The Molecular Chaperone Calnexin Binds GlcManGlcNAc Oligosaccharide as an Initial Step in Recognizing Unfolded Glycoproteins

doi:10.1074/jbc.270.9.4697

The Molecular Chaperone Calnexin Binds GlcManGlcNAc Oligosaccharide as an Initial Step in Recognizing Unfolded Glycoproteins (*)

From the (1)Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, the
(2)Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and the
(3)R. W. Johnson Pharmaceutical Research Institute, La Jolla, California 92121

** To whom correspondence should be addressed.
Dept. of Biochemistry, Medical Sciences Bldg., University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Tel.: 416-978-6034; Fax: 416-978-8548.

Abstract

Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum. Most proteins that calnexin binds are N-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin binding. To test if calnexin is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble calnexin was created. Incubation of soluble calnexin with a mixture of GlcMan₉GlcNAc₂ oligosaccharides resulted in specific binding of the Glc₁Man₉GlcNAc₂ species. Furthermore, Glc₁ManGlcNAc₂ oligosaccharides bound relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residues was important for binding. To assess the involvement of oligosaccharide-protein interactions in complexes of calnexin and newly synthesized glycoproteins, α₁-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without disrupting the association with calnexin. Furthermore, the addition of 1 M α-methyl glucoside or α-methyl mannoside had no effect on complex stability. These findings suggest that once complexes between calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction. However, it is likely that the binding of Glc₁Man₉GlcNAc₂ oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin.

Footnotes

↵^§ Felecia E. Ware and Aikaterini Vassilakos are joint first authors.
↵^¶ Supported by an Ontario Graduate Scholarship.
↵* This work was supported by Grant GM38545 from the National Institutes of Health, by Grant I-1168 from the Robert Welch Foundation (to M. A. L.), by a grant from the National Cancer Institute of Canada, and by funds from the Canadian Cancer Society (to D. B. W.). 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:

ER

endoplasmic reticulum

HA

hemagglutinin

β₂m

β₂-microglobulin

HPLC

high performance liquid chromatography

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

endo H

endoglycosidase H

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonic acid.
↵²A. Vassilakos, unpublished observations.
↵³M. Jannatipour and L. A. Rokeach, submitted for publication.
- Received September 9, 1994.
- Revision received November 22, 1994.