Sequence Flexibility in the Polytropic env gp70-Derived Region of the Membrane Glycoprotein (gp55) of Friend Spleen Focus-Forming Virus Affects Its Biological Activity

We previously reported (N. Watanabe, M. Nishi, Y. Ikawa, and H. Amanuma, J. Virol. 65:132–137, 1991) that the mutant Friend spleen focus-forming virus (F-SFFV_MS), which encodes a mutant gp55 membrane glycoprotein with an ecotropic env gp70 sequence, was nonpathogenic. Here we injected the F-SFFV_MS–Friend murine leukemia virus (F-MuLV) clone 57 complex into newborn DBA/2 mice. We obtained four groups of pathogenic variant F-SFFV complexes, each showing a different degree of pathogenicity in adult mice and a different gp55 profile. Of these, group 1 variant F-SFFV was particularly interesting, because it was the most frequently obtained and because it produced doublet bands of gp55 (59 and 57 kDa), neither of which reacted with the nonecotropic gp70-specific monoclonal antibody, and because its DNA intermediate did not hybridize with the nonecotropic env-specific probe. Cloning and DNA sequence analysis of the env region of one isolate of the group 1 variant F-SFFV revealed that this virus consisted of two distinct F-SFFV genomes; one (clone 117) differed from the other (clone 118) due to the presence of a 39-bp in-frame deletion. Reconstitution to full-length F-SFFV genomes and a pathogenicity assay showed that each reconstituted F-SFFV was pathogenic, with clone 117 showing a higher degree of pathogenicity than clone 118. Both reconstituted F-SFFVs caused activation of the mouse erythropoietin receptor in the factor-independent cell proliferation assay, although much less efficiently than the wild-type polycythemia-inducing isolate F-SFFVp. Clone 118 produced a gp55 of 59 kDa, while clone 117 produced one of 57 kDa. Clone 118 had a substitution by the F-MuLV clone 57 gp70 sequence, indicating that it was derived from the F-SFFV_MS env gene by a homologous recombination with the F-MuLV clone 57 env gene. The site of the 39-bp deletion in clone 117 corresponded to the portion of the clone 118 sequence which was unique to the ecotropic env genes. These results indicated the importance for the biological activity of gp55 of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic env genes.Friend spleen focus-forming virus (F-SFFV), a replication-defective mouse type C retrovirus contained in the Friend virus complex, causes an acute erythroleukemia in adult mice of susceptible strains in the presence of a helper virus, such as the replication-competent Friend murine leukemia virus (F-MuLV) (reviewed in references 8 and 23). F-SFFV does not encode a viral oncogene, but its defective env gene product, gp55, plays a critical role in inducing the disease. In vitro biochemical evidence demonstrated that gp55, a membrane glycoprotein encoded by the polycythemia-inducing isolate of F-SFFV (F-SFFVp), specifically binds to a mouse erythropoietin receptor (EPO-R) and activates it, causing mitogenic signal transduction in the absence of the natural ligand erythropoietin (EPO) (12). Since the EPO-R is expressed in the erythroid progenitor cells, and the interaction between EPO and EPO-R regulates the level of erythropoiesis (15), it is assumed that the continuous expression of F-SFFV gp55 results in an abnormal proliferation of these cells, leading to leukemic transformation after several additional cytogenetic changes (3).gp55, although closely related to the Env protein of MuLV, is not incorporated into the retrovirus particles, but stays inside the cells, mainly in the rough endoplasmic reticulum membrane, with a small fraction of the molecules (3 to 5%) processed through the Golgi apparatus to the cytoplasmic membrane (6, 21, 24). Cell surface-localized gp55 is then shed from the cells, probably after proteolytic cleavage from the transmembrane domain (6, 20, 21).There are three major differences between the primary structures of gp55 of F-SFFVp and the Env protein of ecotropic MuLV (1, 4, 31). These are, from the N terminus, a substitution by the polytropic (dualtropic) env gp70 sequence, a 585-bp deletion which eliminates the proteolytic cleavage site for gp70 and p15E, and a 6-bp duplication and a single base insertion which cause premature termination of translation and loss of a 34-amino-acid peptide at the C terminus. By constructing F-SFFV encoding a mutant gp55, analyzing its pathogenicity in vivo, and also obtaining spontaneous revertant F-SFFVs from the mutant F-SFFV, we demonstrated that each of these three structural differences is essential for the pathogenicity of gp55 (2, 27–29). We previously reported (28) that the constructed F-SFFV (F-SFFV_MS) encoding the mutant gp55, in which the polytropic gp70 sequence had been replaced by the ecotropic F-MuLV clone K-1 gp70 sequence, was nonpathogenic in adult mice. The polytropic gp70-derived sequence in gp55 appears to contain a binding site for EPO-R (32). The purpose of the present study was to obtain spontaneous revertants from mice neonatally injected with F-SFFV_MS and to analyze the structure of their gp55s, with the goal of pinpointing the sequence required for pathogenicity and activation of EPO-R. The results indicated the importance of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic env genes.     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Pathogenicity of a Mutant Friend Spleen Focus-Forming Virus Encoding an Env-Like Membrane Glycoprotein (gp55) with Substitution by a Xenotropic Murine Leukemia Virus Env gp70 Sequence

