Luiz R. Travassos

Biosynthesis ofO-N-Acetylglucosamine-linked Glycans inTrypanosoma cruzi CHARACTERIZATION OF THE NOVEL URIDINE DIPHOSPHO-N-ACETYLGLUCOSAMINE:POLYPEPTIDE N-ACETYLGLUCOSAMINYLTRANSFERASE-CATALYZING FORMATION OFN-ACETYLGLUCOSAMINE α1→O-THREONINE

In this study, we have characterized the activity of a uridine diphospho-N-acetylglucosamine:polypeptide-α-N-acetylglucosaminyltransferase (O-α-GlcNAc-transferase) from Trypanosoma cruzi. The activity is present in microsomal membranes and is responsible for the addition of O-linked α-N-acetylglucosamine to cell surface proteins. This preparation adds N-acetylglucosamine to a synthetic peptide KPPTTTTTTTTKPP containing the consensus threonine-rich dodecapeptide encoded by T. cruzi MUC gene (Di Noia, J. M., Sánchez D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146–24149). Incorporation ofN-[3H]acetylglucosamine is linearly dependent on incubation time and concentration of enzyme and substrate. The transferase activity has an optimal pH of 7.5- 8.5, requires Mn2+, is unaffected by tunicamycin or amphomycin, and is strongly inhibited by UDP. The optimized synthetic peptide acceptor for the cytosolic O-GlcNAc-transferase (YSDSPSTST) (Haltiwanger, R. S., Holt, G. D., and Hart, G. W. (1990)J. Biol. Chem. 265, 2563–2568) is not a substrate for this enzyme. The glycosylated KPPTTTTTTTTKPP product is susceptible to base-catalyzed β-elimination, and the presence ofN-acetylglucosamine α-linked to threonine is supported by enzymatic digestion and nuclear magnetic resonance data. These results describe a unique biosynthetic pathway for T. cruzi surface mucin-like molecules, with potential chemotherapeutic implications.

Cell constituents of mycelia and conidia of Aspergillus fumigatus

Abstract Cell-wall components of mycelia and conidia of Aspergillus fumigatus contain alkali-soluble polysaccharides comprised of d -galacto- d -mannans which coprecipitated with small proportions of a d -glucan, tentatively identified as glycogen. The fine structures of the d -galacto- d -mannans varied as a function of the cell type. In a 5-day-old mycelium, the polysaccharide consisted of a main chain of (1→6)-linked α- d -mannopyranose residues substituted at O-2 by 1 to 3 α- d -mannopyranosyl units that are (1→2)-interlinked. β- d -Galactofuranosyl units are (1→6)-linked to the d -mannan core, being components of side-chains of average length of ∼6 units, which are (1→5)-interlinked. The 10-day-old mycelium had a similar d -galacto- d -mannan, but the proportion of glycogen was smaller. Conidia contain polysaccharides of different structure, as shown by the 13C-n.m.r. spectrum and by methylation analysis. Side chains composed of a single unit of β- d -galactofuranosyl linked (1→6) to adjacent d -mannopyranosyl units were identified with a minor proportion of 6-O-substituted d -galactofuranosyl units. Also present were nonreducing d -galacto-pyranosyl end-groups and 2-amino-2-deoxyglycosyl units. The glucan component was not glycogen. Conidial walls have much less protein than mycelial walls. Predominant amino acids in the latter were aspartic and glutamic acids, tyrosine, alanine, and glycine. Fatty acids C16, C18, C18:1, and C18:2 were present in the mycelial and conidial walls, C18:2 was present in minor amounts in the mycelial wall, but was a major component of the lipid fraction from whole cells.

Further studies on the rhamnomannans and acidic rhamnomannans of Sporothrix schenckii and Ceratocystis stenoceras.

Abstract Studies were continued on the 13 C and proton nuclear magnetic resonance spectra of a series of mannose-containing polysaccharides formed by various strains of the closely related fungal species, Sporothrix schenckii and Ceratocystis stenoceras , and the criteria for their differentiation redefined. Exocellular filtrates of C . stenoceras 1099.40 contain mainly a galactan, with smaller proportions of a rhamnomannan having single-unit α- l -rhamnopyranosyl side-chains ( 1 ), an acidic rhamnomannan ( 2 ) containing glucuronic acid, and amylose. The mono-rhamnomannan was characterized by comparison of its 13 C spectrum with that of authentic polysaccharide. The acidic rhamnomannan was characterized partly with the aid of 13 C n.m.r spectroscopy; in the process, fragments obtained by partial hydrolysis were used to assign signals of C-1 and other O -glycosylated 13 C nuclei. Other 13 C signals in spectra of polysaccharides from C. stenoceras and S. schenckii were assigned following tracer experiments that used media containing glucoses labeled with deuterium and 13 C, and by analogy with 13 C signal-displacement effects that are known to occur on O -glycosylation and O -methylation of hydroxyl groups. Evidence was obtained for the presence of structures 3 , 4 , and 5 in exocellular polysaccharide of S. schenckii . These structures give rise to C-1 signals that were assigned by reference to those of (1→4) linked α- d -mannopyranose di- and tri-saccharides.

