Recombinant alpha 1-antitrypsin Pittsburgh (Met 358----Arg) is a potent inhibitor of plasma kallikrein and activated factor XII fragment.

Schapira, Marc; Ramus, Marie-Andrée; Jallat, S.; Carvallo, D.; Courtney, Michael

doi:10.1172/jci112347

Cited by 38 publications

(33 citation statements)

References 16 publications

(14 reference statements)

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“…Wild-type ␣ 1 -proteinase inhibitor was a recombinant protein previously expressed in baby hamster kidney cells (11). The P1 M 3 R variant of ␣ 1 -proteinase inhibitor was a recombinant protein expressed in Escherichia coli (25) and was a gift from Philip Patston (University of Illinois, Chicago).…”

Section: Methodsmentioning

confidence: 99%

Major proteinase movement upon stable serpin–proteinase complex formation

Stratikos

Gettins

1997

Proc. Natl. Acad. Sci. U.S.A.

134

106

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To determine whether formation of the stable complex between a serpin and a target proteinase involves a major translocation of the proteinase from its initial position in the noncovalent Michaelis complex, we have used f luorescence resonance energy transfer to measure the separation between f luorescein attached to a single cysteine on the serpin and tetramethylrhodamine conjugated to the proteinase. The interf luorophore separation was determined for the noncovalent Michaelis-like complex formed between ␣ 1 -proteinase inhibitor (Pittsburgh variant) and anhydrotrypsin and for the stable complex between the same serpin and trypsin. A difference in separation between the two f luorophores of Ϸ21 Å was found for the two types of complex. This demonstrates a major movement of the proteinase in going from the initial noncovalent encounter complex to the kinetically stable complex. The change in interf luorophore separation is most readily understood in terms of movement of the proteinase from the reactive center end of the serpin toward the distal end, as the covalently attached reactive center loop inserts into ␤-sheet A of the serpin.

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Section: Methodsmentioning

confidence: 99%

Major proteinase movement upon stable serpin–proteinase complex formation

Stratikos

Gettins

1997

Proc. Natl. Acad. Sci. U.S.A.

134

106

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“…The best known example of the gain of inhibitory function against a nontarget protease is a,-antitrypsin Pittsburgh, in which the P1 Met is replaced with an Arg (25). This converts the mutant to an efficient inhibitor of thrombin, plasma kallikrein, and factor XIIa (26,27). The further substitution of the P2 Pro in ca,-antitrypsin Pittsburgh with an Ala (matching the P2 of Ci inhibitor) enhances its inhibition of kallikrein and confers the ability to inhibit Cls (albeit relatively weakly) (28) mutant, however, is of further interest because wild-type Cl inhibitor is a trypsin substrate that is efficiently cleaved at P1 -P1' with catalytic quantities of trypsin (14).…”

Section: Methodsmentioning

confidence: 99%

Unique C1 inhibitor dysfunction in a kindred without angioedema. II. Identification of an Ala443-->Val substitution and functional analysis of the recombinant mutant protein.

Zahedi¹,

Bissler²,

Davis³

et al. 1995

J. Clin. Invest.

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We have determined the cause of an unusual Cl inhibitor abnormality in a large kindred. We previously found that half of serum Cl inhibitor molecules in affected kindred members are normal. The other half complexed with Cls but showed little complex formation with Clr. These molecules also appeared to be relatively resistant to digestion by trypsin. Taken together, the findings suggested that members of this kindred are heterozygous for an unusual Cl inhibitor mutation. Sequencing of genomic DNA from the kindred revealed that thymine has replaced cytosine in the codon for Ala443 (P2 residue) in one Cl inhibitor allele, resulting in substitution with a Val residue. To test the effect of this substitution, a mutant Cl inhibitor containing Ala"3--Val was constructed by site-directed mutagenesis and expressed in COS-1 cells. Both the Ala'3--Val mutant and the wild-type Cl inhibitor complexed completely with Cls, kallikrein, and coagulation Factor XIIa after incubation at 37°C for 60 min. In contrast, the mutant inhibitor failed to complex completely with Clr under the same conditions. Time course analysis showed that the ability of the mutant to complex with Cls is also impaired: although it complexed completely in 60 min, the rate of complex formation during a 0-60-min incubation was decreased compared with wild-type Cl inhibitor. The mutant inhibitor also formed a complex with trypsin, a serine protease that cleaves, and is not inhibited by, wild-type Cl inhibitor. The Ala"3-Val mutation therefore converts Cl inhibitor from a substrate to an inhibitor of trypsin. These studies emphasize the role of the P2 residue in the determination of target protease specificity. (J. Clin. Invest. 1995. 95:1299-1305

