Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study

MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], (1)H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including (15)N and (13)C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)(2). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the (15)N- and (13)C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.     

Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance

The MUC1 mucin represents a prime target antigen for cancer immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas. Attempts to generate strong humoral immunity to MUC1 by immunization with peptides have generally failed partly because of tolerance. In this study, we have developed chemoenzymatic synthesis of extended MUC1 TR glycopeptides with cancer-associated O-glycosylation using a panel of recombinant human glycosyltransferases. MUC1 glycopeptides with different densities of Tn and STn glycoforms conjugated to KLH were used as immunogens to evaluate an optimal vaccine design. Glycopeptides with complete O-glycan occupancy (five sites per repeat) elicited the strongest antibody response reacting with MUC1 expressed in breast cancer cell lines in both Balb/c and MUC1.Tg mice. The elicited humoral immune response showed remarkable specificity for cancer cells suggesting that the glycopeptide design holds promise as a cancer vaccine. The elicited immune responses were directed to combined glycopeptide epitopes, and both peptide sequence and carbohydrate structures were important for the antigen. A MAb (5E5) with similar specificity as the elicited immune response was generated and shown to have the same remarkable cancer specificity. This antibody may hold promise in diagnostic and immunopreventive measures.     

Specificities of anti-sialyl-Tn and anti-Tn monoclonal antibodies generated using novel clustered synthetic glycopeptide epitopes

The fine specificities of MAbs generated using novel synthetic clustered STn and Tn glycopeptides as immunogens were compared with the anti-TAG-72 antibodies B72.3 and CC49. Hapten inhibition experiments demonstrated the specificity of several of the MAbs for STn and Tn expressed on ovine submaxillary mucin and tumor derived MUC-1 mucin. Amongst the STn specific MAbs only the B195.3 MAb shows absolute dependence on the presence of sialic acid and specificity to the simple disaccharide NANAA α2-6-GalNAc. Identification of tumor associated carbohydrate epitopes in cluster and monomer configurations are possible using MAbs detecting the defined structure specificities described herein. This revised version was published online in November 2006 with corrections to the Cover Date.     

Pre-immunotherapy serum CA27.29 (MUC-1) mucin level and CD69+ lymphocytes correlate with effects of Theratope® sialyl-Tn-KLH cancer vaccine in active specific immunotherapy

Patients with metastatic breast, colorectal or ovarian cancers received active specific immunotherapy (ASI) with Theratope® sialyl-Tn-KLH (keyhole limpet hemocyanin) cancer vaccine emulsified in Detox? adjuvant. The median log₂ anti-STn IgG titer generated by ASI, estimated by enzyme-linked immunosorbent assay with solid-phase ovine submaxillary mucin, was 5.322 (range = 0?–?9.322). Following ASI, 51 patients who generated titers higher than the median value for anti-STn⁺ mucin IgG survived longer than 46 patients who generated lower titers below the median. 38 of the patients were phenotyped for CD69 prior to ASI. The patients with lower numbers of CD69⁺ peripheral blood lymphocytes prior to immunotherapy (pre-ASI) also had low serum CA27.29 cancer antigen (MUC-1) levels, and had longer times to disease progression and improved survival following ASI. Elevated pre-ASI serum CA27.29 tumor antigen levels were associated with higher numbers of CD69⁺ PBL, with decreased anti-STn antibody production and decreased survival following ASI. The data are compatible with the hypothesis that elevated serum MUC-1 mucin is specifically immunosuppressive.     

Structure/activity studies of the anti-MUC1 monoclonal antibody C595 and synthetic MUC1 mucin-core-related peptides and glycopeptides

