Structural determinants in the C-terminal domain of apolipoprotein E mediating binding to the protein core of human aortic biglycan.
Apolipoprotein (apo) E-containing high density lipoprotein particles were reported to interact in vitro with the proteoglycan biglycan (Bg), but the direct participation of apoE in this binding was not defined. To this end, we examined the in vitro binding of apoE complexed with dimyristoylphosphatidylcholine (DMPC) to human aortic Bg before and after glycosaminoglycan (GAG) depletion. In a solid-phase assay, apoE.DMPC bound to Bg and GAG-depleted protein core in a similar manner, suggesting a protein-protein mode of interaction. The binding was decreased in the presence of 1 m NaCl and was partially inhibited by either positively (0.2 m lysine, arginine) or negatively charged (0.2 m aspartic, glutamic) amino acids. A recombinant apoE fragment representing the C-terminal 10-kDa domain, complexed with DMPC, bound as efficiently as full-length apoE, whereas the N-terminal 22-kDa domain was inactive. Similar results were obtained with a gel mobility shift assay. Competition studies using a series of recombinant truncated apoEs showed that the charged segment in the C-terminal domain between residues 223 and 230 was involved in the binding. Overall, our results demonstrate that the C-terminal domain contains elements critical for the binding of apoE to the Bg protein core and that this binding is ionic in nature and independent of GAGs.
Klezovitch
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Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA. oklezovi@medicine.bsd.uchicago.edu
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D000911:Antibodies, Monoclonal / Q000276:immunology; D001011:Aorta / Q000378:metabolism*; D001057:Apolipoproteins E / Q000737:chemistry / Q000378:metabolism*; D058578:Biglycan; D001667:Binding, Competitive; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D011509:Proteoglycans / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001057:Apolipoproteins E; C546884:BGN protein, human; D058578:Biglycan; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D004134:Dimyristoylphosphatidylcholine