Deletion mutagenesis of heparin cofactor II: defining the minimum size of a thrombin inhibiting serpin.
Heparin cofactor II (HCII) is a 66 kDa plasma glycoprotein that belongs to the serpin superfamily of protease inhibitors. Its natural target is thrombin. HCII inhibits thrombin in both a progressive reaction, and in an accelerated reaction catalyzed by a glycosaminoglycan, dermatan sulphate (DS). Both modes of inhibition result in the formation of a stable, denaturation-resistant complex. Using a cDNA clone encoding rabbit HCII recently isolated and characterized in our laboratory, we have employed deletion mutagenesis to identify amino-terminal regions of the molecular which are essential to the progressive reaction. PCR was employed to produce four deletion constructs: delta 58, delta 81, delta 106, and delta 169, all in an in vitro transcription vector plasmid background. Transcription of the full-length construct, and of the four deletion constructs, followed by in vitro translation in rabbit reticulocyte lysate, was used to produce the corresponding HCII-related polypeptides. The delta 106 and delta 169 mutants failed to react with thrombin, even in the presence of DS. In contrast, the delta 58 and delta 82 mutants retained the ability to form complexes with thrombin, although the rate of complex formation was decreased for the latter mutant compared to the full-length recombinant HCII; no acceleration of complex formation in the presence of 20 micrograms/ml DS was noted for either truncated recombinant HCII. Alignment of the rabbit HCII primary structure with secondary structural elements found in alpha 1 antitrypsin and other serpins showed that the non-functional delta 106 mutant lacks helix A, while the functional delta 82 mutant contains this element. Our results suggest that helix A is an essential part of a functional serpin, and define the limits of the amino-terminal region of HCII which is not essential for thrombin inhibition.
Sheffield
W P
WP
Canadian Red Cross Blood Services, McMaster University Medical Centre, Hamilton, Ontario.
Blajchman
M A
MA
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D015844:Heparin Cofactor II / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012156:Reticulocytes / Q000378:metabolism; D017384:Sequence Deletion; D013917:Thrombin / Q000037:antagonists & inhibitors*; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D015844:Heparin Cofactor II; D013917:Thrombin