Structure-function analysis of the molecular chaperones calnexin and calreticulin.
Published: 2004
Language: eng
Number of pages: 241
Description: Calnexin (CNX) and calreticulin (CRT) are, respectively, membrane bound and soluble lectins of the endoplasmic reticulum (ER) with specificity for Asn-linked oligosaccharides of the form Glc1Man 9GlcNAC2. CNX and CRT bind transiently to folding intermediates possessing this monoglucosylated glycan and recruit a protein disulfide oxidoreductase, ERp57. In addition, CNX and CRT function as molecular chaperones binding directly to folding intermediates via polypeptide interactions. The objective of this thesis is to localize the sites of interaction on CNX/CRT for oligosaccharide, ERp57 and polypeptide and, further, to examine the functional relevance of the polypeptide binding site. Using deletion mutants, I localized the lectin site of CNX and CRT to their globular beta-sandwich domains. CNX and CRT also possess an arm-like extension and I localized the ERp57-binding site to this domain. In aggregation suppression assays, the globular domain, but not the arm domain, was capable of preventing the aggregation of model substrates, indicating that the polypeptide-binding site resides in the same domain as the lectin site. Six residues of the lectin site of CNX were mutated to alanines and each of these mutants had compromised lectin activity, but wild type levels of ERp57 binding. All six mutants prevented the aggregation of a non-glycosylated substrate, demonstrating that the polypeptide-binding site had not been disturbed. By contrast, the mutants were compromised in preventing the aggregation of a monoglucosylated substrate. To determine whether the lectin site is required for a functional interaction with substrates in vivo, two of the lectin-site mutants of CNX were transfected into a Drosophila  expression system along with a model glycoprotein, the major histocompatibility complex class I heavy chain (MHC I). Like wild-type CNX, lectin-site deficient CNX formed a stable complex with MHC I and was able to delay the degradation of the glycoprotein. Thus, CNX is able to function as a chaperone  in vivo despite lacking the ability to bind oligosaccharide. This demonstrates the importance of polypeptide-based interactions in the assocation of CNX with glycoprotein folding intermediates. The dual-binding ability of CNX allows for both enhanced binding and specificity of binding in its role as a molecular chaperone for glycoproteins in the secretory pathway.
Notes: Adviser:  David B. Williams.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-10, Section: B, page: 5133.