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.     

Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which 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 under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.     

Structural and dynamic consequences of increasing repeats in a MUC1 peptide tumor antigen

MUC1 mucin is a large transmembrane glycoprotein whose extracelluler domain is composed of repeating units of a 20 amino acid sequence. In the cancer associated state, this protein expression becomes upregulated and underglycosylated. Previous studies, which show an enhanced binding of a 5-repeat over a 1-repeat MUC1 peptide to a panel of anti-MUC1 antibodies, have led us to investigate the structural and dynamic consequences of increasing repeat number. Two MUC1 peptides were studied: a 16mer corresponding to slightly less than one full repeat of the MUC1 tandem repeat sequence (GVTSAPDTRPAPGSTA) and a 40mer corresponding to two full repeats of the MUC1 sequence (VTSAPDTRPAPGSTAPPAHG)2. Isotopically labeled versions of these MUC1 peptides were cloned, expressed, purified, and evaluated structurally and dynamically using 15N- and 13C-edited NMR approaches. The data show that MUC1 structure, dynamics, and antibody binding affinity are invariant with increasing repeat number. In light of these results, we conclude that the enhanced antibody affinity of the 5-repeat over the 1-repeat MUC1 peptide is due to multivalency effects, and not due to the development of higher order structure in the longer length peptides. The implications of these results are discussed within the context of a multiple repeat MUC1 breast cancer vaccine design.     

Synthesis and antibody recognition of synthetic antigens from MUC1.

In the altered form of MUC1 mucin associated with breast cancer, the highly immunogenic sequence PDTRPAP is exposed, and may be an immunologically relevant target for the development of diagnostics or cancer immunotherapy. In this study, we report the preparation and antibody binding properties of monomeric and dimeric MUC1 peptides containing the epitope region recognized by monoclonal antibody (mAb) C595. Peptides contained a single or two copies of the whole 20-mer repeat unit (VTSAPDTRPAPGSTAPPAHG) of MUC1 protein. MUC1 40-mer peptides were prepared by the condensation of semi-protected fragments of the repeat unit, in solution or by chemical ligation. In the first case, cyclohexyl-type protecting groups were used for the synthesis of semi-protected fragments by the Boc/Bzl strategy. Unprotected fragments were used in the chemical ligation to produce thioether linkages. In one of the fragments, a Gly residue was replaced by Cys at the C-terminus and the other fragment was chloroacetylated at the N-terminus. In addition, the short peptide APDTRPAPG, and its disulfide dimer, (APDTRPAPGC)(2) were produced. The antibody binding properties of these MUC1 peptide constructs were tested by competition enzyme-linked immunosorbent assay (ELISA). The short epitope region peptide, APDTRPAPG and its dimer (APDTRPAPGC)(2) showed higher IC(50) values (IC(50) = 56.3 and 53.2 micromol/l, respectively). While the 20-mer peptide (IC(50) = 25.9 micromol/l) and more markedly its 40-mer dimers (IC(50) = 0.62 and 0.78 micromol/l) were recognized better. CD data obtained in water or in TFE indicated no significant conformational differences between the 20-mer and 40-mer peptides. We found a high level of similarity between the binding properties of the 40-mer peptides with amide or thioether links, providing a new possibility to build up oligomeric MUC1 peptides by thioether bond formation.     

Structurally defined synthetic cancer vaccines: analysis of structure, glycosylation and recognition of cancer associated mucin, MUC-1 derived peptides

