Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases.

The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.

Processing of the Man(10)GlcNAc (M(10)) oligosaccharide by alpha-mannosidases from Candida albicans.

The hydrolysis of Man(10)GlcNAc (M(10)) by purified alpha-mannosidases and its further processing by a mixed membrane preparation from Candida albicans were studied. Incubation of the oligosaccharide with purified alpha-mannosidases I (E-I) or II (E-II) from C. albicans released 1 and 2 mol of mannose per mol of M(10), respectively. This treatment converted M(10) into an acceptor substrate of further mannose residues from GDP-Man as catalyzed by membrane-bound mannosyltransferases. Elongation of E-I- or E-II-trimmed M(10) yielded a low molecular mass product (14-17 mannose residues added), and in the case of E-II, a minor amount of an additional product of a higher molecular mass. Our results indicate that purified alpha-mannosidases participate in N-glycan processing in C. albicans.

Partial purification and characterization of dolichol phosphate mannose synthase from Entamoeba histolytica.

Dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis, was partially purified from E.histolytica by hydrophobic interaction and affinity chromatography with octyl Sepharose CL-4B and Affi-Gel 501, respectively. Reducing agents, particularly dithiothreitol, positively influenced enzyme activity and stability, indicating a role of sulfhydryl groups on the transferase function. Activity did not depend on phospholipids; however, it was significantly stimulated by phosphatidylethanolamine and to a lower extent by other common phospholipids. Mixtures consisting of activating phospholipids did not exert an additive effect. In vitro phosphorylation with a cAMP-dependent protein kinase resulted in enzyme activation. This alteration was not associated with a change in the K(m) for the substrate but rather with a 2.6-fold increase in V(max). Phosphorylation in the presence of [gamma-(32)P]ATP resulted in strong labeling of two polypeptides, one of which exhibited the molecular mass reported for the enzyme from other organisms. Whether phosphorylation functions in vivo as a mechanism of regulation of dolichol phosphate mannose synthesis in E.histolytica remains to be determined.

Purification and biochemical characterization of two soluble alpha-mannosidases from Candida albicans.

Two soluble alpha-mannosidases, E-I and E-II, were purified from C. albicans yeast cells by a three-step procedure consisting of size exclusion and ion exchange chromatographies in Sepharose CL6B and Mono Q columns, respectively, and preparative nondenaturing electrophoresis. E-I and E-II migrated as monomeric polypeptides of 54.3 and 93.3 kDa in SDS-PAGE, respectively. Some biochemical properties of purified enzymes were investigated by using 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside as substrates. Hydrolysis of both substrates by either enzyme was optimum at pH 6.0 with 50 mM Mes-Tris buffer and at 42 degrees C. Apparent Kmvalues for hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside by E-I were 0.83 microM and 2. 4 mM, respectively. Corresponding values for E-II were 0.25 microM and 1.86 mM. Swansonine and deoxymannojirimicin strongly inhibited the hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside by both enzymes. On the contrary, hydrolysis of p-nitrophenyl-alpha-D-mannopyranoside by E-I and E-II was slightly stimulated or not affected, respectively, by both inhibitors. E-I and E-II did not depend on metal ions although activity of the latter was slightly stimulated by Mn2+and Ca2+in the range of 0.5-2 mM. At the same concentrations, Mg2+was slightly inhibitory of both enzymes. Substrate specificity experiments revealed that both E-I and E-II preferentially cleaved alpha-1,6 and alpha-1,3 linkages, respectively.

Biosynthesis of glycoproteins in Candida albicans: solubilization and partial characterization of dolichol phosphate mannose synthase and protein mannosyl transferases.

Incubation of a mixed membrane fraction isolated from C. albicans yeast cells with Nonidet P-40 at a detergent/protein ratio as low of 0.025 (0.016-0.019%, w/v) yielded a soluble fraction that catalyzed the transfer of mannose from GDP-[14C] Man into dolichol phosphate mannose and from this intermediate into mannoproteins. Over 95% of the sugar in mannoproteins was O-linked as judged from its release after beta-elimination. Mannose was identified as the sole product after this treatment. Transfer activity did not depend on exogenous lipid acceptor indicating that the latter was solubilized along with the mannosyl transferases. Synthesis of mannolipid and mannoproteins occurred at optima temperatures of 20 degrees C, and 37 degrees C, respectively, and at a pH in the range of 7.5-9.5. Mannosyl transfer into the mannolipid was stimulated by Mg2+ and inhibited by Ca2+ and Mn2+ whereas mannoprotein labeling was stimulated by Mn2+ and to a lower extent by Mg2+. When measured as a function of substrate concentration, the synthesis of the mannolipid was a nearly linear function of GDP-Man concentration in the range of 5 to 32 microM whereas protein mannosylation exhibited hyperbolic kinetics with saturation reached at about 10 microM. The solubilized preparation was able to utilize an exogenous source of mannolipid as sugar donor for protein mannosylation. Dinucleotides and, to a higher extent trinucleotides, inhibited mannosyl transfer into the mannolipid and hence into mannoproteins.

