Localization of sites through which C-reactive protein binds and activates complement to residues 14-26 and 76-92 of the human C1q A chain.

Studies were initiated to localize the C-reactive protein (CRP) binding site on the collagen-like region (CLR) of C1q. CRP bound preferentially to the A chain of reduced C1q, in contrast to aggregated immunoglobulin G (Agg-IgG), which reacted preferentially with the C chain. A group of C1q A chain peptides, including peptides identical to residues 81-97, 76-92, and 14-26, respectively, were synthesized from predicted binding regions. Peptide 76-92 contained two proximal lysine groups, and peptide 14-26 contained four proximal arginine groups. CRP-trimers and CRP-ligand complexes did not bind to immobilized peptide 81-97, but bound avidly to immobilized peptides 76-92 and 14-26. Agg-IgG did not bind to any of the peptides. Peptide 76-92 partially, and peptide 14-26 completely, inhibited binding of CRP to intact C1q. Peptide 14-26 also blocked C consumption initiated by CRP, but not by IgG. Replacement of the two prolines with alanines, or scrambling the order of the amino acids, resulted in loss of ability of peptide 14-26 to inhibit C1q binding and C activation by CRP, indicating a sequence specificity, and not a charge specificity alone, as the basis for the inhibitory activity of the peptide. Similar investigations with scrambled peptides showed a sequence specificity for the effects of peptide 76-92 as well. DNA and heparin inhibited binding of CRP trimers to intact C1q, as well as to each peptide 14-26 and 76-92, suggesting involvement of these regions in C1q-CLR binding reactions generally. Collectively, these data identify two cationic regions within residues 14-26 and 76-92 of the C1q A chain CLR as sites through which CRP binds and activates the classical C pathway, and suggest that these residues represent significant regions for C1q CLR binding reactions generally. To our knowledge, this represents the first delineation of sites on C1q through which binding and activation of the classical C pathway can occur.

C-reactive protein mediates the solubilization of nuclear DNA by complement in vitro.

We have studied the interaction of C-reactive protein (CRP)-chromatin complexes with serum. The amount of chromatin solubilized by serum is directly proportional to the amount of CRP present. Serum minus C3 did not appreciably solubilize chromatin within the time allowed in these experiments regardless of the amount of CRP present. This indicates that, in addition to CRP, complement is critical to the solubilization process. Studies using genetically C2-deficient serum and purified C2 indicate that the classical complement pathway is primarily involved in the solubilization, however, there may be minor involvement by the alternative pathway. We used an enzyme-linked immunosorbent assay to determine the amounts of CRP in plasma from eight patients with systemic lupus erythematosus; two of the eight had levels of CRP far lower than previously reported for normal individuals, and an additional sample had antibodies reactive with CRP. Together, these results suggest that one of the functions of CRP is to mediate the removal of exposed nuclear DNA by complement-dependent solubilization of chromatin. A defect in this mechanism could (a) facilitate the production of antibodies against chromatin components exposed due to tissue damage or (b) contribute to immune complexes containing the chromatin components released from damaged tissue because they are not rapidly cleared.

Identification of homologous regions in human immunodeficiency virus I gp41 and human MHC class II beta 1 domain. I. Monoclonal antibodies against the gp41-derived peptide and patients' sera react with native HLA class II antigens, suggesting a role for autoimmunity in the pathogenesis of acquired immune deficiency syndrome.

Homologous regions of five amino acids each, were identified in the NH2-terminal domain of human class II beta chains and the COOH terminus of HIV I envelope protein. The homologous regions are highly conserved among different DR and DQ alleles and also among different isolates of HIV. Septamers containing these sequences were synthesized and used for the generation of murine mAbs. The mAbs selected for this study were raised against the HIV I-derived peptide and reacted strongly not only with the immunizing peptide, but also with the homologous class II-derived peptide. These mAbs also reacted with native MHC class II antigens expressed on human B cell lines and on murine fibroblast L cell lines transfected with the genes coding for the alpha and beta chains of human class II antigens. Furthermore, sera from 36% of AIDS patients tested contained antibodies that reacted with the class II-derived peptide, as well as with intact class II molecule-rich cell extracts. Such antibodies in HIV I-infected individuals may recognize self class II antigens, triggering autoimmune mechanisms that could contribute to the development of immunodeficiency in AIDS patients.

