(1/1393) A hyperstable collagen mimic.

BACKGROUND: Collagen is the most abundant protein in animals. Each polypeptide chain of collagen is composed of repeats of the sequence: Gly-X-Y, where X and Y are often L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp) residues, respectively. These chains are wound into tight triple helices of great stability. The hydroxyl group of Hyp residues contributes much to this conformational stability. The existing paradigm is that this stability arises from interstrand hydrogen bonds mediated by bridging water molecules. This model was tested using chemical synthesis to replace Hyp residues with 4(R)-fluoro-L-proline (Flp) residues. The fluorine atom in Flp residues does not form hydrogen bonds but does elicit strong inductive effects. RESULTS: Replacing the Hyp residues in collagen with Flp residues greatly increases triple-helical stability. The free energy contributed by the fluorine atom in Flp residues is twice that of the hydroxyl group in Hyp residues. The stability of the Flp-containing triple helix far exceeds that of any untemplated collagen mimic of similar size. CONCLUSIONS: Bridging water molecules contribute little to collagen stability. Rather, collagen stability relies on previously unappreciated inductive effects. Collagen mimics containing fluorine or other appropriate electron-withdrawing substituents could be the basis of new biomaterials for restorative therapies.  (+info)

(2/1393) Inhibition of L-selectin-mediated leukocyte rolling by synthetic glycoprotein mimics.

Synthetic carbohydrate and glycoprotein mimics displaying sulfated saccharide residues have been assayed for their L-selectin inhibitory properties under static and flow conditions. Polymers displaying the L-selectin recognition epitopes 3',6-disulfo Lewis x(Glc) (3-O-SO3-Galbeta1alpha4(Fucalpha1alpha3)-6-O-SO3-Glcbeta+ ++-OR) and 3',6'-disulfo Lewis x(Glc) (3, 6-di-O-SO3-Galbeta1alpha4(Fucalpha1alpha3)Glcbeta-OR) both inhibit L-selectin binding to heparin under static, cell-free binding conditions with similar efficacies. Under conditions of shear flow, however, only the polymer displaying 3',6-disulfo Lewis x(Glc) inhibits the rolling of L-selectin-transfected cells on the glycoprotein ligand GlyCAM-1. Although it has been shown to more effective than sialyl Lewis x at blocking the L-selectin-GlyCAM-1 interaction in static binding studies, the corresponding monomer had no effect in the dynamic assay. These data indicate that multivalent ligands are far more effective inhibitors of L-selectin-mediated rolling than their monovalent counterparts and that the inhibitory activities are dependent on the specific sulfation pattern of the recognition epitope. Importantly, our results indicate the L-selectin specificity for one ligand over another found in static, cell-free binding assays is not necessarily retained under the conditions of shear flow. The results suggest that monovalent or polyvalent carbohydrate or glycoprotein mimetics that inhibit selectin binding in static assays may not block the more physiologically relevant process of selectin-mediated rolling.  (+info)

(3/1393) Chlamydia infections and heart disease linked through antigenic mimicry.

Chlamydia infections are epidemiologically linked to human heart disease. A peptide from the murine heart muscle-specific alpha myosin heavy chain that has sequence homology to the 60-kilodalton cysteine-rich outer membrane proteins of Chlamydia pneumoniae, C. psittaci, and C. trachomatis was shown to induce autoimmune inflammatory heart disease in mice. Injection of the homologous Chlamydia peptides into mice also induced perivascular inflammation, fibrotic changes, and blood vessel occlusion in the heart, as well as triggering T and B cell reactivity to the homologous endogenous heart muscle-specific peptide. Chlamydia DNA functioned as an adjuvant in the triggering of peptide-induced inflammatory heart disease. Infection with C. trachomatis led to the production of autoantibodies to heart muscle-specific epitopes. Thus, Chlamydia-mediated heart disease is induced by antigenic mimicry of a heart muscle-specific protein.  (+info)

(4/1393) Crossreactive recognition of viral, self, and bacterial peptide ligands by human class I-restricted cytotoxic T lymphocyte clonotypes: implications for molecular mimicry in autoimmune disease.

The immunodominant, CD8(+) cytotoxic T lymphocyte (CTL) response to the HLA-B8-restricted peptide, RAKFKQLL, located in the Epstein-Barr virus immediate-early antigen, BZLF1, is characterized by a diverse T cell receptor (TCR) repertoire. Here, we show that this diversity can be partitioned on the basis of crossreactive cytotoxicity patterns involving the recognition of a self peptide-RSKFRQIV-located in a serine/threonine kinase and a bacterial peptide-RRKYKQII-located in Staphylococcus aureus replication initiation protein. Thus CTL clones that recognized the viral, self, and bacterial peptides expressed a highly restricted alphabeta TCR phenotype. The CTL clones that recognized viral and self peptides were more oligoclonal, whereas clones that strictly recognized the viral peptide displayed a diverse TCR profile. Interestingly, the self and bacterial peptides equally were substantially less effective than the cognate viral peptide in sensitizing target cell lysis, and also resulted only in a weak reactivation of memory CTLs in limiting dilution assays, whereas the cognate peptide was highly immunogenic. The described crossreactions show that human antiviral, CD8(+) CTL responses can be shaped by peptide ligands derived from autoantigens and environmental bacterial antigens, thereby providing a firm structural basis for molecular mimicry involving class I-restricted CTLs in the pathogenesis of autoimmune disease.  (+info)

(5/1393) Synergism with germ line transcription factor Oct-4: viral oncoproteins share the ability to mimic a stem cell-specific activity.

