Antithrombin Cambridge II, 384 Ala to Ser Further evidence of the role of the reactive centre loop in the inhibitory function of the serpins

Department of Haematology, University of Cambridge, UK.
FEBS Letters (Impact Factor: 3.34). 08/1991; 285(2):248-50. DOI: 10.1016/0014-5793(91)80809-H
Source: PubMed

ABSTRACT Four unrelated individuals have been identified with an identical antithrombin variant, associated in one of them with episodes of recurrent venous thromboses. In each case, the plasma antithrombin concentration was normal and the only function abnormality was a minor but consistent decrease in the heparin-induced thrombin inhibition suggesting a mutation at or near the reactive centre of the molecule. Amplification and direct sequencing of exon 6 showed a G----T mutation at nucleotide 1246, which corresponds to a substitution of a serine for an alanine at residue 384. This is one of a series of conserved alanines that form the stalk to the reactive centre loop. The observed changes in this variant are compatible with recent structural studies that infer that mobility of this stalk with partial re-entry into the A-sheet of the molecule is necessary for optimal inhibitory activity.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Glycosaminoglycans such as e.g. heparin, heparan sulphate and dermatan sulphate display a broad variety of biological activities. Unique, Well-defined domains in some glycosaminoglycans have been characterized that are responsible for the biological activity. For instance, a unique pentasaccharide domain in heparin could be identified which binds and activates the serine protease inhibitor (serpin) anti-thrombin I11 (AT 111). The structure-activity relationships of various synthetic counter-parts of the heparin pentasaccharide fragment reveal the highly specific nature of the pentasaccharide mediated activation of AT 111. With the aid of molecular modelling and the availability of crystal structures of serpins and their target proteases, the activation process of AT I11 by heparin becomes understood at the molecular level. Some attention will also be paid to well-defined domains in heparan sulphate and dermatan sulphate.
    ChemInform 10/1995; 67(10):1663-1672. DOI:10.1351/pac199567101663
  • [Show abstract] [Hide abstract]
    ABSTRACT: For better understanding of the structure-function relationship in serine proteinase inhibitors, a protein engineering approach for converting non-inhibitory chicken ovalbumin (Ova) to the inhibitory form would be a highly useful model system. A prerequisite expression system for the Ova-encoding gene (Ova) was established in this study. The Ova gene was expressed in Escherichia coli with high yield using the T7 phage promoter; the amount of the recombinant Ova (re-Ova) was 29.4% of cellular proteins. SDS-PAGE and Western blotting analysis revealed that re-Ova immunoreacting with the egg ovalbumin antibody is not glycosylated. The re-Ova was purified by anion exchange chromatography into homogeneity, as evaluated by SDS-PAGE. Amino-acid and N-terminal sequence analyses confirmed that the purified product had the correct sequence designed for Ova production. As for secondary structure, re-Ova showed a far-UV circular dichroism spectrum indistinguishable from natural egg Ova. Furthermore, the proteolytic fragmentation pattern that should reflect protein conformation was exactly the same for the natural egg and re-Ova. Using the proteolytic fragments, the identity of the internal sequences for the natural and re- proteins was confirmed.
    Gene 09/1995; 161(2):211-6. DOI:10.1016/0378-1119(95)00234-W · 2.08 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: An expression system for alpha 1-antitrypsin in Escherichia coli was developed using a T7 RNA polymerase promoter. Addition of rifampicin to inhibit the E. coli RNA polymerase after induction of the T7 RNA polymerase gene resulted in about 30% of newly synthesized protein being alpha 1-antitrypsin. This expression system was then used to examine the effect of mutations in the hinge region of alpha 1-antitrypsin on its activity. The mutations were based on ones in antithrombin III that had previously been shown to have adverse effects on activity. Mutation of Ala347 to threonine in alpha 1-antitrypsin did not affect the kinetic behavior of the protein with trypsin or human leukocyte elastase. In contrast, mutation of Gly349 to proline converted the majority of the protein into a substrate for both proteinases. The small fraction of this mutant that was active, however, had kinetic parameters that were indistinguishable from wild-type alpha 1-antitrypsin. Cleavage within the reactive-site loop of wild-type alpha 1-antitrypsin causes a conformational change in the molecules (the S-to-R transition) and results in a marked increase in heat stability. This increase in heat stability was also seen upon cleavage within the reactive-site loops of both of the alpha 1-antitrypsin mutants. The results are discussed in terms of a kinetic mechanism for serpin-proteinase interactions, in which after the formation of an initial complex the serpin partitions between the formation of a stable complex and a cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
    Biochemistry 09/1993; 32(30):7650-7. DOI:10.1021/bi00081a008 · 3.19 Impact Factor