Friend spleen focus-forming virus (F-SFFV) is a replication-defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)-like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus-inducing (MCF) murine leukemia virus (MuLV) Env gp70-derived sequence in its amino-terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino-terminal region. XE gp55 displayed almost the same biological activities as the wild-type gp55, excluding the above possibility.     

N-Glycosylation of membrane glycoproteins in retinol-deficient rat liver

The effect of vitamin A deficiency onN-linked oligosaccharides of membrane glycoproteins was studied in rat liver in order to evaluate the suggested role of retinol in proteinN-glycosylation. First, oligosaccharides of newly synthesized glycoproteins from rough endoplasmic reticulum of vitamin A deficient liver were compared with that of pair-fed controls. Oligosaccharides were metabolically labelled withd-[2-³H]mannose, released from the glycoproteins with endoglycosidase H, purified by reversed phase HPLC and ion exchange chromatography, and were reduced with sodium borohydride. HPLC fractionation of the oligosaccharide alditols showed that the glycoproteins carried mainly four oligosaccharide species, Glc₁Man₉GlcNAc₂, Man₉GlcNAc₂, Man₈GlcNAc₂ and Man₇GlcNAc₂, in identical relative amounts in the vitamin A deficient and the control tissue. In particular, no increase in the proportion of short chain oligosaccharides was noted in vitamin A deficient liver. Second, the number ofN-linked oligosaccharides was estimated in dipeptidylpeptidase IV (DPP IV), a major glycoprotein constituent of the hepatic plasma membrane, comparing the newly synthesized glycoprotein from rough endoplasmic reticulum and the mature form of DPP IV from the plasma membrane. No evidence was obtained that retinol deficiency caused incomplete glycosylation of this membrane glycoprotein. From these data, the suggested role of retinol as a cofactor involved in the synthesis ofN-linked oligosaccharides of glycoproteins must be questioned.Abbreviations DolP Dolichyl phosphate - DolPP dolichyl pyrophosphoryl - RetPMan retinyl phosphate mannose - DPP IV dipeptidyl peptidase IV (EC 3.4.14.5) - endo H endo--N-acetylglucosaminidase H (EC 3.2.1.96) - endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Effect of deglycosylation on the properties of β-fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524

Summary Most of the carbohydrate moiety of -fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524 was removed by endo--N-acetylglucosaminidase F. A subunit of 94000 Da was observed in SDS-PAGE after deglycosylation. TheK _m value for sucrose was not changed by deglycosylation but the stability at pH 4–5 and 50°C was decreased. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is considered that the carbohydrate moiety of -fructofuranosidaseP-1 contributes to the stability of the enzyme but is not essential in its catalytic function.     

