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Vol. 19, Issue 6, 2413-2423, June 2008

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Lectin-deficient Calreticulin Retains Full Functionality as a Chaperone for Class I Histocompatibility Molecules

Breanna S. Ireland^*,, Ulf Brockmeier^*,, Christopher M. Howe, Tim Elliott, and David B. Williams^*

*Departments of Biochemistry and Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada; and Cancer Sciences Division and CRC Medical Oncology Unit, University of Southampton School of Medicine, Southampton SO16 6YD, United Kingdom

Submitted October 19, 2007; Revised February 19, 2008; Accepted March 5, 2008
Monitoring Editor: Benjamin Glick

Calreticulin is a molecular chaperone of the endoplasmic reticulum that uses both a lectin site specific for Glc₁Man_5-9GlcNAc₂ oligosaccharides and a polypeptide binding site to interact with nascent glycoproteins. The latter mode of substrate recognition is controversial. To examine the relevance of polypeptide binding to protein folding in living cells, we prepared lectin-deficient mutants of calreticulin and examined their abilities to support the assembly and quality control of mouse class I histocompatibility molecules. In cells lacking calreticulin, class I molecules exhibit inefficient loading of peptide ligands, reduced cell surface expression and aberrantly rapid export from the endoplasmic reticulum. Remarkably, expression of calreticulin mutants that are completely devoid of lectin function fully complemented all of the class I biosynthetic defects. We conclude that calreticulin can use nonlectin-based modes of substrate interaction to effect its chaperone and quality control functions on class I molecules in living cells. Furthermore, pulse-chase coimmunoisolation experiments revealed that lectin-deficient calreticulin bound to a similar spectrum of client proteins as wild-type calreticulin and dissociated with similar kinetics, suggesting that lectin-independent interactions are commonplace in cells and that they seem to be regulated during client protein maturation.

These authors contributed equally to this work.

Address correspondence to: Dr. David B. Williams (david.williams{at}utoronto.ca)

Abbreviations used: β₂m, β₂-microglobulin; Cnx, calnexin; Crt, calreticulin; ER, endoplasmic reticulum; MHC, major histocompatibility complex; PLC, peptide loading complex; TAP, transporter associated with antigen processing; UGGT, UDP-glucose:glycoprotein glucosyltransferase.