PROTEIN O-MANNOSYLATION IN CANDIDA-ALBICANS - DETERMINATION OF THE AMINO-ACID-SEQUENCES OF PEPTIDE ACCEPTORS FOR PROTEIN O-MANNOSYLTRANSFERASE

A protein-O-D-mannosyltransferase (PMT) assay was optimised using a mi crosomal membrane preparation from Candida albicans and a peptide acce ptor, YNPTSV. [C-14]Mannose was transferred from dolichyl phosphate [C -14]mannose to the threonine or serine residues of the peptide. During the assay, the peptide was highly susceptible to proteolysis. A block ed peptide Ac-YNPTSV-NH2 was resistent to proteolysis and was apparent ly a better acceptor for O-mannosylation. This peptide had a K(m) valu e of 4.3 mM in the assay. A number of other peptides were tested with altered sequences. Maximum incorporation of [C-14]mannose was obtained with a pentapeptide YATAV (K(m) 2.2 mM) which was further improved by blocking both ends: Ac-YATAV-NH2 (K(m) 0.25 mM). Finally, and unexpec tedly, an improvement was noted if the acetyl group on the N terminus was replaced by a biotin residue. Biotin-YATAV-NH2 had a K(m) of 0.075 mM. The biotin residue may be important in increasing the lipophilici ty of the peptide and thus aid its adhesion to the Candida membranes. The simplest peptide that could act as an efficient mannose acceptor w as Ac-ATA-NH2, whilst no incorporation was observed with Ac-GTG-NH2.