The glycoprotein nature of A1 adenosine receptors

A1 adenosine receptors from different tissues and species were photoaffinity labelled and then the carbohydrate content was examined by both enzymatic and chemical treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labelled membrane receptors shows that neuraminidase treatment alters the electrophoretic mobility of the receptor band indicating the presence of terminal neuraminic acids. Neuraminidase digestion does not influence the binding characteristics of the receptor. The totally deglycosylated receptor protein obtained by chemical treatment has an apparent molecular weight of 32,000.     

Cytokinins regulate calcium binding to a glycoprotein from fungal cells

A glycoprotein that binds about 20 atoms of Ca per mole has been purified from the osmotic shock fluid of some unicellular coenocytic water-molds, $\text{Achlya}$ spp. and $\text{Blastocladiella emersonii}$. The binding of calcium is allosterically regulated by N⁶-(substituted)adenine derivatives, cytokinins. Pyrimidines, purine and pyrimidine nucleosides, auxins, and benzimidazole derivatives are ineffective in inhibiting calcium binding. Lysozyme partially inactivates the molecule so that a high affinity calcium binding site is destroyed. Trypsin and pronase inactivate the molecule so that Ca⁺⁺ binding to both high and low affinity sites is affected. Cytokinins inhibit calcium binding to both sites.     

Dematiaceous fungal pathogens isolated from nature

This study was conducted to demonstrate the presence of pathogenic dematiaceous fungi in nature. Using hamster and mouse inoculation techniques, 43 isolates of dematiaceous fungi were recovered from 39 samples of woody plant material and soil from the Virginia environment. Thirteen species were identified and included 4 Phialophora spp., 3 Cladosporium spp., 2 Exophiala spp., Sporothrix sp., Wangiella dermatitidis, Bispora betulina, and Scytalidium lignicola. Evidence is presented for the first isolations of C. trichoides from nature in the United States; these isolates proved to be pathogenic for mice in which they produced disease and death in a course similar to that seen in man. Natural isolates of Phialophora verrucosa, Phialophora repens, Exophiala jeanselmei, and Wangiella dermatitidis were identical to those species isolated from man using the following criteria: morphology, 12% gelatin reaction, and survival in laboratory animals.     

Evidence for the glycoprotein nature of retina glycogen

Incubation of a bovine retina membrane preparation with micromolar amounts of UDP-[14C]glucose resulted in the incorporation of [14C]glucose into endogenous (1----4)-alpha-glucan, insoluble in trichloroacetic acid, and acid-soluble ethanol-insoluble glycogen. The trichloroacetic-acid-insoluble glucan fraction of retina migrated in 2.6-3% acrylamide gels when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and was rendered acid-soluble by digestion with pronase. The solubility of the acid-insoluble glucan in acidified organic solvent was different from that of amylose or glycogen and similar to membrane proteins and glycoproteins. The glycogen fraction of retina contained 1.5-2.0 micrograms protein/100 micrograms glucose. When this fraction was analyzed by SDS-PAGE only one band, which moved near the top of 3% acrylamide gels, was stained with periodic acid Schiff reagent and Coomassie blue. The protein nature of the Coomassie-blue-stainable material was demonstrated by iodination of the glycogen fraction with [131I]iodide and identification of labeled monoiodotyrosine and diiodotyrosine. The bulk of the label comigrated with carbohydrate near the top of gels in SDS-PAGE and treatment with alpha- amylse decreased the molecular size of both labeled and stainable material. Physical dissociative conditions (7.5 M urea/0.83% SDS/0.83% mercaptoethanol) and the following chemical treatments failed to dissociate the iodinated protein from glycogen: (a) 0.1 M NaOH/0.1 M NaBH4 at room temperature for 24 h; (b) 1 M HCl in methanol at 50 degrees C for 10 min; (c) trifluoroacetic acid at 50 degrees C for 6 min. 131I-labeled glycogenpeptide was isolated after 131I-labeled protein-bound glycogen had been subjected to digestion with papain/pronase and passed through a Sepharose column. The results suggest that at least part of glycogen in bovine retina is firmly combined to protein as a single proteoglycogen molecule. Furthermore some of the proteoglycogen might be present as a trichloroacetic-acid-precipitable proteoglucan owing to its lower glucose content.     

The glycoprotein nature of solubilized muscarinic acetylcholine receptors from bovine cerebral cortex

Muscarinic acetylcholine receptors were solubilized from bovine cerebral cortex with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The so-obtained receptors could be precipitated by Wheat Germ (73%), Concanavalin A (55%), Lens Culinaris (36%) and Ricinus Communis (26%), but not by Peanut, Dolichus Biflorus and Ulex Europaeus. On Wheat Germ- and Concanavalin A-affinity chromatography, the solubilized muscarinic receptors were retained on both columns and subsequently eluted with N-acetylglucosamine and alpha-methyl-D-mannoside, respectively. A high concentration (100 micrograms/ml) of Wheat Germ or Concanavalin A did not interfere with Z-[3H]quinuclidinyl benzilate binding, thereby suggesting that the lectin binding sites are not directly involved in the receptor binding function. These solubilized muscarinic receptors are postulated to contain carbohydrate residues, N-acetyl-glucosamine, mannose and galactose, as glycoprotein.     

Structure‐antigenicity of the V3 region of SIVmac envelope glycoprotein

The objective of this study was to analyze the immunogenicity and antigenicity of the V3 domain (Cys313–Cys346) of the external envelope glycoprotein gp125 of SIVmac251. The corresponding peptide was synthesized and characterized as linear and cyclic peptides. Our results showed that this region, as for HIV‐1, contained an immunodominant epitope. The antigenicity was similar for the linear and cyclic peptides when tested against a panel of 15 sera from SIV infected macaques. Similarly, both peptide structures presented similar immunogenicity as shown by the characterization of the anti‐peptide antibodies produced in rabbits against the cyclic and linear forms. But, unexpectedly, the antibodies produced against linear peptides recognized with a relatively higher intensity the native envelope gp140 than those produced against the cyclic structure. Furthermore, we showed that these antibodies recognized better the deglycosylated form of the glycoprotein. But, in contrast to the neutralizing activity obtained with anti‐V3 peptides from HIV‐1, no antiviral activity was obtained with antibodies generated against linear or cyclic SIVmac V3 peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Chemical nature, ligand denticity and quantification of fungal siderophores

Thirtyfive siderophore producing fungi were categorized for their hydroxamate, catecholate or carboxylate nature by chemical and bioassays. Out of 35 fungi, 30 were hydroxamates and 5 showed carboxylate nature. However, none of the fungi produced catecholate type of siderophores. Eighteen out of 29 fungi were trihydroxamate and the rest 11 fungi were dihydroxamates. Twenty-three fungi were hexadentate and 6 were tetradentate in nature. Quantification of siderophores using standard compounds deferrioxamine mesylate and rhizoferrin revealed that Phanerochaete chrysosporium produced maximum among the hydroxamate producing fungi and Mycotypha africana resulted maximum among the carboxylate producing fungi.     

Structure,receptor recognition,and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein

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Evidence for the glycoprotein nature of kidney renin.

The glycoprotein nature of renin isolated from either rabbit or human kidney has been demonstrated by affinity chromatography on concanavalin A-Sepharose. The bulk of rabbit renin activity bound to concanavalin A is released by 20 to 50 mM alpha-methyl-D-mannoside. Adsorption of renin is prevented by periodate oxidation prior to chromatography. Mild acid treatment (pH 2.5) prior to chromatography does not alter the concanavalin A binding profile although the pI values of native rabbit renin (5.1-5.6) are shifted into a broader distribution (4.7-6.4). The molecular weight values of rabbit renin obtained by gel filtration and those from zone centrifugation are identical (37000 +/- 1000), consistent with a low percent of carbohydrate in the glycoprotein. A hydrophobic contribution to the binding of renin by concanavalin A is evident since, in the presence of mM Ca2+ and Mn2+, higher concentrations of alpha-methyl-D-mannoside are required to affect the same release of renin at 23 degrees C compared to that at 4 degrees C. Furthermore, 25% ethylene glycol releases renin in the absence of alpha-methyl-D-mannoside. It is concluded that renin contains a small number of carbohydrate residues in relatively close proximity to a hydrophobic surface which enhances the interaction with concanavalin A.     

Evidence for the glycoprotein nature of radish beta-fructosidase.

Concanavalin A (Con A) was utilized free, bound to Sepharose 4 B or cross-linked to glutaraldehyde to investigate the possibility of binding this lectin to radish beta-fructosidase (E.C.3.2.1.26). The choice of cross-linked Con A as affinoadsorbent is discussed and standard conditions for binding are defined. Specificity of precipitation of this enzyme by the lectin was especially investigated. Thus, the possibility of binding was tested in the presence of high ionic strength, ethylene glycol, alpha-methyl mannoside, alpha-methyl glucoside and during periodate oxidation of the enzyme. Based on the interactions observed between beta-fructosidase and Con A under these conditions it is concluded that the saccharide binding site of the lectin is primarily involved with a secondary contribution from the hydrophobic site. The specificity of binding and the complete precipitation of beta-fructosidase activity by the insolubilized lectin imply that all beta-fructosidase activity measured in Raphanus sativus seedling extracts is linked to (a) glycoprotein form(s) of this enzyme.     

