A protein-0-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-NH, was resistent to proteolysis and was apparently a better acceptor for 0-mannosylation. This peptide had a K,,, 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 (K, 2.2 mM) which was further improved by blocking both ends: Ac-YATAV-NH, (K, 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-NH, had a K, 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-NH,, whilst no incorporation was observed with Ac-GTG-NH,.Both Candida and Saccharomyces species contain carbohydrate chains which are 0-linked to the hydroxyl group of threonine and serine residues of polypeptide chains, giving rise to mannoproteins. The sequence of reactions for the formation of 0-linked mannoproteins has been described previously [l -31 and involves the transfer of mannose from a dolichyl phosphate mannose (Dol-P-Man) to the protein which is catalysed by an enzyme, protein O-mannosyltransferase (PMT), that is believed to be unique to fungi. Several attempts to purify the enzyme from Saccharomyces cerevisiae [4, 51 have been partially successful but it has proved difficult because the enzyme is membrane-bound. Little is known about the enzyme or its assay requirements, although some information is available on the use of different peptide acceptors for PMT in Saccharomyces [3, 4, 61.PMT has been even less well studied in Candida albicans; hence, we have developed an assay for the enzyme which utilises microsomal membranes as enzyme source and exogenously added peptide as the mannose acceptor. We were interested to study the peptide sequence requirements for mannosylation by the enzyme and have investigated a number of variables. In particular, we have examined the effects of peptide length, preference for a serine or threonine residue and the influence of the amino acids immediately flanking the 0-mannosylation site.Correspondence to A. Weston, Chemotherapy Department, Microbiology, Division, Glaxo Group Research Ltd, Greenford Road, Greenford, Middlesex, England, UB6 OHE Abbreviations. PMT, protein 0-D-mannosyltransferase; Dol-PMan, dolichyl phosphate mannose ; Me,SO, dimethylsulphoxide ; Abu, 2-aminobutyric acid.Enzymes. Protein 0-D-mannosyltransferase (EC 2.4.1.109). MATERIALS AND METHODSAll chemicals were analar grade unless otherwise stated. GDP-['4C]mannose (specific activity 272 Ci/mol) ...
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