Calnexin Influences Folding of Human Class I Histocompatibility Proteins but Not Their Assembly with -Microglobulin

doi:10.1074/jbc.270.33.19638

Calnexin Influences Folding of Human Class I Histocompatibility Proteins but Not Their Assembly with -Microglobulin (*)

From the Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

^§ To whom correspondence should be addressed:
Dept. of Pathology, 733 Scaife Hall, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
Fax: 412-648-1916; Tel.: 412-648-9471.

Abstract

Class I major histocompatibility complex heavy chains bind to calnexin before associating with β₂-microglobulin (β₂m) and peptides. Calnexin has been shown to retain in the endoplasmic reticulum those class I heavy chains which have not assembled properly and, thus, to serve as a quality control mechanism. In addition, calnexin may direct the folding of class I subunits or their subsequent assembly. We asked whether calnexin plays a role in the initial folding of HLA-B*0702 heavy chains by assessing disulfide bond formation in vivo. Our results show that class I heavy chains form intrachain disulfide bonds very soon after translation, and that calnexin is bound to both reduced and oxidized forms during this process. When a cell-permeable reducing agent, dithiothreitol, was added to cells, disulfide bond formation in newly synthesized heavy chains was substantially blocked, as was their association with calnexin. The reducing agent appeared to affect calnexin directly, since binding was similarly abolished to a subset of proteins which do not contain internal disulfide bonds. Addition of the glucosidase inhibitor castanospermine to cells, shown previously to disrupt calnexin binding to ligands, slowed formation of disulfide bonds but did not decrease the amount of assembled heavy chain-β₂m complexes that formed. Our data suggest that calnexin can promote disulfide bond formation in class I heavy chains but does not directly facilitate subsequent binding to β₂m.

Footnotes

↵* This study was supported by American Cancer Society Grant IM-668 and by a student fellowship from the Pathology Education and Research Foundation at the University of Pittsburgh. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MHC

major histocompatibility complex

ER

endoplasmic reticulum

PAGE

polyacrylamide gel electrophoresis

β₂m

β₂-microglobulin

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

DTT

dithiothreitol.
↵² M. Tector and R. D. Salter, unpublished data.
- Received March 27, 1995.
- Revision received June 8, 1995.