Frank A. Robey

Autoimmune disease of the ovary induced by a ZP3 peptide from the mouse zona pellucida.

We describe a novel experimental system in mice for the study of ovarian autoimmune disease, a condition encountered in women with premature ovarian failure. The ovarian autoimmune disease is induced in B6AF1 mice by a 15-amino acid peptide (Cys-Ser-Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln-Ile-His-Gly-Pro-Arg) from mouse ZP3, the sperm-binding component of the zona pellucida that surrounds growing and mature oocytes. Whereas the peptide induces both T cell and antibody responses, adoptive transfer of CD4+ T cell lines derived from affected animals causes oophoritis without observable antibodies to the zona pellucida peptide. The primacy of the T cell response in the pathogenesis of disease is further substantiated by defining oophoritogenic peptides as small as eight amino acids (Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln) that do not elicit an antibody response to the full-length ZP3 peptide. The identification of a well characterized peptide as a causative agent of autoimmune oophoritis should facilitate understanding of the pathogenesis of this T cell-mediated autoimmune disease. Because the proteins of the zona pellucida are conserved among mammals (the mouse and human ZP3 proteins are 67% identical), this murine model may lead to better understanding of the pathogenesis of human autoimmune oophoritis.

Protection against mucosal simian immunodeficiency virus SIV (mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting

ABSTRACT Whereas several recent AIDS vaccine strategies have protected rhesus macaques against a pathogenic simian/human immunodeficiency virus (SHIV)89.6P challenge, similar approaches have provided only modest, transient reductions in viral burden after challenge with virulent, pathogenic SIV, which is more representative of HIV infection of people. We show here that priming with replicating adenovirus recombinants encoding SIV env/rev, gag, and/or nef genes, followed by boosting with SIV gp120 or an SIV polypeptide mimicking the CD4 binding region of the envelope, protects rhesus macaques from intrarectal infection with the highly pathogenic SIVmac251. Using trend analysis, significant reductions in acute-phase and set point viremia were correlated with anti-gp120 antibody and cellular immune responses, respectively. Within immunization groups exhibiting significant protection, a subset (39%) of macaques have exhibited either no viremia, cleared viremia, or controlled viremia at the threshold of detection, now more than 40 weeks postchallenge. This combination prime-boost strategy, utilizing replication competent adenovirus, is a promising alternative for HIV vaccine development.

Automated synthesis of N-bromoacetyl-modified peptides for the preparation of synthetic peptide polymers, peptide−protein conjugates, and cyclic peptides

A method to incorporate N-bromoacetyl moieties at the amino termini of synthetic peptides using a standard program with an automated peptide synthesizer has been developed. The N-bromoacetyl-derivatized peptides react well with sulfhydryl-containing proteins and with peptides containing cysteine residues. Autopolymerization or cyclization occurs by reaction of the free sulfhydryl of cysteine in a peptide with the bromoacetyl group and reactions can generally be controlled by controlling the concentrations of starting peptide in neutral pH buffers. Analytical methods for evaluating the polymers or cyclized peptides include gel filtration chromatography, reverse phase HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis where the degree of reaction can be evaluated by quantifying the amount of S-carboxymethylcysteine formed after HCl hydrolysis. N-Bromoacetyl-derivatized peptides may be useful as reagents for potential peptide immunogens, vaccines, and therapeutics and as intermediates in the production of solid supports with peptide surfaces.

Bone Sialoprotein Mediates Human Endothelial Cell Attachment and Migration and Promotes Angiogenesis

