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Purification and characterization of two high-density-lipoprotein-binding proteins from rat and human liver.

ABSTRACT: The liver plays a major role in the metabolism of plasma high-density lipoprotein (HDL). Several groups have postulated, but others refuted, the existence of a classical membrane receptor which recognizes HDL. In the present study, we identified and purified two liver HDL-binding proteins of 120 kDa (HB1) and 100 kDa (HB2), with apparent specificity for HDL3 devoid of E apolipoprotein. The plasma membrane was the richest source of the HDL-binding protein. Both proteins bound A-I and A-II apolipoproteins and retained HDL-binding activity after final purification. HB1 activity, but not that of HB2, was lost after treatment with beta-mercaptoethanol, but reduction did not change the apparent molecular mass of either protein. Antibodies against HB1 or HB2 did not cross-react, and preliminary structural investigations provide evidence to suggest that HB1 and HB2 are not structurally related. We thus provide evidence for at least two liver plasma-membrane proteins which bind HDL apolipoproteins, suggesting that protein-protein interaction participates to some degree in the mechanism of HDL recognition by cells.

SUBMITTER: Tozuka M

PROVIDER: S-EPMC1138806 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

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Project description: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.

Project description:Background:Metabolic diseases such as obesity and diabetes are associated with changes in high-density lipoprotein (HDL) particles, including changes in particle size and protein composition, often resulting in abnormal function. Recent studies suggested that patients with non-alcoholic fatty liver disease (NAFLD), including individuals with non-alcoholic steatohepatitis (NASH), have smaller HDL particles when compared to individuals without liver pathologies. However, no studies have investigated potential changes in HDL particle protein composition in patients with NAFLD, in addition to changes related to obesity, to explore putative functional changes of HDL which may increase the risk of cardiovascular complications. Methods:From a cohort of morbidly obese females who were diagnosed with simple steatosis (SS), NASH, or normal liver histology, we selected five matched individuals from each condition for a preliminary pilot HDL proteome analysis. HDL particles were enriched using size-exclusion chromatography, and the proteome of the resulting fraction was analyzed by liquid chromatography tandem mass spectrometry. Differences in the proteomes between the three conditions (normal, SS, NASH) were assessed using label-free quantitative analysis. Gene ontology term analysis was performed to assess the potential impact of proteomic changes on specific functions of HDL particles. Results:Of the 95 proteins identified, 12 proteins showed nominally significant differences between the three conditions. Gene ontology term analysis revealed that severity of the liver pathology may significantly impact the anti-thrombotic functions of HDL particles, as suggested by changes in the abundance of HDL-associated proteins such as antithrombin III and plasminogen. Conclusions:The pilot data from this study suggest that changes in the HDL proteome may impact the functionality of HDL particles in NAFLD and NASH patients. These proteome changes may alter cardio-protective properties of HDL, potentially contributing to the increased cardiovascular disease risk in affected individuals. Further validation of these protein changes by orthogonal approaches is key to confirming the role of alterations in the HDL proteome in NAFLD and NASH. This will help elucidate the mechanistic effects of the altered HDL proteome on cardioprotective properties of HDL particles.

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Purification and characterization of two high-density-lipoprotein-binding proteins from rat and human liver.

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