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Journal of Lipid Research, Vol. 42, 399-406, March 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

Vesicle-binding properties of wild-type and cysteine mutant forms of ₁ domain of apolipoprotein B

Correspondence to: Gregory S. Shelness, To whom correspondence should be addressed., gshelnes{at}wfubmc.edu (E-mail)

Previous studies demonstrated that structural perturbation of the 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 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 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 ₁ domain may reflect an essential role required for the initiation of lipoprotein formation, some other aspect of 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 ₁ domain of apolipoprotein B. J. Lipid Res. 2001. 42: 399;–406.

Supplementary key words: lipoprotein assembly, recombinant lipoproteins, disulfide bonds, apoB-17, vesicle-binding assay

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