Bone sialoprotein (BSP) is a secreted glycoprotein primarily found in sites of biomineralization. Recently, we demonstrated that BSP is strongly upregulated in osteotropic cancers and particularly those that exhibit microcalcifications. BSP contains an Arg-Gly-Asp (RGD) motif found in other adhesive molecules that interact with cellular integrins. In bone, BSP has been shown to mediate the attachment of osteoblasts and osteoclasts via alpha(v)beta(3) integrin receptors. Ligands for alpha(v)beta(3) integrin are considered to play a central role during angiogenesis. Therefore, we used human umbilical vein endothelial cells (HUVECs) to study the potential role of BSP in angiogenesis. We found that purified eukaryotic recombinant human BSP (rhBSP) is able to promote both adhesion and chemotactic migration of HUVECs in a dose-dependent manner. These interactions involve HUVEC alpha(v)beta(3) integrin receptors and the RGD domain of BSP. Indeed, HUVECs attach to a recombinant BSP fragment containing the RGD domain, whereas this response is not observed with the same fragment in which RGD has been mutated to Lys-Ala-Glu (KAE). A cyclic RGD BSP peptide inhibits both adhesion and migration of HUVECs to rhBSP. Moreover, anti-alpha(v)beta(3) but not anti-alpha(v)beta(5) monoclonal antibodies also prevent BSP-mediated adhesion and migration of HUVECs. We observed that both rhBSP and the RGD BSP recombinant fragment stimulated ongoing angiogenesis on the chorioallantoic chick membrane assay. BSP angiogenic activity was inhibited by anti-alpha(v)beta(3) antibody, and the KAE BSP fragment was inactive. Our findings represent the first report implicating BSP in angiogenesis. BSP could play a critical role in angiogenesis associated with bone formation and with tumor growth and metastatic dissemination.

Use of Capillary Zone Electrophoresis for the Analysis of DNA-BINDING to a Peptide Derived from Amyloid P Component

Abstract Capillary zone electrophoresis was used to characterize the binding interactions between oligonucleotides and synthetic peptides derived from human serum amyloid P component. From a solution containing free and peptide-bound oligonucleotides, free nucleotide was separated from the complex by means of electrophoresis. In this way, both qualitative and quantitative aspects of the binding could be assessed rapidly using minute amounts of unlabelled samples. Minimal structures and sequence specificity of binding of nucleotides and peptides could be characterized and, based on the quantitative output of the electrophoretic analysis, binding constants were estimated. In theory, the approach is applicable for any molecular interaction where the charge/mass ratio of complexes differ from the free molecules and where at least one of the interacting components is quantitatively recoverable and detectable in the capillary electrophoresis system. As such, it is the only method available for simple and fast e...

A capillary electrophoresis-based assay for the binding of Ca2+ and phosphorylcholine to human C-reactive protein

Affinity capillary electrophoresis was performed to quantitate the binding of Ca2+ and phosphorylcholine to human C-reactive protein (CRP). The assay requires no modifications of any of the molecules involved, uses minuscule amounts of protein (8.5 x 10(-15) mol per analysis, i.e., less than 1 pmol for 15 triplicate data points), and the binding could be examined under conditions of physiological ionic strength and pH. The values for the dissociation constants obtained here (KD = 59 microM for Ca(2+)-CRP and 18 microM for the phosphorylcholine-CRP interaction) were in close agreement with previous studies using gel filtration and equilibrium dialysis. As long as one of the reactants can be detected and recovered quantitatively in the capillary electrophoresis system, the method is generally useful to study interactions where complexed molecules display an electrophoretic mobility that is different from that of unbound molecules and where the rates of association and dissociation are sufficiently fast.

Rat Brain Protein Kinase C: Purification, Antibody Production, and Quantification in Discrete Regions of Hippocampus

Abstract: Protein kinase C (PKC), a calcium‐ and phospholipid‐dependent kinase, is highly enriched in rat brain, where it may function in signal transduction processes. We purified rat brain PKC to homogeneity by a three‐column procedure of diethylaminoethyl‐cellulose, phenyl‐Sepharose, and protamine‐agarose with a yield of 16% and a final specific activity of 9,600 pmol of [3H]phorbol‐12,13‐dibutyrate bound/mg of protein. The pure protein consisted of a doublet of 80 and 78 kilodaltons. Rabbit antibodies prepared against a β‐type PKC synthetic peptide sequence (RAKIGQGTKAPEEKTANTISK) showed high specificity and sensitivity for PKC and recognized only the 78‐kilodalton form of PKC. Micropunches (300 μm in diameter) of rat hippocampal subregions were solubilized in sodium dodecyl sulfate (SDS) sample buffer, electrophoresed on SDS–10% polyacrylamide gels, and transferred to nitrocellulose. PKC was visualized by 125I‐protein A autoradiography and quantified by densitometry. The highest concentrations of PKC were found in the CA1 pyramidal cell layer (0.43 ± 0.04 OD), with the lowest amounts in the CA3 and CA4 pyramidal cell layers (0.11 ± 0.02 and 0.085 ± 0.006 OD, respectively). These results demonstrate a simple way of preparing antibodies against domains of PKC. We also describe a procedure for quantifying the relative amounts of PKC in discrete brain regions.

