Isolation and sequence characterization of a cDNA clone of human antithrombin III.

Abstract

A human liver cDNA library was constructed by using poly(A)-containing RNA isolated from a human liver biopsy specimen. This library is comprised of 40,000 independent transformants with an average inserted DNA length of 1,200 base pairs. By using the previously cloned baboon antithrombin III cDNA as a specific hybridization probe, >30 human antithrombin mII cDNA clones were identified from this library. The Antithrombin III is a plasma protease inhibitor synthesized in the liver. The glycoprotein has a molecular weight of 55,000 and its entire amino acid sequence has almost been completed (1). It is a natural anticoagulant in that it specifically inhibits a number of serine proteases that participate in the blood coagulation cascade, including thrombin, factors IXa, Xa, XIa, and XIIa (2-5). The mechanism of inhibition involves the stoichiometric formation of protease-antiprotease complexes and the rate of complex formation is greatly enhanced in the presence of heparin, which is a well-known anticoagulant used clinically in myocardial infarction and surgery (6, 7). Deficiency of antithrombin III is a hereditary disorder that is associated with recurrent thrombophlebitis, acute aortic thrombosis, and thromboembolism (8-10). Heterogeneity of the classical antithrombin III deficiency has been observed (11). Abnormal antithrombin III also has been isolated from deficient patients and partially characterized (12), suggesting that the deficiency could be the result of mutations in the antithrombin III gene itself. Therefore, the genetic deficiency can be analyzed in molecular detail if the human antithrombin III gene can be isolated and characterized. We recently have reported the purification of antithrombin III mRNA from a baboon liver by polysome immunoprecipitation and the cloning of its cDNA (13). In this paper, we report the construction of a human liver cDNA library and the identification and sequence of a full-length human antithrombin III cDNA clone. MATERIALS AND METHODSConstruction of a Human Liver cDNA Library. A human liver biopsy specimen was kindly provided by Bill Williams (University of Texas Medical School in Houston). Total nucleic acid was extracted from the frozen tissue with phenol (14), and poly(A)-containing RNA was prepared by oligo(dT)-cellulose column chromatography (15). The RNA preparation was used for cDNA synthesis under conditions that favored full-length cDNA production (16). The cDNA preparation was sedimented through an alkaline sucrose gradient (16) and only fractions containing cDNA species of 1,000 nucleotides or more were pooled. The single-stranded cDNA was subsequently made double-stranded with reverse transcriptase (17). After nuclease S1 treatment, tracts of poly(dC) were added to the 3' termini of the DNA molecules by using terminal transferase (18). The enzymatically synthesized cDNA was rehybridized with Pst I-linearized pBR322 DNA that had been tailed with poly(dG) and the DNA was used for transformation of Escherichia coli RR1 (18). Bacterial transformants we...

Probing the heparin‐binding domain of human antithrombin III with V8 protease

“…The heparin -antithrombin-111 complex is apparently formed by the electrostatic interaction between the acidic groups (mostly sulfate) of heparin [9, 211 and the Lys/ Arg residues of antithrombin 111 122 -281. The complete sequence of human antithrombin 111 has been worked out on both protein [29] and cDNA levels [30]. So far, the heparinbinding domain of antithrombin I11 has been inferred from the works on chemical characterization of the two genetically abnormal antithrombin 111, i.e.…”

“…3 was eventually sequenced by both manual and automatic methods and results are shown in Table 1. The position of each peptide in the antithrombin I11 sequence was assigned on the basis of the known primary structure of human antithrombin I11 [30]. Peak 1 consists of four hydrophilic peptides and two of them, Val-Asn-Glu-Glu and Gly-Ser-Glu, happen to be in the vicinity of the reactive site (Arg393-Ser394) of antithrombin 111.…”

A novel member of the serpin superfamily is encoded on a circular plasmid‐like DNA species isolated from rabbit cells

“…It should [21], chicken ovalbumin [22], chicken gene Y [ 131 and rat angiotensinogen [ 151 have been aligned to minimize the insertion of gaps within regions of secondary structure based on that of al-AT. There are 18 residues which are conserved through all the serpins shown and in 133 positions the amino acid is conserved in at least 50% of the proteins.…”

Complete amino acid sequence of human thyroxine-binding globulin deduced from cloned DNA: close homology to the serine antiproteases.

“…A human liver cDNA library of =1.2 x 106 independent recombinants was constructed in Xgtll (9) and kindly provided by S. L. C. Woo (Department of Cell Biology, Baylor College of Medicine, Houston, TX). The library was screened with anti-TBG antibodies (Calbiochem) essentially as described by Davis and Young (5).…”