Structure determination of Cryptococcus neoformans serotype A-variant glucuronoxylomannan by 13C-n.m.r. spectroscopy

A series of polysaccharides was derived by physical and chemical methods from an antigenic, O-acetyl-containing, glucuronoxylomannan (GXM), isolated from the growth medium of Cryptococcus neoformans (CDC B2550) serotype A-variant having composition ratios of Man:Xyl:GlcA:OAc = 10:4:3:6. 13C-N.m.r. spectra of derivatives provided new structural evidence for GXM. Treatment of GXM with Li in ethylenediamine gave a xylomannan (XM, with Man:Xyl = 5:2). Smith degradation of XM gave a mannan (M). Ultrasonic treatment of GXM gave GXM-sonicated (GXMS). Treatment of GXM with 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide.HCl and then with NaBH4 gave reduced GXMS (RGXMS), or with aq. trifluoroacetic acid gave partially acid-hydrolyzed GXMS. Periodate oxidation of GXM and NaBH4 reduction of the product gave a polyalcohol-mannan (PM). Treatment of GXMS, RGXMS, and PM with NH4OH at pH 11 gave the respective O-deacetylated analogs. Comparison among the 13C-n.m.r. spectra of GXM, the various derivatives, and reference monosaccharides allowed the following conclusions: M is (1----3)-alpha-D-mannopyranan; XM consists of the M backbone with 91% of the Xyl on nonadjacent Man residues as 2-O-beta-D-Xylp substituents and with 9% as 4-O-D-Xylp substituents on other Man residues. GXM consists of the XM structure, but with non-D-xylosylated Man residues substituted with 2-O-beta-D-GlcpA substituents and with 6-O-acetyl groups distributed approximately equally on Man residues that have other substituents and those that have none. The molecular mechanics program MM2 was used to estimate the relative energies of anomeric orientations of the typical glycosidic linkage in M. The results suggest that 6'-OH----O-2 H-bonding is significant in the minimal-energy orientation of M, with phi = -36 degrees and psi = 51 degrees, and that two other glycosidic orientations may be important in the 2-O- or 6-O-substituted derivatives of M.     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

1H- and 13C-n.m.r.-spectral study of synthetic methyl d-manno-oligosaccharides

¹H- and ¹³C-n.m.r. spectra for 16 synthetic methyl manno-oligosaccharides were recorded, and the signals for the anomeric protons and anomeric carbon atoms in branched manno-pentaosides and -hexaosides were assigned, based on the data for methyl manno-biosides and -triosides. These n.m.r. data identified the branching pattern of high-mannose types of glycans of glycopeptides with those of unambiguously synthesized manno-oligosaccharides, and confirmed the structures proposed for such glycans.     

DL-apiose substituted with stable isotopes: synthesis, n.m.r.-spectral analysis, and furanose anomerization

The branched-chain pentose DL-apiose has been synthesized in good yield by a new and simple chemical method that can be adapted to prepare (1-13C)-, (2-13C)-, (1-2H)- and/or (2-2H)-enriched derivatives. N.m.r. spectra (1H- and 13C-) have been interpreted with the aid of selective (13C)- and (2H)-enrichment, and 2D and 13C[13C]-n.m.r. spectra. The solution composition of DL-(1-13C)apiose in 2H2O, determined by 13C-n.m.r. spectroscopy, has been found to differ from that determined previously by 1H-n.m.r. spectroscopy. Several 13C-1H and 13C-13C couplings have been measured and interpreted in terms of apiofuranose ring conformation. Ring-opening rate-constants of the four apiofuranoses [3-C-(hydroxymethyl)-alpha- and -beta-D-erythrofuranose, and 3-C-(hydroxymethyl)-alpha- and -beta-L-threofuranose] have been determined by 13C-saturation-transfer n.m.r. spectroscopy, and compared to those obtained previously for the structurally related tetrofuranoses.     

Facile syntheses of the hexasaccharide repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A

Two hexasaccharides, beta-D-Xylp-(1-->2)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)-]alpha-D-Manp-(1-->3)-[beta-D-GlcpA-(1-->2)-]alpha-D-Manp and beta-D-GlcpA-(1-->2)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)-]alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)-]alpha-D-Manp, the repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A, were synthesized as their methyl glycosides in a regio- and stereoselective manner.     