Biosynthesis of glycoproteins in Candida albicans: biochemical characterization of dolichol phosphate glucose synthase.

A mixed membrane fraction isolated from C. albicans yeast cells catalyzed the transfer of glucose from UDP-Glc into three classes of endogenous acceptors: glucolipid, glycoprotein and lipid-linked oligosaccharides. About 80% of the total radioactivity transferred into these products corresponded to the glucolipid which was identified as dolichol phosphate glucose by several criteria. The remainder was detected in about equal proportions in the other two fractions. Conditions that stimulated or inhibited glucolipid synthesis did not affect the extent of glycoprotein labeling. The synthesis of dolichol phosphate glucose exhibited a K(m) of 104 microM UDP-Glc and was stimulated by Mg2+ but not by Mn2+ or Ca2+. The latter cations were, however, better stimulators of glycoprotein labeling than Mg2+. Most nucleotides strongly inhibited the synthesis of dolichol phosphate glucose, UMP being a competitive inhibitor with a Ki of 100 microM. The dolichol phosphate glucose synthase reaction was reversed about 57% by 0.62 mM UDP but not by UMP.

Entamoeba histolytica: solubilization and biochemical characterization of dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis.

Sequential treatment of trophozoite membranes with the nonionic detergents Brij 35 and Igepal CA-630 released a soluble fraction that efficiently catalyzed the transfer of mannose from GDP-Man into a mannolipid that was identified as dolichol phosphate mannose (Dol-P-Man) by several criteria. The transfer reaction occurred only in the presence of exogenously added dolichol monophosphate (Dol-P). Plots of enzyme velocity versus Dol-P and GDP-Man concentrations revealed sigmoidal and hyperbolic kinetics, respectively. Values of S0.5 for Dol-P and K(m) for GDP-Man were 15 micrograms/ml and 4.1 microM, respectively. The solubilized fraction failed to transfer the label into other products such as lipid-linked oligosaccharides and glycoproteins. The optimum pH was 7.5-8.0 in potassium phosphate or Tris/HCl buffers and the enzyme required either Mg2+ or Mn2+. The latter was more effective but in a narrower range of concentrations. The transferase was inhibited by a number of nucleotides the strongest being GMP, GDP, and GTP. When assayed in the reverse direction, however, the enzyme catalyzed the transfer of mannose from Dol-P-Man back into GDP-Man as a function of increasing concentrations of GDP. Mg2+ was a better activator of the reverse reaction than Mn2+, which reached up to 60% at 2 mM GDP. These results suggest that some of the enzyme catalytic properties may change depending on the direction of the transfer reaction.

Partial purification and characterization of ornithine decarboxylase from Entamoeba histolytica.

Multiplication of E. histolytica was accompanied by a parallel increase in ornithine decarboxylase (ODC) specific activity up to 72 h of cultivation in TYI-S-33 medium. Thereafter, activity rapidly decayed whereas growth continued for another 24 h before entering into the stationary growth phase. ODC was very unstable. Partial purification (14-fold) of the enzyme was achieved by a three-step procedure involving high-speed centrifugation, gel filtration and adsorption to hydroxylapatite. The partially purified enzyme (Mr 211 kDa) revealed maximum activity at pH 8.5-9.0 and a sigmoidal response to substrate concentration. An S0.5 value of 1.0 mM ornithine was estimated. Although ODC did not exhibit an absolute dependence on pyridoxal phosphate (PLP), addition of PLP increased catalytic activity about 4-fold, with an S0.5 value of 45 microM. Evolution of 14CO2 from ornithine was markedly inhibited by polyamines in the following increasing order of effectiveness: putrescine > spermidine > spermine. The substrate analogs alpha-methylornithine and alpha-difluoromethylornithine had no effect on enzyme activity and cell growth. In contrast, 1,3-diaminopropane and 2,4-diamino-2-butanone, 2 putrescine analogs, severely inhibited both enzyme activity and amoeba multiplication. Results are discussed in terms of the role of ODC in the amoeba proliferation.

Biosynthesis of glycoproteins in Candida albicans: activity of dolichol phosphate mannose synthase and protein mannosylation in a mixed membrane fraction.