Soluble and insoluble glucans from different cell types of the human pathogen Sporothrix schenckii

The structures of soluble and insoluble glucans from isolated cell walls of yeast, mycelial, and conidial forms of Sporothrix schenckii were compared. Methylation analysis showed the presence of linear structures and did not reveal any qualitative variation of glycosidic linkage type. Periodate oxidation methods demonstrated that soluble and insoluble glucans were chemically very similar in all cell types. Soluble glucans from S. schenckii yeast forms have proportions of 44, 28, and 28% of, (1→3), (1→6), and (1→4) linkages, respectively. Insoluble glucans from yeast forms also contain (1→3), (1→6), and (1→4) linkages but their proportions are 66, 29, and 5% respectively. Proportions of these linkages in the mycelial and conidial glucans were similar to those in yeast glucans. The β -configuration of the d -glucopyranose units was shown by the specific rotations of the soluble glucans or the methylated derivatives of the insoluble ones. It was also determined by enzymolysis of both types of glucan with β -glucanases. The disruptive action of these enzymes on the fibrillar network of walls from all cell types was observed by electron microscopy. 13 C nuclear magnetic resonance spectra of soluble glucans are consistent with the configuration and position of substitution of glycosidic linkages indicated by other methods.

Location and biochemical nature of surface components reacting with concanavalin A in different cell types of Sporothrix schenckii

Surface components of cell walls of hyphae, conidia, and yeast-like forms of Sporothrix schenckii react with concanavalin A (Con A). By use of fluorescein-conjugated Con A, and a cytochemical method using the horseradish peroxidase technique, it was observed that the yeast-like forms reacted more strongly with Con A as compared with the mycelium. These studies demonstrated the presence of a thick Con A-binding layer in the cell wall of the yeast-like forms. In some instances, this layer was resolved into a basal and an outer sublayer. The reactivity of the latter with Con A differed in the two strains examined: In strain 1099.12, all yeast cells reacted uniformly with Con A, whereas, in strain 1099.18, the reactivity of the outer sublayer in different yeast cells ranged from weak or negative to strongly positive. The reactivity with Con A was probably due to peptido-rhamnomannans since such complexes extracted from both strains precipitated Con A and agglutinated a mannose-sensitive fimbriated Escherichia coli K12. The corresponding rhamnomannans obtained by hot alkali extraction of S. schenckii , which did not contain terminal nonreducing α- d -mannopyranose units, were unreactive with Con A. Reaction of the peptido-polysaccharides with Con A probably reflects the presence in variable proportions, depending on strain and growth conditions, of serine- or threonine-linked mannose-containing short oligosaccharide chains in addition to the long-chain rhamnomannans.

Cell wall composition in different cell types of the dimorphic species Sporothrix schenckii

Cell wall components from the yeast, mycelial, and conidial forms of Sporothrix schenckii were compared. Mycelial and conidial walls were qualitatively and quantitatively similar. Minor differences included slightly higher levels of protein and carbohydrate and lower levels of lipids and phosphate in the conidial as compared to the mycelial cell walls. In contrast, yeast walls had higher contents of total carbohydrate, mainly water- and alkali-soluble polysaccharides, and lower contents of proteins and lipids. The lipid fraction from the yeast walls contained a different proportion of saturated and unsaturated fatty acids, including a C 18:3 acid which was absent in the acid-extractable lipid fraction from mycelial and conidial walls. The amino acid composition of proteins from the yeast walls also differed from that of the other cell types mainly by containing more threonine, serine, and alanine and less lysine, arginine, and glutamic acid.

Surface polysaccharides of phytopathogenic strains of Ceratocystis paradoxa and Ceratocystis fimbriata isolated from different hosts

Surface polysaccharides from three strains of Ceratocystis paradoxa and two strains of Ceratocystis fimbriata were isolated from cells grown either at 25 or 37°C and their structures were analyzed by chemical methods and by nuclear magnetic resonance spectroscopy. All strains were phytopathogenic and were isolated from tropical fruits, sugar cane, and the shrub Crotolaria juncea . Mannans, glucomannans, and galactomannans were identified and their partial structures determined. Isolates of Ceratocystis within the same species synthesized different polysaccharides depending on their original hosts. Temperature variation influenced the synthesis of polysaccharides in one strain of C. paradoxa and also influenced the incorporation of N -acetylglucosamine units into several mannans. No correlation was observed between synthesis of a particular polysaccharide and host specialization or host specificity in the Ceratocystis isolates.