Transfer of Man9GlcNAc tol-fucose by endo-β-N-acetylglucosaminidase fromArthrobacter protophormiae

We have reported that transglycosylation activity of endo--N-acetylglucosaminidase fromArthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995)J Biol Chem 270: 17723–29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man₉GlcNAc, tol-Fuc using Man₉GlcNAc₂Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2m l-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence ofl-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man₉GlcNAc-l-Fuc-PA, revealed that a -anomeric configuration linkage was formed between GlcNAc andl-Fuc. The GlcNAc was found to be 1,2-linked tol-Fuc by two methods; i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man₉GlcNAc-l-Fuc; and ii) preparation of Man₉GlcNAc-l-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man₉GlcNAc(1,2)-l-Fuc. Methyl -l-fucopyranoside,l-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation failed when methyl -l-fucopyranoside,d-Fuc andd-Gal were used. These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a⁴C₁-conformation or equivalent conformation of the acceptor to perform transglycosylation.Abbreviations endo-A endo--N-acetylglucosaminidase fromArthrobacter protophormiae - PA pyridyl-2-amino- - AP aminopyridine - GlcNAc N-acetyl-d-glucosamine - Man mannose - Gal galactose - Fuc fucose - Glc glucose - PA-C₂ PA-glycolaldehyde - PA-C₃ PA-l-glyceraldehyde - PA-C₄ PA-d-threose - HPAEC-PAD high performance anion exchange chromatography with pulsed amperometric detector - HPLC high performance liquid chromatography - ODS octadecylsilyl - ES-MS electrospray mass spectrometry - CID collision-induced decomposition     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Properties of the deglycosylatedβ-fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524

Summary The carbohydrate moiety of -fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524 was largely removed by exposure of the enzyme to endo--N-acetylglucosaminidase F; the total carbohydrate content of the enzyme was decreased from 53% (w/w) to 15% (w/w). The stability of the deglycosylated enzyme at pH 4 to 7 and 40 to 50°C was decreased and theK _m value for sucrose was increased from 0.65 to 1.43 M. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is concluded that the carbohydrate moiety of -fructofuranosidaseP-2 contributes to the stability of the enzyme as well as its affinity for sucrose.     

Two-dimensional gel electrophoresis analysis of the abundance of virulent exoproteins of group A streptococcus caused by environmental changes

Group A streptococci regulate the expression of virulence factors in response to environmental change. In order to investigate this mechanism, the growth of group A streptococci and the abundance of virulent exoprotein production in culture supernatant were analyzed by two-dimensional gel electrophoresis (2-D electrophoresis) under several culture conditions. Judging from alterations in their growth, group A streptococci were affected by various environmental stresses. Under high O₂ and low CO₂ concentrations, streptococcal pyrogenic exotoxin B (SpeB) and streptococcal pyrogenic exotoxin F (SpeF) significantly decreased, and the streptococcal inhibitor of complement (Sic) increased . At 30 °C, increases in endo--N-acetylglucosaminidase (EndoS) and -amylase were also detected, while at 41 °C EndoS became undetectable and SpeB and SpeF decreased. Sic, SpeF and mitogenic factor 3 (Mf3) decreased when cells were cultured in higher NaCl concentrations, and EndoS disappeared following culture of the cells in high glucose concentration. An increase in acid phosphatase and a decrease in several other proteins were detected when the cells were cultivated in high iron concentrations. These results suggest that group A streptococci have a versatile adaptation system that responds to several environmental stresses by altering the level of exoprotein production.Abbreviations SpeB Streptococcal pyrogenic exotoxin B - SpeF Streptococcal pyrogenic exotoxin F - Sic Streptococcal inhibitor of complement - EndoS Endo--N-acetylglucosaminidase     

Use of resorufin-labelled N-glycopeptide in a high-performance liquid chromatography assay to monitor endoglycosidase activities during cultivation ofFlavobacterium meningosepticum

