Expression and release of the a and b subunits for human coagulation factor XIII in baby hamster kidney (BHK) cells.
The a subunit of coagulation factor XIII lacks a hydrophobic signal sequence for secretion from cells, while the b subunit has a typical signal sequence. To determine whether the a subunit can be synthesized and released, expression vectors containing the cDNA for either subunit were transfected into baby hamster kidney (BHK) cells. Western blotting analysis and gel filtration chromatography demonstrated that the recombinant a and b subunits (rXIIIa and rXIIIb) had the same molecular weights and subunit structures (a2, b2, and a2b2) as the native molecules. rXIIIa was enzymatically active when activated by thrombin. Most rXIIIb was secreted as measured by ELISA, while most rXIIIa was detected in the cytosol by subcellular fractionation. Co-expression with rXIIIb in the same cells did not promote the release of rXIIIa. Treatment of the cells with brefeldin A, a potent inhibitor of protein transportation, blocked the secretion of rXIIIb, although it had no effect on the release of rXIIIa. Several drugs and heat stress induced the release of rXIIIa, which correlated directly with that of cytoplasmic lactate dehydrogenase. These results suggest that the a subunit is released from cells as a consequence of cell injury, which is independent of the classical secretory pathway.
Kaetsu
H
H
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Hashiguchi
T
T
Foster
D
D
Ichinose
A
A
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D020126:Brefeldin A; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D002850:Chromatography, Gel; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D004790:Enzyme Induction; D004797:Enzyme-Linked Immunosorbent Assay; D005176:Factor XIII / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection
D003517:Cyclopentanes; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C064015:factor XIII subunit A; D020126:Brefeldin A; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D005176:Factor XIII; D008985:Monensin; C054380:factor XIIIb; D013917:Thrombin; D019297:2,4-Dinitrophenol