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J. Biol. Chem., Vol. 279, Issue 10, 9072-9079, March 5, 2004

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Lectin-deficient Calnexin Is Capable of Binding Class I Histocompatibility Molecules in Vivo and Preventing Their Degradation^*

Michael R. Leach and David B. Williams

From the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc₁Man₉GlcNAc₂, calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, we have generated six calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc₁ Man₉GlcNAc₂ oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean -mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K^b and D^b as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with K^b and D^b heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions.

Received for publication, September 30, 2003 , and in revised form, December 15, 2003.

^* This work was supported by the Canadian Institutes of Health Research and by the National Cancer Institute of Canada with funds from the Canadian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a studentship from the Canadian Institutes of Health Research.

To whom correspondence should be addressed: Dept. of Biochemistry, Medical Sciences Bldg., University of Toronto, Toronto, ON M5S 1A8, Canada. Tel.: 416-978-2546; Fax: 416-978-8548; E-mail: david.williams{at}utoronto.ca.

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