Peptide-N ⁴-(N-acetyl--glucosaminyl) asparagine amidase F (PNGase F) and endo--N-acetyl glucosaminidase F (Endo F) activities were monitored during cultivation ofFlavobacterium meningosepticum using a new fluorescence-HPLC procedure based on a commercially available substrate. The PNGase F activity reached a maximum level at the end of the log phase and remained constant during the stationary phase, while Endo F continuously increased until late stationary phase. PNGase F obtained at the end of the log phase was less contaminated by other proteins compared with late stationary phase.Abbreviations Con A concanavalin A - Endo F endo--N-acetyl glucosaminidase F (EC 3.2.1.96) - GlcNAc N-acetylglucosamine - PNGase F peptide-N ⁴-(N-acetyl--glucosaminyl) asparagine amidase F (EC 3.5.1.52).     

N-andO-alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl lodide

O-Methylation of simple neutral oligosaccharides is readily accomplished in dimethyl sulphoxide containing solid sodium hydroxide and methyl iodide [Cincanu I, Kerek F (1984) Carbohydr Res 131209-17]. This procedure has been extended to 2-acetamido-2-deoxy sugars and sialic acid-containing oligosaccharides. CompleteO-andN-methylation was in most cases achieved in 15 min. Esterification of carboxylic groups in uronic acids was fast and resulted in concomitant -elimination. The method is also suitable for methylation of glycoproteins and glycosphingolipids. Polysaccharides can also be methylated by this technique. Analysis of the products by gas-liquid chromatography and mass spectrometry showed no degradation products.Abbreviations lacto-N-tetraose LcOse₄, Gal3GlcNAc3Gal4Glc - lacto-N-fucopentaose III III³Fuc-nLcOse₄, Gal4[Fuc3]GlcNAc3Gal4Glc - trihexosylceramide GbOse₃Cer, Gal4Gal4Glc1-1Cer - globoside GbOse₄Cer, GalNAc3Gal4Glc1-1Cer - FAB-MS fas atom bombardment mass spectrometry     

Cloning and expression of the Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in Escherichia coli

A gene coding for the endo--1,3-1,4-glucanase of B. circulans ATCC21367 was cloned into Escherichia coli. The cloned enzyme hydrolyzed lichenan or barley -glucan to produce 3-O--cellobiosyl-d-glucose as a main product but was inactive with carboxymethyl cellulose, laminarin and xylan. The enzyme, M _r=28 kDa, remained within the cytoplasm of E. coli. A 771 bp open reading frame was in the 2 kb PstI fragment of the recombinant plasmid pLL200K. The deduced protein sequence consists of 257 amino acids and has a putative signal peptide of 26 amino acids. The amino acid sequence of the endo--1,3-1,4-glucanase showed 68 and 51% homology to previously reported endo--1,3-1,4-glucanases from Bacillus strain N-137 and B. brevis, respectively.     

Different susceptibilities of complex-, hybrid- and high-mannose-type α1- inhibitor and α1-acid glycoprotein to endo-β-N-acetylglucosaminidase F and peptide:N-glycosidase F

Endo--N-acetylglucosaminidase F (endo F, EC 3.2.1.96) and peptide:N-glycosidase F (PNGase F, EC 3.2.2.18) fromFlavobacterium meningosepticum were used for the deglycosylation of ₁-proteinase inhibitor and ₁-acid glycoprotein carrying oligosaccharide side chains of the complex-, high-mannose- and hybrid-type. High-mannose-and hybrid-type glycoproteins were obtained by the incubation of rat hepatocyte primary cultures with 1-deoxymannojirimycin or swainsonine, respectively. It was found that endo F cleaves hybrid- and high-mannose-type ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 as well as at pH 8.5 in the presence or absence of 1% octyl--d-glucopyranoside. Complex-type ₁-proteinase inhibitor or ₁-acid glycoprotein were not cleaved by endo F even in the presence of octyl--d-glucopyranoside.PNGase F was found to cleave complex-, hybrid- and high-mannose-type oligosaccharide side chains of ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 and pH 8.5 in the presence of 0.75% octyl--d-glucopyranoside. The deglycosylation of both protein substrates was very poor without detergents.Abbreviations Endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - PNGase F peptide:N-glycosidase F (EC 3.2.2.18) Dedicated to Prof. Dr. Wolfgang Gerok on the occasion of his 60th birthday     

