title: Affinity purification of the hepatic high-density lipoprotein receptor identifies two acidic glycoproteins and enables further characterization of their binding properties.
abstract: Several high-density lipoprotein (HDL)-binding proteins, candidates for the putative HDL receptor, have recently been identified, including two membrane proteins: HB1 of 120 kDa and HB2 of 100 kDa, present in rat and human liver plasma membranes respectively. Further insights into their function however, have been hampered by poor recoveries of these hydrophobic peptides, and the present work was undertaken to improve yields and enable a more detailed investigation of their properties. A significant improvement has been achieved using two affinity chromatographic procedures, one exploiting the glycoprotein nature of the proteins and the other exploiting their ligand properties, which in combination resulted in considerable enrichment of HB1 and HB2. Thus DEAE-Sephacel fractionation (0.05-0.2 M-NaCl) of CHAPS-solubilized plasma membranes yielded active HDL-binding proteins which bound to concanavalin A-Sepharose or wheat-germ-lectin-Sepharose columns and retained their binding activity after eluting with methyl-alpha-D-mannoside or N-acetylglucosamine respectively. These glycoproteins were further purified by affinity chromatography using apo-HDL-Sepharose columns. Final purification required preparative SDS/PAGE. Investigation of the carbohydrate moieties of the proteins using glycosidases and two-dimensional gel electrophoresis revealed pI values ranging from 4.6 to 4.9 and from 4.5 to 4.7 for HB1 and HB2 respectively, which after treatment with neuraminidase shifted towards basic pH (5.4-5.7 and 5.3-5.5 respectively). The molecular masses were decreased to 115 kDa and 95 kDa respectively, demonstrating that sialic acid residues contributed significantly to the negative charge of the glycosylated peptides. Treatment with the enzyme peptide N-glycosidase F (N-glycanase) resulted in a decrease in molecular mass of HB1 and HB2 to 105 kDa and 80 kDa respectively, but endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment was not effective. Neither neuraminidase nor N-glycanase treatment destroyed activity, suggesting that sialic acids or N-linked oligosaccharides are not important determinants of HDL binding. Digestion of plasma membranes with trypsin or Pronase resulted in a loss of activity of both HB1 and HB2 that was not influenced by prior treatment with neuraminidase, suggesting that sialic acid residues play no protective role against proteolytic cleavage of HDL receptor proteins.
authors.affiliation: Protein Chemistry and Molecular Biology Unit, Baker Medical Research Institute, Prahran, Victoria, Australia.
authors.forename: H
authors.initials: H
authors.lastname: Hidaka
authors.forename: N H
authors.initials: NH
authors.lastname: Fidge
mesh_terms: D000818:Animals; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002793:Cholic Acids; D003636:DEAE-Cellulose / Q000031:analogs & derivatives; D006031:Glycosylation; D006801:Humans; D008075:Lipoproteins, HDL; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure*; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D016601:RNA-Binding Proteins; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D018110:Receptors, Lipoprotein; D012685:Sepharose / Q000031:analogs & derivatives
chemical_list: D002352:Carrier Proteins; D002793:Cholic Acids; D008075:Lipoproteins, HDL; D008562:Membrane Glycoproteins; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D018110:Receptors, Lipoprotein; C028326:concanavalin A-sepharose; C031616:high density lipoprotein receptors; C075197:high density lipoprotein binding protein; D012685:Sepharose; D003636:DEAE-Cellulose; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate