Molecular Cloning and Expression of Rabbit Heparin Cofactor II: A Plasma Thrombin Inhibitor Highly Conserved between Species

Abstract

SummaryHeparin cofactor II (HCII), a circulating plasma protein that inhibits thrombin, is a member of the serine proteinase (serpin) family of proteins. The extent to which HCII structure is conserved actross species lines was investigated, by obtaining cDNA clones encoding rabbit HCII. Overlapping clones corresponding to rabbit HCII were obtained by the combined use of hybridization screening of a rabbit liver cDNA library, and by rapid amplification of cDNA ends (RACE). The consensus sequence obtained spans 2178 nucleotides, and is comprised of a 5' untranslated region of 77 nucleotides, an open reading frame of 1440 nucleotides, and 3' untranslated region of 661 nucleotides that concludes with a poly A tract. The open reading frame is subdivided into a secretory signal sequence of 19 amino acids, and a mature protein of 461 amino acids. Within the region comprising the mature protein, 87% of the amino acid residues are identical to those seen in human HCII. Expression of an appropriately modified form of the rabbit HCII clone in an in vitro reticulocyte expression system yielded two major polypeptides, of 60 and 56 kD respectively, both of which were able to form SDS-stable complexes with human α-thrombin, in a reaction accelerated by dermatan sulphate. The remarkable degree of homology observed between rabbit HCII and its human conterpart, indicating a high degree of conservation of structure through evolution, suggests an important function of HCII in hemostatis.

Deletion mutagenesis of heparin cofactor II: defining the minimum size of a thrombin inhibiting serpin

“…We have chosen to use deletion mutagenesis to map the minimum essential form of HCII that is sufficient for complex formation with thrombin. HCII was selected over AT because of its lack of disulphide bonding, and because we have recently demonstrated that its expression in rabbit reticulocyte lysate systems yields a more functional product than that of AT [14]. Our results suggest that the helix A structural element is an essential part of a functional serpin.…”

“…Transcription plasmids were transcribed in vitro from phage promoters; the A169, 106, and 58 constructs using T 7 polymerase, and the A81 construct using SP 6 polymerase, as described previously [14]. Translation was performed as described [14,18], in a messenger-dependent reticulocyte lysate supplemented with [35S]methionine.…”

“…In order to generate plasmid transcription templates encoding amino-terminal truncations of rabbit HCII, PCR was employed, using the previously described plasmid pMHC3 as template [14], and the M 13 universal primer (Pharmacia) and a mutagenic HCII-specific oligonucleotide as primers. Constructs, and the truncated HCII protein they encode, are named for the number of amino acids deleted.…”

“…SSDI 0014-5793(95)00468-8 Blajchman/FEBS Letters 365 (1995) [189][190][191][192] (5'-TCATGACGCATCGCCTCTTCAGGA-3'); for the AI06 construct, primer 4114 was used (5'-CCATGGCCAACGCTTTTGATA-ACATC-3'); and for the A58 construct, primer 3839 was used (5'-TCATGATCATCGACGCTGTTTCCC-3'). PCR conditions were exactly as previously described [14]. After PCR, each product was made blunt-ended with Klenow fragment, gel-purified from agarose, and introduced into the EcoRV site of pGEM3zf+ by standard methods of ligation and transformation [15].…”

“…These related proteins share approximately 30% amino acid identity, as well as a common mechanism of action. This involves attack by target proteases at the reactive site of the inhibitor, followed by formation of a stable, 1:1 complex in which both protease and inhibitor are inactive [14].…”