Translation of an immune response into therapy is probably the toughest task in designing vaccines for cancer due to the heterogeneity of the cell surface antigens which display tremendous variations in glycoforms. Consequently, a small segment (antigen) of the cancer-associated mucin, in spite of generating antigen-specific immune responses, may be limited in therapeutic value. It is important that the synthetic segment resembles the native cancer-associated mucin in both structure and conformation. Synthetic cancer associated mucin derived 16 amino acid peptide GVTSAPDTRAPAPGSTA and its partially glycosylated forms have demonstrated specific binding to two monoclonal antibodies, B27.29 and BCP8, raised against the native cancer associated mucin, MUC-1 and a MUC-1 derived synthetic peptide, respectively. In spite of the structural similarities at the core peptide level of both glycosylated and unglycosylated peptides, it appears that partial glycosylation does not inhibit and even slightly enhances binding to the MAb B27.29 indicating that the glycosylated synthetic peptide more closely resembles the native mucin epitope recognized by MAb B27.29. From molecular dynamic simulations using NMR derived distance constraints, both glycosylated and unglycosylated peptides have shown a type I turn involving the same amino acids in both glycosylated and unglycosylated peptides. The GalNAc attached to the threonine (T³) and serine (S⁴) in the 16 amino acid sequence has not imposed any conformational changes to the peptide backbone nor has offered severe steric resistance to the binding of either antibody to the glycopeptides as indicated by hapten inhibition studies. Nevertheless, all peptides have displayed glycosylation dependent specificities in binding to these antibodies, i.e. the glycosylated peptides demonstrated relative higher affinities to the native mucin antibody B27.29 while the unglycosylated peptide is more specific to the MAb BCP8. Immune responses generated by these synthetic glycopeptides are highly specific in recognizing the native cancer associated mucin.     

Synthesis and antibody recognition of mucin 1 (MUC1)-alpha-conotoxin chimera.

We synthesized and characterized new chimera peptides by inserting an epitope of the mucin 1 glycoprotein (MUC1) as a 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The Pro-Asp-Thr-Arg (PDTR) sequence of MUC1 selected for these studies is highly hydrophilic and adopts a beta-turn conformation. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, which is stabilized by two disulphide bridges in positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, Arg9-His-Tyr-Ser12, has been replaced by PDTR, comprising the minimal epitope for MUC1 specific monoclonal antibodies (MAbs) HMFG1 (PDTR) and HMFG2 (DTR). Synthesis of the chimera peptide was carried out by Fmoc strategy on (4-(2',4'-dimethoxyphenyl-aminomethyl)phenoxy) (Rink) resin and either 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) or air oxidation was applied for the formation of the first Cys3-Cys13 or Cys2-Cys7 disulphide bridge, respectively. For the second disulphide bridge, three different oxidation procedures (iodine in acetic acid, 10% DMSO/1 M HCl or tallium trifluoroacetate (Tl(tfa)3) in TFA) were utilized. The HPLC purified peptides were characterized by electrospray mass spectrometry (ES-MS) and amino acid analysis. The CD spectra of the bicyclic MUC1-alpha-[Tyr1]-conotoxin chimera peptide showed partially ordered conformation with turn character. In antibody binding studies, the RIA data showed that both the linear and the bicyclic forms of MUC1-alpha-[Tyr1]-conotoxin chimera were recognized by MAb HMFG1 specific for PDTR sequence, while no binding was observed between MAb HMFG2 and various forms of the chimera. MAb HMFG1, using synthetic epitope conjugates or native MUC1 as target antigens, recognizes the PDTR motif more efficiently in the linear than in the bicyclic compound, but no reactivity was found with the monocyclic forms of MUC1-alpha-[Tyr1]-conotoxin chimera, underlining the importance of certain conformers stabilized by double cyclization.     

Immunoreactivity of Lewis blood group and mucin peptide core antigens: correlations with grade of dysplasia and malignant transformation in the colorectal adenoma-carcinoma sequence

Previous studies on the immunoreactivity of various mucin peptide and carbohydrate antigens in neoplastic colorectal tissues led to at least in part contradictory results. Therefore, we investigated a series of 42 adenomas and 44 carcinomas applying monoclonal antibodies (mabs) directed against Lewis blood group antigens (sialyl-Le(a), Le(x), sialyl-Le(x), Le(y)) as well as mucin peptide cores (MUC1, MUC2 and MUC5AC) by immunohistochemistry. A statistically significant positive correlation between the development of high-grade dysplasia in colorectal adenomas and the immunoreactivity of Le(y) and MUC1 epitopes was observed, whereas MUC2 exhibited a significant negative correlation. The reactivity of the other epitopes did not show an association with the progression of malignant transformation. Colorectal carcinomas were subdivided according to their histopathological subtype. The immunohistochemical staining resulted in a significantly stronger MUC2 reactivity of mucinous vs. tubular adenocarcinomas. Immunoreactivity of the MUC1-specific mab, which does not react with the fully glycosylated peptide core, showed a statistically non-significant inverse tendency, whereas all carbohydrate antigens displayed a strong expression in both tumor subtypes. Furthermore, correlations between mucin peptide and carbohydrate epitope labelling were evaluated. Progression of the adenoma-carcinoma sequence was accompanied by an increase of Le(y) as well as MUC1 antigen and an increase of all Lewis antigens compared to MUC2 immunoreactivity. On the other hand, mucinous carcinomas exhibited an inverse pattern. In conclusion, these results demonstrate that Le(y) and MUC1 immunoreactivity correlate with malignant transformation in the colorectum, whereas MUC2 represents a marker for low-grade dysplasia and the subtype of mucinous carcinomas.     

