ProteinC-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

Abstract

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 proteinsugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-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 dolichylphosphate-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 -Ͼ -Ͼ GDP-Man -Ͼ Dol-P-Man -Ͼ (C 2 -Man-)Trp.

The Four Terminal Components of the Complement System AreC-Mannosylated on Multiple Tryptophan Residues

“…In Vitro C-Mannosylation-C-Mannosylation in human RNase 2 and IL-12 can be reproduced in vitro using peptides containing the motif WXXW and a microsome-associated transferase (8,12). The modification in these cases is completely restricted to the first Trp, and replacement of the second by Ala completely abolishes the modification in vitro and in vivo (8,9).…”

“…After centrifugation the upper, aqueous, phase contained the peptide. The radioactivity in 0.2 ml of the upper phase was determined by scintillation counting (8). For preparative purposes, 10 reactions were performed for 42 h at room temperature (12).…”

“…It involves the C-glycosidic attachment of an ␣-mannopyranosyl residue to the C-2 atom of the tryptophan side chain (5-7) (Scheme 1). This modification has been shown to be catalyzed by a microsomeassociated transferase, which C-mannosylates the first Trp residue in the recognition sequence -WXXW- (8,9). The enzyme uses dolichylphosphate mannose as the sugar donor, and its activity has been found in mammals, birds, amphibians, and fish.…”

Golgi Glycosylation

“…1). The addition of C-linked Man to tryptophan is thought to occur in the ER (Doucey et al 1998) and no further modification in the Golgi has been observed to date (Wang et al 2009). The O-fucose glycan of TSP repeats is also initiated in the ER, but it is extended in the Golgi with a single Glc (Kozma et al 2006).…”

Glycoprotein Folding in the Endoplasmic Reticulum

“…One example is C-mannosylation of tryptophan residues within a WXXW motif. This modification also utilizes dolichol phosphate as the sugar donor (Doucey et al, 1998) and has thus far been found in RNase 2 (Loffler et al, 1996), IL-12, properdin, and other members of the complement protein family (Hofsteenge et al, 1999) (Hartmann andHofsteenge, 2000). C-mannosylation almost certainly occurs in the ER, but its timing and consequences for protein folding and activity are not known.…”