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 peptide, TTSAPTTS (in one-letter code), was achieved with human gastric microsomal homogenates. The analyses using capillary electrophoresis online coupled with electrospray mass spectrometry and further Edman degradation of the purified products (obtained by capillary electrophoresis at preparative scale) allowed us to distinguish two components at close masses: the addition of a mass of 202 corresponded to an N-terminal elongation of the peptide TTSAPTTS with the dipeptide (TT) and the addition of a mass of 203 corresponded to an N-acetylgalactosamine O-linkage. Using different peptidase inhibitors, a dipeptidyl peptidase/transferase activity was further characterized. A thiol dependence and an inhibition by H-Gly-PheCHN2 (specific to cathepsin C activity) were found. Moreover, besides TTSAPTTS, other MUC5AC motif peptides (GTTPSPVP, 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 vitro GalNAc incorporation rates when using our selected MUC5AC motif peptides. Our study therefore shows that caution must be taken to prevent peptidic substrate elongation while performing in vitro O-glycosylation with microsomal preparations as the enzyme source. In fact, the results of the N-acetylgalactosamine incorporation rates and thus the microsomal N-acetylgalactosamine transferase affinity can be misinterpreted if dipeptidyl peptidase/transferase activity is not inhibited by the thiol inhibitor E-64 or the cathepsin C inhibitor H-Gly-PheCHN2.
Soudan
B
B
INSERM Unité 377, Biologie et Physiopathologie des Cellules Mucipares, Lille, France.
Tetaert
D
D
Hennebicq
S
S
Briand
G
G
Zerimech
F
F
Richet
C
C
Demeyer
D
D
Gagnon
J
J
Petillot
Y
Y
Degand
P
P
D020789:Cathepsin C; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry* / Q000378:metabolism; D005753:Gastric Mucosa / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008861:Microsomes / Q000378:metabolism*; D055271:Mucin 5AC; D009077:Mucins / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013270:Stomach / Q000648:ultrastructure; D013379:Substrate Specificity
C524678:MUC5AC protein, human; D055271:Mucin 5AC; D009077:Mucins; D010446:Peptide Fragments; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C