Search

HIV-1 has maximized its utilization of syndecans. It uses them as in cis receptors to infect macrophages and as in trans receptors to infect T-lymphocytes. In this study, we investigated at a molecular level the mechanisms that control HIV-1-syndecan interactions. We found that a single conserved arginine (Arg-298) in the V3 region of gp120 governs HIV-1 binding to syndecans. We found that an amine group on the side chain of this residue is necessary for syndecan utilization by HIV-1. Furthermore, we showed that HIV-1 binds syndecans via a 6-O sulfation, demonstrating that this binding is not the result of random interactions between basic residues and negative charges, but the result of specific contacts between gp120 and a well defined sulfation in syndecans. Surprisingly, we found that Arg-298, which mediates HIV-1 binding to syndecans, also mediates HIV-1 binding to CCR5. We postulated that HIV-1 recognizes similar motifs on syndecans and CCR5. Supporting this hypothesis, we obtained several lines of evidence that suggest that the 6-O sulfation recognized by HIV-1 on syndecans mimics the sulfated tyrosines recognized by HIV-1 in the N terminus of CCR5. Our finding that CCR5 and syndecans are exploited by HIV-1 via a single determinant echoes the mechanisms by which chemokines utilize these two disparate receptors and suggests that the gp120/chemokine mimicry may represent a common strategy in microbial pathogenesis.

We have used an antisynthetic peptide antiserum to a murine recombinant virus gp70 to probe normal mouse tissues for immunologically related proteins. In addition to cognate gp70s, this antiserum reacts with the heterogeneous nuclear ribonucleoparticle protein A1 by virtue of a 5-amino acid epitope, PRNQG. Further structural similarity is evident both 5' and 3' of this epitope. Since the function of the heterogeneous nuclear ribonucleoprotein particles in the cell is to aid in the stabilization and processing of newly synthesized RNA, we have investigated whether this retroviral sequence exhibits any nucleic acid-binding properties by the same criteria established for the identification of heterogeneous nuclear ribonucleoprotein particles. Analysis of the peptide in a poly(eA) binding assay shows this retroviral sequence to bind with high affinity to single-stranded nucleic acid. This binding occurs in a salt-sensitive manner characteristic of single-stranded nucleic acid-binding proteins. Flanking peptides not containing this sequence generated from either the A1 or gp70 show no ability to bind single-stranded nucleic acids by this assay.

A method is described for radioiodination to high specific activity of fixed and stained proteins within sodium dodecyl sulfate-polyacrylamide gels, without elution of the proteins from the gel. Following radioiodination, the proteins can be removed from the gel by trypsin treatment and the peptides analyzed. This procedure offers a means to structurally compare the proteins of multicomponent systems when purification of each component to homogeneity is unfeasible. Using this technique, we have compared the tryptic peptides of all the major protein components of Moloney and Rauscher leukemia viruses using only 50 to 100 microgram of total protein from each virus. Additionally, we have analyzed the membrane proteins of Dictyostelium discoideum at various stages in development. The validity of the technique and its value as a tool for comparative studies and identification of precursor-product relationships is discussed.

Endo-beta-N-acetylglucosaminidase F preparations from Flavobacterium meningosepticum have been found to contain peptide:N-glycosidase activity. Only the second activity, designated as peptide:N-glycosidase F, readily cleaves the beta-aspartylglycosylamine linkage of a fetuin triantennary complex glycopeptide, as shown by the isolation of the corresponding carbohydrate-free peptide containing aspartic acid and of an intact oligosaccharide with a di-N-acetylchitobiosyl moiety at the reducing end. Both activities in the mixture will hydrolyze a high mannose octaglycopeptide from ovalbumin, with the type of product formed being influenced by pH. At pH 4.0, only the endo-beta-N-acetylglucosaminidase F activity is functional, releasing octapeptide-GlcNAc and oligosaccharide-GlcNAc. At pH 9.3, the predominant cleavage is by peptide:N-glycosidase F at the glycosylamine bond, releasing octapeptide and oligosaccharide-GlcNAc-GlcNAc. This latter oligosaccharide is then hydrolyzed by endo-beta-N-acetylglucosaminidase F to oligosaccharide-GlcNAc plus GlcNAc.

We have purified a reduced and alkylated tryptic fragment of von Willebrand factor (vWF) which migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 52/48-kDa doublet, but behaved as a single 46-kDa species after partial deglycosylation. After extensive treatment with denaturants, the 52/48-kDa polypeptide retained its ability to inhibit ristocetin-induced platelet aggregation in the presence of native vWF, as well as aggregation induced by desialylated vWF alone. Therefore, the 52/48-kDa polypeptide interacts with the platelet glycoprotein Ib receptor even in the absence of ristocetin. Both the 52/48- and the 46-kDa species inhibited ristocetin-induced binding of the intact molecule to platelets, but did not affect thrombin-induced binding. Determination of the NH2-terminal sequence of both members of the doublet gave identical results: VTLNPSDPEHCQ. This provided additional evidence that differences between the doublet constituents were only of carbohydrate composition and established the position of this peptide within the vWF polypeptide chain of approximately 2050 amino acid residues as beginning with the residue tentatively designated 449. These studies suggest that native conformation is not necessary for binding of vWF to platelets at the glycoprotein Ib receptor and that a linear amino acid sequence following residue 449 defines a domain responsible for this interaction.