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.

Localization of oxidized HDL in atheromatous plaques and oxidized HDL binding sites on human aortic endothelial cells

“…However, the intimal distributions of oxidized HDL particles and oxidized LDL particles were somewhat distinct compared with the data of Itabe et al 27 120 kDa in human placenta 28 and 120 kDa or 100 kDa in rat and human liver plasma membranes. 21 Recently, it was reported that the class B scavenger receptor SR-BI, another native HDL receptor, had an amino acid sequence homology of about 30% to CD36 and a molecular mass of about 82 kDa. 9 Native HDL binding proteins found in the liver or peripheral tissues therefore appear to have distinct structures and different roles.…”

Cloning and Characterization of HB2, a Candidate High Density Lipoprotein Receptor

“…Protein Sequencing-HB 2 , a glycoprotein of M r 100,000 was purified from rat liver plasma membrane as described previously (12). Attempts at NH 2 -terminal sequencing were unsuccessful.…”

“…We have identified two liver plasma membrane proteins named HB 1 and HB 2 (HDL-binding proteins 1 and 2) which are candidate HDL receptors (8,12). Although present in low abundance, HB 2 was purified in sufficient quantities to provide amino acid sequence data.…”

The Heparin/Heparan Sulfate-binding Site on Apo-serum Amyloid A

“…Identification of the basic residues of the m27-mer, within the GAG-binding consensus sequence (A) and outside the GAG-binding consensus sequence (B), which are important for heparin binding. Nine synthetic peptides were applied to heparinSepharose 4B, the m27-mer, R83A, H84A, R86A, R83A/H84A/R86A (triple mutant), K89A, R95A, Lys-102 Ϫ (mouse apoSAA2 [77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94][95][96] ) which lacked the carboxyl-terminal Lys-102, and the h27-mer (human apoSAA2 78 -104 ). The elution profile of the Lys-102 Ϫ peptide on heparin/ Affi-Gel is also shown.…”