Partial purification and characterization of a mannosyl transferase involved in O -linked mannosylation of glycoproteins in Candida albicans.
Incubation of a mixed membrane fraction of C. albicans with the nonionic detergents Nonidet P-40 or Lubrol solubilized a fraction that catalyzed the transfer of mannose either from endogenously generated or exogenously added dolichol-P-[14C]Man onto endogenous protein acceptors. The protein mannosyl transferase solubilized with Nonidet P-40 was partially purified by a single step of preparative nondenaturing electrophoresis and some of its properties were investigated. Although transfer activity occurred in the absence of exogenous mannose acceptors and thus depended on acceptor proteins isolated along with the enzyme, addition of the protein fraction obtained after chemical de-mannosylation of glycoproteins synthesized in vitro stimulated mannoprotein labeling in a concentration-dependent manner. Other de-mannosylated glycoproteins, such as yeast invertase or glycoproteins extracted from C. albicans, failed to increase the amount of labeled mannoproteins. Mannosyl transfer activity was not influenced by common metal ions such as Mg(2+), Mn(2+) and Ca(2+), but it was stimulated up to 3-fold by EDTA. Common phosphoglycerides such as phosphatidylglycerol and, to a lower extent, phosphatidylinositol and phosphatidylcholine enhanced transfer activity. Interestingly, coupled transfer activity between dolichol phosphate mannose synthase, i.e., the enzyme responsible for Dol-P-Man synthesis, and protein mannosyl transferase could be reconstituted in vitro from the partially purified transferases, indicating that this process can occur in the absence of cell membranes.
Arroyo-Flores
Blanca L
BL
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apdo. Postal No. 187, Guanajuato, Gto. 36000, Mexico.
Calvo-Méndez
Carlos
C
Flores-Carreón
Arturo
A
López-Romero
Everardo
E
D002176:Candida albicans / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D004288:Dolichol Phosphates / Q000378:metabolism; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D008364:Mannosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism
D003902:Detergents; D004288:Dolichol Phosphates; D005656:Fungal Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; D010743:Phospholipids; D008364:Mannosyltransferases; D008358:Mannose