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“…Thus, a given serpin can often form SDS-stable complexes with many different proteinases that differ greatly in size and shape. The Pittsburgh variant used here is a good example, in that it can inhibit trypsin, elastase, thrombin, C1s, factor XIIa, plasmin, and urokinase (18,19). Similarly a given proteinase may be able to form complexes with different serpins.…”

Section: Figmentioning

confidence: 99%

Mapping the Serpin-Proteinase Complex Using Single Cysteine Variants of α1-Proteinase Inhibitor Pittsburgh

Stratikos¹,

Gettins

1998

Journal of Biological Chemistry

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To probe the covalent serpin-proteinase complex, we used wild-type and 4 new single cysteine variants (T85C, S121C, D159C, and D298C) of ␣ 1 -proteinase inhibitor Pittsburgh. Cysteines in each variant could be labeled both in native and proteinase-complexed ␣ 1 -proteinase inhibitors. Pre-reaction with 7-nitrobenz-2-oxa-1,3-diazole-chloride or fluorescein prevented complex formation only with the D298C variant. Label at Cys 121 greatly increased the stoichiometry of inhibition for thrombin and gave an emission spectrum that discriminated between native, cleaved, and proteinase-complexed serpin and between complexes with trypsin and thrombin, whereas fluorophore at residue 159 on helix F was almost insensitive to complex formation. Fluorescence resonance energy transfer measurements for covalent and non-covalent complexes were consistent with a location of the proteinase at the end of the serpin distal from the original location of the reactive center loop. Taken together, these findings are consistent with a serpin-proteinase complex in which the reactive center loop is fully inserted into ␤-sheet A, and the proteinase is at the far end of the serpin from its initial site of docking with the reactive center loop close to, but not obscuring, residue 121.Serpins are a family of widely distributed, structurally homologous proteins (1), many of which are inhibitors of serine proteinases (2). Whereas the many other families of protein inhibitors of serine proteinases, such as the Bowman-Birk, Kazal, and Kunitz families, inhibit target proteinases by forming tight non-covalent 1:1 complexes in which neither the proteinase nor the inhibitor undergoes significant structural change in most cases (3), serpins differ not only by apparently forming covalent 1:1 acyl enzyme complexes with their target proteinases (4), but by undergoing a major conformational change during, and as an essential part of, the inhibition process (5). Because of the requirement for conformational change as part of the inhibition mechanism, knowledge of the structure of the serpin-proteinase complex is critical for an understanding of how serpins inhibit their target proteinases through kinetic trapping of a normal covalent acyl enzyme intermediate on the proteinase substrate cleavage pathway.A previous proposal that a major movement of the proteinase occurs following cleavage of the scissile bond (6) has been supported by two recent studies (7,8). In one study (8) chemical cross-linking between the proteinase and the serpin in the complex, together with a measurement of the separation between P3 and P1Ј residues of the serpin in the complex by fluorescence resonance energy transfer, was consistent with a location of the proteinase half-way down the flank of the serpin (Fig. 1) and in contact with helix F. The other study (7), from this laboratory, used fluorescence resonance energy transfer between fluorophores on the serpin ␣ 1 -proteinase inhibitor (␣ 1 PI) 1 Pittsburgh and the proteinase to compare the interfluorophore separation in the normal ...

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Recombinant alpha 1-antitrypsin Pittsburgh (Met 358----Arg) is a potent inhibitor of plasma kallikrein and activated factor XII fragment.

Cited by 38 publications

References 16 publications

Major proteinase movement upon stable serpin–proteinase complex formation

Major proteinase movement upon stable serpin–proteinase complex formation

Unique C1 inhibitor dysfunction in a kindred without angioedema. II. Identification of an Ala443-->Val substitution and functional analysis of the recombinant mutant protein.

Mapping the Serpin-Proteinase Complex Using Single Cysteine Variants of α1-Proteinase Inhibitor Pittsburgh

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