Apolipoprotein B Binding to Microsomal Triglyceride Transfer Protein Decreases with Increases in Length and Lipidation: Implications in Lipoprotein Biosynthesis ² M. Mahmood Hussain,* ,‡ Ahmed Bakillah, ‡,§ and Haris Jamil | Departments of Pathology and Biochemistry, Allegheny UniVersity of the Health Sciences, Philadelphia, PennsylVania 19129, and Bristol-Myers Squibb, Pharmaceutical Research Institute, Princeton, New Jersey 08543 ReceiVed June 11, 1997; ReVised Manuscript ReceiVed August 13, 1997 X ABSTRACT: Microsomal triglyceride transfer protein (MTP), a heterodimer of 97 kDa and protein disulfide isomerase, is required for the assembly of apolipoprotein B (apoB)-containing triglyceride-rich lipoproteins. These proteins have been shown to interact with each other during early stages of lipoprotein biosynthesis. Our studies indicated that binding between apoB and heterodimeric MTP was of high affinity (K d 10-30 nM) due to ionic interactions. In contrast to MTP, protein disulfide isomerase alone interacted very poorly with lipoproteins, indicating the importance of the heterodimer in these bindings. Preincubation of lipoproteins with detergents enhanced their interaction with MTP. Native VLDL bound poorly to MTP, but its preincubation with Tween-20 resulted in significantly increased binding to MTP. Furthermore, binding of LDL was enhanced by preincubation with taurocholate, indicating that partial delipidation of apoB-containing lipoproteins results in increased binding to MTP. Subsequently, attempts were made to study interactions between C-terminally truncated apoB polypeptides and MTP. Binding of all the polypeptides to MTP was enhanced in the presence of taurocholate. Comparisons revealed that the binding of different apoB polypeptides to MTP was in the order of apoB18 > apoB28 > apoB42 > apoB100. These studies indicated that optimum interactions occur between apoB18 and MTP, and that the increase in apoB length beyond apoB18 has a negative effect on these interactions. Since apoB18 does not assemble triglyceride-rich lipoproteins, these studies suggest that apoB may interact with MTP before its lipidation. It is proposed that steps in lipoprotein biosynthesis may be dictated by the sequential display of different functional domains on the apoB polypeptide. Apolipoprotein B (apoB) 1 is an essential structural protein required for the assembly of triglyceride-rich lipoproteins. It is synthesized as a single polypeptide of 4536 (apoB100) or 2152 (apoB48) amino acids by the liver and intestine, respectively. Unlike other secretory proteins, which are co- translationally inserted into the lumen of endoplasmic reticulum (ER), apoB is co-translationally integrated, at least transiently, into the ER membranes in a transmembrane orientation [for reviews, see Innerarity et al. (1996), Hussain et al. (1996), Davis (1991), Sparks and Sparks (1994), Yao and McLeod (1994); Gibbons (1990), Vance and Vance (1990), and Dixon and Ginsberg (1993)]. Translocation of apoB across the ER membrane is inefficient and probably determines lipoprotein production (Bonnardel & Davis, 1995; Sakata et al., 1993). It is thought that apoB can be lipidated even before the completion of peptide synthesis. The cotranslational lipidation of apoB is supported by the observations that incompletely synthesized apoB polypep- tides are secreted as lipoprotein particles by cells incubated with puromycin, which stops protein synthesis by releasing peptides from ribosomes (Spring et al., 1992; Boren et al., 1992). Lipidation of apoB results in the release of apoB from the ER membrane and in the formation of primordial lipoproteins. The early lipidation of nascent apoB polypeptides requires the microsomal triglyceride transfer protein (MTP) [for reviews, see Wetterau et al. (1997) and Gregg and Wetterau (1994)]. The purified MTP activity consists of two subunits of 58 and 97 kDa (Wetterau & Zilversmit, 1985). The 97 kDa subunit is essential for lipid transfer activity and is defective in abetalipoproteinemia patients who lack apoB- containing lipoproteins in their plasma (Gregg & Wetterau, 1994; Wetterau et al., 1992; Sharp et al., 1993). The 58 kDa protein disulfide isomerase (PDI) is required to keep the larger subunit in solution and to retain it in the ER (Ricci et al., 1995; Wetterau et al., 1991). A direct correlation between MTP activity and lipoprotein assembly has been obtained in vitro by co-expressing apoB and MTP in cells that do not secrete lipoproteins (Patel & Grundy, 1996; Wang et al., 1996; Gretch et al., 1996; Leiper et al., 1994; Gordon et al., 1994). Expression of apoB cDNAs in most studies resulted in the intracellular synthesis of apoB polypeptides, but no lipoprotein secretion. In contrast, cotransfection of apoB with MTP resulted in increased secretion of apoB polypeptides (Patel & Grundy, 1996; Wang et al., 1996; Gretch et al., 1996; Leiper et al., 1994; Gordon et al., 1994). It has been suggested that MTP most likely assists in the increased translocation of nascent apoB from the ER ² Supported by grants from the National Institutes of Health (DK- 46900 and HL-22633) and American Heart Association, National Center and Southeastern Pennsylvania Affiliate. * Address correspondence to this author. Phone: 215-991-8497. Fax: 215-843-8849. E-mail: Hussain@auhs.edu. ‡ Allegheny University of the Health Sciences. § Visiting scientist from Universite ´ Chouaib Doukkali, Faculte ´ des Sciences, Laboratoire de Biochimie Appliquee, El Jadida, Morocco. | Bristol-Myers Squibb, Pharmaceutical Research Institute. X Abstract published in AdVance ACS Abstracts, October 1, 1997. 1 Abbreviations: ApoB, apolipoprotein B; ER, endoplasmic reticu- lum; HDL, high-density lipoproteins; LDL, low-density lipoproteins; MTP, microsomal triglyceride transfer protein; PBS, phosphate-buffered saline; PBS-Tween, PBS containing 0.05% Tween-20; PDI, protein disulfide isomerase; VLDL, very low density lipoproteins. 13060 Biochemistry 1997, 36, 13060-13067 S0006-2960(97)01395-0 CCC: $14.00 © 1997 American Chemical Society