity. The combined observations indicate that we have isolated a mimic of the L2/HNK-1 carbohydrate that is able to act as its functional substitute.     

Structural similarities of micelle-bound peptide YY (PYY) and neuropeptide Y (NPY) are related to their affinity profiles at the Y receptors

Here, we investigate the structure of porcine peptide YY (pPYY) both when unligated in solution at pH 4.2 and when bound to dodecylphosphocholine (DPC) micelles at pH 5.5. pPYY in solution displays the PP-fold, with the N-terminal segment being back-folded onto the C-terminal alpha-helix, which extends from residue 17 to 31. In contrast to the solution structure of Keire et al. published in the year 2000 the C-terminal helix does not display a kink around residue 23-25. The root mean square deviation (RMSD) for backbone atoms of the NMR ensemble of conformers to the mean structure is 0.99(+/-0.35) Angstrom for residues 14-31. The back-fold is supported by values of 0.60+/-0.1 for the (15)N(1)H-NOE and by generalized order parameters S(2) of 0.74+/-0.1 for residues 5-31 which indicate that the peptide is folded in that segment. We have additionally used DPC micelles as a membrane model and determined the structure of pPYY when bound to it. Therein, an alpha-helix occurs in the segment comprising residues 17-31 and the N terminus freely diffuses in solution. The hydrophobic side of the amphipathic helix forms the micelle-binding interface and hydrophobic side-chains extend into the micelle interior. A significant stabilization of helical conformation occurs in the C-terminal pentapeptide, which is important for receptor binding. The latter is supported by positive values of the heteronuclear NOE in that segment (0.52+/-0.1 compared to 0.08+/-0.4 for the unligated form) and by values of S(2) of 0.6+/-0.2 (versus 0.38+/-0.2 for the unligated form). The structures of micelle-bound pPYY and pNPY are much more similar than those of pPYY and bPP with pairwise RMSDs of 1.23(+/-0.21)A or 3.21(+/-0.39) Angstrom, respectively. In contrast to the conformational similarities in the DPC-bound state their structures in solution are very different. In fact pPYY is more similar to bPP, which with its strong preference for the Y(4) receptor displays a completely different binding profile. Considering the high degree of sequence homology of pNPY and pPYY (>80%) and the fact, that their binding affinities at all receptor subtypes are high and, more importantly, rather similar, it is much more likely that PYY and NPY are recognized by the Y receptors from the membrane-bound state. As a consequence of the latter the PP-fold is not important for recognition of PYY or NPY at the Y receptors. To our knowledge this work provides for the first time strong arguments derived from structural data that support a membrane-bound receptor recognition pathway.     

The human epithelial carcinoma antigen recognized by monoclonal antibody AE3 is expressed on a sulfoglycolipid in addition to neoplastic mucins

The term human epithelial carcinoma antigen (HCA) has been applied collectively to mucin-type high molecular weight (>1000 kDa) glycoproteins that are over-expressed in epithelial cancers. Since the 1990s, over 40 monoclonal antibodies have been raised that recognize HCA. There has been evidence that the antigenic determinants are mostly carbohydrates, but details have been elusive. Here we have carried out carbohydrate microarray analyses of one of the monoclonal antibodies, AE3, that has been regarded the ‘most carcinoma specific’ in respect to its ability to detect HCA in sera of patients with epithelial cancers. The microarrays encompassed a series of 492 sequence-defined glycan probes in the form of glycolipids and neoglycolipids. We have thus established that the antigen recognized by antibody AE3 is a carbohydrate sequence distinct from the A, B, H, Lewis^a/b, Lewis^x/y and T antigens, but that it is strongly expressed on the monosulfated tetra-glycosyl ceramide, SM1a, Galβ1-3GalNAcβ1-4(3-O-sulfate)Galβ1-4GlcCer. This is the first report of an anti-HCA to be characterized with respect to its recognition sequence and of the occurrence of the antigen on a glycolipid as well as on glycoproteins. Knowledge of a discrete glycan sequence as target antigen now opens the way to its exploration as a serologic cancer biomarker, namely to determine if the antigen elicits an autoantibody response in early non-metastatic cancer, or if it is shed and immunochemically detectable in more advanced disease.