Roberto O. Couso

Further characterization and partial amino acid sequence of a cysteine proteinase from Trypanosoma cruzi

A cysteine proteinase from epimastigotes of Trypanosoma cruzi, Tul 2 stock, has been purified to homogeneity from cell-free extracts obtained by freezing and thawing, by a procedure involving ammonium sulfate fractionation, DEAE-Sephacel chromatography, and gel filtration on Sephadex G-200; when necessary, further purification was attained by fast protein liquid chromatography on Mono Q and Superose 6 columns. The purified enzyme was strongly inhibited by leupeptin, antipain and chymostatin (I50 values of 0.25, 0.75 and 1 microM, respectively), little inhibited by elastatinal, and unaffected by pepstatin A. The enzyme is a glycoprotein, as shown by binding to ConA-Sepharose and elution with alpha-methyl-D-mannopyranoside and alpha-methyl-D-glucopyranoside. Partial amino acid sequences were obtained from the N-terminal end (32 amino acids) of the carbamidomethylated enzyme, and from a tryptic peptide (14 amino acids) of the pyridylethylated enzyme. Both regions show considerable homology with papain and some cathepsins, such as cathepsin L, thus showing that the enzyme belongs to the cysteine proteinase family.

Lipid-linked intermediates in the biosynthesis of xanthan gum

Pyruvic acid acetal and O-acetyl groups are also present in differing proportions [ 1,2]. Although thousands of tons of xanthan gum are produced in a year, because of its industrial applications [2], nothing has been reported about its biosynthesis. Evidence on the mechanism of the in vitro assembly of this polysaccharide is described in this communication: UDP-Glc, GDP-Man and UDP-GlcUA sequentially donate their sugar moieties to a lipidic acceptor to form a pentasaccharide-P-P-lipid which is subsequently polymerized into xanthan gum. The role of polyprenyl phosphosugars was first described in bacterial lipopolysaccharide [3] and murein [4] biosynthesis, and later on extended to capsular polysaccharides [S ,6], teichoic acids [7,8] and other polysaccharides closely associated to the bacterial cell body [9,10]. This is the first report on the participation of lipidlinked sugars in the biosynthesis of an exopolysaccha. ride freely liberated into the culture medium.

Amino acid and carbohydrate composition of a lysosomal cysteine proteinase from Trypanosoma cruzi. Absence of phosphorylated mannose residues.

The size of a lysosomal cysteine proteinase from epimastigotes of Trypanosoma cruzi decreased from 60 to 54 kDa upon treatment with endo beta-N-acetylglucosaminidase H. A lower-molecular weight component (30-35 kDa), which usually accompanies the 60-kDa protein also increased its electrophoretic mobility, and seems to consist of a mixture of degradation products of the enzyme, since both the larger and the smaller components had the same N-terminal sequence as the 60-kDa protein. The amino acid composition of the protein moiety and the composition of the oligosaccharide chains have been determined. The oligosaccharide chains are of the high-mannose type, and contain 6, 7, 8 or 9 mannose residues, as shown both by in vivo labeling with [U-14C]glucose, and by labeling the endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides of the purified enzyme with tritiated sodium borohydride. The oligosaccharide chains did not contain phosphate residues. Further studies with [U-14C]-labeled total glycoproteins of T. cruzi and enzyme assays, suggest that T. cruzi and other trypanosomatids do not target their lysosomal enzymes to the organelle through the mannose-6-phosphate marker pathway.

A xanthan-gum-like polysaccharide from Acetobacter xylinum

SUMMARY: A new exopolysaccharide, secreted in addition to cellulose, has been isolated from the culture medium of Acetobacter xylinum NRRL B42. This polysaccharide, for which the name acetan is proposed, contains glucose, mannose, glucuronic acid and rhamnose in a molar ratio of 4:1:1:1. On the basis of methylation, thin-layer, paper and gas-liquid chromatography, paper electrophoresis and mass spectrometry studies of the degradation products obtained by total and partial hydrolysis and acetolysis of acetan, the following structure is proposed for its repeating unit: Since our previous work with this strain demonstrated the in vitro synthesis of a lipid-linked heptasaccharide with the same structure, the possibility of acetan being the result of its polymerization is discussed. One to two O-acetyl residues per repeating unit are also present in positions not yet determined.

Xanthan cum biosynthesis pyruvic acid acetal residues are transferred from phosphoenolpyruvate to the pentasaccharide-P-P-lipid

Summary Xanthan gum is a complex polysaccharide produced by Xanthomonas campestris , composed of pentasaccharide repeating units containing mannose, glucose and glucuronic acid (in the ratio 2:2:1) and pyruvic acid acetal and 0-acetyl residues in differing proportions Jansson et al. (1975) Carbohyd.Res. 45, 275–282). Previous work (Ielpi et al. FEBS Lett., in press) has shown that the pentasaccharide is first assembled on a pyrophosphate lipid and then polymerized to xanthan gum. It is now shown, using EDTA-treated cells and different combinations of the adequate [ 14 C]labeled donors, that phosphoenol pyruvate provides the acetal residues and that the transfer occurs on the terminal mannose of the pentasaccharide-P-P-lipid, as judged by the solubility properties and DEAE-cellulose column chromatography of the compounds formed as well as by the behaviour of the substances liberated by mild acid and alkaline treatments and Smith degradation. The subsequent polymerization process leads to pyruvylated xanthan gum.

Synthesis of mannosyl cellobiose diphosphate prenol in Acetobacter xylinum

Abstract The enzymatic synthesis of a β-mannosyl (1 → 3) β-glucosyl (1 → 4) α-glucose-1-pyrophosphate-prenol (allylic) by Acetobacter xylinum preparations is described. Glucose pyrophosphate lipid, already known to be formed from UDP-glucose and endogenous phosphate lipid, is demonstrated to accept another glucose from UDP-glucose to give a cellobiose pyrophosphate lipid. The latter in turn accepts mannose from GDP-mannose to form a mannosyl cellobiose pyrophosphate lipid. The structure of the trisaccharide and the way it is linked to the lipid moiety were established by enzymatic and chemical methods such as mild alkaline and acid hydrolysis, phenol treatment, partial acid hydrolysis and acetolysis, periodate oxidation, borohydride reduction, and treatments with glycosidases. The α-unsaturated, polyprenolic nature of the lipid was inferred from and confirmed by the reaction between UDP-glucose and ficaprenol monophosphate to give glucose pyrophosphate ficaprenol, which had the same properties as the glucose pyrophosphate lipid formed from the endogenous acceptor. The allylic structure proposed for the endogenous acceptor is suggested by the lability to phenol treatment and catalytic reduction of its glycosylated derivatives. The enzyme preparation also synthesizes a β-mannose phosphate prenol (allylic), which does not seem to participate in the trisaccharide synthesis. The possible role of these sugar prenols in the synthesis of exopolysaccharides is considered.

Thermal-ultraviolet method for differential detection and recovery of organic compounds in thin-layer chromatography

Abstract Extending work on the detection of organic compounds in thin-layer chromatography using the thermal—ultraviolet method, the differential detection of sugars based on a temperature-dependent characteristic pattern revealed under UV and visible light is reported. Specific colours related to structural changes have been observed under UV light. High recoveries of 14 C- and also 3 H-labelled compounds after thermal—UV detection have been obtained. The detection of lipids and the determination of the sensitivity of the method for these compounds and carbohydrates are described. Results comparable to those in sulphuric acid-based methods have been obtained. In addition, the method has the advantage of a very low background even after prolonged periods of heating at high temperatures. The detection, differentiation and recovery of several methyl glucosides is also reported.