Calnexin is associated with and induced by overexpressed human complement protein C2.
C2 is a serum glycoprotein that is essential for activation of the classical and lectin pathways of the complement system. We reported previously that in transiently transfected COS cells, C2 accumulates in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). Transfection with a cDNA corresponding to a variant C2 mRNA in which exon 17 is spliced out, C2Delta(17), resulted in retention of the mutant polypeptide in the ER. We now show that calnexin, a lectin-like chaperone, colocalizes with wild-type (wt) C2 and C2Delta(17). Biosynthetic labeling and sequential immunoprecipitation experiments indicated that colocalization is due to a physical association between calnexin and C2. Immunofluorescence analysis indicated that calnexin was upregulated in cells transfected with either C2 species. Upregulation of calnexin was not affected by castanospermine, which inhibits glucosidases I and II. However, castanospermine inhibited translocation of calnexin to the ERGIC in wt C2 transfected cells. Upregulation of calnexin was also observed in cells transfected with the complement protein factor B, a glycoprotein with extensive structural and functional similarities to C2, but not in cells transfected with complement proteins C3 or factor D, which have no structural similarity to C2, and low or no glycan content, respectively. Calnexin upregulation by transfection with C2 or factor B, but not factor D, was also demonstrated by quantitative analysis of calnexin immunoprecipitates from biosynthetically labeled cells. Increased calnexin expression by overexpressed C2 and factor B appears to be triggered either by the high glycan content of these proteins or, since it also occurs in the presence of castanospermine, by shared features of the structure of these two proteins.
Tsukamoto
Hiroshi
H
Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama-Birmingham, Birmingham, Alabama 35294-0005, USA.
Tousson
Albert
A
Circolo
Antonella
A
Marchase
Richard B
RB
Volanakis
John E
JE
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D019556:COS Cells / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D037281:Calnexin; D002522:Chlorocebus aethiops; D003175:Complement C2 / Q000378:metabolism*; D011415:Complement Factor B / Q000378:metabolism; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D006801:Humans; D018832:Molecular Chaperones / Q000032:analysis / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation
D002135:Calcium-Binding Proteins; D003175:Complement C2; D018832:Molecular Chaperones; D037281:Calnexin; D011415:Complement Factor B