Vaccination with a synthetic zona pellucida peptide produces long-term contraception in female mice.

The zona pellucida surrounding mouse oocytes is an extracellular matrix composed of three sulfated glycoproteins, ZP1, ZP2, and ZP3. It has been demonstrated that a monoclonal antibody to ZP3 injected into female mice inhibits fertilization by binding to the zona pellucida and blocking sperm penetration. A complementary DNA encoding ZP3 was randomly cleaved and 200- to 1000-base pair fragments were cloned into the expression vector lambda gt11. This epitope library was screened with the aforementioned contraceptive antibody, and the positive clones were used to map the seven-amino acid epitope recognized by the antibody. Female mice were immunized with a synthetic peptide containing this B cell epitope coupled to a carrier protein to provide helper T cell epitopes. The resultant circulating antibodies to ZP3 bound to the zona pellucida of immunized animals and produced long-lasting contraception. The lack of ovarian histopathology or cellular cytotoxicity among the immunized animals may be because of the absence of zona pellucida T cell epitopes in this vaccine.

YIGSR, a synthetic laminin pentapeptide, inhibits experimental metastasis formation.

The invasion of tumor cells through basement membranes is a critical step in the formation of metastases. The binding of the malignant cells to laminin in the basement membranes allows their attachment and activates their invasiveness. Recently a synthetic nonapeptide from the B1 chain sequence of laminin was identified as a major site for cell binding. A pentapeptide within the nonapeptide sequence was found to reduce the formation of lung colonies in mice injected with melanoma cells and also to inhibit the invasiveness of the cells in vitro.

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.

Bone sialoprotein peptides are potent inhibitors of breast cancer cell adhesion to bone.

Bone and bone marrow are important sites of metastasis formation in breast cancer. Extracellular matrix proteins with attachment properties are generally believed to play a key role in tumorigenesis and metastasis formation. We have investigated whether mammary carcinoma cells (MDA-MB-231) can recognize constructs of the fairly bone-specific human bone sialoprotein, which encompass the RGD sequence (EPRGD-NYR). Exogenously added bone sialoprotein peptides with this amino acid sequence in their backbone structure, but not the more common fibronectin-derived GRGDS peptide, strongly inhibited breast cancer cell adhesion to extracellular bone matrix at micromolar concentrations. Most cyclic derivatives with the EPRGDNYR sequence were more effective inhibitors of tumor cell adhesion to bone than their linear equivalents. Furthermore, changes in the RGD-tripeptide of the backbone structure of the constructs, removal of the NYR flanking sequence, or a different tertiary cyclic structure significantly decreased their inhibitory potencies. In addition, the RGE-analogue EPRGENYR was capable of inhibiting breast cancer cell adhesion to bone, albeit to a lesser extent. We conclude therefore, that the inhibitory potency of the bone sialoprotein-derived peptides on breast cancer cell adhesion to bone is not solely due to a properly positioned RGD-motif alone but is also determined by its flanking regions, together with the tertiary structure of the EPRGDNYR peptide. Synthetic cyclic constructs with the EPRGDNYR sequence may, therefore, be potentially useful as antiadhesive agents for cancer cells to bone in vivo.

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.

A new sensitive assay for the calcium-dependent binding of C-reactive protein to phosphorylcholine.

