Effect of glycosylation of a synthetic MUC1 mucin-core-related peptide on recognition by anti-mucin antibodies

Spencer, Daniel I. R.; Price, Michael R.; Tendler, Saul J. B.; Matteis, Cristina I. De; Stadie, Tanja R. E.; Hanisch, Franz‐Georg

doi:10.1016/0304-3835(95)04055-2

Cited by 27 publications

(23 citation statements)

References 10 publications

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“…Most of these antibodies are non-reactive to the glycoform of MUC1 expressed by the lactating mammary gland but are strongly binding to the carcinoma-associated glycoform of the mucin. As previously shown, this effect partly results from glycosylation at vicinal sites (10,14). However, a direct effect by glycosylation of the PDTR motif cannot be ruled out according to the results of this study.…”

Section: Discussioncontrasting

confidence: 44%

“…The naked peptide motif is known to be highly immunogenic and plays a crucial role in the non-MHC-restricted cytotoxic T-cell response in cancer patients (32). Murine monoclonal antibodies reactive to this motif have been demonstrated to bind to MUC1 in a glycosylation-dependent manner (10,11). Most of these antibodies are non-reactive to the glycoform of MUC1 expressed by the lactating mammary gland but are strongly binding to the carcinoma-associated glycoform of the mucin.…”

Section: Discussionmentioning

confidence: 99%

“…Site specificity of O-glycosylation strongly affects the antigenicity of a protein as has been revealed for the highly immunogenic peptide motif PDTR within MUC1 tandem repeats (10). Most antibodies reactive to the PDTR sequence are more or less influenced by GalNAc incorporation into threonine residues of the flanking motifs VTSA and GSTA by showing a significant enhancement or decrease of their binding activities (10,11).…”

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confidence: 99%

“…Most antibodies reactive to the PDTR sequence are more or less influenced by GalNAc incorporation into threonine residues of the flanking motifs VTSA and GSTA by showing a significant enhancement or decrease of their binding activities (10,11). In vitro studies have established that preferentially Thr residues within VTSA and GSTA are glycosylated (12)(13)(14)(15), whereas only Ser within GSTA serves as a substrate to a minor degree (14,15).…”

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confidence: 99%

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Localization of O-Glycosylation Sites on Glycopeptide Fragments from Lactation-associated MUC1

Müller¹,

Goletz²,

Packer³

et al. 1997

Journal of Biological Chemistry

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136

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Since there is no consensus sequence directing the initial GalNAc incorporation into mucin peptides, Oglycosylation sites are not reliably predictable. We have developed a mass spectrometric sequencing strategy that allows the identification of in vivo O-glycosylation sites on mucin-derived glycopeptides. Lactation-associated MUC1 was isolated from human milk and partially deglycosylated by trifluoromethanesulfonic acid to the level of core GalNAc residues. The product was fragmented by the Arg-C-specific endopeptidase clostripain to yield tandem repeat icosapeptides starting with the PAP motif. PAP20 glycopeptides were subjected to sequencing by post-source decay matrix-assisted laser desorption ionization mass spectrometry or by solid phase Edman degradation to localize the glycosylation sites. The masses of C-or N-terminal fragments registered for the mono-to pentasubstituted PAP20 indicated that GalNAc was linked to the peptide at Ser 5 ,Thr 6 (GSTA) and Thr 14 (VTSA) but contrary to previous in vitro glycosylation studies also at Thr 19 and Ser 15 located within the PDTR or VTSA motifs, respectively. Quantitative data from solid phase Edman sequencing revealed no preferential glycosylation of the threonines. These discrepancies between in vivo and in vitro glycosylation patterns may be explained by assuming that O-glycosylation of adjacent peptide positions is a dynamically regulated process that depends on changes of the substrate qualities induced by glycosylation at vicinal sites.Post-translational modification of proteins by glycosylation has extensively been studied in the case of N-linked glycans, and accordingly, rules for the substitution of asparagine residues by dolichol phosphate-linked glycans are well established (1). While N-glycosylation is directed by the consensus peptide motif Asn-X-(Ser/Thr), no strict sequence dependence is known for the initiation of O-glycosylation. Currently, there are several approaches to the identification of O-glycosylation sites: studies on in vitro O-glycosylation of synthetic peptides (2-4) or studies on in vivo processed mucin-type glycoproteins (5-7).The relative merits of both approaches have been discussed (8).The results obtained so far agree with the statement that there are no clear-cut motifs for the addition of GalNAc at Ser or Thr residues; however, the non-random patterns of O-glycosylation suggest influences of flanking sequences (2). Wang et al. (3) proposed different motifs for threonine and serine glycosylation, and the studies of O'Connell et al. (2) revealed critical positions in the vicinity of putative O-glycosylation sites. The obviously less specific GalNAc addition to Ser/Thr residues compared with N-glycosylation is reflected also in the existence of at least four distinct species of UDP-GalNAc/peptide Nacetylgalactosaminyltransferase(s) (GalNAc-transferase) for which different substrate specificities have been established (9). Accordingly, the differentiation and organ localization of a cell should determine its characteristic equipment ...