MUC1 mucin is a large complex glycoprotein expressed on normal epithelial cells in humans and overexpressed and under or aberrantly glycosylated on many malignant cancer cells which consequently allows recognition of the protein core by antibodies. In order to understand how glycosylation may modulate or regulate antibody binding of mucin protein core epitopes, we have analyzed the antibody C595 (epitope RPAP) for its structure, stability, and its binding to a series of synthetic peptides and glycopeptides by a number of spectroscopic methods. Thermal and pH denaturation studies followed by changes in the CD spectrum of the antibody indicate critical involvement of specific residues to the stability of the antibody. Fluorescence binding studies indicate that alpha-N-acetylgalactosamine (GalNAc) glycosylation of a MUC1 mucin synthetic peptide TAPPAHGVT9SAPDTRPAPGS20T21APPA at threonine residues 9 and 21 and serine residue 20 enhanced the binding of antibody. The structural effects of GalNAc glycosylation on the conformation of the MUC1 peptide were studied. CD of the peptides and glycopeptides in a cryogenic mixture cooled to approximately -97 degrees C revealed that a left-handed polyproline II helix (PPII) is adopted by the peptides in solution, which appears to be further stabilized by addition of the GalNAc residues. Consistent with the PPII helical structure, which has no intra-amide hydrogen bonds, high-field NMR spectroscopy of the glycopeptide revealed no sequential dNN, medium-range, or long-range nuclear Overhauser effect (NOE) connectivities. These studies indicate that stabilization of the PPII helix by GalNAc glycosylation present the epitope of C595 antibody with a favorable conformation for binding. Furthermore, they illustrate that glycosylation of the MUC1 tumor marker protein with a simple O-linked saccharide expressed in many cancers, can enhance the binding of the clinically relevant C595 antibody.     

Development and characterization of an antibody directed to an α-N-acetyl-D-galactosamine glycosylated MUC2 peptide

In an attempt to raise anti-Tn antibodies, an -N-acetyl-D-galactosamine glycosylated peptide based on the tandem repeat of the intestinal mucin MUC2 was used as an immunogen. The MUC2 peptide (PTTTPISTTTMVTPTPTPTC) was glycosylated in vitro using concentrated -N-acetylgalactosaminyltransferases activity from porcine submaxillary glands which resulted in the incorporation of 8–9 mol of Ga/NAc. Rabbits and mice developed specific anti-MUC2-GalNAc glycopeptide antibodies and no detectable anti-Tn antibodies. Anti-glycopeptide antibodies did not show reactivity with the unglycosylated MUC2 peptide or with other GalNAc glycosylated peptides. A mouse monoclonal antibody (PMH1) representative of the observed immune response was generated and its immunohistological reactivity analysed in normal tissues. PMH1 reacted similarly to other anti-MUC2 peptide antibodies. However, in some cells the staining was not restricted to the supranuclear area but extended to the entire cytoplasm. In addition, PMH1 reacted with purified colonic mucin by Western blot analysis suggesting that PMH1 reacted with some glycoforms of MUC2. The present work presents a useful approach for development of anti-mucin antibodies directed to different glycoforms of individual mucins.     

Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid, Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Glycosylated analogs of formaecin I and drosocin exhibit differential pattern of antibacterial activity

The synthetic glycopeptides are interesting model systems to study the effect of O-glycosylation in modulating their function and structure. A series of glycosylated analogs of two antibacterial peptides, formaecin I and drosocin, were synthesized by varying the nature of sugar and its linkage with bioactive peptides to understand the influence of structure variation of glycosylation on their antibacterial activities. Higher antibacterial activities of all glycopeptides compared to their respective non-glycosylated counterparts emphasize in part the importance of sugar moieties in functional implications of these peptides. The consequences of the unique differences among the analogs were apparent on their antibacterial activities but not evident structurally by circular dichroism studies. We have shown that differently glycosylated peptides exhibit differential effect among each other when tested against several Gram-negative bacterial strains. The change of monosaccharide moiety and/or its anomeric configuration in formaecin I and drosocin resulted into decrease in the antibacterial activity in comparison to that of the native glycopeptide, but the extent of decrease in antibacterial activity of glycosylated drosocin analogs was less. Probably, the variation in peptide conformation arising due to topological dissimilarities among different sugars in the same peptide resulting in possible modulation in binding properties appears to be responsible for differences in their antibacterial activities. Indeed, these effects of glycosylation are found to be sequence-specific and depend in the milieu of amino acid residues. Interestingly, none of the carbohydrate variants affected the basic property of these peptides, which is non-hemolytic and non-toxicity to eukaryotic cells.     

Solid-phase synthesis of human salivary mucin-derived O-linked glycopeptides

Summary Short glycopeptides derived from salivary mucin have been synthesized in order to delineate the O-glycosylation pattern that is important in the biological activity of mucin. Two glycopoptides, APPETT*AAP-OMe and PAPPSS*SAP-OMe (*=-d-GalNAc), were prepared by solid-phase peptide synthesis integrating the Fmoc and Boc strategies. Since these peptides contain a C-terminal proline, we devised an efficient strategy using facile Boc chemistry, where the glycosylation at the desired position in the sequence was achieved using corresponding Fmoc-glycoamino acid esters A and B as building blocks. The transformation of the 2-azido group into the acetamido derivative was performed with thioacetic acid on the polymer-bound glycopeptides. Corresponding nonglycosylated peptides were also synthesized to study the influence of -d-GalNAc on peptide backbone conformation.     

