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A Brass

Publications and source records attributed to A Brass.

At least 55 records · Page 3Linked to original sources
PubMed1994

Identification of a key integrin-binding sequence in VCAM-1 homologous to the LDV active site in fibronectin.

The integrin adhesion receptor alpha 4 beta 1 binds two ligands, the extracellular matrix glycoprotein fibronectin and the immunoglobulin superfamily member VCAM-1. Ligand-binding sites are contained with the HepII/IIICS domain of fibronectin, and within the homologous immunoglobulin domains 1 and 4 of VCAM-1. Previous studies have shown that the binding of each ligand to alpha 4 beta 1 is mutually exclusive, suggesting that they may employ similar mechanisms to bind receptor. Fibronectin contains at least three distinct peptide sequences that are active sites for alpha 4 beta 1 binding, two homologous sequences Leu-Asp-Val-Pro (LDVP) and Ile-Asp-Ala-Pro (IDAP), and a third related to Arg-Gly-Asp (RGD). Using a combination of site-directed mutagenesis and synthetic peptide approaches in conjunction with VCAM-1-dependent cell adhesion assays, we now report the identification of a key alpha 4 beta 1-binding sequence in both domains 1 and 4 of VCAM-1 as the tetrapeptide Ile-Asp-Ser-Pro (IDSP). Mutagenesis studies also suggest that an additional sequence in domain 1, KLEK, participates in receptor binding. Since IDSP is homologous to the LDVP and IDAP fibronectin peptides, this therefore provides a molecular explanation for the promiscuity of ligand binding by alpha 4 beta 1 and has implications for the design of synthetic VCAM-1 antagonists. The extrapolation of these findings to other integrin-binding immunoglobulin ligands is also discussed.

Amino Acid Sequence↗
PubMed1994

A secondary structure model of the integrin alpha subunit N-terminal domain based on analysis of multiple alignments.

The integrins are alpha/beta heterodimeric proteins which mediate cell-matrix and cell-cell interactions. Current data indicate that the N-terminal moiety of the alpha subunit is involved in ligand binding. This region of the receptor is made up of a seven-fold repeated sequence of unknown structure which contains EF-hand-like putative divalent cation-binding sites. Recent studies have shown that multiple sequence alignments can be analysed to yield secondary structure predictions. Therefore, to obtain a model structure for the integrin alpha subunit N-terminal domain repeat, a large alignment of the seven repeats from sixteen integrin sequences was generated. Two methods of analysis were used: First, Chou and Fasman and Garnier, Osguthorpe and Robson predictions were carried out for individual sequences and the consensus predictions derived. Consensus hydrophobicity and chain flexibility data were also used to provide additional data. Second, sites of conservation and variation were analysed by a computer program STAMA (STructure After Multiple Alignment) to yield a secondary structure prediction. The two analyses gave essentially the same predicted structure: undefined region, loop, alpha-helix, beta-strand, divalent cation-binding loop, beta-strand, putative turn, loop, beta-strand. This is the first model structure to be presented for an integrin domain. Its implications for integrin function are discussed.

Amino Acid Sequence↗
PubMed1993

Epitopes on the CB-11 peptide of type II collagen recognized by antibodies from patients with rheumatoid arthritis.

Using the technique of epitope scanning, the binding sites of antibodies to the CB-11 peptide of type II collagen, which is known to induce an inflammatory arthritis in animal models, have been investigated. Sera from seven patients have been tested against 272 peptides representing the CB-11 sequence, each patient produced a different profile of antibody binding and a number of epitopes have been identified. The specific nature of this binding has been established by the reduction in binding by absorption with type II collagen. The reactivity of sera from a further 13 patients, to peptides representing the two most frequently recognized epitopes, has been assessed.

Amino Acid Sequence↗
PubMed1992

Type I procollagens containing substitutions of aspartate, arginine, and cysteine for glycine in the pro alpha 1 (I) chain are cleaved slowly by N-proteinase, but only the cysteine substitution introduces a kink in the molecule.

Type I procollagen was purified from the medium of dermal fibroblasts cultured from four individuals with osteogenesis imperfecta (OI) type II who had mutations in the COL1A1 gene of type I procollagen. The procollagens were mixtures of normal molecules and molecules that contained substitutions of aspartate for glycine 97, arginine for glycine 550, cysteine for glycine 718, and aspartate for glycine 883 in one or both of the alpha 1 (I) chains of the molecule. The procollagens were cleaved more slowly than control type I procollagen by procollagen N-proteinase. Double-reciprocal plots of initial relative velocities and initial substrate concentrations indicated that the OI procollagens were all cleaved slowly by N-proteinase because of decreased Vmax, rather than increased Km. This suggested that slow cleavage of the OI procollagens by N-proteinase was the result of slow conversion of the N-proteinase-procollagen complex. Further experiments showed that the vertebrate collagenase A fragment of the aspartate for glycine alpha 1(I) 883 OI procollagen that contained the N-proteinase cleavage site but not the site of the substitution was also cleaved more slowly by N-proteinase than the normal vertebrate collagenase A fragments in the samples. These data show, for the first time, that an altered triple-helical structure is propagated from the site of a substitution of a bulky residue for glycine to the amino-terminal end of the procollagen molecule and disrupts the conformation of the N-proteinase cleavage site. Rotary shadowing electron microscopy of molecules in the preparation of cysteine for glycine alpha 1(I)-718 showed the presence of a kink in approximately 5% of a population of molecules in which 60% were abnormal and 20% contained a disulfide bond. In contrast, procollagens containing aspartate and arginine for glycine were indistinguishable by rotary shadowing electron microscopy from those in control samples. The results here confirm previous suggestions that substitution of cysteine for glycine in the alpha 1(I) chain of type I collagen can introduce a kink near the site of the substitution. However, the presence of a kink is not a prerequisite for delayed cleavage of abnormal procollagens by N-proteinase.

