Sophie Jallat

cDNA cloning and expression of a hamster α‐thrombin receptor coupled to Ca2+ mobilization

The serine protease α‐thrombin (thrombin) potently stimulates G‐protein‐coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G‐protein‐coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C‐coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N‐terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N‐terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion‐binding exosite of thrombin.

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

In normal plasma, the serine protease inhibitor alpha 1-antitrypsin (alpha 1-AT) plays little or no role in the control of plasma kallikrein or activated Factor XII fragment (Factor XIIf), this function being performed by Cl-inhibitor. Recently, an alpha 1-AT variant was described with a Met----Arg mutation at the reactive center P1 residue (position 358) which altered the specificity of inhibition from the Met- or Val-specific protease neutrophil elastase to thrombin, an Arg-specific protease. We have now examined the inhibition of plasma kallikrein and Factor XIIf, both Arg-specific enzymes, with recombinant alpha 1-AT(Met358----Arg) produced by an Escherichia coli strain carrying a mutated human alpha 1-AT gene. The engineered protein was a very efficient inhibitor of both enzymes. It was more effective than Cl-inhibitor by a factor of 4.1 for kallikrein and 11.5 for Factor XIIf. These results suggest that recombinant alpha 1-AT(Met358----Arg) has therapeutic potential for disease states where activation of the plasma kinin-forming system is observed, for example in hereditary angioedema or septic shock.

RNA structural elements for expression in Escherichia coli: α1-antitrypsin synthesis using translation control elements based on the cII ribosome-binding site of phage λ

Analysis of a series of λ cII::α1‐antitrypsin (α1AT) gene fusions of different sizes showed that increased α1AT expression correlated with the stabilisation of a particular computer‐predicted RNA secondary structure. Moreover, significant synthesis of unfused α1AT was achieved by reconstruction of this conformation to permit interaction between the upstream region of the ribosome‐binding site and the first part of the α1 AT coding sequence. This high‐level expression was dependent upon certain silent point mutations in the coding sequence, indicating that RNA primary and secondary structure determinants can operate in concert to dictate the efficiency of protein synthesis.

Protection by recombinant alpha 1-antitrypsin Ala357 Arg358 against arterial hypotension induced by factor XII fragment.

The specificity of serpin superfamily protease inhibitors such as alpha 1-antitrypsin or C1 inhibitor is determined by the amino acid residues of the inhibitor reactive center. To obtain an inhibitor that would be specific for the plasma kallikrein-kinin system enzymes, we have constructed an antitrypsin mutant having Arg at the reactive center P1 residue (position 358) and Ala at residue P2 (position 357). These modifications were made because C1 inhibitor, the major natural inhibitor of kallikrein and Factor XIIa, contains Arg at P1 and Ala at P2. In vitro, the novel inhibitor, alpha 1-antitrypsin Ala357 Arg358, was more efficient than C1 inhibitor for inhibiting kallikrein. Furthermore, Wistar rats pretreated with alpha 1-antitrypsin Ala357 Arg358 were partially protected from the circulatory collapse caused by the administration of beta-Factor XIIa.

Sulphation of hirudin in BHK cells

Hirudin, a thrombin inhibitor of the leech, was expressed in BHK cells; the α1‐antitrypsin signal peptide was used to direct secretion into the culture medium. The recombinant hirudin so produced inhibited thrombin and was shown by labelling experiments with [35S]sulphate to have been post‐translationally modified.