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Protein C-mannosylation is enzyme-catalysed and uses dolichyl-phosphate-mannose as a precursor.

  • 1998-04-04
Published in:
  • Molecular biology of the cell. - 1998
Animals
CHO Cells
Carbon
Cell Membrane
Cricetinae
Dolichol Monophosphate Mannose
Endoribonucleases
Glycosylation
Guanosine Diphosphate Mannose
Humans
Mannose
Mannosyltransferases
Microsomes, Liver
Models, Chemical
Peptide Fragments
Rats
Substrate Specificity
Tryptophan
English C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence -x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man -> -> GDP-Man -> Dol-P-Man -> (C2-Man-)Trp.
Language
  • English
Open access status
green
Identifiers
Persistent URL
https://sonar.ch/global/documents/93972
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