Partial purification and characterization of a mannosyl transferase involved in O-linked mannosylation of glycoproteins in Candida albicans

Abstract

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.

Biosynthesis of glycoproteins in the pathogenic fungusCandida albicans: Activation of dolichol phosphate mannose synthase by cAMP-mediated protein phosphorylation

“…Yeast cells were collected, broken and the homogenate was subjected to differential centrifugation according to previously described protocols [14] except that cells were disrupted in the presence of 1 μM trans ‐epoxysuccinyl‐ l ‐leucylamido‐(4‐guanidino)butane (E‐64) to minimize proteolytic activity. The high‐speed pellet consisting of a mixed membrane fraction was resuspended in 8–9 ml of 50 mM Tris–HCl buffer, pH 7.5 (Tris buffer) and used for DPMS solubilization and purification in the conditions described previously [16]. Briefly, the fraction solubilized with Nonidet P‐40 at a detergent/protein ratio of 0.3 (0.25%), routinely 3 ml, was freeze‐dried, resuspended in 300 μl of electrophoresis loading buffer and applied onto a preparative 5% non‐denaturing polyacrylamide minigel that was run at 80 V for 4 h at 4 °C according to standard protocols [17].…”

“…DPMS solubilized under these conditions was, however, very unstable thus difficulting enzyme purification. A slight modification of this protocol (see below) rendered more stable enzyme preparations that still catalyzed the preferential labeling of the mannolipid and could be used for the partial purification of DPMS using a single step of preparative non‐denaturing electrophoresis [16]. Using this preparation, we investigated some properties of the mannosyl transfer reaction with emphasis on enzyme regulation by phosphorylation and dephosphorylation.…”

Biosynthesis of Glycoproteins in the Human Pathogenic Fungus Sporothrix Schenckii: Synthesis of Dolichol Phosphate Mannose and Mannoproteins by Membrane-Bound and Solubilized Mannosyl Transferases

“…Over 90% of the sugar transferred to proteins was O-linked and consisted of a sole mannose residue (Arroyo-Flores et al 1995). Later, Dpms and Pmt were solubilized, partially purified and some of their properties investigated (Arroyo-Flores et al 1998, 2004, 2005.…”