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.

Selective and facile cyclization of N-chloroacetylated peptides from the C4 domain of HIV Gp120 in LiCl/DMF solvent systems.

Lithium salts have been reported to mediate the solubilization of peptides in organic solvents in 1989 (Seebach, D., Thaler, A. & Beck, A. K. Helv. Chim. Acta 1989; 72, 857-867). The use of Li salts in an organic solvent to influence cyclization of a reactive peptide that only polymerizes in an aqueous solvent, has not been reported. Here, the selective and facile cyclization of N-chloroacetylated, C-cysteine amide peptides from the C4 domain of HIV-1 gp120 in LiCl/DMF solvent systems is demonstrated. The addition of stoichiometric amounts of Tris base to 1 mg/mL peptide in LiCl/DMF solutions was sufficient to drive the cyclization to completion within 3 h at ambient temperatures. Cyclic peptides were the only detectable reaction products and these were confirmed using reversed-phase HPLC and mass spectrometric analyses of the final products. In aqueous solutions at pH 7.4, only polymers were obtained as judged by HPLC and SDS-PAGE. The method of using Li salts in an organic solvent to enhance the cyclization of unprotected amphipathic peptides may be useful in many situations beyond those described here.

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.

Immunization of mice with peptomers covalently coupled to aluminum oxide nanoparticles.

Subunit vaccines generally require adjuvants to elicit immune responses, but adjuvants may alter the conformation of critical epitopes and reduce vaccine efficacy. We therefore tested an immunization strategy in which antigen is covalently coupled to aluminum oxide nanoparticles using a method that favors preservation of the native conformation. The test antigen consisted of "peptomers" (head-to-tail-linked peptide homopolymers) derived from the 4th conserved region (C4) of HIV-1 gp120 which is believed to be in an alpha-helical conformation prior to binding to CD4. Immune responses in mice to peptomer-nanoparticle conjugates were compared to responses elicited by free C4 peptide and C4 peptomers, with and without the hydrophilic adjuvant muramyl dipeptide (MDP). Highest peptomer-specific serum antibody responses were induced by peptomer-particles without MDP. Serum antibodies induced by peptomer-particles also showed highest reactivity towards recombinant, glycosylated gp120 and HIV-1 infected T cells. The results suggest that this novel vaccine approach could be useful for induction of immune responses against conformation-sensitive viral antigens without the need for additional adjuvants.

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.

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.

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.

Peptomer aluminum oxide nanoparticle conjugates as systemic and mucosal vaccine candidates: synthesis and characterization of a conjugate derived from the C4 domain of HIV-1MN gp120.

Peptomers are polymers composed of peptides that are specifically cross-linked in a head-to-tail fashion. Recently, a peptomer composed of an amphipathic peptide from the C4 domain of HIV-1MN gp120 was shown to display a prominent alpha-helical conformation that, as an immunogen, elicited rabbit antibodies recognizing native and recombinant gp120 [Robey et al. (1995) J. Biol. Chem. 270, 23918-23921]. For the present study, we synthesized a conjugate composed of the C4 peptomer covalently linked to calcinated aluminum oxide nanoparticles. The nanoparticles were first reacted with (3-aminopropy])-triethoxysilane to provide an amine load of 15.9 mmol of R-NH2/g of solid. The amine-modified aluminum oxide nanoparticles then were reacted with N-acetylhomocysteine thiolactone at pH 10 to place a reactive thiol on the nanoparticles. A bromoacetylated C4 peptomer, modified at the epsilon-amines of lysine residues, then was reacted with the thiolated nanoparticles to give the peptomer covalently linked to aluminum oxide via a thioether bond. The peptomer load was determined to be 16 mg of peptomer/g of particles, a 55% theoretical yield. Particle shape and size of the peptomer-conjugated alumina were analyzed by electron microscopy and displayed a mean maximum diameter of 355 nm and a mean minimum diameter of 113 nm, well within the desired size range of 300 nm believed to be optimal for mucosal immunization purposes. Experimentally determined values of mean particle diameters, specific surface area, and specific peptomer load provided the information necessary to calculate the mean antigen load, which was determined to be 53000 +/- 42000 peptomer epitopes per particle. Peptomer-alumina conjugates, such as that described here, could form the basis of a new class of biomaterial that combines a chemically defined organic immunogen with a nontoxic chemically defined inorganic adjuvant.

Effective use of free thiols as scavengers for HF cocktails to deprotect bromo- and chloroacetylated synthetic peptides.

A variety of thiol-containing compounds, in combination with m-cresol, were tested as scavengers in hydrogen fluoride (HF) cocktails that are used to deprotect haloacetylated peptidyl resins. Our results indicate that brome and chloroacetyl moieties on a synthetic peptide remain intact following HF treatment when the HF cocktail contains m-cresol along with either thiophenol, m-thiocresol or 1,2-ethanedithiol. The free thiols prevent the formation of a number of impurities in the preparation of bromo- and chloroacetylated peptides that contain amino acids that could be oxidized in a nonreducing HF environment. Ethvimethylsulfide, however, could not be used with bromoacetylated peptides, but it could be used with chloroacetylated peptides.

Ligand-binding sites in human serum amyloid P component.

Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes. AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly and specifically to certain glycosaminoglycans that are components of amyloid deposits, AP may play an important role in the maintenance of amyloid. In the present work, we isolated and identified two proteolytic fragments of AP that are responsible for its heparin-binding activity. Neither fragment corresponds to published heparin-binding sequences. The structural requirements for activity of the peptides (amino acid residues 27-38 and 192-203 of AP) were examined by means of solid-phase inhibition assays with synthetic peptides. AP-(192-203)-peptide inhibits the Ca(2+)-dependent binding of AP to heparin with an IC50 of 25 microM, while the IC50 of AP-(27-38)-peptide and AP-(33-38)-peptide are 10 microM and 2 microM, respectively. The understanding of the structure and function of active AP peptides will be useful for development of amyloid-targeted diagnostics and therapeutics.

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 alpha-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% alpha-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 microM. These results indicate that C4 constructs from HIV-1 and HIV-2 do bind CD4, but the constructs must display an alpha-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.

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.

Long-term sun exposure alters the collagen of the papillary dermis. Comparison of sun-protected and photoaged skin by northern analysis, immunohistochemical staining, and confocal laser scanning microscopy.

BACKGROUND: Long-term solar irradiation produces both morphologic and functional changes in affected skin. Because collagen is the major structural component of skin, any alteration in its production or degradation could have profound effects on cutaneous functional integrity. OBJECTIVE: Our purpose was to investigate alterations in the production and morphology of collagen fibers brought about by long-term sun exposure. METHODS: We compared collagen and collagenase gene expression and collagen immunohistochemical staining and used confocal laser scanning microscopy for morphologic examination of dermal collagen fibers in photodamaged compared with sun-protected skin from the same persons. RESULTS: Despite a large increase in elastin messenger RNA in sun-damaged skin, collagen and collagenase gene expression remained essentially unchanged. However, striking alterations in the papillary dermis of photoaged skin were found, which revealed large, abnormally clumped elastic fibers and deformed collagen fibers of various diameters, replacing the normal architecture of the papillary dermis. CONCLUSION: Our data provide evidence for normal collagen gene expression in sun-damaged skin and suggest that degradation and remodeling of collagen take place in the papillary dermis accompanied by deposition of other matrix components, predominantly abnormal elastic fibers.

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 C4's 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-1MN was synthesized for use as a conformationally constrained immunogen. Circular dichroism studies disclosed that the polymerized peptide, peptomer-(419-436), in 0.01 M Na2HPO4 buffer, pH 7.4, at 25 degrees C contained a dominant alpha-helical structure (53 +/- 1%) compared with 2 +/- 4% alpha-helical content for the monomeric peptide-(419-436). The peptomer in Ribi's 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 alpha-helix in gp120.