A Synthetic Conformational Epitope from the C4 Domain of HIV Gp120 That Binds CD4

The fourth conserved domain of the human immunodeficiency virus type 1 (HIV-1) envelope, the C4 region of glycoprotein 120 (gp120), is believed to be a major part of gp120 that is necessary for binding to CD4. Recently, we found that C4 in gp120 is probably an α-helix, because antibodies made against helical constructs of C4 react with native and recombinant gp120 but antibodies against linear C4 constructs do not. For the present study, we performed experiments to determine, first, if CD4 could bind to the helical C4 constructs and, second, if the binding was comparable with CD4 binding to gp120. Immobilized helical constructs derived from the C4s from HIV-1 and HIV-2 bound biotinylated recombinant CD4 with Kd values of 8.59 nM and 14.59 nM, respectively. Recombinant soluble CD4 inhibited the binding of biotinylated CD4 to the C4 construct from HIV-1 with a Kd of 9.88 nM, and recombinant gp120 blocked the binding of CD4 to the immobilized helical construct from C4 of HIV-1 with a Kd of 8.08 nM. The C4 peptide-(419-436) from HIV-1 (KIKQIINMWQEVGKAMYA-NH2) blocked CD4 binding to gp120 but only in a buffer containing 0.03% Brij 35 where the peptide displayed 17 ± 1% α-helix; without the Brij 35, peptide-(419-436) displayed no helical content. The Kd for the peptide-(419-436) blocking CD4 binding to gp120 in Brij 35-containing buffer was found to be 42 μM. These results indicate that C4 constructs from HIV-1 and HIV-2 do bind CD4, but the constructs must display an α-helical conformation to do so. In addition, the results reported here will provide answers to key questions about structural requirements for HIV vaccines and therapeutics that hinge on understanding the molecular nature of the gp120-CD4 interaction as the first step in the HIV infection process.

Conjugation of synthetic peptides to proteins: Quantitation from S-carboxymethylcysteine released upon acid hydrolysis

A method described here for conjugating synthetic peptides to carrier proteins provides a convenient method for determining peptide-to-carrier protein ratios. N-Bromoacetyl-containing peptides are reacted in situ with carrier proteins in which the disulfide bonds were reduced with tri-n-butylphosphine. At pH 7-8 and ambient temperature, the newly formed sulfhydryl groups of the carrier protein react exclusively with the bromoacetyl mokiety of the peptide to form conjugates having stable thio ether linkages. Acid hydrolyses of these conjugates release S-carboxymethylcysteine in amounts proportional to the amounts of peptides conjugated and thus allow determination of peptide-to-protein ratios.

A helical epitope in the C4 domain of HIV glycoprotein 120.

The fourth conserved domain of the human immunodeficiency virus type 1 (HIV-1) envelope, the C4 region of glycoprotein 120 (gp120), is an amphipathic stretch of amino acids that, based on mutational analyses, constitutes a major component of the CD4 binding region in gp120. In the absence of crystallographic and NMR data on C4 in intact gp120, we sought to gain insight into C4s conformation and accessibility in gp120 by taking an immunochemical approach. In this study, a peptomer composed of a repeat peptide of C4 amino acids 419-436 from gp120 of HIV-1 was synthesized for use as a conformationally constrained immunogen. Circular dichroism studies disclosed that the polymerized peptide, peptomer-(419-436), in 0.01 M NaHPO buffer, pH 7.4, at 25°C contained a dominant α-helical structure (53 ± 1%) compared with 2 ± 4% α-helical content for the monomeric peptide-(419-436). The peptomer in Ribis adjuvant induced the production of rabbit antibodies that recognized recombinant and native gp120 but, consistent with the literature, the C4 peptide having no conformational constraints did not. The experimental results show that only those antibodies formed against a helical immunogen from C4 will recognize recombinant and native gp120, and, therefore, the results support the notion that C4 is an α-helix in gp120.