Comparison of serological and chemical characteristics of capsular polysaccharides of Cryptococcus neoformans var. neoformans serotype A and Cryptococcus albidus var. albidus

The antigenic formula and chemical structure of capsular polysaccharide (CPS) of Cryptococcus albidus var. albidus (C. albidus) were studied in relation to those of C. neoformans var. neoformans serotype A (C. neoformans A). The results of slide agglutination tests with factor sera and reciprocal adsorption experiments showed that antigenic formula of C. albidus was the same as that of C. neoformans A. The soluble CPSs from the two species were obtained from culture supernatants by precipitation with ethanol followed by purification by chromatography on DEAE-cellulose column. The structural analyses of such CPSs from the two species showed that the antigenic CPS fractions consisted of a backbone of alpha(1-3)-linked D-mannopyranosyl residues with a single branch of beta(1-2)-xylose or glucuronic acid, and mostly with O-acetyl groups, in which side chains and O-acetyl groups were responsible for antigenic specificity. It was found that there was a minor difference between the CPS of C. neoformans A and that of C. albidus; in the former, unsubstituted mannose residues existed in a low frequency, but in the latter none. Moreover, the 1H-nuclear magnetic resonance spectra of partially hydrolyzed acidic fragments of the two CPSs indicated that two xylose side chains were present between glucuronic acid side chains. Taken together, it was suggested that these two species of C. neoformans A and C. albidus are closely related to each other in their CPSs.     

1H n.m.r. study of geletin gels

Proton spin-lattice and spin-spin relaxation times have been measured in the gelatin-water system during gelatin at various concentrations and quenching temperatures. The spin-spin relaxation time is shown to be a good parameter for following the kinetics of gelation and can be related to the amount of helices measured by polarimetry. The results are analysed within the framework of the Zimmerman-Brittin theory.     

13C n.m.r. study of L-alanine peptides

The di-, tri-, and tetrapeptides of L-alanine have been studied in aqueous solution by 13C n.m.r. spectroscopy at 25 and 50 MHz. By using selectively 13C enriched analogs containing either 90% 13C methyl or carbonyl carbons and measurements as a function of pH, assignment of the chemical shifts of the peptides has been made. T1 and NOE measurements of the peptides in their cationic, anionic, and zwitterionic states have been recorded as a function of concentration. The results show considerable segmental motion along the backbone carbons of the peptides, with only small changes occurring in the dynamic motions of the peptides as their charge states are altered. The lack of concentration dependence of the chemical shift and T1 values, as well as the similarity of T1 values for individual peptides in the three charge states, indicate that the peptides do not self-associate in aqueous solution.     

Oligosaccharides obtained from a blood-group-Sd(a+) Tamm-Horsfall glycoprotein. An n.m.r. study.

Treatment of Tamm-Horsfall urinary glycoprotein with Bacteroides fragilis endo-beta-galactosidase over a range of enzyme concentrations, pH and temperature resulted in the release of a small but constant proportion of the terminal sugars, which indicates the presence in the glycoprotein of relatively few enzyme-susceptible -GlcNAc beta 1-3Gal beta 1-4GlcNAc- units. Three oligosaccharides were isolated from the enzyme digest and characterized as Gal beta 1-4GlcNAc beta 1-3Gal, NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4GlcNAc beta 1-3Gal by methylation analysis and exo-glycosidase digestion. The alditols of these oligosaccharides and related structures were examined by 500 MHz 1H-n.m.r. spectroscopy aided by spin-spin decoupling and two-dimensional correlated spectroscopy. An almost complete assignment of proton shifts was possible, and significant differences between the signals of some of the protons in the blood-group-Sda-active oligosaccharide III and literature values for the corresponding signals in the structurally related Cad-blood-group determinant are noted.     

Proton n.m.r. of ferredoxin from Clostridium pasteurianum

Proton magnetic resonance spectra at 500 MHz are reported for the oxidized and reduced forms of the 2[4Fe-4S]-ferredoxin from Clostridium pasteurianum. The reduced protein showed additional peaks in the 10–60 ppm region, which were previously unobserved, and there were significant differences between oxidized and reduced states in the whole region. The electron exchange rate in partially reduced ferredoxin is slow on the n.m.r. time scale when reduced with sodium dithionite, but fast when zinc reduced methyl viologen is used as reducing agent. We explain the difference between fast and slow exchange as being due to the different chemical properties of the two reducing agents.     

13C-n.m.r.-spectral study of galactosyltransferase specificity with regard to the carbohydrate side-chain of native ovalbumin

As a prelude to studies using bovine N-acetylglucosaminide-β-(1→4)-galactosyltransferase to label membrane-surface glycoproteins with isotopically enriched d-galactose, the structural specificity of the enzymic reaction with water-soluble, hen ovalbumin has been examined. The enzyme-catalyzed transfer of d-galactose from UDP-d-galactose requires a (nonreducing) terminal 2-acetamido-2-deoxy-d-glucosyl group and exhibits selectivity towards saccharide chains containing d-mannose. This study considers the structural specificity of the enzyme with regard to the anomeric linkage between 2-acetamido-2-deoxy-d-glucose and d-mannose in the carbohydrate chains of hen ovalbumin. Uniformly ¹³C-enriched d-galactose was enzymically attached to the ovalbumin carbohydrate chain (which exhibits microheterogeneity in its structure), the protein was hydrolyzed, and separate glycopeptide fractions were chromatographically isolated. The ¹³C-n.m.r. spectra (60.5 MHz) of the fractions revealed two peaks for the anomeric carbon atom of d-galactose. The two peaks, at 104.20 and 104.39 p.p.m., were ascribed to d-galactosyl groups attached to 2-acetamido-2-deoxy-d-glucose respectively linked β-(1→4) and β-(1→2), to d-mannose in the glycopeptide chains. Quantifying of the spectral data revealed no specificity of d-galactosyltransferase towards the linkage from the terminal 2-acetamido-2-deoxy-d-glucosyl group to the penultimate d-mannosyl residue.     