Synthesis and use of a new bromoacetyl-derivatized heterotrifunctional amino acid for conjugation of cyclic RGD-containing peptides derived from human bone sialoprotein.

A new amino acid derivative, N alpha-(tert-butyloxycarbonyl)-N beta-(bromoacetyl)diaminopropionic acid (BBDap), has been synthesized as a reagent for introducing side-chain bromaocetyl groups into any position of a peptide sequence during solid-phase peptide synthesis. By using minor modifications to the protocol of the automated peptide synthesizer and a two-step in situ neutralization procedure, the syntheses of (bromoacetyl)diaminopropionic acid (BDap) in Arg-Gly-Asp-containing peptides from human bone sialoprotein were optimized and completed. Following HPLC purification, the BDap-derivatized peptides were cyclized or/and conjugated to carrier protein or to glass cover slips. In addition, a new procedure for site-specific conjugation of cyclic peptides to protein carriers or to glass was developed. The cell attachment activity of the peptide derivatives and conjugates was tested in cell adhesion assays with human osteoblasts, and the specificity of the binding was confirmed by competition with linear and/or cyclic forms of GRGDS. The results show that conjugates containing the linear and cyclic derivatives of the peptide EPRGDNYR supported cell attachment and spreading in a dose-dependent manner when the peptides were immobilized as described. Cell attachment to the intact bone sialoprotein and to conjugates containing the linear peptides was abolished by competition with linear and cyclic RGD-containing peptides, whereas the attachment to conjugates containing the cyclic peptide was inhibited only partially, and the cell spreading was preserved even in the presence of RGD-peptides.

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.

DNA binds and activates complement via residues 14-26 of the human C1q A chain.

The mechanism by which DNA activates the classical complement pathway was investigated, with emphasis upon the C1q binding sites involved. DNA bound to both the collagen-like and globular regions of C1q. Binding reactivity with DNA was retained after reduction/alkylation and sodium dodecyl sulfate treatment of C1q. DNA bound preferentially to the A chain of C1q. Binding sites for DNA were localized by using synthetic C1q A chain peptides to two cationic regions within residues 14-26 and 76-92, respectively. Peptides 14-26 and 76-92 avidly bound DNA in enzyme-linked immunosorbent and gel shift assays. Peptide 14-26 also precipitated with DNA and blocked its ability to bind C1q and activate C. 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 complement activation by DNA; similar investigations showed a sequence specificity for peptide 76-92 as well. These experiments identify C1q A chain residues 14-26 as the major site, and residues 76-92 as a secondary site, through which DNA binds C1q and activates the classical complement pathway, and demonstrate that a peptide identical to residues 14-26 can modulate C1q binding and complement activation by DNA.

Use of capillary zone electrophoresis to evaluate the binding of anionic carbohydrates to synthetic peptides derived from human serum amyloid P component.

Capillary zone electrophoresis was used to study interactions between anionic carbohydrates and synthetic peptides derived from the heparin-binding region of human serum amyloid P component. The method involves quantitation of unbound peptides after a charge-dependent electrophoretic separation of the peptide-carbohydrate mixture. The concentrations of free peptide were determined by extrapolating the obtained peak areas of the peptide in the presence of ligand to a standard curve. Dissociation constants in the 10(-5) M range were determined, and differences in binding affinity of various peptide modifications were illustrated. The assay requires minute amounts of material (sample volume is 7-15 nL), and as long as the reactants are soluble at the chosen conditions, no modifications or special characteristics of the interacting molecules are needed for their identification. It should be possible to use electrophoretic separation in capillaries to evaluate the binding of peptides to any ligand as long as the differences in charge/mass ratio between free and complexed peptide are of a sufficient magnitude as in the peptide-heparin binding demonstrated here.

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.