STRUCTURAL BIOLOGY--EARLY EVENTS IN LIPOPROTEIN ASSEMBLY

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The process of assembly of triacylglycerol rich lipoproteins (TAG-LP) is a complicated process involving the initial formation of a small primordial (120-200 AD) particle with a neutral lipid (TAG and CE) core and a later process which adds TAG and phospholipid (PL) to increase the size to that of a nascent VLDL (300-600 A D). Once synthesized apoB can follow 2 pathways: if lipid is available, apoB is secreted on nascent TAG rich particles-if lipid is deficient, apoB is degraded. Mammary derived C27 cells make no apolipoproteins, secreted no lipid and have no microsomal TAG transfer protein (MTP). These cells are used to study assembly directed only by the primary sequence of apoB. When C127 cells are transfected with cDNA for C-terminal truncated apoB forms, they efficiently secrete the N-terminal 17% of apoB (B17) in a lipid poor state but secrete apoB29, B32.5, B37, and B41 with progressively increasing amounts of lipids. We show that B29 binds PL and DAG, B32 binds PL and TAG, while the sequences between B32 and B41 bind mainly TAG. Thus, specific sites for PL and DAG binding appear in the sequence between B20 and B29 while specific TAG sites occur from B32 to B41. Structural analysis of B37 and B41 particles indicated that apoB must interact directly with the core. These truncated forms are secreted more efficiently when oleate is supplied and degraded if lipids are deficient. Several intermediate folding forms have been identified in the assembly process and these forms bind a variety of chaperones. Some chaperones appear to be involved in early folding events and others in targeting unlipidated, misfolded forms toward the degradation path/ A search for potential lipid binding sequences in B41 indicates that there are amphipathic beta strands (AbetaS) located between B21 and B41 probably organized in sheets of 2 to 4 strands. A consensus 27 aa amphipathic beta sheet was synthesized and bound avidly to a hydrocarbon/water interface lowering the interfacial tension from 50 to 22 Mn/M. The sheet bound elastically and could not be displaced from the oil/water interface when compressed. An ideal property for apoB binding to the hydrophobic core of TAG-LP.