title: A novel amino acid substitution in the reactive site of a congenital variant antithrombin. Antithrombin pescara, ARG393 to pro, caused by a CGT to CCT mutation.
abstract: Antithrombin is a plasma protein inhibitor that can be grouped within a serine proteinase inhibitor superfamily. Antithrombin Pescara is a functional variant of antithrombin found in a family with a high incidence of thrombosis. Preliminary functional analysis has suggested that the abnormality resides in the reactive site rather than in the heparin binding domain of the molecule. Accordingly, we have isolated the variant from plasma using heparin-Sepharose chromatography, followed by chromatography upon thrombin-Sepharose to remove the normal antithrombin that is present (the propositus is heterozygous for the variant). The variant protein was reduced, S-carboxy-methylated, and fragmented with CNBr. A pool ("CNBr pool 4") containing the reactive site region was isolated by reverse-phase high performance liquid chromatography and sequentially treated with trypsin and V8 protease. Fast atom bombardment-mass spectrometric analysis of this subdigest identified a novel peptide of mass 1708. Four steps of Edman degradation together with further analysis by fast atom bombardment-mass spectroscopy identified the NH2-terminal sequence of this peptide as Ala-Ala-Ala-Ser. The mass of the novel peptide and its changing mass in response to Edman degradation are only compatible with its identity as Ala382-Arg399, with the reactive site Arg393 replaced by Pro. Using specific oligonucleotide hybridization, we demonstrated that the molecular defect of antithrombin Pescara is caused by a CGT to CCT mutation in codon 393. These findings may be of broad interest, as other members of the serine protease inhibitor superfamily contain arginine at their reactive sites and may be expected to undergo a similar mutation.
authors.affiliation: Department of Haematology, Charing Cross and Westminster Medical School, Hammersmith, Great Britain.
authors.forename: D A
authors.initials: DA
authors.lastname: Lane
authors.forename: H
authors.initials: H
authors.lastname: Erdjument
authors.forename: E
authors.initials: E
authors.lastname: Thompson
authors.forename: M
authors.initials: M
authors.lastname: Panico
authors.forename: V
authors.initials: V
authors.lastname: Di Marzo
authors.forename: H R
authors.initials: HR
authors.lastname: Morris
authors.forename: G
authors.initials: G
authors.lastname: Leone
authors.forename: V
authors.initials: V
authors.lastname: De Stefano
authors.forename: S L
authors.initials: SL
authors.lastname: Thein
mesh_terms: D000595:Amino Acid Sequence; D058833:Antithrombin Proteins; D000991:Antithrombins / Q000235:genetics* / Q000302:isolation & purification; D001120:Arginine; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D011392:Proline; D012333:RNA, Messenger / Q000235:genetics*; D012016:Reference Values
chemical_list: D058833:Antithrombin Proteins; D000991:Antithrombins; D003062:Codon; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; C059998:antithrombin Pescara; D001120:Arginine; D011392:Proline