Abstract 14476: Glycosylation-Dependent Lipoprotein Binding of Low-Density Lipoprotein Receptor-Like Domain of Perlecan and Implications for Atherosclerosis
Lipoprotein retention by proteoglycans in the arterial wall is a critical step for atherosclerosis. Perlecan is the major proteoglycan in the arterial wall, and various studies have linked it to atherosclerosis, but it has been poorly understood how perlecan contributes to the development of atherosclerosis. Perlecan is a gigantic molecule containing a core protein with five domains (I-V) and three heparan sulfate (HS) side chains. Domain I contains three attachment sites for HS. Domain II has five repeats and four of them are cysteine-rich, and highly homologous to the ligand-binding portion of LDL receptor, but the binding activity has never been investigated. In this study, we hypothesized that perlecan domain II is functional in LDL binding and its coordinated activity with the HS of domain I plays a significant role in the initial LDL retention. Using tagged expression system we expressed and purified the recombinant domain II. We found that the domain is heavily modified and the modification is only present in the secreted, not cellular, form. The modification is O-linked glycosylation containing N-acetyl-glucosamine, fucose, sialic acid, and galactose. In vitro binding assay shows that only the secreted form strongly interacts with LDL, indicating that the interaction is glycosylation-dependent. Two repeats of domain II, the second and fifth, are glycosylated, and most of the domain II glycosylation is from the second, which is serine/threonine rich, no cysteine residue. The mutation of all the glycosylated sites resulted in a mutant whose secreted form is glycosylation defective. We validate that the mutant is inactive in ApoB binding confirming the above glycosylation-dependent interaction. We prove that there is synergistic effect for domain II with the HS of domain I on the ApoB binding. The ligand-binding portion of LDL receptor has 7 cysteine-rich repeats, in which 5 were shown to be required for binding. Our results define the unique feature of perlecan domain II in LDL binding, namely the glycosylation enables the non-cysteine-rich domain to participate in LDL interaction. Thus our data clarify the molecular basis for perlecan in lipoprotein binding suggesting an important role for perlecan in the early LDL retention.
- © 2012 by American Heart Association, Inc.