Polypeptide substrate recognition by calnexin requires specific conformations of the calnexin protein.
Calnexin is an endoplasmic reticulum chaperone that binds to substrates containing monoglucosylated oligosaccharides. Whether calnexin can also directly recognize polypeptide components of substrates is controversial. We found that calnexin displayed significant conformational lability for a chaperone and that heat treatment and calcium depletion induced the formation of calnexin dimers and higher order oligomers. These conditions enhanced the chaperone activity of calnexin toward glycosylated and non-glycosylated major histocompatibility complex (MHC) class I heavy chains, and enhanced calnexin binding to MHC class I heavy chains. In contrast to these observations, calnexin binding to oligosaccharide substrates has been reported to be impaired under calcium-depleting conditions. Calnexin dimers were induced in HeLa cells upon heat shock and under calcium-depleting conditions, and heat shock enhanced calnexin binding to MHC class I heavy chains in HeLa cells. Virus-induced endoplasmic reticulum stress also resulted in the appearance of calnexin dimers. Tunicamycin treatment of HeLa cells induced a slow accumulation of calnexin dimers, the appearance of which correlated with enhanced calnexin binding to deglycosylated MHC class I heavy chains. In vitro, the presence of calnexin-specific oligosaccharides inhibited the formation of calnexin dimers and higher order structures. Together, these data indicate that polypeptide binding is favored by conditions that induce partial unfolding of calnexin monomers, whereas oligosaccharide binding is favored by conditions that enhance the structural stability (folding) of calnexin monomers. Conditions that induce the calnexin "polypeptide-binding" conformation also induce self-association of calnexin if the concentration is sufficiently high; however, calnexin dimerization/oligomerization per se is not essential for polypeptide substrate binding.
Thammavongsa
Vilasack
V
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620, USA.
Mancino
Laura
L
Raghavan
Malini
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D004285:Dogs; D004533:Egtazic Acid / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D008968:Molecular Conformation; D009844:Oligosaccharides / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020673:Protein Renaturation; D012995:Solubility; D013379:Substrate Specificity; D014415:Tunicamycin / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D015395:Histocompatibility Antigens Class I; D018832:Molecular Chaperones; D009844:Oligosaccharides; D010455:Peptides; D014415:Tunicamycin; D037281:Calnexin; D004533:Egtazic Acid; D002118:Calcium