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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.

SUBMITTER: Hidaka H

PROVIDER: S-EPMC1132711 | biostudies-other | 1992 May

REPOSITORIES: biostudies-other

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Project description:In contrast to water-soluble fuels such as glucose or ketone bodies, the use of lipids as an energy source for tissues has required the development of complex structures for their transport through the aqueous plasma. In the case of endogenously synthesized triacylglycerol this is achieved by the assembly and secretion of hepatic VLDL which provides the necessary stability in an aqueous medium. An essential component of this assembly process is apo B. Dietary changes which require an increase in hepatic VLDL secretion appear to be accompanied by increases in the availability of functional apo B. Interesting questions relate to: (a) the intracellular site(s) of triacylglycerol association with apo B, and (b) the mechanism(s) by which the availability of functional apo B at this site responds to metabolic and hormonal signals which reflect dietary status and, thus, the need to secrete triacylglycerol. As regards the latter, although in some cases changes in apo B synthesis occur in response to VLDL secretion hepatic apo B mRNA levels appear to be quite stable in vitro. Intracellular switching of apo B between the secretory and degradative pathways may be important in controlling VLDL assembly and post-translational modifications of the apoprotein may also play a role by influencing its ability to bind to triacylglycerol. Transport is not the only problem associated with the utilization of a concentrated energy source such as triacylglycerol and the complex problems of waste product disposal and recycling have to be dealt with. In the case of triacylglycerol, potentially toxic waste products include atherogenic remnants and LDL. The overall problem, then, in the long-term, involves the development of a 'safe' means of utilizing triacylglycerol and this requirement accounts for much of the complexity of plasma lipoprotein metabolism. In this area, the rat could teach the human a few tricks. One of these appears to be the utilization of hepatic apo B48 rather than apo B100 for VLDL assembly in response to increases in the extrahepatic utilization of hepatically synthesized triacylglycerol. Under these conditions, the remnants of hepatic triacylglycerol utilization by peripheral tissues are cleared from the plasma much more readily via a process which seems to involve the cycling of more triacylglycerol back to the liver than that which occurs in humans. The means by which this is achieved, though, are obscure and may involve a chylomicron remnant receptor, the nature of which, itself, remains controversial.(ABSTRACT TRUNCATED AT 400 WORDS)

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Affinity purification of the hepatic high-density lipoprotein receptor identifies two acidic glycoproteins and enables further characterization of their binding properties.

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