Novel hirudin variants from the leech Hirudinaria manillensis

Abstract

Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described. Isolation of antithrombin polypeptides was performed by ion-exchange chromatographies followed by an affinity chromatography step on immobilized thrombin. The major active component, antithrombin polypeptide peak 2 (HM2), and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined. The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions. Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C-terminal region of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the Cterminal portion of the molecule which is replaced by aspartic acid.Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin-like polypeptides. The cDNA clones coding for the two variants indicate the expression of pre-hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported.The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20-amino-acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides.HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene.Hirudin, the most potent thrombin inhibitor known, is a small polypeptide discovered over 100 years ago in the saliva of the medicinal leech Hirudo medicinalis [l]. It was first characterized biochemically by Markwardt [2, 31 and its structure was lately elucidated by Dodt et al. [4]. It is a single-chain polypeptide composed of 65 amino acids, including 6 cysteine residues, and a molecular mass of about 7000 Da. Hirudin produces its action by binding directly to thrombinCorrespondence to E. Scacheri,

Probing the Structure of Hirudin from Hirudinaria manillensis by Limited Proteolysis

“…Recombinant hirudin HM2 from H. manillensis was produced at the Bioscience Centre of the Pharmacia-Farmitalia and stored at -20°C (Scacheri et al, 1993). A sample of recombinant hirudin variant HV1 from H. medicinalis was purchased from Sigma Chem.…”

“…Amino acid composition of hirudin HM2 from H. manillensis and its proteolytic fragments. Theoretical values are givtn in parenthesis and were calculated from the amino acid sequence of hirudin variant HM2 (Scacheri et al, 1993). The various proteolytic fragments of hirudin HM2 were prepared and isolated to homogeneity as described in the text.…”

“…Limited proteolysis of recombinant hirudin HM2 from H. manillensis (Scacheri et al, 1993) was conducted using several proteases of known properties and substrate specificity. Since the aim of these experiments was to probe the structure and dynamics of the hirudin molecule, proteolytic enzymes of restricted specificity such as the Glu-C-specific protease from S. aureus V8 (Houmard and Drapeau, 1972;Drapeau, 1977) and trypsin (Walsh, 1970), as well as of broad specificity, such as subtilisin (Ottesen ans Svendsen, 1970) and thermolysin (Heinrikson, 1977), were employed.…”

“…Hirudin from H. manillensis was found to be glycosylated and not sulphated (Steiner et al, 1992). A 64-residue hirudin variant, HM2, from H. manillensis was expressed in Escherichia coli utilizing a synthetic gene (Scacheri et al, 1993). The recombinant HM2, lacking post-translational modifications, pos-sessed an inhibitory activity for thrombin comparable to that of other recombinant hirudins.…”

“…In recent studies (Electricwala et al, 1991 ;Steiner et al, 1992;Scacheri et al, 1993), hirudin variants have been isolated from Hirudinaria manillensis, called buffalo leech, a species which is significantly more specialized for mammalian parasitism than H. medicinalis (Sawyer, 1986). The primary structures of hirudin isoforms from H. manillensis (Steiner et al, 1992) show significant (60-75%) sequence similarity to those of H. medicinalis (Dodt et al, 1985;Scharf et al, 1989), and, in particular, contain six cysteine residues at highly conserved positions, thus signifying the same pattern of disulfide formation (Thornton, 1981).…”