NOVEL HIRUDIN VARIANTS FROM THE LEECH HIRUDINARIA-MANILLENSIS - AMINO-ACID-SEQUENCE, CDNA CLONING AND GENOMIC ORGANIZATION

Novel hirudin variants isolated from the leech Hirudinaria manillensis , a leech more specialized for mammalian parasitism, are described. Is olation of antithrombin polypeptides was performed by ion-exchange chr omatographies followed by an affinity chromatography step on immobiliz ed thrombin. The major active component, antithrombin polypeptide peak 2 (HM2), and a second polypeptide, named HM1, were purified to homoge neity and their complete amino acid sequences were determined. The pro tein 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 di fferences mainly in the central part and in the C-terminal region of t he polypeptides. Particularly significant is the lack of a sulfated ty rosine residue in the C-terminal portion of the molecule which is repl aced by aspartic acid. Polymerase chain reaction cloning techniques we re used to isolate and characterize the cDNAs and determine the genomi c structures of these hirudin-like polypeptides. The cDNA clones codin g for the two variants indicate the expression of pre-hirudins of 84 a mino acids where the first 20 residues constitute the signal peptide r equired for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity w ith the partial hirudin variant 2 (HV2) signal peptide sequence previo usly reported. The HM1 and HM2 gene fragments show the presence of fou r exons: the first one corresponding to a 20-amino-acid signal peptide while the other three exons share the full primary structure of the a ntithrombin polypeptides. HM2 was also efficiently produced in recombi nant Escherichia coli by expressing a periplasmic construction contain ing the synthetic gene.