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

Chandra, T.S.; Stackhouse, Robin; Kidd, Vincent J.; Woo, Savio L. C.

doi:10.1073/pnas.80.7.1845

Cited by 138 publications

(78 citation statements)

References 29 publications

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“…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.…”

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confidence: 99%

“…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.…”

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

Liu

Chang

1987

European Journal of Biochemistry

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From structural analysis on genetically abnormal and chemically modified human antithrombin Ill [Koide, T., Odani, S., Takahashi, K., Ono, T. and Sakuragawa, N. (1984) Proc. Natl Acad. Sci. USA 81,[289][290][291][292][293] Chang, J.-Y. and Tran, T. H., (1986) J. Biol. Chem. 261, 1174-1176Blackburn, M. N., Smith, R. L., Carson, J. and Sibley, C. C. (1984) J . Biol. Chem. 259,, the heparin-binding site of antithrombin 111 has been suggested to be in the region of Pro-41, Arg-47 and Trp-49. In this study the heparin-binding site was probed by preferential cleavage of V8 protease on heparin-treated and non-treated native antithrombin 111. The study has been based on the presumption that the heparin-binding site of antithrombin 111 is situated at exposed surface domain and may be preferentially attacked during limited proteolytic digestion. Partially digested antithrombin I11 samples were monitored by quantitative amino-terminal analysis and amino acid sequencing to identify the preferential cleavage sites. 1-h-digested antithrombin 111 was separated on HPLC and peptide fragments were isolated and characterized both qualitatively and quantitatively. The results reveal that Glu-Gly (residues 34 -39, Glu-Ala (residues 42-43) and Glu-Leu (residues 50-51) are three preferential cleavage sites for V8 protease and their cleavage, especially the Glu-Ala and the Glu-Leu sites, was drastically inhibited when antithrombin 111 was preincubated with heparin. Both high-affinity and low-affinity antithrombin-111-binding heparins were shown to inhibit the V8 protease digestion of native antithrombin 111, but the high-affinity sample exhibited a higher inhibition activity than the low-affinity heparin. These findings (a) imply that the segment containing residues 34-51 is among the most exposed region of native antithrombin I11 and (b) support the previous conclusions that this region may play a pivotal role in the heparin binding.The plasma glycoprotein, antithrombin 111, regulates the blood-blotting system by inhibiting a number of hemostatic proteases, such as thrombin and factors IXa, Xa, XIa and XIIa. These reactions are greatly accelerated by the polysaccharide heparin [l -31. Elucidation of the molecular basis for the inhibitory action of antithrombin I11 in the presence of heparin has been complicated by the fact that heparin binds not only to antithrombin 111 but also with the cognate enzymes like thrombin and factor IXa [l, 41. Different models have been proposed to account for the mechanism of heparin function [5 -141. One model that has been studied in detail showed that heparin combines with antithrombin 111 to induce conformational change for more competent inhibitory function [15 -201. 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 region including Pro-41, Arg-47 and Trp-49 could be the potential heparin-binding site of antithrombin 111.In this paper ...

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confidence: 99%

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confidence: 99%

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

Liu

Chang

1987

European Journal of Biochemistry

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“…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.…”

Section: Resultsmentioning

confidence: 99%

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

1986

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A novel member of the serpin family of serine protease inhibitors is presented. A plasmid-like DNA was isolated from rabbit cells by its homology to the genome of Shope fibroma virus (SFV), a tumorigenic poxvirus of rabbits, and was shown elsewhere to encode a serpin-like protein ) Mol. Cell. Biol. 6, 265-2761 Although significant DNA homology exists between the rabbit plasmid serpin open reading frame and the SFV terminal inverted repeat DNA there is no intact serpin counterpart encoded by this region of the SFV genome. The alignment of the novel plasmid-borne polypeptide with the serpin family of proteins confirms its status within this group.

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“…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).…”

Section: Methodsmentioning

confidence: 99%

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

Flink¹,

Bailey²,

Gustafson³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

162

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Antibodies directed against thyroxine-binding globulin (TBG) have been used to screen a human liver Xgtll expression library. A 1.46-kilobase clone was identified which encodes nearly the complete amino acid sequence, beginning at amino acid 17 of the mature protein. To complete the protein sequence, the cDNA clone was used to identify a genomic clone coding for TBG in a human X chromosome library. The overlapping recombinant clones contained an open reading frame coding for 415 amino acids followed by a polyadenylylation signal (AATAAA) located 275 nucleotides from a TAG termination codon. Beginning at residue 21, the deduced amino acid sequence agrees closely with the known NH2-terminal sequence of the mature peptide. The preceding 20 amino acid residues are hydrophobic in character and presumably represent a leader sequence. Four glycosylation sites were identified, corresponding to the number determined for the purified protein. DNA blot hybridization revealed a single-copy gene, which by chromosomal analysis was found to be located on the long arm of the X chromosome. Unexpectedly, the nucleotide sequence of TBG is closely homologous to those encoding the plasma serine antiproteases a1-antichymotrypsin and a1-antitrypsin. However, there is little overall homology between TBG and transthyretin (prealbumin), the other major thyroxine-binding protein of human plasma.In most vertebrate species, thyroxine-binding globulin (TBG) has been reported to be the major thyroxine-binding protein (1). It is a glycoprotein (molecular mass about 54 kDa) that is synthesized in the liver as a polypeptide of 45 kDa (2). TBG represents a relatively rare translation product of total liver mRNA, accounting for only about 0.018% of total acidprecipitable radioactivity (2). Efforts to obtain detailed information about the structure of TBG and its thyroxinebinding site have been hampered by the presence of multiple electrophoretic forms and the inability to crystallize the protein (3, 4).In this report, we describe the use of serum containing polyclonal antibodies directed against human TBG to isolate cDNA clones coding for TBG from a human liver Xgtll expression library (5). To complete the coding sequence, one of these clones was used to identify a genomic clone from a human X chromosome library. Residues 21-40 of the deduced amino acid sequence correspond closely to the NH2-terminal amino acid sequence of mature TBG reported by Cheng (6). Amino acids 1-20 are very hydrophobic in nature and presumably represent the leader sequence of the propeptide. Unexpectedly, TBG shows a high degree of homology to a1-antichymotrypsin and a1-antitrypsin, and appears to belong to the serpin superfamily, most of which are serine proteinase inhibitors (7,8

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

Cited by 138 publications

References 29 publications

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

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

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

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

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