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

Abstract

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 ...

High Density O-Glycosylation on Tandem Repeat Peptide from Secretory MUC1 of T47D Breast Cancer Cells

“…In vitro glycosylation studies had shown that the Ser within VTS and the Thr within DTR were neither target sites for the ppGalNAc-Ts from human cancer cells (12,13) or from milk (13), nor for the recombinant enzymes rGalNAc-T1 to rGalNAc-T3 (14). Conclusions based on in vivo studies, however, using recombinant glycosylation probes (15) or calculations based on sequence data of glycoproteins (16) were finally confirmed by direct chemical evidence obtained for ex vivo isolated MUC1 from milk (17), and they agreed with the finding that all five putative sites within the tandem repeat of MUC1 were glycosylation targets.…”

“…The remaining, partially deglycosylated mucin was dried in a speedvac and digested by addition of 10 g of activated clostripain (Sigma) as described previously (17). PAP20 glycopeptides were isolated from the crude digest by reversed phase (RP) HPLC on a Beckmann C18 column (2.0 ϫ 150 mm) using previously described conditions (17). PAP20 glycopeptides were detected by enzyme-linked immunosorbent assay using monoclonal antibody M38 (Table I and Ref.…”

“…To demonstrate the extent of sialic acid and galactose hydrolysis, the enzyme treated mucin was desalted on Sephadex G25 (NAP5) column, and a fraction was subjected to monosaccharide composition analysis as described below. The remaining, partially deglycosylated mucin was dried in a speedvac and digested by addition of 10 g of activated clostripain (Sigma) as described previously (17). PAP20 glycopeptides were isolated from the crude digest by reversed phase (RP) HPLC on a Beckmann C18 column (2.0 ϫ 150 mm) using previously described conditions (17).…”

“…MALDI mass spectrometry in the reflectron mode was performed on a Bruker Reflex III under conditions described previously (17).…”

“…Enzyme Immunoassays-Immunochemical detection of PAP20 glycopeptides in eluates from RP-HPLC was performed by enzyme-linked immunosorbent assay as described previously (17,22). mAbs M38 and VA-1 were available as pure antibodies at 1 mg/ml from the ISOBM TD-4 International Workshop on Monoclonal Antibodies against MUC1 (see Ref.…”