Antithrombin III Utah: proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site

Abstract

A dysfunctional antithrombin III (ATIII) gene encoding a qualitatively and quantitatively abnormal anticoagulant molecule is responsible for hereditary thrombosis in a Utah kindred [Bock et al. (1985) Am. J. Hum. Genet. 37, 32-41]. Nucleotide sequencing of the entire protein-encoding portion of the cloned ATIII-Utah gene revealed a C to T transitional mutation which converts proline-407 to leucine. Proline-407 is located 14 amino acids C-terminal to the reactive site arginine of ATIII in a core region of the molecule that has been highly conserved during evolution of the serine protease inhibitor (serpin) gene family. The location of this proline in the crystal structure of the homologous serpin alpha 1-antitrypsin suggests that the leucine substitution in ATIII-Utah may interfere with correct folding of the mutant gene product, leading to its rapid turnover and the low antithrombin levels observed in patient plasmas. The Pro-407 to Leu mutation does not interfere with binding of antithrombin III to heparin. Patient antithrombin III, isolated by affinity chromatography on heparin-Sepharose, was reacted with purified thrombin. ATIII encoded by the patient's normal gene formed protease-inhibitor complexes with thrombin, whereas the product of the ATIII-Utah gene did not. The Pro-407 to Leu mutation destroys a restriction site for the enzyme StuI, permitting rapid diagnosis of affected members of the Utah kindred by Southern blotting of genomic DNA.

Pleiotropic effects of antithrombin strand 1C substitution mutations.

“…Plasma samples from all seven families were subjected to CIE with heparin in the first dimension to investigate this possibility (Fig. 1 ) (31 ).…”

“…Decreased heparin affinity was confirmed for antithrombins Rosny and Torino by heparin-Sepharose chromatography (Fig. 2 ), but not for antithrombin Utah ( 31 ). Elements of the heparin-binding site are found on the lower face of the molecule shown in Fig.…”

“…Furthermore, the circulating concentrations ofthe proteins encoded by the normal and variant alleles in the heterozygous patients are usually similar, and the plasma antithrombin antigen concentration is near normal. In contrast, a total of eight different substitution mutants leading to antithrombin deficiency have now been identified in five positions of the 402-407 region (this report; [31,64] ), see Fig. 7, and these exhibit pleiotropic defects.…”

Molecular characterization of antithrombin III (ATIII) variants using polymerase chain reaction. Identification of the ATIII Charleville as an Ala 384 Pro mutation.

“…One of the variants of our series, ATIII Avranches, was found to bear such a mutation (393 Arg to His), identical to those found in ATIII Sheffield (30) and ATIII Glasgow (32,33 Antiprotease activity was also abolished by mutations located in the peptide sequences adjacent to the cleavage site. The Pro 407 Leu and Ala 382 Thr mutations observed in ATIII Utah and Hamilton (34,35) are associated with a loss of antiprotease activity. These two amino acids are extremely conserved in the serpin family; thus the nature or the location (respectively in P14 and P12) ofthe substitution may modify the presentation or the structure of the loop, so that it can no longer react with the catalytic site of thrombin (Fig.…”

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

“…The amino acid and cDNA sequences of antithrombin, as well as the intron structure of the antithrombin gene, have been determined (Petersen et al, 1979;Bock et al, 1982;Prochownik et al, 1985;Bock et al, 1988), and the protein has been expressed in yeast, insect cells and mammalian cell lines (Zettlmeissl et al, 1987(Zettlmeissl et al, , 1988(Zettlmeissl et al, , 1989Stephens et al, 1987;Wasley et al, 1987;Br6ker et al, 1987;Gillespie et al, 1991). It has thus become feasible to design studies, using site-directed mutagenesis, of the structure and localization of the heparinbinding site of antithrombin.…”