A new sensitive assay for the calcium-dependent binding of rabbit C-reactive protein to phosphorylcholine has been developed. The assay involves coating the wells of polyvinylchloride microtiter plates with a bovine serum albumin-phosphorylcholine conjugate followed by the addition of C-reactive protein. The quantity of C-reactive protein is determined by indirect enzyme-linked immunoadsorbent assay using first, affinity purified goat anti-C-reactive protein immunoglobulin followed by a commercial rabbit anti-goat immunoglobulin-alkaline phosphatase conjugate. The binding of rabbit C-reactive protein to the bovine serum albumin-phosphorylcholine conjugate is completely inhibited by free phosphorylcholine, by pneumococcal C-polysaccharide and by calcium chelators but not by high concentrations of neutral, cationic or zwitterionic detergents. The assay is sensitive to 2 ng C-reactive protein.

Synthesis and study of a spin-labeled cyclophosphamide analogue, 3-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide.

3-(1-Oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (7) was isolated in 36% yield following H2O2-Na2WO4 oxidation of 3-(2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (6), which was synthesized in three steps (25% yield) starting with 4-amino-2,2,6,6-tetramethylpiperidine. Binding of 7 to mouse liver microsomes was investigated by optical and electron spin resonance spectroscopy. Compared with the mouse liver microsomal metabolism of 1, separate incubations of 6 and an ca. 1:1 mixture of 1 and 6 gave approximately 90 and 60% less acrolein, respectively. A spin-labeled metabolite of 7, viz., N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)phosphoramide mustard (9), was synthesized and its intramolecular O-alkylation at pH 7.4, 37 degrees C, was studied by 31P NMR spectroscopy. Compounds 7 and 9 were inactive in screening tests against L1210 lymphoid leukemia in mice.

Specific interaction of conformational polypeptides derived from HIV gp120 with human T lymphocyte CD4 receptor.

Specifically cross-linked peptides (peptomers) have been prepared from the repeating sequences of the C4 domains of glycoproteins 120 present in different isolates of human immunodeficiency virus (HIV). In order to investigate if the HIV C4 peptomers could function as gp120 protein, we have used a novel protein-binding assay to examine if and which components of the peptomers could interact with CD4 receptor in vitro. Here, we demonstrate that all the polymeric components of the HIV-1 C4 peptomer could bind to recombinant soluble CD4 protein. A similar result was also obtained with HIV-2 C4 peptomer except that the binding occured only in those of constituents having molecular weights higher than that of trimer. Remarkably, the CD4-binding was demonstrated to be specific to the HIV C4 peptomers as it did not occur with control peptomers such as Poly V3 MN and Poly NINA whose peptide sequences bore no homology to those of the HIV C4 peptomers. Furthermore, consistent with previous findings, no interaction of HIV-1 C4 monomeric peptide (419-436) with CD4 was detected under the same conditions. Since it is known that the HIV C4 peptomers have much higher contents of alpha-helical conformation than those of their monomeric peptides, we conclude that the secondary structure is a pivotal determinant for the successful CD4-binding by the peptomers. Our finding reveals a more defined molecular nature of the gp120-CD4 interaction and may be important for designing HIV vaccines and therapeutics which target the first step in the virus infection.

A defined conformational epitope from the C4 domain of HIV type 1 glycoprotein 120: anti-cyclic C4 antibodies from HIV-positive donors magnify glycoprotein 120 suppression of interleukin 2 produced by T cells.

The C4 domain of HIV gp120 plays a functionally vital role in the binding of gp120 to CD4 receptors on target cells. Antibodies to an 11-amino acid cyclic C4 peptide were obtained from immunized rabbits and from the serum of an HIV-positive human and were found to recognize gp120 bound to CD4. Anti-cyclic C4 antibodies magnified gp120-induced suppression of IL-2 produced by T cells in vitro. Rabbit antibodies to the 11-amino acid linear C4 peptide did not recognize gp120 in the free state or when bound to CD4. These results indicate that a conformationally defined, highly conserved epitope in the gp120 C4 region remains exposed on CD4 binding. Naturally occurring antibodies to this epitope can augment gp120-induced immunosuppression and may contribute to disease progression.

A conformational C4 peptide polymer vaccine coupled with live recombinant vector priming is immunogenic but does not protect against rectal SIV challenge.

