[Molecular mechanisms of antithrombin deficiency caused by T98I and A404T mutation].
OBJECTIVE
To study the molecular mechanisms of antithrombin (AT) deficiency caused by AT gene mutations T98I and A404T.


METHODS
Wild-type and mutant AT cDNA expression plasmids (ATwt, AT T98I and AT A404T) were constructed and transfected into the monkey fibroblast of the line COS-7 or Chinese hamster ovary (CHO) cells. NH4Cl. ALLN and brefeldin A were added. ELISA was used to detect the AT: Ag. Pulse-chase experiment and immunofluorescence assay were used to detect the radioactivity of the 35S-labeled AT. Fluorescence real-time PCR was used to detect the expression of AT mRNA, protein degradation inhibition was used to elucidate the mutant T98I degradation pathway inside the cells.


RESULTS
AT T98I was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of AT A404T, but most of the molecule was not secreted but was degraded intracellularly. Fluorescence real-time PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Protein degradation inhibition experiment showed that mutant AT T98I was degraded intracellularly through the proteasome pathway. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT T98I mutant were stained diffusely without perinuclear enhancement and cells expressing AT A404T mutant mainly in the whole cytoplasm with weaker perinuclear enhancement.


CONCLUSIONS
Impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular mechanism of AT deficiency caused by T98I and A404T mutation.
Zhou
Rong-fu
RF
Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao tong University, Shanghai 200025, China.
Dai
Jing
J
Fu
Qi-hua
QH
Wang
Wen-bin
WB
Xie
Shuang
S
Ding
Qiu-lan
QL
Wang
Xue-feng
XF
Wang
Hong-li
HL
D000818:Animals; D020152:Antithrombin III Deficiency / Q000235:genetics*; D000991:Antithrombins / Q000235:genetics* / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000991:Antithrombins; D050505:Mutant Proteins; D012333:RNA, Messenger