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J Whisstock

Publications and source records attributed to J Whisstock.

10 recordsLinked to original sources
PubMed2000

Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling.

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing the PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive with MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.

Amino Acid Sequence↗
PubMed1999

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes.

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

Algorithms↗
PubMed1999

Characterization of epitope regions of thyrotropin beta-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach.

This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.

Amino Acid Sequence↗
PubMed1998

An atlas of serpin conformations.

The serpins are a family of proteins that inhibit chymotrypsin-like serine proteinases, with an unusual mechanism involving a large conformational change known as the stressed-->relaxed (S-->R) transition. This article is a guide to the known serpin conformations and their biological significance.

Amino Acid Sequence↗
PubMed1997

Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.

Amino Acid Sequence↗
PubMed1996

Conservation within the myosin motor domain: implications for structure and function.

BACKGROUND: Myosins are motors that use energy supplied by ATP to travel along actin filaments. The structure of myosin is known, but the actin-binding site is not well defined, and the mechanisms by which actin activates ATP hydrolysis by myosin, and myosin moves relative to the actin filament, developing force, are not fully understood. Previous phylogenetic analyses of the motor domain of myosins have identified up to twelve classes. We set out to analyse the positions of conserved residues within this domain in detail, and relate the conserved residues to the myosin structure. RESULTS: Our analysis indicates that there are at least thirteen myosin classes. Conserved residues in the motor domain have been positioned within the framework provided by the recent crystal structures, thus helping to define those residues involved in actin and ATP binding, in hydrolysis and in conformational change. This has revealed remarkably poor overall conservation at the site thought to be involved in actin binding, but several highly conserved residues have been identified that may be functionally important. CONCLUSIONS: Information from such a sequence analysis is a useful tool in the further interpretation of X-ray structures. It allows the position of crucial residues from other members of a superfamily to be determined within the framework provided by the known structures and the functional significance of conserved or mutated residues to be assessed.

Actins↗
PubMed1996

Modeling of serpin-protease complexes: antithrombin-thrombin, alpha 1-antitrypsin (358Met-->Arg)-thrombin, alpha 1-antitrypsin (358Met-->Arg)-trypsin, and antitrypsin-elastase.

Based on the most recent available crystal structures and biochemical studies of protease complexes of normal and mutant serine protease inhibitors (serpins), we have built models of the complexes: alpha 1-antitrypsin + human neutrophil elastase; alpha 1-antitrypsin Pittsburgh (358Met-->Arg) (Scott et al., J. Clin. Invest. 77:631-634, 1986) + tyrpsin; alpha 1-antitrypsin Pittsburgh (358Met-->Arg) + thrombin; and antithrombin + thrombin. All serpin sequences correspond to human molecules. The models show correct stereochemistry and no steric clashes between protease and inhibitor. The main structural differences in the serpins from the parent structures are: (1) the reactive center loop is inserted into the A-sheet as far as P12; (2) strand s1C is removed from the C-sheet; and (3) the C-terminus has changed conformation and interacts with the protease. In the absence of an X-ray structure determination of a serpin-protease complex, the demonstration that insertion of the reactive center loop into the A-sheet as far as P12 is stereochemically feasible provides structures of a protease-bound conformation of intact serpins with which to rationalize the properties of mutants, guide the design of experiments, and form a basis for further modeling studies, such as the investigation of the interaction of heparin with serpin-protease complexes.

Antithrombin III↗
PubMed1994

Function of maspin.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed1994

Conformational changes in serpins and the mechanism of alpha 1-antitrypsin deficiency.

alpha 1-Antitrypsin is a member of the serine proteinase inhibitor, serpin, family of protease inhibitors, which have their reactive centers situated on a mobile peptide loop. This reactive loop can adopt varied conformations and perturbations of molecular structure to allow the pathological linking of the loop of one molecule to a beta-pleated sheet of another. This linkage has been shown to be the cause of the polymerization and aggregation within the hepatocyte of the common Z mutant of antitrypsin. The occurrence of loop-sheet polymerization has been confirmed with other deficiency variants of antitrypsin that accumulate in the liver (Mmalton, Siiyama) and also shown to occur in pathological mutants of C1-inhibitor and antithrombin. Deductive evidence indicates that the loop is inserted into the A-sheet of the next molecule, but recent structural findings raise the possibility of insertion into the C-sheet. This detail of loop-sheet polymerization is important for the design of strategies to interfere with insertion and hence lesson the accumulation of Z antitrypsin that is responsible for associated liver damage.

Humans↗