PROTEIN C-MANNOSYLATION IS ENZYME-CATALYZED AND USES DOLICHYL-PHOSAHATE-MANNOSE AS A PRECURSOR

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 establishe d that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-manno sylated. Here we investigated the reaction with respect to the mannosy l donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cell s, which are deficient in dolichylphosphate-mannose (Dol-P-Man) syntha se 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 vi tro, 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 previou sly. The C-C linkage between the indole and the mannosyl moiety was de monstrated by tandem electrospray mass spectrometry analysis of the pr oduct. 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 contr ast, 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 -> (C-2-Man-)Trp.