Separation and characterization of the complexes constituting the cellulolytic enzyme system of Clostridium thermocellum

Clostridium thermocellum, strain JW20 (ATCC 31449) when growing in cellulose produces a cellulolytic enzyme system, that at the early stage of the fermentation is largely bound to the substrate. As cellulose is consumed the bound enzyme is released as free enzyme to the culture fluid. The bound enzyme fraction extracted with distilled water from the cellulose contains two major components, a large complex (M_r100×10⁶) and a small complex M_r4.5×10⁶) which were separated by gel filtration and sucrose solved by affinity chromatography into a complex that binds to the column and into a non-bindable mixture of proteins. All four fractions have endo--glucanase activity but only the two bound complexes and the free bindable complex hydrolyze crystalline cellulose with cellobiose as the main product. These three complexes are qualitatively similar in that they each contain about 20 different polypeptides (M_r values from 45,000 to 200,000) of which about ten are major components. However, the relative amounts of some of the peptides in the complexes differ. At least four polypeptides of the complexes have endo--glucanase activity.Abbreviations CM cellulose, carboxymethyl cellulose - CMCase carboxymethyl cellulase cosidered endo--1,4-glucanase - SDS sodium dodecyl sulfate - YAS yellow affinity substance - YAS-cellulose yellow affinity substance-cellulose complex     

High-mannose oligosaccharides from human Tamm-Horsfall glycoprotein

The present communication reports the occurrence of high-mannose oligosaccharides on Tamm-Horsfall glycoprotein prepared from human pooled urine. The Pronase digest of the glycoprotein was fractionated by gel filtration and a high-mannose glycopeptide species was separated from complex-type glycopeptides. When high-mannose glycopeptides were digested with endo--N-acetylglucosaminidase H, followed by reduction with [³H]KBH₄, three oligosaccharides were resolved by thin-layer chromatography. On the basis of chromatographic mobility and exoglycosidase digestions the composition Man₇-, Man₆-, and Man₅-GlcNAc was assigned to the three oligosaccharides. Man₆GlcNAc is by far the major component.     

N-andO-glycosylation of glycoprotein C synthesized by Herpes simplex virus type 1-infected ricin resistant cells

The progeny of Herpes simplex virus type 1 (HSV-1) grown in ricin-resistant 14 cells (Ric^R14) lackingN-acetylglucosaminyltransferase I was released in the extracellular medium at a very low rate. By using a monoclonal antibody immobilized on Sepharose we purified from HSV-1-infected Ric^R14 cells a viral glycoprotein (gC), which carries bothN-andO-linked oligosaccharides. Glycopeptides obtained from [³H]mannoselabeled gC by Pronase digestion were entirely susceptible to endo--N-acetylglucosaminidase H, and the major oligosaccharide released was Man₄GlcNAc. The accumulation of this high-mannose species was related to the enzymic defect of the host cells and to the long retention of the viral glycoprotein within the cells. The extent ofO-glycosylation evaluated in [¹⁴C]glucosamine-labeled gC from Ric^R14 cells as compared to that of gC from wild type cells did not appear to be significantly modified.Abbreviations Con A concanavalin A - BHK cells baby hamster kidney cells - HSV Herpes simplex virus     

α1-Acid glycoprotein binds human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein viaN-linked glycans

