Molecular cloning and expression of rabbit antithrombin III.
A cDNA containing the complete open-reading frame encoding rabbit antithrombin III (AT-III) was isolated from a rabbit liver cDNA expression library, using a specific antibody as a probe. Sequence analysis showed 84% identity between the deduced amino acid sequences of the rabbit and human proteins. A previously described cell-free expression system was used to verify the identity of the clone. The full-length cDNA was inserted into an expression vector, and messenger RNA (mRNA) transcripts generated. In vitro translation of these transcripts, in the presence of [35S]methionine, in an mRNA-dependent rabbit reticulocyte lysate system resulted in the synthesis of a 51-Kd polypeptide, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This nonglycosylated protein was capable of forming SDS-stable complexes with human alpha-thrombin. Complex formation was significantly enhanced following the deletion of nucleotides encoding the signal peptide, and the resultant generation of a 47-Kd nonglycosylated mature protein product. When the template DNA giving rise to this product was internally truncated, two rabbit AT-III deletion mutants were generated that lacked the ability to interact with thrombin, but retained the ability to bind heparin. Cell-free expression plasmids encoding the human and rabbit AT-III mature molecules were manipulated to produce two interspecies fusion proteins. For the first, human codons were used to replace rabbit codons from residue 369-433, while in the second human codons replaced rabbit codons from residue 217-433. Both fusion proteins exhibited less efficient thrombin-complexing ability than the original cell-free-derived mature rabbit AT-III. Thus, portions of AT-III molecules from the two species, despite their high degree of homology, are not interchangeable. Knowledge of the structure of rabbit AT-III, combined with the availability of the rabbit cDNA, will permit defined experimentation aimed at understanding antithrombin III structure relative to its function in vivo.
Sheffield
W P
WP
Canadian Red Cross Blood Transfusion Service, McMaster University, Hamilton, Ontario, Canada.
Brothers
A B
AB
Wells
M J
MJ
Hatton
M W
MW
Clarke
B J
BJ
Blajchman
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000990:Antithrombin III / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D002474:Cell-Free System; D003001:Cloning, Molecular; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits; D013917:Thrombin / Q000378:metabolism
D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin