Intracellular events determine the fate of antithrombin Utah.
We sought to determine whether intracellular or extracellular events contribute to the decrease in circulating antithrombin (AT) levels that is seen in subjects with the Utah mutation (Pro 407 to Leu). Site-directed mutagenesis was used to recreate this mutation within a previously characterized rabbit AT cDNA. Cell-free expression of the mutated cDNA yielded an AT protein that failed to react with thrombin. Expression of the rabbit AT-Utah protein in transiently transfected Cos cells resulted in a 10-fold decrease in the amount of AT antigen detected in the conditioned media, as compared with that seen with the wild-type recombinant AT. This effect was not caused by variations in transfection efficiency, because AT levels were normalized to the product of a cotransfected plasmid, chloramphenicol acetyl transferase. Moreover, on Northern blot analysis, AT mRNA levels were comparable in cells expressing either the rabbit AT-Utah or wild-type recombinant rabbit AT. Immunoblots of conditioned media from the two populations of transfected cells showed that the recombinant AT-Utah protein was intact. The results obtained with Cos cells were reproduced using permanently transfected Chinese hamster ovary (CHO) cells. Pulse-chase experiments with the CHO lines showed that both initial levels of rabbit AT-Utah after the pulse labeling and the rate of subsequent secretion during the chase period were reduced compared with that seen with cells expressing the wild-type AT. The observed reduction in AT secretion was also observed for the AT-Oslo mutation (Ala 404 to Thr) when recreated in the rabbit AT background, and expressed in Cos cells. In these experiments, the media levels of mutant AT were reduced by 50%, compared with wild-type. These results show that intracellular events, as opposed to accelerated clearance or other extracellular causes, contribute to the paucity of AT secretion seen in these strand 1C AT mutants.
Sheffield
W P
WP
Canadian Red Cross Society Blood Services, Hamilton, Ontario, Canada.
Castillo
J E
JE
Blajchman
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D058833:Antithrombin Proteins; D000991:Antithrombins / Q000235:genetics; D001483:Base Sequence; D016466:CHO Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D058833:Antithrombin Proteins; D000991:Antithrombins; D011993:Recombinant Fusion Proteins; C057784:antithrombin III Utah; C096224:antithrombin Oslo; D000990:Antithrombin III