Synthesis and CD structural studies of CD52 peptides and glycopeptides

The syntheses of five natural and N-terminal acetylated peptides and glycopeptides of the CD52 antigen are described. Solid phase peptide synthesis was employed in the construction of the target compounds from Fmoc-protected commercial amino acids and synthetic glycan-asparagine conjugates. Circular dichroism studies of the synthetic targets showed that they exist as random coils in solution, and no significant change in secondary structure was observed when the CD52 peptide was either acetylated at the N-terminus or glycosylated at the Asn³ residue with a disaccharide or a fucose-containing branched trisaccharide.     

Glycopeptide microarray for autoantibody detection in cancer

Evaluation of: Pedersen JW, Blixt O, Bennett EP et al. Seromic profiling of colorectal cancer patients with novel glycopeptide microarray. Int. J. Cancer 128(8), 1860-1871 (2011). Autoantibodies to cancer-associated antigens hold promise as sensitive biomarkers for cancer detection. Based on this hypothesis, and knowing that O-glycans on proteins constitute a source of possible epitopes recognized by autoantibodies, Pedersen and colleagues have generated a glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from human mucins. The profiling of sera immunoreactivity of colon cancer patients allowed the identification of cancer-associated autoantibodies to various mucin (MUC)1 and MUC4 glycopeptides carrying aberrant glycosylation. This article provides evidence for the value of glycopeptides displaying cancer-associated glycans in diagnostic applications, and opens new avenues for the expansion to other protein glycoforms, as well as to further applications of such a microarray strategy for other post-translational modifications of proteins in the search for cancer biomarker.     

MUC-6 mucin is a major component of "blood group substance" from human ovarian cyst fluid

Ovarian cyst fluid has been a valuable source of the mucins (traditionally termed "blood group substances") that were used for the elucidation of the structures of the ABO Lewis blood group determinants, but the identity of the mucin peptide core(s) carrying these carbohydrate specificities is not known. An ovarian cyst fluid mucin was purified, deglycosylated with HF and digested with trypsin or chymotrypsin to yield a number of peptides. Amino acid sequencing of these peptides yielded five different sequences which showed complete or partial homology to the MUC-6 apomucin deduced from DNA sequencing. As no other sequences were identified, it is concluded that MUC-6 is the major mucin core structure of ovarian cyst fluid mucin.     

Reactivity of anti-human milk fat globule antibodies with synthetic peptides

The nucleotide sequence of partial cDNA clones coding for the core protein of a human polymorphic epithelial mucin has recently been obtained, this mucin consists of a highly conserved 60 bp tandem repeat and the amino acids commonly found are PDTRPAPGSTAPPAHGVTSA. We synthesized three peptides, 1) P1.24 containing the 20 amino acids and four amino acids (PDTR) of the adjoining repeat; 2) P1.15 consisting of the first fifteen (PDTRPAPGSTAPPAH) and P1.09 the second nine amino acids (GVTSAPDTR) of peptide P1.24. The reactivities of the synthetic peptides with mAb known to react with breast cancer (BC1, BC2, BC3, HMFG-1, 3E1.2, and RCC-1) were studied. The synthetic peptide, P1.24, corresponding to the antigenic sequence predicted from the tandem repeat reacted with antibodies BC1, BC2, and BC3 (known to react with human milk mucin and mucin expressed in breast cancer) and the antibody HMFG-1 which was used to select the cDNA clones. In addition, the epitopes recognized by BC1, BC2, and BC3 appear to be in the same region of the molecule represented by their reactions with the nine amino acids in peptide P1.09 (GVTSAPDTR). By contrast, other antibodies such as 3E1.2 which reacts only weakly with components of human milk, and RCC-1 that detects a low Mr component (95 kDa) in breast cancer, had no specific reaction with the synthetic peptides, indicating that their epitopes are distinct from those of BC1, BC2, BC3, and HMFG-1. Inasmuch as the antibodies HMFG-1, BC1, BC2, and BC3 react with the fully processed milk mucin, it is likely that some of the peptide is exposed, even in the fully glycosylated molecule. Identification of the different epitopes could lead to the development of "second generation" mAb with enhanced specificity for breast carcinoma using the appropriate synthetic peptides as immunogens.     

Immunogenicity of synthetic peptides related to the core peptide sequence encoded by the human MUC1 mucin gene: Effect of immunization on the growth of murine mammary adenocarcinoma cells transfected with the human MUC1 gene

The immune response of CAF1 mice to various synthetic peptides (SP) related to the amino acid sequence (PDTRPAPGSTAPPAHGVTSA) of the tandem repeat of the MUC1 human breast mucin core peptide was evaluated. The most immunogenic preparations of the synthetic peptides were those conjugated to keyhole limpet hemocyanin (KLH) or clustered in a dendritic multiple antigenic peptide (MAP-4) configuration. The mice were immunized subcutaneously with synthetic peptides emulsified in RIBI adjuvant, employing various immunization protocols. Equivalently high IgG responses were induced using SP-KLH conjugates (GVTSAPDTRPAPGSTA-KLH) or an SP — MAP-4 chimeric configuration (SP1-6), which also included a universal malarial CST-3 T-helper epitope (SP1-6 = SAPDTRPAEKKIAKMEKASSVFNVVNS — MAP-4). These IgG antibodies bound both the appropriate MUC1 synthetic peptides and the cell surface expressed MUC1 mucin on murine mammary cells that had been transfected with the human MUC1 gene and a human breast cancer cell line that expresses cell-surface MUC1. A MAP-4 molecule, which included the entire 20-aminoacid sequence of the MUC1 tandem repeat (SP1-5 = PDTRPAPGSTAPPAHGVTSA—MAP-4) induced a poor IgG response. In contrast, all three types of molecule: SP-KLH, SP1-6 and SP1-5, were found to be good immunogens for the induction of specific delayedtype hypersensitivity (DTH) reactions measured using either synthetic peptides or MUC1-transfected cells. In addition, immunization with irradiated MUC1-transfected cells induced strong DTH reactions measured using synthetic peptides that expressed the PDTRP sequence, which has been shown to be, or to overlap, a T cell epitope in humans and a B cell epitope in mice. Finally, it was demonstrated that synthetic MUC1 peptide vaccines could be used both prophylactically and therapeutically to inhibit the growth of MUC1-transfected tumor cells and prolong the survival of tumor-bearing mice.     

Synthesis of peptides and glycopeptides with polyproline II helical topology as potential antifreeze molecules

A library of peptides and glycopeptides containing (4R)-hydroxy-l-proline (Hyp) residues were designed with a view to providing stable polyproline II (PPII) helical molecules with antifreeze activity. A library of dodecapeptides containing contiguous Hyp residues or an Ala-Hyp-Ala tripeptide repeat sequence were synthesized with and without α-O-linked N-acetylgalactosamine and α-O-linked galactose-β-(1→3)-N-acetylgalactosamine appended to the peptide backbone. All (glyco)peptides possessed PPII helical secondary structure with some showing significant thermal stability. The majority of the (glyco)peptides did not exhibit thermal hysteresis (TH) activity and were not capable of modifying the morphology of ice crystals. However, an unglycosylated Ala-Hyp-Ala repeat peptide did show significant TH and ice crystal re-shaping activity suggesting that it was capable of binding to the surface of ice. All (glyco)peptides synthesized displayed some ice recrystallization inhibition (IRI) activity with unglycosylated peptides containing the Ala-Hyp-Ala motif exhibiting the most potent inhibitory activity. Interestingly, although glycosylation is critical to the activity of native antifreeze glycoproteins (AFGPs) that possess an Ala-Thr-Ala tripeptide repeat, this same structural modification is detrimental to the antifreeze activity of the Ala-Hyp-Ala repeat peptides studied here.     

Glycopeptide microarray for autoantibody detection in cancer

Evaluation of: Pedersen JW, Blixt O, Bennett EP et al. Seromic profiling of colorectal cancer patients with novel glycopeptide microarray. Int. J. Cancer 128(8), 1860–1871 (2011).

