Abstract
Synthetic peptides (30 and 20 residues long) corresponding to the native MUC1 tandem repeat sequence (20 residues long) were glycosylated in vitro using UDP-[3H]GalNAc and lysates from the human breast tumor cell line MCF7. Purified glycopeptides were sequenced on a gas-phase sequenator, and glycosylated positions were determined by measuring the incorporated radioactivity in fractions collected following each round of Edman degradation. The results showed that 2 of 3 threonines on the MUC1 tandem repeat peptides were glycosylated at the following positions: GVTSAPDTRPAPGSTAPPAH (underlined Thr residues indicate positions of GalNAc attachment); no glycosylation of serine residues was detected. Determination of the mass of the glycopeptides by mass spectrometry showed that a maximum of two molecules of GalNAc were covalently linked to each 20-residue repeat unit in the peptides. The influence of substrate primary amino acid sequence in determining the substrate specificity of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl-transferase activity was evaluated using as acceptor substrates a series of overlapping 9-residue peptides that represent a moving set through the tandem repeat of the MUC1 mucin. In addition, the influence of primary amino acid sequence on acceptor substrate activity was evaluated using several peptides that contained single or double amino acid substitutions (relative to the native human MUC1 sequence). These included substitutions in the residues that were glycosylated and substitutions in the surrounding primary amino acid sequence. This study demonstrates that primary amino acid sequence, length, and relative position of the residue to be glycosylated dramatically affect the ability of peptides to serve as acceptor substrates for UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase.
Highlights
Synthetic peptides (30 and 20 residues long) corre- The addition of N-acetylgalactosamine by an 0-glycosidic sponding to the native MUC1 tandem repeat sequence linkage to serine and threonine residues is an importapnotst
The results showed that 2 of 3 lation sites areidentified on the tandem repeatdomain of the threonines on the MUCl tandem repeat peptides were glycosylated at the following positions: GmSAPDTRF”
Thesiencludedsubstitutions in strate specificity of human GalNAc transferase was investithe residues that were glycosylatedand substitutions in thesurrounding primary amino acid sequence.This study demonstrates that primary amino acid sequence, length, and relative position of the residue to be glycosylated dramatically affect the ability of peptides to serve as acceptor substrates for UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase
Summary
(Receivedfor publication, February 17, 1994, and in revised form, March 17, 1994). Isao NishimoriM, Ronald L. There is evidence strates a series of overlapping 9-residue peptides that that the two glycosylated sites were glycosylated a t different represent a moving set through the tandem repeat of the relative rates. This finding supports the hypothesis that sub-. Thesiencludedsubstitutions in strate specificity of human GalNAc transferase was investithe residues that were glycosylatedand substitutions in thesurrounding primary amino acid sequence.This study demonstrates that primary amino acid sequence, length, and relative position of the residue to be glycosylated dramatically affect the ability of peptides to serve as acceptor substrates for UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase. Fltuheence of primaryamino acid sequenceonacceptor substrate activity was evaluated using several peptides thatcontained isosteric substitutions in the residues that were glycosylated and various other amino acid substitutions in the primaryamino acid
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