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 microsomal membrane preparation from Candida albicans and a peptide acceptor, YNPTSV. [14C]Mannose was transferred from dolichyl phosphate [14C]mannose to the threonine or serine residues of the peptide. During the assay, the peptide was highly susceptible to proteolysis. A blocked peptide Ac-YNPTSV-NH2 was resistant to proteolysis and was apparently a better acceptor for O-mannosylation. This peptide had a Km value of 4.3 mM in the assay. A number of other peptides were tested with altered sequences. Maximum incorporation of [14C]mannose was obtained with a pentapeptide YATAV (Km 2.2 mM) which was further improved by blocking both ends: Ac-YATAV-NH2 (Km 0.25 mM). Finally, and unexpectedly, an improvement was noted if the acetyl group on the N terminus was replaced by a biotin residue. Biotin-YATAV-NH2 had a Km of 0.075 mM. The biotin residue may be important in increasing the lipophilicity of the peptide and thus aid its adhesion to the Candida membranes. The simplest peptide that could act as an efficient mannose acceptor was Ac-ATA-NH2, whilst no incorporation was observed with Ac-GTG-NH2.
Weston
A
A
Chemotherapy Department, Glaxo Group Research, Greenford, Middlesex, England.
Nassau
P M
PM
Henly
C
C
Marriott
M S
MS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001710:Biotin; D002176:Candida albicans / Q000201:enzymology* / Q000254:growth & development; D002462:Cell Membrane / Q000201:enzymology; D008358:Mannose / Q000378:metabolism*; D008364:Mannosyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D000596:Amino Acids; D009842:Oligopeptides; D013912:Threonine; D012694:Serine; D001710:Biotin; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase; D008358:Mannose