title: Effects of glycosylation on the structure and function of the extracellular chaperone clusterin.
abstract: Clusterin is the first well characterized, constitutively secreted extracellular chaperone that binds to exposed regions of hydrophobicity on non-native proteins. It may help control the folding state of extracellular proteins by targeting them for receptor-mediated endocytosis and intracellular lysosomal degradation. A notable feature of secreted clusterin is its heavy glycosylation. Although carbohydrate comprises approximately 20-25% of the total mass of the mature molecule, its function is unknown. Results from the current study demonstrate that deglycosylation of human serum clusterin had little effect on its overall secondary structure content but produced a small increase in solvent-exposed hydrophobicity and enhanced the propensity of the molecule to aggregate in solution. These changes were associated with increased binding to a variety of ligands but did not substantially impact the ability of clusterin to inhibit heat-induced precipitation of citrate synthase. Evidence suggesting that the normally conjugated sugars are important in the interaction of secreted clusterin with a lectin-type receptor on liver cells is also presented. Bulk expression of fully processed, glycosylated clusterin in mammalian cells is difficult, often producing inappropriately disulfide-bonded high molecular weight aggregates; this has hampered previous studies aimed at identifying those regions of the molecule important in its chaperone action. The current results suggest that it may be possible in the future to study the structure and chaperone function of clusterin using recombinant protein (lacking sugars) conveniently bulk-expressed in bacteria.
authors.affiliation: School of Biological Sciences and the Institute for Biomolecular Sciences, University of Wollongong, Northfields Avenue, Wollongong 2522, NSW, Australia.
authors.forename: Elise M
authors.initials: EM
authors.lastname: Stewart
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Aquilina
authors.forename: Simon B
authors.initials: SB
authors.lastname: Easterbrook-Smith
authors.forename: Danielle
authors.initials: D
authors.lastname: Murphy-Durland
authors.forename: Christian
authors.initials: C
authors.lastname: Jacobsen
authors.forename: Soren
authors.initials: S
authors.lastname: Moestrup
authors.forename: Mark R
authors.initials: MR
authors.lastname: Wilson
mesh_terms: D002241:Carbohydrates / Q000494:pharmacology; D002950:Citrate (si)-Synthase / Q000378:metabolism; D051152:Clusterin / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018832:Molecular Chaperones / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011975:Receptors, Mitogen / Q000378:metabolism
chemical_list: C495242:CLU protein, human; D002241:Carbohydrates; D051152:Clusterin; D018832:Molecular Chaperones; D011975:Receptors, Mitogen; D002950:Citrate (si)-Synthase