Vesicle-binding properties of wild-type and cysteine mutant forms of alpha(1) domain of apolipoprotein B.
Previous studies demonstrated that structural perturbation of the alpha(1) domain of apolipoprotein B (apoB) blocked the initiation of lipoprotein assembly. We explored the hypothesis that this domain may interact with the inner leaflet of the endoplasmic reticulum membrane in a manner that may nucleate microsomal triglyceride transfer protein-dependent lipid sequestration. ApoB-17 (amino-terminal 17% of apoB), which contains most of the alpha(1) domain, was expressed stably in rat hepatoma cells and recovered from medium in lipid-poor form. On incubation with phospholipid vesicles composed of 1-myristol-2-myristoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-gylycero-3-phosphocholine, apoB-17 underwent vesicle binding and was recovered in the d < 1.25 g/ml gradient fraction. To determine whether vesicle binding is disrupted by the same structural perturbations that block lipoprotein assembly in vivo, apoB-17 was subjected to partial and complete chemical reduction. Although normally a soluble peptide, mild reduction of apoB-17 caused its precipitation, suggesting that hydrophobic, solvent-inaccessible domains within the alpha(1) domain of apoB are stabilized by intramolecular disulfide bonds. In contrast to apoB-17 chemically reduced in vitro, forms of apoB-17 bearing pairwise cysteine-to-serine substitutions were recovered in soluble form from transiently transfected COS-1 cell extracts. Although individual disruption of disulfide bond 2 or 4 in apoB-28 and apoB-50 was previously shown to block lipoprotein assembly in vivo, these alterations had no impact on the ability of apoB-17 to bind to phospholipid vesicles in vitro or on its capacity to form recombinant lipoprotein particles. These results suggest that while the vesicle/lipid-binding property of the alpha(1) domain may reflect an essential role required for the initiation of lipoprotein formation, some other aspect of alpha(1) domain function is perturbed by disruption of native disulfide bonds. -- DeLozier, J. A., J. S. Parks, and G. S. Shelness. Vesicle-binding properties of wild-type and cysteine mutant forms of alpha(1) domain of apolipoprotein B. J. Lipid Res. 2001. 42: 399--406.
Department of Pathology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA.
J A
JA
DeLozier
J S
JS
Parks
G S
GS
Shelness
D000818:Animals; D001055:Apolipoproteins B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002021:Buffers; D019556:COS Cells; D002846:Chromatography, Affinity; D003545:Cysteine / Q000235:genetics; D004134:Dimyristoylphosphatidylcholine; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006358:Hot Temperature; D008081:Liposomes / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D016296:Mutagenesis; D011489:Protein Denaturation; D051381:Rats; D011994:Recombinant Proteins; D012995:Solubility; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured
D001055:Apolipoproteins B; D002021:Buffers; D008081:Liposomes; D011994:Recombinant Proteins; C416027:apolipoprotein b-17; D003545:Cysteine; D004134:Dimyristoylphosphatidylcholine