DIPEPTIDYL AMINOTRANSFERASE ACTIVITY AND IN-VITRO O-GLYCOSYLATION OF MUC5AC MUCIN MOTIF PEPTIDES BY HUMAN GASTRIC MICROSOMAL PREPARATIONS

The in vitro O-glycosylation reaction of the MUC5AC mucin motif peptid e, TTSAPTTS (in one-letter code), was achieved with human gastric micr osomal homogenates. The analyses using capillary electrophoresis onlin e coupled with electrospray mass spectrometry and further Edman degrad ation of the purified products (obtained by capillary electrophoresis at preparative scale) allowed us to distinguish two components at clos e masses: the addition of a mass of 202 corresponded to an N-terminal elongation of the peptide TTSAPTTS with the dipeptide (TT) and the add ition of a mass of 203 corresponded to an N-acetylgalactosamine O-link age. Using different peptidase inhibitors, a dipeptidyl peptidase/tran sferase activity was further characterized. A thiol dependence and an inhibition by H-Gly-PheCHN(2) (specific to cathepsin C activity) were found. Moreover, besides TTSAPTTS, other MUCSAC motif peptides (GTTPSP VP, TSAPTTS) were also dipeptide donors (GT and TS, respectively) and our results suggested the involvement of a single dipeptidyl peptidase /transferase activity. Finally, this latter activity modified the in v itro GalNAc incorporation rates when using our selected MUCSAC motif p eptides. Our study therefore shows that caution must be taken to preve nt peptidic substrate elongation while performing in vitro O-glycosyla tion with microsomal preparations as the enzyme source. In fact, the r esults of the N-acetylgalactosamine incorporation rates and thus the m icrosomal N-acetylgalactosamine transferase affinity can be misinterpr eted if dipeptidyl peptidase/transferase activity is not inhibited by the thiol inhibitor E-64 or the cathepsin C inhibitor H-Gly-PheCHN(2). (C) Munksgaard 1998.