Activation of transcription by Oct-4 from remote binding sites requires a cofactor that is restricted to embryonal stem cells. The adenovirus E1A protein can mimic the activity of this stem cell-specific factor and stimulates Oct-4 activity in differentiated cells. Here we characterize the Oct-4-E1A interaction and show that the E1A 289R protein harbors two independent Oct-4 binding sites, both of which specifically interact with the POU domain of Oct-4. Furthermore, we demonstrate that, like E1A, the human papillomavirus E7 oncoprotein also specifically binds to the Oct-4 POU domain. E7 and Oct-4 can form a complex both in vitro and in vivo. Expression of E7 in differentiated cells stimulates Oct-4-mediated transactivation from distal binding sites. Moreover, Oct-4, but not other Oct factors, is active when expressed in cells transformed by human papillomavirus. Our results suggest that different viruses have evolved oncoproteins that share the ability to target Oct-4 and to mimic a stem cell-specific activity.  (+info)

(6/1393) A kinetic mechanism for the polymerization of alpha1-antitrypsin.

The mutation in the Z deficiency variant of alpha1-antitrypsin perturbs the structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepatocytes to form inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The process of polymer formation has been investigated here by intrinsic tryptophan fluorescence, fluorescence polarization, circular dichroic spectra and extrinsic fluorescence with 8-anilino-1-naphthalenesulfonic acid and tetramethylrhodamine-5-iodoacetamide. These biophysical techniques have demonstrated that alpha1-antitrypsin polymerization is a two-stage process and have allowed the calculation of rates for both of these steps. The initial fast phase is unimolecular and likely to represent temperature-induced protein unfolding, while the slow phase is bimolecular and associated with loop-sheet interaction and polymer formation. The naturally occurring Z, S, and I variants and recombinant site-directed reactive loop and shutter domain mutants of alpha1-antitrypsin were used to demonstrate the close association between protein stability and rate of alpha1-antitrypsin polymerization. Taken together, these data allow us to propose a kinetic mechanism for alpha1-antitrypsin polymer formation that involves the generation of an unstable intermediate, which can form polymers or generate latent protein.  (+info)

(7/1393) C1qRP is a heavily O-glycosylated cell surface protein involved in the regulation of phagocytic activity.

C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) interact with human monocytes and macrophages, resulting in the enhancement of phagocytosis of suboptimally opsonized targets. mAbs that recognize a cell surface molecule of 126,000 Mr, designated C1qRP, have been shown to inhibit C1q- and MBL-mediated enhancement of phagocytosis. Similar inhibition of the SPA-mediated enhancement of phagocytosis by these mAbs now suggests that C1qRP is a common component of a receptor for these macromolecules. Ligation of human monocytes with immobilized R3, a IgM mAb recognizing C1qRP, also triggers enhanced phagocytic capacity of these cells in the absence of ligand, verifying the direct involvement of this polypeptide in the regulation of phagocytosis. While the cDNA for C1qRP encodes a 631 amino acid membrane protein, Chinese hamster ovary cells transfected with the cDNA of the C1qRP coding region express a surface glycoprotein with the identical 126,000 Mr in SDS-PAGE as the native C1qRP. Use of glycosylation inhibitors, cleavage of the mature C1qRP with specific glycosidases, and in vitro translation of C1qRP cDNA demonstrated that both posttranslational glycosylation and the nature of the amino acid sequence of the protein contribute to the difference between its predicted m.w. and its migration on SDS-PAGE. These results verify that the cDNA cloned codes for the mature C1qRP, that C1qRP contains a relatively high degree of O-linked glycoslyation, and that C1qRP cross-linked directly by monoclonal anti-C1qRP or engaged as a result of cell surface ligation of SPA, as well as C1q and MBL, enhances phagocytosis.  (+info)

(8/1393) Endothelial cytotoxicity mediated by serum antibodies to heat shock proteins of Escherichia coli and Chlamydia pneumoniae: immune reactions to heat shock proteins as a possible link between infection and atherosclerosis.

BACKGROUND: Growing evidence suggests that an immunological reaction against heat shock proteins (HSPs) may be involved in atherogenesis. Because HSPs show a high degree of amino acid sequence homology between different species, from prokaryotes to humans, we investigated the possibility of "antigenic mimicry" caused by an immunological cross-reaction between microorganisms and autoantigens. METHODS AND RESULTS: Serum antibodies against the Escherichia coli HSP (GroEL) and the 60-kDa chlamydial HSP (cHSP60) from subjects with atherosclerosis were purified by use of affinity chromatography. Western blot analyses and competitive ELISAs confirmed the cross-reaction of the eluted antibodies with human HSP60 and the bacterial counterparts. The cytotoxicity of anti-GroEL and anti-cHSP60 antibodies was determined on human endothelial cells labeled with 51Cr. A significant difference (40% versus 8%) was observed in the specific 51Cr release of heat-treated (42 degrees C for 30 minutes) and untreated cells, respectively, in the presence of these anti-HSP antibodies and complement. This effect was blocked by addition of 100 microg/mL recombinant GroEL. In addition, seropositivity against specific non-HSP60 Chlamydia pneumoniae antigens is more prominent among high-anti-HSP titer sera than low-titer sera. CONCLUSIONS: Serum antibodies against HSP65/60 cross-react with human HSP60, cHSP60, and GroEL; correlate with the presence of antibodies to C pneumoniae and endotoxin; and mediate endothelial cytotoxicity. These findings suggest that humoral immune reactions to bacterial HSPs, such as cHSP60 and GroEL, may play an important role in the process of vascular endothelial injury, which is believed to be a key event in the pathogenesis of atherosclerosis.  (+info)