Properties of potato lectin and the nature of its glycoprotein linkages

1. Potato lectin is a glycoprotein that contains about 47% (by weight) l-arabinose, 3% d-galactose and 11% hydroxyproline. It has a monomeric molecular weight of about 50000 and probably exists as a monomer-dimer system in aqueous solution, with the monomer predominating. It has a very high viscosity, which would indicate either that the molecule is very expanded or that it is an elongated ellipsoid. 2. After prolonged proteolytic digestion of a reduced and carboxymethylated derivative of the lectin, a glycopeptide was isolated (of mol.wt. 32000-34000) that included all the carbohydrate and hydroxyproline of the original glycoprotein but less than 30% of the total original amino acid residues. 3. The arabinose of the glycoprotein is present exclusively as the beta-arabinofuranoside and this includes those residues that are directly linked to the hydroxyproline residues of the polypeptide chain. All the arabinose of the glycoprotein is linked to the polypeptide chain through the hydroxyproline residues; the ratio of arabinose to hydroxyproline is 3.4:1. Although alpha-arabinofuranosides are known to be present in arabinans and arabinogalactans, the natural occurrence of beta-arabinofuranosides has not previously been reported. 4. Nine or ten serine residues of the polypeptide chain are substituted with single alpha-galactopyranoside residues that can be removed by the action of alpha-galactosidase from coffee beans but not by a beta-galactosidase. This is the first report of an alpha-galactoside linkage to serine. The effect of alpha-galactosidase is much greater on a glycopeptide from which the arabinose has been already removed, which indicates a steric hindrance of the galactosidase action by adjacent chains of arabinosides. 5. In 0.5m-NaOH (pH13.7), galactose residues were removed from the serine residues of the glycopeptide by a process of beta-elimination. This reaction took place very slowly in the intact glycopeptide but much more rapidly when the arabinofuranoside residues had been removed. This inhibitory effect of the arabinofuranoside residues on the beta-elimination reaction is likely to be due to a negative charge on the hydroxy groups of the adjacent arabinofuranoside residues, which would be ionized at this high pH value. 6. It is suggested that potato lectin may be representative of a class of soluble plant glycoproteins that would include precursors of the cell-wall glycoprotein extensin. If this is the case, extensin should also contain beta-l-arabinofuranosides linked to hydroxyproline and alpha-d-galactopyranosides linked to serine residues of the polypeptide chain.     

The contribution of cysteine residues to antigenicity and extent of processing of herpes simplex virus type 1 glycoprotein D.

Glycoprotein D (gD) is an envelope component of herpes simplex virus types 1 (gD-1) and 2 (gD-2). The gD-1 polypeptide contains seven cysteine residues among its 369 amino acids; six are located on the N-terminal or luminal portion of the glycoprotein, and a seventh is located in the transmembrane region. Previous studies used a panel of monoclonal antibodies (MAbs) to define gD epitopes as continuous or discontinuous. Purified gD, denatured by reduction and alkylation, loses discontinuous epitopes, whereas continuous epitopes are retained. The contribution of disulfide bonds to maintenance of discontinuous epitopes is, therefore, significant. In the present study, our objective was to determine the contribution of individual cysteine residues to folding of gD-1 into its native conformation. Site-directed oligonucleotide mutagenesis was used to create seven mutants, each with a serine residue replacing a cysteine. The mutated genes were cloned into a eucaryotic expression vector and transfected into COS-1 cells, and the proteins were separated by nondenaturing polyacrylamide gel electrophoresis, followed by immunoblotting. Replacement of cysteine 7 (residue 333) had only a minimal effect on the antigenic properties of gD-1. In contrast, replacement of any one of the other six cysteine residues resulted in either a major reduction or a complete loss of binding of those MAbs that recognize discontinuous epitopes, with no effect on the binding of MAbs which recognize continuous epitopes. These mutations also had profound effects on the extent of oligosaccharide processing of gD-1. This was determined by digestion of the expressed proteins with various endoglycosidases, followed by electrophoresis and Western blotting (immunoblotting) to observe any mobility changes. Three mutant gD proteins which did not express discontinuous epitopes contained only high-mannose-type oligosaccharides, suggesting that processing had not proceeded beyond the precursor stage. Two mutant forms of gD exhibited reduced binding of MAbs to discontinuous epitopes. A small proportion of the molecules which accumulated at 48 h posttransfection contained complex oligosaccharides. One mutant exhibited reduced binding of MAbs to discontinuous epitopes, but was present at 48 h posttransfection only in the precursor form. The cysteine 7 mutant was processed to the same extent as wild-type gD. We conclude that the first six cysteine residues are critical to the correct folding, antigenic structure, and processing of gD-1, and we speculate that they form three disulfide-bonded pairs.     

Occurrence of a D-glucosyltransferase in germinating pea seeds and isolation of an endogenous acceptor.

A $\underset{=}{D}$-glucosyltransferase has been found in soluble extracts of germinating pea seeds. The enzyme transfers$\underset{=}{D}$-glucose from uridine diphosphate $\underset{=}{D}$-glucose to a substance which also occurs in the seed extracts. The substance was the only active acceptor of$\underset{=}{D}$-glucose found for this enzymatic reaction. The enzyme has been separated from the acceptor by several purification steps. The acceptor has been purified to near homogeneity. It appears to be a complex glycoside which contains $\underset{=}{L}$-rhamnose, $\underset{=}{D}$-galactose, and $\underset{=}{D}$-glucuronic acid. The aglycone portion of the acceptor has not been characterized, but preliminary findings suggest that it is phenolic in nature.     

Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles

Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2(660)) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2(660) with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2(660) but not VLPs or FL E1E2 complex. The antigenic differences between E2(660,) FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.