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. 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 C-terminal 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.
Scacheri
E
E
Biotechnology Department, Farmitalia Carlo Erba, Nerviano, Milan, Italy.
Nitti
G
G
Valsasina
B
B
Orsini
G
G
Visco
C
C
Ferrera
M
M
Sawyer
R T
RT
Sarmientos
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D006629:Hirudins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013917:Thrombin / Q000037:antagonists & inhibitors
D006629:Hirudins; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D013917:Thrombin