Autoantibodies to cancer-associated antigens hold promise as sensitive biomarkers for cancer detection. Based on this hypothesis, and knowing that O-glycans on proteins constitute a source of possible epitopes recognized by autoantibodies, Pedersen and colleagues have generated a glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from human mucins. The profiling of sera immunoreactivity of colon cancer patients allowed the identification of cancer-associated autoantibodies to various mucin (MUC)1 and MUC4 glycopeptides carrying aberrant glycosylation. This article provides evidence for the value of glycopeptides displaying cancer-associated glycans in diagnostic applications, and opens new avenues for the expansion to other protein glycoforms, as well as to further applications of such a microarray strategy for other post-translational modifications of proteins in the search for cancer biomarker.     

Blood peptidome-degradome profile of breast cancer

Background

Cancer invasion and metastasis are closely associated with activities within the degradome; however, little is known about whether these activities can be detected in the blood of cancer patients.

Methodology and Principal Findings

The peptidome-degradome profiles of pooled blood plasma sampled from 15 breast cancer patients (BCP) and age, race, and menopausal status matched control healthy persons (HP) were globally characterized using advanced comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down peptidomic analysis. The BCP pool displayed 71 degradome protein substrates that encompassed 839 distinct peptidome peptides. In contrast, the HP 50 degradome substrates found encompassed 425 peptides. We find that the ratios of the peptidome peptide relative abundances can vary as much as >4000 fold between BCP and HP. The experimental results also show differential degradation of substrates in the BCP sample in their functional domains, including the proteolytic and inhibitory sites of the plasmin-antiplasmin and thrombin-antithrombin systems, the main chains of the extracellular matrix protection proteins, the excessive degradation of innate immune system key convertases and membrane attack complex components, as well as several other cancer suppressor proteins.

Conclusions

Degradomics-peptidomics profiling of blood plasma is highly sensitive to changes not evidenced by conventional bottom-up proteomics and potentially provides unique signatures of possible diagnostic utility.     

Synthesis,conformation and T-helper cell stimulation of an O-linked glycopeptide epitope containing extended carbohydrate side-chains

To answer the question whether or not T cells to immunodominant protein fragments recognize glycosylated antigens, we synthesized a series of glycopeptides corresponding to peptide 31D, a major T-helper cell epitope of the rabies virus nucleoprotein. Thr4 of the epitope is known to allow mono- or disaccharide side-chain substitutions in either - or β-anomeric configuration without interfering with MHC-binding. To model naturally occurring glycoprotein fragments that carry extended sugar chains, we prepared Fmoc-Ser/Thr-OPfp building blocks containing - and β-linked linear tri- and heptasaccharides. Peptide 31D was synthesized with the complex carbohydrates attached to Thr4, and the T-helper cell activity of the glycopeptides was determined. Addition of -linked carbohydrates, that mimic most of the natural O-linked glycoproteins, resulted in a major drop in the T-cell stimulatory ability in a sugar length-dependent manner. In contrast, the cytosolic glycoprotein mimicking β-linked glycopeptides retained their T-cell stimulatory activity, with the trisaccharide-containing analogue being almost as potent as the unglycosylated peptide. When the peptides were preincubated with diluted human serum, all peptides lost their ability to stimulate the 9C5.D8-H hybridoma. These findings indicated that (i) in contrast to cytosolic glycosylation, incorporation of long O-linked carbohydrates into T-helper cell epitopes abrogates the antigenicity of these protein fragments, and (ii) glycosylation is not a viable alternative to improve the immunogenic properties of subunit peptide vaccines. Glycosylation with all four carbohydrate moieties similarly destroyed the inducible -helical structure of peptide 31D as detected by CD, indicating that the differences in the T-cell activity were not due to different peptide conformations.     

A new monoclonal antibody (3D3) generated with human respiratory mucins and directed against Lewis determinants

We have prepared a monoclonal antibody (MAb), 3D3, raised againstpurified human respiratory mucins. This antibody recognizedmucins and proteolytically derived glycopeptides. The epitoperecognized by the antibody was destroyed by -L-fucosidase, indicatingthat it was present on the carbohydrate moieties. Structuralspecificity was determined by adsorption on a variety of synthetic,insolubilized oligosaccharides. Several lines of evidence indicatethat the 3D3 MAb reacted strongly with the Lewis (Le^b) antigen,but also recognized Le^a and Le^y determinants. This antibodymight be useful to study mucin secretion. human bronchial mucins Lewis b