Heparin cofactor II: cDNA sequence, chromosome localization, restriction fragment length polymorphism, and expression in Escherichia coli

• Published in: Biochemistry (Easton) Vol. 27; no. 2; pp. 752 - 759
• Main Authors: Blinder, Morey A, Marasa, Jayne C, Reynolds, Craig H, Deaven, Larry L, Tollefsen, Douglas M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 26.01.1988
• Subjects: Amino Acid Sequence; Antithrombins - genetics; Base Sequence; Biological and medical sciences; Chromosome Mapping; Chromosomes, Human, Pair 22; Cloning, Molecular; DNA - genetics; DNA Restriction Enzymes; DNA, Recombinant - metabolism; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genes; Genes. Genome; Glycoproteins - genetics; Heparin Cofactor II; Humans; Leukocytes - metabolism; Liver - metabolism; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Protein Biosynthesis; Transcription, Genetic; Human; Complementary DNA; Gene; Liver; Primary structure; Thrombin; Inhibitor; Heparin; Gene expression; Polymorphism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00402a039

Heparin cofactor II (HCII) is an inhibitor of thrombin in plasma that is activated by dermatan sulfate or heparin. An apparently full-length cDNA for HCII was isolated from a human liver lambda gt11 cDNA library. The cDNA consisted of 2215 base pairs (bp), including an open-reading frame of 1525 bp, a stop codon, a 3'-noncoding region of 654 bp, and a poly(A) tail. The deduced amino acid sequence contained a signal peptide of 19 amino acid residues and a mature protein of 480 amino acids. The sequence of HCII demonstrated homology with antithrombin III and other members of the alpha 1-antitrypsin superfamily. Blot hybridization of an HCII probe to DNA isolated from sorted human chromosomes indicated that the HCII gene is located on chromosome 22. Twenty human leukocyte DNA samples were digested with EcoRI, PstI, HindIII, KpnI, or BamHI, and Southern blots of the digests were probed with HCII cDNA fragments. A restriction fragment length polymorphism was identified with BamHI. A slightly truncated form of the cDNA, coding for Met-Ala instead of the N-terminal 18 amino acids of mature HCII, was cloned into the vector pKK233-2 and expressed in Escherichia coli. The resultant protein of apparent molecular weight 54,000 was identified on an immunoblot with 125I-labeled anti-HCII antibodies. The recombinant HCII formed a complex with 125I-thrombin in a reaction that required the presence of heparin or dermatan sulfate.