A mixed membrane fraction (MMF) was isolated from yeast cells of Candida albicans with the ability to synthesize dolichol phosphate mannose (Dol-P-Man) from GDP-Man and dolichol phosphate (Dol-P) and transfer the sugar to proteins. Temperature of incubation (20-37 degrees C) did not affect the synthesis of Dol-P-Man but protein mannosylation occurred better at physiological temperatures (28 degrees C and 37 degrees C). Most of the sugar (87-93%) in the mannoproteins was O-linked as judged by its release by beta-elimination. Mannose was identified as the sole product after this treatment. Following incubation of MMF with the sugar donor, parallel levels of Dol-P-Man and mannosylated proteins were detected up to 30 min. Thereafter, Dol-P-Man levels reached a steady value whereas mannoproteins rapidly accumulated. Lipid-linked oligosaccharides were also detected in incubation mixtures, though in much lower amounts than those of Dol-P-Man or mannoproteins. Dol-P-Man synthase activity increased proportionally in response to increasing concentrations of either of the two enzyme substrates. A Km value of 0.36 microM for GDP-Man was calculated. MMF failed to use exogenous Dol-P-Man for protein glycosylation. Specific inhibition of Dol-P-Man synthesis with amphomycin was concomitant with a parallel decrease in protein mannosylation, indicating that most of the sugar is transferred to protein via the carrier lipid. Results are discussed in terms of the role of Dol-P-Man in protein glycosylation in C. albicans.

Ornithine decarboxylase activity in Entamoeba invadens.

Growth of E. invadens was paralleled by a concomitant increase in ornithine decarboxylase activity which peaked after 5 days of cultivation in TYI-S-33 medium. Over this period, enzyme activity increased about nine-fold with respect to that present at the start of incubation. Thereafter and coinciding with the onset of the stationary growth phase, enzyme activity started to decline reaching trace levels after 8 days of cultivation. Most of the enzyme remained soluble following centrifugation of amoeba homogenates at 105,000 g. alpha-Difluoromethylornithine failed to affect ornithine decarboxylase activity in vitro and amoeba growth. The enzyme was markedly inhibited by polyamines (putrescine, spermidine and spermine) and 1,4-diamino-2-butanone, a putrescine-analog. The latter arrested proliferation of cells, an effect that could not be reversed by polyamines which by themselves also inhibited growth to a low but significant extent. Our results indicate that polyamine biosynthesis from ornithine is required for growth of E. invadens and that this function is rapidly abolished following entry into the stationary growth phase.

The role of polyamine metabolism in dimorphism of Yarrowia lipolytica.

We have devised a convenient procedure to induce the yeast-to-mycelium transition of Yarrowia lipolytica in conditions which avoid the occurrence of the reverse process during the period of study. Yeast cells in late exponential phase were resuspended in water and cooled down to 4 degrees C for at least 15 min, then heat-shocked by inoculation into a pre-warmed (30 degrees C) medium containing N-acetyl-D-glucosamine. Under these conditions, yeast cells developed into large branching filaments which continued elongating for more than 24 h. Further, ornithine decarboxylase (ODC) activity and polyamine cell pools increased compared to those of cells maintained in glucose medium, which continued yeast-like growth. Addition of ODC inhibitors blocked mycelial development, but only if added during a critical initial period after which they had no effect. At effective concentrations, ODC inhibitors had no significant effect on cell growth. Comparative studies of intact and permeabilized cells suggest that this selective effect is probably due to the location of ODC in more than one cell compartment, one of them being inaccessible to the drugs. Blocking of the morphological transition by ODC inhibitors was specifically reversed by putrescine, and by growing the cells in the presence of 5-azacytidine. It is suggested that the effect of the latter compound is related to its capacity to inhibit DNA methylation, indicating a relationship between polyamines and DNA methylation at the onset of the differentiation process.

Chitinase activity in encysting Entamoeba invadens and its inhibition by allosamidin.

Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of encystation in axenic conditions using 4-MU(Ch)3 as substrate. Encystment was paralleled by chitinase activity which showed a peak after about 72 h of cultivation where cysts accounted for 63% of cell population. Thereafter, activity fell off rapidly, whereas encystment continued, reaching 80% at the end of the experiment (96 h). Comparison of activity between cysts and the total cell population in 48- and 72-h-old encysting cultures suggested that chitinase may start to accumulate in the pre-cyst forms. About 70% of the enzyme was recovered in the supernatant following low-speed centrifugation of whole extracts. Most of this activity represented soluble chitinase since it was not sedimented by further centrifugation at 105,000 x g. A minor proportion of enzyme activity remained associated to the buffer-washed, high-speed sediment. In addition to 4-MU(Ch)3, chitinase activity was also measured following the hydrolysis of other substrates such as nascent, preformed or colloidal chitin. Like other chitinases, the cyst enzyme preferred nascent over preformed chitin as substrate. Digestion of the former yielded GlcNAc and minor amounts of (GlcNAc)2 as products. Allosamidin strongly inhibited hydrolysis of the fluorogenic substrate by the amebic chitinase in vitro with a Ki of 0.065 microM. IC50 values were 0.085 microM and 0.16 microM at 5 microM and 10 microM 4-MU(Ch)3, respectively. When added to the axenic medium, the drug markedly retarded encystment though it was partially recovered after longer periods of incubation.

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone.

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.