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Section: Discussioncontrasting

confidence: 44%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Localization of O-Glycosylation Sites on Glycopeptide Fragments from Lactation-associated MUC1

Müller¹,

Goletz²,

Packer³

et al. 1997

Journal of Biological Chemistry

Self Cite

136

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“…Their staining characteristics led to the false assumption that normal colorectal epithelia do not express MUC1 at all. An interesting case is #172 (C595) whose binding to a synthetic MUC1 peptide of 25 amino acids was increased after glycosylation in the vicinity of the epitope [10]. This behaviour, which was not shared by an otherwise similar MAb, HMFG-1 (#169), is explained by a conformational effect of the glycans on the peptide.…”

Section: Group Bmentioning

confidence: 93%

Immunohistochemical Characterization of a Panel of 56 Antibodies with Normal Human Small Intestine, Colon, and Breast Tissues

Cao

Karsten²,

Hilgers³

1998

Tumor Biol

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The epithelial mucin MUC1 is heavily but differently glycosylated depending on the origin and developmental status of the tissue, which greatly influences the reactivity of monoclonal antibodies (MAbs). A partial characterization of their epitopes is possible by mild, carbohydrate-specific periodate oxidation of tissue sections prior to immunostaining. Using this strategy, we have evaluated 56 MAbs submitted to the ISOBM TD-4 (MUC1) Workshop. Paraffin sections from normal human small intestine, colon and breast were immunostained at different defined antibody concentrations either directly or after oxidation with 20 mM periodate at pH 5 for 30 min (PO). In addition, monolayers of T-47D breast cancer cells without PO treatment were examined in immunofluorescence. The array of observed reactivities allowed us to classify the MAbs as follows. Fourteen antibodies were found to detect MUC1 largely independent of the degree of glycosylation, and are therefore classified as pan-MUC1 MAbs (Group A). Twenty-four MAbs were nonreactive with one or more types of the examined epithelia, but became reactive after PO of the tissue sections. We have called these differentiation-dependent MUC1 MAbs (Group B). They might be especially valuable in histological tumour diagnosis. According to their differential staining behaviour towards untreated small intestine, colon, and breast tissue sections, we divided these MAbs into 4 subtypes (Group B1 through Group B4). A further group of six MAbs detected PO-sensitive carbohydrate epitopes (Group C). A seventh antibody apparently also belongs to Group C by immunohistological criteria, although its corresponding epitope was not PO-sensitive. Three further MAbs are still unclear in their specificity, and another 2 are not MUC1-specific (Group D). Six preparations were found nonreactive with the examined tissues; 4 of these were also negative with T-47D cells. Generally, a broad spectrum of different immunohistological patterns has emerged which appears to be widely independent of the type of epitope (sequence versus conformational, length of sequence) and the relative affinities determined in vitro.

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

et al. 1999

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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 α‐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°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. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 79–91, 1999

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Effect of glycosylation of a synthetic MUC1 mucin-core-related peptide on recognition by anti-mucin antibodies

Cited by 27 publications

References 10 publications

Localization of O-Glycosylation Sites on Glycopeptide Fragments from Lactation-associated MUC1

Localization of O-Glycosylation Sites on Glycopeptide Fragments from Lactation-associated MUC1

Immunohistochemical Characterization of a Panel of 56 Antibodies with Normal Human Small Intestine, Colon, and Breast Tissues

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

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