The physical properties of the capsular polysaccharides from Cryptococcus neoformans suggest features for capsule construction

The most distinctive feature of the human pathogenic fungus is a polysaccharide capsule that is essential for virulence and is composed primarily of glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). GXM mediates multiple deleterious effects on host immune function, yet relatively little is known about its physical properties. The average mass of Cryptococcus neoformans GXM from four antigenically different strains ranged from 1.7 to 7 x 10(6) daltons as calculated from Zimm plots of light-scattering data. GalXM was significantly smaller than GXM, with an average mass of 1 x 10(5) daltons. These molecular masses imply that GalXM is the most numerous polysaccharide in the capsule on a molar basis. The radius of gyration of the capsular polysaccharides ranged between 68 and 208 nm. Viscosity measurements suggest that neither polysaccharide altered fluid dynamics during infection since GXM behaved in solution as a polyelectrolyte and GalXM did not increase solution viscosity. Immunoblot analysis indicated heterogeneity within GXM. In agreement with this, scanning transmission electron microscopy of GXM preparations revealed a tangled network of two different types of molecules. Mass per length measurements from light scattering and scanning transmission electron microscopy were consistent and suggested GXM molecules self-associate. A mechanism for capsule growth is proposed based on the extracellular release and entanglement of GXM molecules.     

C-n.m.r. spectral study of C reductively methylated glycopeptides derived from glycophorin A

¹³C-n.m.r. spectral data for ¹³C reductively methylated intact homozygous and heterozygous glycophorins A were compared with the ¹³C-n.m.r. spectral data for the ¹³C reductively methylated homozygous and heterozygous N-terminal glycopeptides derived from the trypsin digest of glycophorin A. The results indicate that pronounced aggregation of this glycoprotein in solution does not affect the structural differences that we have previously observed for glycophorins A^M and A^N at and/or near the N-terminal amino acid. Moreover, the data suggest that two structural states exist for glycophorin A^M.     

Synthesis and n.m.r.-spectral analysis of unenriched and [1-13C]-enriched 5-deoxypentoses and 5-O-methylpentoses

Chemical methods are described for preparing unenriched and [1-13C]-enriched 5-deoxy- and 5-O-methyl-pentoses in the D or L configuration. The 1H-n.m.r. spectra of these compounds have been interpreted, and the 13C-n.m.r. spectra assigned with the aid of 2-D 13C-1H chemical-shift correlation spectroscopy. Tautomeric forms (furanoses, hydrate, and aldehyde) in solution in 2H2O have been quantified with the aid of [1-13C]-enriched derivatives. Spectra of 5-deoxypentoses, 5-O-methylpentoses, and methyl pentofuranosides have been compared, in order to assess the effect of 5-C-deoxygenation and 5-O-methylation on chemical shifts and coupling constants (1H-1H, 13C-1H, and 13C-13C) and on the pentofuranose conformations.     

Application of new, high-sensitivity, 1H-13C-n.m.r.-spectral techniques to the study of oligosaccharides

Four n.m.r. methods that are especially useful for characterization of oligosaccharides are applied to the trisaccharide alpha-Neu5Ac-(2----3)-beta-Gal-(1----4)-Glc (1). Three of these are two-dimensional, heteronuclear methods that provide chemical-shift correlation maps having much higher sensitivity than was previously possible, because they rely on indirect observation of 13C via 1H detection. These methods are used to assign, completely, the 1H- and 13C-n.m.r. spectra of both anomers of the trisaccharide. In addition to these two-dimensional methods, a one-dimensional method is used to measure 1H-1H coupling-constants accurately within each sugar ring. The values of the coupling constants thus measured for 1 are evidence that the conformations of the individual sugar rings are not affected by linkage into the trisaccharide.     

新生隐球菌共孵育后血管内皮细胞的蛋白质组学研究

目的研究新生隐球菌共孵育后血管内皮细胞与正常细胞的差异蛋白质谱,推测蛋白质表达改变在孵育过程中的作用。方法利用二维凝胶电泳获得新生隐球菌孵育后血管内皮细胞与正常细胞的差异表达蛋白点,对部分差异蛋白点进行质谱鉴定分析,并以实时荧光定量PCR对其mRNA表达进行定量比较。结果Peroxiredoxin I及Calpactin I lightchain等13个蛋白的表达水平发生明显改变,Peroxiredoxin I及Calpactin Ilightchain的mRNA表达明显改变,其mRNA含量的改变趋势与相应的蛋白质的变化趋势相同。结论Peroxiredoxin I及Calpactin Ilightchain等蛋白表达量的改变可能与新生隐球菌侵袭血管内皮细胞屏障有关。