A Novel Abetalipoproteinemia Genotype

Abstract

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35 S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His 297 3 Gln, Asp 384 3 Ala, and Arg 540 3 His. Transient expression of each mutant showed that only the Arg 540 3 His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg 540 3 His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg 540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation. The microsomal triglyceride transfer protein (MTP)1 is an endoplasmic reticulum resident protein that catalyzes the transfer of lipids between phospholipid surfaces. Although MTP can transfer most classes of lipids, it shows a distinct preference for triglyceride and cholesteryl esters. MTP is a heterodimer composed of the multifunctional protein, protein disulfide isomerase (PDI) and a unique large subunit of 97 kDa (1). PDI is a ubiquitous protein found at high levels in many different tissues (2). The 97-kDa subunit of MTP is expressed primarily in hepatocytes and intestinal enterocytes (3). Thus, the active MTP complex is found predominantly in the liver and small intestine. The subcellular localization, tissue distribution, and activity of MTP all suggest a role for this protein in the assembly and secretio...

Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein

“…The fraction containing soluble MTP was tested for lipid transfer activity and was found to be active (results not shown). These results reproduce the findings of Lamberg et al [16] and Rehberg et al [6], reporting the expression of non-tagged MTP and PDI in insect cells. We conclude that the modifications to the Ctermini of MTP and PDI did not impair the production of the MTP-PDI heterodimer in a soluble and active conformation.…”

“…This was made possible by the co-infection of insect cells with two different viruses and the subsequent purification of the resulting heterodimer by two affinity chromatography steps. Although the dual infection of two recombinant viruses into Sf9 cells has been described by us [16] and others on an analytical scale [6,7,29], this is the first description of a simple and robust methodology for the largescale purification of the expressed heterodimer. This protocol supersedes the more traditional approach of isolating liverderived MTP-PDI, involving a series of five column chromatography steps [3,30], and provides a means for isolating human, as opposed to bovine, material for crystallization studies.…”

“…This process has an absolute requirement for a microsomal triglyceride transfer protein (MTP) complexed to an ER-resident chaperone and disulphide-bond forming enzyme, protein disulphide isomerase (PDI) [1][2][3]. Defects in the apoB gene and the MTP gene cause hypobetalipoproteinaemia [4] and abetalipoproteinaemia [5][6][7][8][9] respectively. Affected individuals secrete no normal apoB-containing lipoproteins and have malabsorption of dietary fat and of the fat-soluble vitamins [10].…”

Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins

“…MTP is present in the endoplasmic reticulum due in part to association with protein disulfide isomerase (PDI) (24,25) and has also been localized to the Golgi apparatus (26,27). The subcellular distribu- A, MTP-FLAG chimeras were affinity-purified from transiently expressing COS cells.…”

Anderson’s Disease

“…It was necessary to demonstrate that the MTP protein was active because we have detected the presence of a normal-sized inactive MTP protein with a false sense mutation in a case of abetalipoproteinemia. 54 The linkage analysis definitively ruled out the MTP gene as well as any other genes located in a 30 cM region around the gene.…”