Stabilization of the T1 fragment of glycophorin AN through interactions with N- and O-linked glycans

Pieper, Jörg; Ott, Karl‐Heinz; Meyer, Bernd

doi:10.1038/nsb0396-228

Cited by 13 publications

(6 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, to date, structural definition of mucins at high resolution has been elusive, which is largely due to the molecular size and microheterogeneity in glycosylation of samples isolated from natural sources. Structural information pertaining to mucins that has been gathered thus far has relied primarily on NMR analysis and, in some cases, has involved computer-generated modeling based on the limited NMR data. − The results of such studies have been expressed in largely qualitative terms, and the information that has been gleaned generally suggests that native mucin glycoforms have a stiffened, extended structure and do not prefer conventional globular forms. − Under such circumstances, intramolecular interactions of sequentially remote segments in the native glycoprotein are unlikely. Consequently, we wondered whether more tractable glycopeptide segments of suitable complexity levels could serve as realistic models for the full mucin structure.…”

Section: Introductionmentioning

confidence: 99%

Principles of Mucin Architecture: Structural Studies on Synthetic Glycopeptides Bearing Clustered Mono-, Di-, Tri-, and Hexasaccharide Glycodomains

et al. 2002

View full text Add to dashboard Cite

The structural characteristics of a mucin glycopeptide motif derived from the N-terminal fragment STTAV of the cell surface glycoprotein CD43 have been investigated by NMR. In this study, a series of molecules prepared by total synthesis were examined, consisting of the peptide itself, three glycopeptides having clustered sites of alpha-O-glycosylation on the serine and threonine side chains with the Tn, TF, and STF carbohydrate antigens, respectively, and one with the beta-O-linked TF antigen. Additionally, a glycopeptide having the sequence SSSAVAV, triglycosylated with the Le(y) epitope, was investigated. NMR data for the tri-STF-STTAV glycopeptide were used to solve the structure of this construct through restrained molecular dynamics calculations. The calculations revealed a defined conformation for the glycopeptide core rooted in the interaction of the peptide and the first N-acetylgalactosamine residue. The similarity of the NMR data for each of the alpha-O-linked glycopeptides demonstrates that this structure persists for each construct and that the mode of attachment of the first sugar and the peptide is paramount in establishing the organization of the core. The core provides a common framework on which a variety of glycans may be displayed. Remarkably, while there is a profound organizational effect on the peptide backbone with the alpha-linked glycans, attachment via a beta-linkage has little apparent consequence.

show abstract

Section: Introductionmentioning

confidence: 99%

Principles of Mucin Architecture: Structural Studies on Synthetic Glycopeptides Bearing Clustered Mono-, Di-, Tri-, and Hexasaccharide Glycodomains

et al. 2002

View full text Add to dashboard Cite

show abstract

“…The differences between amino acids are less pronounced than in case of the unglycosylated peptide indicating that either the glycopeptide is less flexible or that the binding contributions are more evenly distributed within the glycopeptide. It has been established in literature that the O‐type glycosylation in peptides introduces a stabilization of that particular peptide fragment [27,28].…”

mentioning

confidence: 99%

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

Möller

Serttas

Paulsen

et al. 2002

European Journal of Biochemistry

View full text Add to dashboard Cite

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-a-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.

show abstract

“…At the outset, we were mindful that, to date, mucin proteins have resisted detailed NMR analysis and that spectral resonances of highly glycosylated proteins lack the dispersion necessary to permit sequence assignment (16). The main difficulties arise from the presence of tandemly repeated peptide segments rich in serine and threonine.…”

mentioning

confidence: 99%

“…We have surmounted the synthetic challenges of constructing polypeptides bearing clustered glycodomains (7,8), and one such construct is currently in human clinical trials. Given this access, we probed the effects of clustered glycosylation patterns on peptide conformation and recognition (9)(10)(11)(12)(13)(14)(15)(16)(17)(18). We have conducted extensive NMR and restrained molecular dynamics calculations (19,20) on fully synthetic clustered carbohydrate tumor antigens (1)(2)(3)(4) corresponding to a fragment of CD43, a glycoprotein aberrantly expressed on the surface of acute myelogenous leukemia cells (21)(22)(23)(24).…”

mentioning

confidence: 99%

Probing cell-surface architecture through synthesis: An NMR-determined structural motif for tumor-associated mucins

Live

Williams

Kuduk

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Cell-surface mucin glycoproteins are altered with the onset of oncogenesis. Knowledge of mucin structure could be used in vaccine strategies that target tumorassociated mucin motifs. Thus far, however, mucins have resisted detailed molecular analysis. Reported herein is the solution conformation of a highly complex segment of the mucin CD43. The elongated secondary structure of the isolated mucin strand approaches the stability of motifs found in folded proteins. The features required for the mucin motif to emerge are also described. Immunocharacterization of related constructs strongly suggests that the observed epitopes represent distinguishing features of tumor cell-surface architecture.The exterior of most cells is dominated by glycolipids, proteoglycans, and glycoproteins, including mucin-like proteins. These display polyvalent ␣-O-linked carbohydrates on proximal serine and threonine residues (1-4). Altered expression of cell-surface mucin character is often characteristic of malignant cells (1,3,5). Accordingly, tumor-associated mucins are good targets for a vaccination strategy (3, 5-9). We have surmounted the synthetic challenges of constructing polypeptides bearing clustered glycodomains (7,8), and one such construct is currently in human clinical trials. Given this access, we probed the effects of clustered glycosylation patterns on peptide conformation and recognition (9-18). We have conducted extensive NMR and restrained molecular dynamics calculations (19, 20) on fully synthetic clustered carbohydrate tumor antigens (1-4) corresponding to a fragment of CD43, a glycoprotein aberrantly expressed on the surface of acute myelogenous leukemia cells (21-24). Our findings demonstrate how clustered glycosylation induces the peptide backbone into an unprecedented rigid scaffold corresponding to a polypeptide secondary conformation, which is consistent with elongated mucin glycoprotein structure and function (1,(25)(26)(27)(28)(29). Remarkably, the glycosylation-induced structure approaches the stability of motifs found in globular proteins.The CD43, or leukosialin, protein presented an ideal candidate from which to select a substructure for synthesis. In consequence of its possible role in inducing immunologic response, the system is relatively well characterized (22-24). The sequence STTAV is a glycosylation locus found in the amino terminus of the protein. We have accessed through chemical synthesis the clustered 2,6-STF trisaccharide 1 (7), which is present on CD43 when expressed on acute myelogenous leukemia cells (24). In addition, we have synthesized the TF disaccharide 2 and the monosaccharide Tn antigen 3. Through our synthetic methods, we also gained access to the ␤-linked stereoisomer of the TF antigen 4 (Fig.

show abstract

Stabilization of the T1 fragment of glycophorin AN through interactions with N- and O-linked glycans

Cited by 13 publications

References 15 publications

Principles of Mucin Architecture: Structural Studies on Synthetic Glycopeptides Bearing Clustered Mono-, Di-, Tri-, and Hexasaccharide Glycodomains

Principles of Mucin Architecture: Structural Studies on Synthetic Glycopeptides Bearing Clustered Mono-, Di-, Tri-, and Hexasaccharide Glycodomains

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

Probing cell-surface architecture through synthesis: An NMR-determined structural motif for tumor-associated mucins

Contact Info

Product

Resources

About