A novel abetalipoproteinemia genotype - Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase

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, [S-35]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) --> Gln, Asp(384) --> Ala, and Arg(540) --> His. Transient expression of each mutant showed that only the Arg(540) 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) --> 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.