Maurice C. Owen

Mutation of antitrypsin to antithrombin: α1-antitrypsin Pittsburgh (358 Met→Arg), a fatal bleeding disorder

Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant proteins being an acute-phase reactant, the level of which increased several-fold after trauma.

Mutation of antitrypsin to antithrombin. alpha 1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder.

Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant proteins being an acute-phase reactant, the level of which increased several-fold after trauma.

Human α1-antitrypsin: carbohydrate attachment and sequence homology

Human err-antitrypsin (or-AT) has 3 carbohydrate sidechains [ 11. The attachment point of one of these sidechains has been determined in conjunction with the amino acid sequence of the C-terminal third of the molecule [2]. We now provide further sequence data which define the attachment points of the other 2 carbohydrate sidechains. These data also show the extent of the sequence homology with antithrombinIII and ovalbumin, and provide support for a single reactive centre situated near the C-terminus. tryptic digestion in 2 M guanidine hydrochloride in 0.1 M NI&HCOa buffer, pH 8.0 at 37’C for 15 h with an enzyme to substrate ratio of 1: 10.

Carboxy terminal fragment of human α-1-antitrypsin from hydroxylamine clevage: Homology with antithrombin III

Abstract Human α-1-antitrypsin (AT) was reacted with hydroxylamine at pH 9.0 giving cleavage at an Asn-Gly bond. A fragment of molecular weight 8,500 was released and this was isolated and sequenced. The fragment had the same carboxy terminal amino acid sequence as intact AT. The 80 residue polypeptide contained the Z variant mutation site and a portion of sequence identical to that found by others for the reactive site, inferring the presence in AT of two active sites. This sequence combined with prviously published work gives a continuous sequence of 152 amino acid residues from the carboxy terminal end of the AT molecule, including the mutation site of the S variant. The sequence shows strong homology with human antithrombin III.

Circulating proalbumin associated with a variant proteinase inhibitor

The unique finding of normal proalbumin in human plasma provides an insight into the mechanism of propeptide cleavage. Proalbumin, present as 1-5% of the total albumin, was found in a boy whose prime problem was the presence of a mutant proteinase inhibitor, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) [2]. The inferred structure of human proalbumin was confirmed as Arg-Gly-Val-Phe-Arg-Arg-Alb. On incubation with various enzymes (trypsin, tryptase, thrombin, chymotrypsin, chymase and cathepsin B), only trypsin was capable of converting proalbumin to albumin. There was no conversion when proalbumin was incubated with whole blood, plasma or serum. However, intravenous injection of proalbumin into a rat resulted in complete conversion to albumin, the half-life of this process being 6 h. We conclude that propeptide cleavage is dependent on a serine proteinase which is inhibited intracellularly, by the mutant inhibitor, and that all the albumin in the boy was secreted as proalbumin, but was subjected to a separate cleavage process after export from the hepatocyte.

The abnormality of the S variant of human α-1-antitrypsin

Abstract The tryptic peptide map of the S variant of α -1-antitrypsin differs in only one peptide from that of the normal M map. The amino acid sequence of this fifteen residue peptide is reported, the S peptide having a valine in position 5 compared to a glutamic acid in the same position in the M peptide. The abnormality has been confirmed in two unrelated donors with the SS phenotype.

Active site of α1-antitrypsin: Homologous site in antithrombin-III

Abstract Examination of peptides resulting from reaction of bovine trypsin and human α 1 -antitrypsin in near-equimolar amounts showed anomalous cleavage of antitrypsin at a Met-Ser bond 37 residues from the C-terminus, giving evidence that this is the active site for trypsin inhibition. Alignment of the C-terminal 141 residues of α 1 -antitrypsin with the C-terminal 147 residues of human antithrombin-III showed homology with 30% identity and allowed the identification of a homologous active site in antithrombin.

Antithrombin Glasgow, 393 arg to his: A P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41‐year‐old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin‐Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C‐terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.

Mutation of Antitrypsin to Antithrombin

Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant proteins being an acute-phase reactant, the level of which increased several-fold after trauma.

A new approach to haemoglobin variant identification haemoglobin christchurch β71 (E15) phenylalanine å serine

Abstract 1. 1. The identification of the abnormality in pathological haemoglobin variants poses technical difficulties since many of these abnormalities are due to amino acid substitutions involving no change in electrophoretic charge. Identification of this type of substitution is dependent on the detection of slight changes in peptide chromatographic mobility. The use of 14 C-labelled Hb A as a control to the abnormal haemoglobin with comparison of ninhydrin maps and autoradiographs or peptide counts greatly assists identification of the abnormal peptide. It lessens the dependence on an established normal peptide map and enables quick confirmation that a peptide of changed chromatographic mobility is an aberrant peptide from an abnormal haemoglobin rather than a normal peptide which has undergone an artefact change. 2. 2. The technique has been used to identify a new unstable haemoglobin (Hb Christchurch) causing a Heinz body haemolytic anaemia. The identified substitution, serine for phenylalanine at position β71 (E15), would be expected to result in increased haem release with resultant instability and haemolysis.