Amino Acid Sequence↗
PubMed1992

Secondary and tertiary structures involving chondroitin and chondroitin sulphates in solution, investigated by rotary shadowing/electron microscopy and computer simulation.

Rotary shadowing/electron microscopy of chondroitin 6-sulphate (CS6) and 4-sulphate (CS4) showed that the former, but not the latter, aggregated to mesh works. Preparations made from salt (ammonium acetate) solutions showed enhanced aggregation. Computer modelling, using molecular mechanics and dynamics, was applied to secondary structures (twofold helices) derived from NMR studies, to determine geometric and energetic constraints on duplex and higher-aggregate formation. The calculations suggested that chondroitin, CS6 and undersulphated CS4 could form duplexes, while CS4 could not, thus bridging the gap between atomic dimensions (NMR) and high polymer scale (electron microscopy). Calculations suggested that water structure helped to stabilise the twofold helix. It is proposed that the twofold helical, flat, tape-like molecules aggregate via hydrophobic bonding between the very extensive hydrophobic patches (9 CH units) repeated on alternating sides of the polymers. The negative charge of the polyanions opposes aggregate formation. Calculations showed that duplexes were formed with decreasing stability as the charge density increased, and as the charge was concentrated towards the centre line of the polymer (i.e. in CS4). The unsulphated polymer chondroitin could form duplexes and higher aggregates as readily as hyaluronan. Hyaluronan was calculated to form stable heteroduplexes with CS6 and CS4. The frequency and positioning of the sulphate-ester group within the polymer thus determines whether the molecule participates in duplex formation.

Animals↗
PubMed1992

Homology modelling of integrin EF-hands. Evidence for widespread use of a conserved cation-binding site.

Integrin alpha-subunits contain three or four peptide sequences that are similar to the EF-hand, a 13-residue bivalent cation-binding motif found in calmodulin and parvalbumin. The integrin sequences differ from classical EF-hands in that they lack a co-ordinating residue at position 12. One hypothesis to explain integrin-ligand binding is that aspartate-containing recognition sequences in integrin ligands, which bind at or near to the EF-hand-like sequences, may take the place of the missing residue and co-ordinate directly to the bound cation. In this report, homology modelling of integrin EF-hand-like sequences has been performed using the X-ray structure of calmodulin as a template in order to assess the functional activity of the integrin sequences. In the calmodulin-integrin hybrid structures, integrin EF-hand-like sequences were able to retain cations whereas control sequences did not. Structural analyses demonstrated that the integrin sequences in the hybrid proteins closely resembled conventional EF-hands. The integrin sequences are therefore highly likely to bind Ca2+ ions in vivo, a prerequisite for the ligand-binding model. Database searching with a matrix derived from known integrin EF-hand-like sequences has been used to identify other proteins containing the integrin EF-hand-like motif. Annexin V (anchorin CII), atrial natriuretic peptide receptors and the 70 kDa heat-shock protein were identified by the matrix; the functions of these proteins are known from previous studies to be bivalent cation-dependent. These findings suggest that the integrin EF-hand-like sequence may be a more common motif than originally thought.

Amino Acid Sequence↗
PubMed1992

The fibrillar collagens, collagen VIII, collagen X and the C1q complement proteins share a similar domain in their C-terminal non-collagenous regions.

A sequence comparison of the C-termini of collagens X, VIII, the collagen-like complement factor C1q, and the fibrillar collagens showed a conserved cluster of aromatic residues. This conserved cluster was in a domain of approximately 130 amino acids that exhibited marked similarities in hydrophilicity profiles between the different collagens, despite a low level of sequence similarity. These data suggest that the 'collagen X-like family' and the fibrillar collagens contain a domain within their C-termini that adopts a common tertiary structure, and that a conserved cluster of aromatic residues in this domain may be involved in C-terminal trimerization.

Amino Acid Sequence↗
PubMed1992

Identification of an immunodominant B-cell epitope in bovine type II collagen and the production of antibodies to type II collagen by immunization with a synthetic peptide representing this epitope.

Using epitope scanning of 272 short, synthetic peptides representing the amino acid sequence of the CB-11 peptide of type II collagen, we have shown that five strains of rat, immunized with type II collagen, produce antibodies to a region 37-45 amino acids from the amino end of CB-11 peptide. Antibodies to this region always gave the highest binding values suggesting that it is an immunodominant region. Wistar rats immunized with a synthetic peptide representing this region, coupled to keyhole limpet haemocyanin, produced antibodies to this peptide which could still be detected at 1:4000 to 1:8000 dilution but none developed clinical arthritis. All sera also showed binding of antibodies to denatured bovine type II collagen but not to native type II collagen, keyhole limpet haemocyanin or to bovine serum albumin by ELISA. Sera from peptide-immunized rats were examined for antibody binding to the 272 short peptides of the CB-11 peptide and to the synthetic peptides representing shortened forms of the immunodominant region and forms of it with substituted amino acids. These results showed that the antibodies in the peptide-immunized rats were not identical to those produced to that peptide by rats immunized with type II collagen but may represent subpopulations of them. These findings suggest caution in interpreting the role of antibodies to individual peptides in arthritis induction without knowledge of their fine specificity.

Animals↗
PubMed1991

Inhibition of glucose transport in human erythrocytes by ubiquinone Q0.

Searches of the protein data bases revealed limited homologies between several regions of the human erythrocyte glucose transporter containing a relative abundance of hydrogen-bonding amino-acid side chains, and proteins of the NADH-ubiquinone oxidoreductase family. This raised the possibility the binding sites for glucose and ubiquinone may be similar in the respective proteins. Experimental studies demonstrated that ubiquinone Q0 does in fact inhibit both glucose entry and glucose exit in human erythrocytes with kinetics consistent with the existence of ubiquinone binding sites at both the exofacial and endofacial sides of the transporter. Glucose transport was also inhibited by the water-soluble tryptophan-inactivating agent, dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide, and this is consistent with the presence of tryptophan residues in two of the exofacial amino-acid sequences proposed as candidates for involvement in glucose binding sites.

Amino Acid Sequence↗
PubMed1991

Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation. Hyaluronan is a very efficient network-forming polymer.

1. Hyaluronan from mesothelioma fluid, rooster comb and streptococci was examined by rotary shadowing and electron microscopy. All preparations showed extensive branched networks, but high-viscosity hyaluronan networks were essentially infinite, with no individual 'molecules' that were not integrated via multiple branched points into the meshwork. Low-viscosity hyaluronan, recovered after papain digestion of mesothelioma fluid, showed occasional single filaments that were independent of the main aggregates, some of which were themselves independent of other aggregates. 2. Hyaluronan is a polymer with a very marked capability to form meshworks at very low dilution (less than 1 microgram/ml). The longer the hyaluronan molecule, the more branching is potentially possible, and the more extensive and coherent is the network, with every hyaluronan molecule in contact with every other in the solution, via the network. This behaviour accounts for the mechanical properties of the soft tissues (e.g. vitreous humour) and fluids (e.g. synovial fluid) of which hyaluronan is a major component. 3. The hyaluronan twofold helix, previously demonstrated to be present in solution [Heatley & Scott (1988) Biochem. J. 254, 489-493] was shown by computer simulation and energy calculations to be sterically capable of extensive duplex formation, probably driven by interactions between the large hydrophobic patches on alternate sides of the tape-like polymer, forming stable aggregates at biological temperatures in water. This 'stickiness' is postulated to be the basis of the network-forming and laterally aggregating behaviour of hyaluronan. 4. The tertiary structures formed by hyaluronan may not be possible in the case of chondroitin 4-sulphate.

Animals↗
PubMed1991

Identification of antibody epitopes within the CB-11 peptide of type II collagen. I: Detection of antibody binding sites by epitope scanning.

Using epitope scanning, the precise location of antibody binding sites on the CB-11 peptide of bovine type II collagen have been identified for the first time. Two hundred and seventy two peptides (8 amino acids in length and overlapping by seven amino acids), representing the complete CB-11 sequence, were synthesised on solid phase supports, in duplicate, and were screened with sera from arthritic and non-arthritic, bovine type II collagen-immunised rats. A total of twenty one different antibody binding sites were identified with no epitope being uniquely recognised by sera from arthritic, as compared to non-arthritic, rats although differences in the relative amount of antibody binding were seen. Individual sera identified between two and thirteen epitopes with one epitope being recognised by all sera. Some of the amino acid sequences, of the CB-11 region of bovine type II collagen, recognised by the rat sera are identical to the sequences in human type II collagen and thus these epitopes may be relevant to autoimmunity to type II collagen in patients with rheumatoid arthritis.

Amino Acid Sequence↗
PubMed1991

Identification of antibody epitopes within the CB-11 peptide of type II collagen. II. Computer modelling studies of peptides and the interpretation of epitope scanning results.

Computer modelling techniques were used to investigate the structure of 8-mers from the CB-11 peptide of bovine type II collagen which were recognised by sera from rats which had previously been injected with bovine type II collage. It was discovered that all the hydrophobic peptides recognised by the rat sera were predicted to have collagenous-like secondary structures. The primary structure of the 8-mers which were recognised was also compared against the sequences in the OWL protein sequence database. The combined results of the computer modelling and sequence analysis suggested that the sequence Gly-Pro-Gly-Phe-Pro is a minimal B cell epitope of the CB-11 fragment of bovine type II collagen.

Amino Acid Sequence↗