High density lipoprotein-binding proteins in porcine liver. Isolation and histological localization.

Abstract

The antiatherogenic properties of high density lipoproteins (HDLs) are thought to reside in their involvement hi the reverse cholesterol transport pathway. Specific HDL-binding proteins could play a key role in this process. Two HDL-binding proteins of approximately SKI and 180 kd were identified in porcine liver by ligand blotting and were purified to apparent homogeneity by a combination of protein extraction, DEAE-cellulose chromatography, Con A-Sepharose chromatography, and preparative sodium dodecyl sulfate-poJyacryiamide gel electrophoresis. Binding of IZ5 I-HDL by these proteins could be actively competed for by unlabeled HDL but not by low density lipoprotein. Polyclonal antisera have been raised against these two proteins. Each antiserum recognized only one of the HDL-binding proteins, indicating that they are not immunologically related. Moreover, striking differences in localization were observed in Immunohistochemical studies. The 90-kd protein is located within the hepatocellular plates, while the 180-kd protein is present along the lining of the sinusoids. These results suggest functional differences between the two HDL-binding proteins described. ( This hypothesis is based on epidemiological data, which show a strong inverse correlation between plasma HDL cholesterol level and the prevalence of CHD, 2 -4 and on intervention studies, which indicate that elevation of HDL cholesterol level is effective in the primary prevention of CHD. 5 The antiatherogenic properties of HDL are probably based on the ability of HDL to promote efflux of cholesterol from peripheral cells and to deliver it to the liver for excretion, a concept termed reverse cholesterol transport. 6 -9 The binding characteristics of HDL to cultured cells and to purified plasma membrane fractions strongly suggest the existence of a specific, high-affinity HDL receptor.10 -13 Because such a receptor could have a key position in the process of reverse cholesterol transport, several groups have sought to identify HDL-binding proteins by using ligand blot studies. Their results differ in the molecular masses of HDL-binding proteins found, 14 -19 which may be attributable to species and/or tissue specificity.It is conceivable that in the reverse cholesterol transport pathway, the tissues involved in cholesterol efflux (peripheral tissues) have a different HDL metabolism than the liver, which is involved in cholesterol clearance. A peripheral HDL-binding protein has been described and characterized by Oram and coworkers. 17-20 * 21 We investigated HDL-binding proteins in the liver. In the present study, two HDL-binding proteins in porcine liver are described. Their histological localization is notably different, presumably reflecting their involvement in different steps in the reverse cholesterol transport pathway. Methods LipoproteinsLow density lipoproteins (LDLs) (d= 1.019-1.063 g/ml) and HDL (d= 1.063-1.21 g/ml) were isolated from human plasma of healthy volunteers by sequential ultracentrifugation. 22 The same procedure was followed fo...

Structural relation between HDL-binding proteins in porcine liver.

“…16 Fractions containing HDL-binding proteins (the 250 mmol/L-NaCl eluate), as established in the HDL-binding assay, were pooled and dialyzed against ConA buffer as described in "Methods" and subjected to ConA-Sepharose chromatography. Retained proteins were eluted with methyl-a-D-glucopyranoside.…”

“…These are localization sites that have been previously shown to be specifically labeled with antisera recognizing either the 90-or 180-kDa HDL-binding protein. 16 The reactivity of these antisera was tested with purified preparations of HDL-binding proteins used in this study (Fig 10). The antiserum directed against the 90-kDa HDL-binding protein reacted with the purified 90-kDa protein preparation under nonreducing conditions but hardly reacted under reducing conditions.…”

“…16 In brief, protein fractions were subjected to SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes (0.45 fim, Schleicher & Schuell). 21 These membranes were incubated with blocking buffer (10 …”

“…The antibodies were eluted by a procedure adapted from Smith Proteins were solubilized from a plasma membrane-enriched fraction of either porcine or human liver and applied to DEAE-cellulose. 16 Elution was performed with a discontinuous salt gradient. Fractions containing HDL-binding activity were applied to concanavalin A (ConA)-Sepharose.…”

“…We have also found evidence for two HDL-binding proteins of 110 and 130 kDa in porcine liver, which appear to be present in minor quantities. 16 In this article we describe a structural relation between the 90-, 110-, and 180-kDa proteins.…”