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.     

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.     

Structure-based design of a protein immunogen that displays an HIV-1 gp41 neutralizing epitope

Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K_d of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.     

Solution structure of the all L- and D-amino acid-substituted mucin 2 epitope peptides

High-molecular-weight mucin 2 (MUC2) glycoproteins show an aberrant glycosylation pattern when expressed in human colon carcinoma: the oligosaccharide chains are shorter and some are missing. In our ongoing effort of MUC2 vaccine development, we have solved the NMR structure of the all L-amino acid and various D-amino acid-substituted derivatives of the peptide TPTPTGTQTPT, previously identified as an epitope within the tandem repeat unit of the MUC2 glycoprotein. In the all L-amino acid containing peptide and in peptide tpTPTGTQtpt (where lowercase letters mark the position of D-amino acids) we identified a type I beta-turn spanning through residues (3)TPTG(6) and (5)TGTQ(8), respectively. Our structural findings are in good agreement with the antibody recognition properties of the investigated peptides and demonstrate that peptides with good stability against enzymatic degradation can be designed with good antibody binding characteristics.     

Divergent solid-phase synthesis and candidacidal activity of MUC7 D1, a 51-residue histidine-rich N-terminal domain of human salivary mucin MUC7.

Domain 1 of the low-molecular-weight human salivary mucin, designated MUC7 D1, spans the 51 N-terminal amino acid residues. This domain contains a 15-residue basic histidine-rich subdomain (R3-Q17) which has 53% sequence similarity to histatin 5 (Hsn-5), a salivary molecule known to exert potent in vitro cidal activity against Candida albicans and many other medically important fungi. The MUC7 D1-15mer and its derivatives have previously been synthesized in our laboratory and their candidacidal activities have been found to be inferior to that of Hsn-5. We were therefore intrigued to explore the candidacidal potency of the full-length MUC7 D1 (51-mer). Linear solid-phase synthesis of this domain has been accomplished following standard Fmoc chemistry. The problems of partial coupling, owing to the peptide chain length, at several stages of the solid-phase step-by-step synthesis were circumvented either by double-coupling techniques or efficient coupling procedures. The MUC7 D1 peptide was purified to homogeneity by conventional reverse-phase HPLC using two columns connected in series. Secondary structure of the purified peptide was assessed by circular dichroism (CD) spectroscopy in phosphate buffer and trifluoroethanol and compared to that of MUC7 D1-15mer and Hsn-5. The MUC7 D1 candidacidal activity was assessed against azole-sensitive and azole-resistant C. albicans strains and was found, unlike that of the MUC7 D1-15mer, to be comparable with that of Hsn-5, indicating that in addition to Hsn-5, MUC7 D1 could provide an attractive alternative to the classical antifungal agents. The candidacidal potency of MUC7 D1, like that of MUC7 D1-15mer, and of Hsn-5, appears to be largely dependent on peptide charge, irrespective of alpha-helical structure.     

Production of MUC1 and MUC2 mucins by human tumor cell lines.

A mucus secreting, clonal derivative (HT29-SB) of the human colonic adenocarcinoma cell line HT29, and the LS174T colon cancer cell line, secrete mucin into the culture medium as a viscoelastic gel. Mab BC2, which defines a peptide epitope present in the variable number of tandem repeats (VNTR) of the MUC1 core protein, reacted with this material after deglycosylation. Two high molecular weight bands were detected in TFMSA treated gel-formed mucin from HT29-SB and LS174T by western blotting (Mr 580 kDa and 420 kDa). A similar pattern of reactivity was seen with the culture supernatants from HT29-SB, the ovarian tumor cell line COLO-316, and the breast cancer cell line MCF-7. Mab CCP58 (anti-MUC2 VNTR) reacted with a 580 kDa band in gel-formed mucin produced by LS174T, but was not reactive with mucin produced by the other cell lines. The findings indicate that human colonic cell lines, in addition to breast and ovarian cell lines, may both express and secrete the MUC1 protein core, and that the LS174T cell line expresses and secretes both the MUC1 and MUC2 core proteins.     

Characterization of a novel weak interaction between MUC1 and Src-SH3 using nuclear magnetic resonance spectroscopy

Breast cancer causes death through cancer cell migration and subsequent metastasis to distant organs. In vitro, the MUC1 mucin can mediate breast cancer cell migration by binding to intercellular adhesion molecule-1 (ICAM-1). This migration is dependent on MUC1 cytoplasmic domain (MUC1-CD) activation of the non-receptor tyrosine kinase, Src, possibly through competitive displacement of an inhibitory Src intramolecular SH3 binding. Therefore, we characterized the binding site and affinity of the MUC1-CD for Src-SH3 using multidimensional nuclear magnetic resonance (NMR) spectroscopy to monitor the titration of the (15)N labeled Src-SH3 domain with synthetic native and mutant peptides of MUC1-CD. The results revealed that the dissociation constant (K(d)) for the interaction of the native MUC1-CD peptides and Src-SH3 domain was weak with a K(d) of 2-3 mM. Notably, the SH3 residues most perturbed upon peptide binding were located outside the usual hydrophobic binding cleft in a previously described alternate binding site on the Src-SH3, suggesting that MUC1-CD binds to a non-canonical site. The binding characteristics outlined here suggest that the interaction between Src-SH3 and MUC1-CD represents a novel weak electrostatic interaction of the type which is increasingly recognized as important in transient and dynamic protein complexes required for cell migration and signal transduction. As such, this study forms the foundation for the design of specific inhibitors of this interaction which may target breast cancer metastases with exquisite specificity.     

Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles.

The proline-rich tandem repeat domain of human mucin MUC1 forms an extended structure containing large repeating loops that are crested by a turn. We show that the repeating-loop structure of MUC1 can be replaced by an antibody complementarity-determining region loop of a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing antibody to create a chimeric, multivalent, mucin-like, anti-HIV-1 compound. We used 8 residues of an antibody molecule to replace 8 of 20 residues of the MUC1 tandem-repeat sequence. The antiviral peptide discussed here contains three copies of a 20-residue tandem repeat, (IYYDYEEDPAPGSTAPPAHG)3, for a total of 60 residues. We demonstrate that the mucin-antibody chimera retains the binding specificity of the parent antibody (monoclonal antibody F58), GPGR of the HIV-1 gp120 V3 neutralizing epitope, and the ability to neutralize virus particles. In inhibition enzyme-linked immunosorbent assay, the mucin-antibody chimeric peptide could inhibit 71 to 84% of binding to a V3 loop peptide by monoclonal antibodies known to be specific for GPGR in the V3 loop. The mucin-antibody chimeric peptide could also inhibit monoclonal antibody binding to native gp120 captured from virus particles. In addition, the chimeric peptide neutralized the homologous HIV-IIIB virus in a standard neutralization assay. The methods of antiviral peptide design and construction presented here are general and theoretically limited only by the size of the antibody repertoire. This approach could be used to synthesize peptides for a variety of therapeutic applications.     

Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. I. Stomach. A relationship to gastric carcinoma.

Studies were undertaken to provide information regarding cell-specific expression of mucin genes in stomach and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of eight mucin genes (MUC1-4, MUC5AC, MUC5B, MUC6, MUC7) in stomach of 13 human embryos and fetuses (8-27 weeks' gestation), comparing these with normal, metaplastic, and neoplastic adult tissues. These investigations have demonstrated that MUC1, MUC4, MUC5AC, MUC5B, and MUC6 are already expressed in the embryonic stomach at 8 weeks of gestation. MUC3 mRNA expression can be observed from 10.5 weeks of gestation. MUC2 is expressed at later stages, concomitant with mucous gland cytodifferentiation. Normal adult stomach is characterized by strong expression of MUC1, MUC5AC, and MUC6, less prominent MUC2, and sporadic MUC3 and MUC4, without MUC5B and MUC7. Intestinal metaplasia is characterized by an intestinal-type pattern with MUC2 and MUC3 mRNA expression. Gastric carcinomas exhibit altered mucin gene expression patterns with disappearance of MUC5AC and MUC6 mRNAs in some tumor glands, abnormal expression of MUC2, and reappearance of MUC5B mRNAs. In conclusion, we have observed that patterns of mucin gene expression in embryonic and fetal stomach could show similarities with some gastric carcinomas in adults. Differences in mucin gene expression in developmental, metaplastic, and neoplastic stomach compared to normal adult stomach suggest a possible regulatory role for their products in gastric epithelial cell proliferation and differentiation.     

Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O‐glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O‐glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn‐antigen (GalNAc‐O‐Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding‐competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution‐state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre‐selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.     

HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.     

NMR-based determination of the binding epitope and conformational analysis of MUC-1 glycopeptides and peptides bound to the breast cancer-selective monoclonal antibody SM3.

Mucin glycoproteins on breast cancer cells carry shortened carbohydrate chains. These partially deglycosylated mucin 1 (MUC-1) structures are recognized by the monoclonal antibody SM3, which is being tested for its diagnostic utility. We used NMR spectroscopy to analyze the binding mode and the binding epitope of peptide and glycopeptide antigens to the SM3 antibody. The pentapeptide PDTRP and the glycopentapeptide PDT(O-alpha-D-GalNAc)RP are known ligands of the monoclonal antibody. The 3D structures of the ligands in the bound conformation were determined by analyzing trNOESY build-up rates. The peptide was found to adopt an extended conformation that fits into the binding pocket of the antibody. The binding epitopes of the ligands were determined by saturation transfer difference (STD) NMR spectroscopy. The peptide's epitope is predominantly located in the N-terminal PDT segment whereas the C-terminal RP segment has fewer interactions with the protein. In contrast, the glycopeptide is interacting with SM3 utilizing all its amino acids. Pro1 shows the strongest binding effect that slightly decays towards Pro5. The GalNAc residue interacts mainly via the N-acetyl residue while the other protons show less interactions similar to that of Pro5. The glycopeptide in the bound state also has an extended conformation of the peptide with the carbohydrate oriented towards the N-terminus. Docking studies showed that peptide and glycopeptide fit the binding pocket of the mAb SM3 very well.     

NMR analysis of human salivary mucin (MUC7) derived O-linked model glycopeptides: comparison of structural features and carbohydrate-peptide interactions.

Two series of glycopeptides with mono- and disaccharides, [GalNAc and Galbeta (1-3)GalNAc] O-linked to serine and threonine at one, two or three contiguous sites were synthesized and characterized by 1H NMR. The conformational effects governed by O-glycosylation were studied and compared with the corresponding non-glycosylated counterparts using NMR, CD and molecular modelling. These model peptides encompassing the aa sequence, PAPPSSSAPPE (series I) and APPETTAAPPT (series II) were essentially derived from a 23-aa tandem repeat sequence of low molecular weight human salivary mucin (MUC7). NOEs, chemical shift perturbations and temperature coefficients of amide protons in aqueous and nonaqueous media suggest that carbohydrate moiety in threonine glycosylated peptides (series II) is in close proximity to the peptide backbone. An intramolecular hydrogen bonding between the amide proton of GalNAc or Galbeta (1-3)GalNAc and the carbonyl oxygen of the O-linked threonine residue is found to be the key structure stabilizing element. The carbohydrates in serine glycosylated peptides (series I), on the other hand, lack such intramolecular hydrogen bonding and assume a more apical position, thus allowing more rotational freedom around the O-glycosidic bond. The effect of O-glycosylation on peptide backbone is clearly reflected from the observed overall differences in sequential NOEs and CD band intensities among the various glycosylated and non-glycosylated analogues. Delineation of solution structure of these (glyco)peptides by NMR and CD revealed largely a poly L-proline type II and/or random coil conformation for the peptide core. Typical peptide fragments of tandem repeat sequence of mucin (MUC7) showing profound glycosylation effects and distinct differences between serine and threonine glycosylation as observed in the present investigation could serve as template for further studies to understand the multifunctional role played by mucin glycoproteins.