In the present study, we demonstrate a specific low-affinity interaction between recombinant precursor gp160 (rgp160) or surface unit gp 120 (rgp 120) of human immunodeficiency virus type 1 (HIV-1) and ₁-acid glycoprotein (AGP), a human glycoprotein displaying complex typeN-glycans. Binding of rgp 160/rgp 120 to agarose-coupled AGP was dose-dependent, saturable, calcium-, pH- and temperature-dependent. Binding was inhibited by soluble AGP, asialo-AGP, fetuin, -d-GlcNAc₄₇-BSA, -d-Man₂₀-BSA, mannan, complex-type asialo-agalacto-tetraantenary precursor oligosac-charide from human AGP and oligomannose 9 from porcine thyroglobulin; fully deglycosylated AGP was not inhibitory. The three AGP glycoforms separated on immobilized ConA bound rgp 160 to the same extent as did unfractionated AGP. These findings extend our previous results on the carbohydrate-binding properties of HIV-1 envelope (Env) glycoprotein in that they demonstrate the involvement of AGP glycan moieties in the binding to rgp 160/rgp 120. Preincubation of rgp 160 with AGP or mannan significantly reduced its binding to monocyte-derived macrophages (MDM), suggesting that AGP may play a role in preventing binding of soluble or virus-boundEnv glycoprotein to CD4⁺ monocytic cells.     

The purification of the three trimannosyl-N-acetylchitobiose pentasaccharides from the urine of swainsonine-intoxicated sheep

A simple method for the preparative resolution of three Man₃GlcNAc₂ isomers called Ia, Ib and II has been designed. It consists mainly of the use of concanavalin A-Sepharose which allowed the total purification of Man₃GlcNAc₂-Ia, and then of anion-exchange resin in borate buffer-gradient to separate the Ib and II isomers. The purity of each oligosaccharide was checked by two HPLC methods. The use of these oligosaccharides for different analytical and biosynthetic purposes is discussed, and the unexpected resistance of one of the Man₃GlcNAc₂ alditols to the action of endo--N-acetylglucosaminidase H is noted.     

Purification and partial characterization of an acid β-fructosidase from sweet-pepper (Capsicum annuum L.) fruit

The present study describes the biochemical characteristics of an acid -fructosidase (EC 3.2.1.26) purified from the fruit of sweet pepper (Capsicum annuum L.). The soluble form, which constitutes more than 95% of the total activity at pH 4.5, hydrolyzes sucrose, raffinose, and stachyose. Its pH and temperature optima are 4.5 and 55 °C, respectively. Metal cations such as Ag⁺ and Hg²⁺ strongly inhibit its activity, suggesting the presence of at least one sulfhydryl group at the catalytic site. After purification of the enzyme by means of ammonium sulfate fractionation, gel chromatography (diethyl-aminoethyl-Sephacel, hydroxylapatite, concanavalin A-Sepharose), and preparative gel electrophoresis, the purified enzyme was shown to be a 42 kDa glycoprotein interacting specifically with concanavalin A. After complete chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight of the constitutive polypeptide was estimated to be 39 kDa. The enzyme glycans were characterized using both affino- and immunodetection. The enzyme has at least two N-linked oligosaccharide sidechains, one of the high-mannose type, and the other of the complex type. The high-mannose glycan has a low molecular weight (1 kDa), and is responsible for the interaction between the enzyme and concanavalin A. The complex-type glycan has an estimated molecular weight of 2 kDa. It contains one 1 2-linked xylose residue, probably one fucose residue 1 3-linked to the chitobiose unit, and no terminal galactose residue. The two glycans, associated to the 39 kDa polypeptide, constitute the acid -fructosidase of the sweet-pepper fruit.Abbreviations F -fructosidase - ConA concanavalin A - DEAE diethylaminoethyl - DTNB dithionitrobenzoic acid - endo F endo--N-acetylglucosamidase F - endo H endo--N-acetylglucosamidase H - NEM N-ethylmaleimide - PCMB parachloromercurobenzoate - PNGase glycopeptide-N-glycosidase - TFMS trifluoromethane sulfonic acid This work was partly supported by a grant from the Commission Permanente de Coopération Franco-Québécoise to L. Faye, and S. Yelle. D. Michaud was a recipient of a graduate scholarship from the Natural Science and Engineering Research Council of Canada.