The conserved, immunogenic CD4 binding site on the viral envelope is an attractive HIV or SIV vaccine candidate. Polymerization of an 18 amino acid segment derived from the C4 domain of SIV gp120 produced a peptide polymer or "peptomer," having an alpha-helical conformation possibly mimicking a proposed structure of the C4 domain in the unbound native protein. The SIV peptomer and native gp120 were compared as subunit boosts following two adenovirus type 5 host range (Ad5hr)-SIVenv recombinant priming immunizations. Both vaccine regimens successfully elicited SIV-specific CTL responses in five of six immunized macaques. Peptomer-boosted macaques exhibited significantly higher envelope-specific T cell proliferative responses than either the gp120-boosted macaques or controls. Peptomer immunization also elicited peptomer and SIV gp120-specific binding antibodies, but only native gp120 boosting elicited SIV neutralizing antibodies. Upon intrarectal challenge with SIVmac32H, all nine macaques became infected. The solely envelope-based vaccine conferred no protection. However, changing the boosting immunogen to the C4 peptomer did not improve protective efficacy in spite of its elicitation of humoral and cellular immune responses, including robust T-helper activity. In spite of the peptomer's strong immunogenicity and potential for induction of broadly protective immune responses, it was not effective as a subunit vaccine.

Tyrosine phosphorylation induced by C4 peptide constructs from HIV Gp120.

Treatment of HUT78 cells with CD4-binding peptide constructs derived from the C4 domain of HIV-1 gp120 results in autophosphorylation of a src-related kinase, p56lck. This leads to p56lck activation and the subsequent phosphorylation of tyrosine residues in several intracellular proteins. The phosphorylation is specific to the C4 peptides as no new phosphorylation occurs when the cells are treated with control peptides or polymers. The induction of tyrosine phosphorylation by the C4 peptide constructs depends on the capability of the peptide to assume a helical conformation because similar peptide constructs that were not able to form helices did not induce cellular tyrosine phosphorylation.

Synthetic integrin-binding peptides promote adhesion and proliferation of human periodontal ligament cells in vitro.

Periodontal ligament (PDL) cells have been shown to express several integrins (alphav, alpha5, beta1, beta3) that use RGD (arginine-glycine-aspartic Acid)-dependent mechanisms for the recognition and binding of their ligands. The objective of this study was to evaluate the effects of certain integrin-binding cyclic and linear synthetic RGD-containing peptides on PDL cells' adhesion, proliferation, and de novo protein synthesis in vitro. Fifth passages of normal human PDL cells established from teeth extracted from patients (ages 12 to 14) for orthodontic reasons were used for all experiments. Synthetic peptides containing the EPRGDNYR sequence in two different spatial conformations (linear and cyclic) were covalently attached to bovine serum albumin (BSA). Type I collagen, EPRGDNYR-BSA conjugates, 1:1 mixtures of type I collagen and conjugates, as well as BSA (a negative control) were coated on bacteriological plastic and evaluated for their attachment-promoting activities. In addition, the effects of these substrates on cell proliferation were evaluated by [3H]thymidine incorporation by the PDL cells. For attachment and spreading, the cyclic forms of EPRGDNYR-BSA conjugate and type I collagen were most potent, followed by linear EPRGDNYR-BSA conjugate. The effects of all collagen/conjugate mixtures were equivalent to that of type I collagen except for the collagen/linear EPRGDNYR-BSA mixture, which was less potent. The cyclic EPRGDNYR-BSA conjugate was the most effective substrate to stimulate cell proliferation, and it was followed in potency by the linear peptide-BSA conjugate. Collagen alone did not stimulate [3H]thymidine incorporation above the control level. Mixtures of collagen with all of the conjugates showed stimulatory effects similar to that of the cyclic peptide-BSA conjugate. No significant differences in de novo protein synthesis were detected. These results suggest that the synthetic RGD-containing peptides attached to a carrier are potent ligands for the human PDL cells, and that they could provide a basis for the development of new strategies aimed at the regeneration of the periodontium.