A NOVEL ABETALIPOPROTEINEMIA GENOTYPE - IDENTIFICATION OF A MISSENSE MUTATION IN THE 97-KDA SUBUNIT OF THE MICROSOMAL TRIGLYCERIDE TRANSFERPROTEIN THAT PREVENTS COMPLEX-FORMATION WITH PROTEIN DISULFIDE-ISOMERASE

The microsomal triglyceride transfer protein (MTP) is a heterodimer co mposed of the ubiquitous multifunctional protein, protein disulfide is omerase, and a unique 97-kDa subunit, Mutations that lead to the absen ce of a functional 97-kDa subunit cause abetalipoproteinemia, an autos omal recessive disease characterized by a defect in the assembly and s ecretion 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 sh owed that this tissue was capable of synthesizing the 97-kDa MTP subun it, Electrophoretic analysis showed two bands, one with a molecular ma ss of the wild type 97-kDa subunit and the other with a slightly lower molecular weight, Sequence analysis of cDNAs from additional intestin al biopsies showed this patient to be a compound heterozygote. One all ele contained a perfect in-frame deletion of exon 10, explaining the l ower molecular weight band, cDNAs of the second allele were found to c ontain 3 missense mutations: His(297) --> Gln, Asp(384) --> Ala, and A rg(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 b aculovirus system of the wild type 97-kDa subunit or the Arg(540) --> His mutant along with human protein disulfide isomerase showed that th e wild type was capable of forming an active MTP complex while the mut ant was not, Biochemical analysis of lysates from these cells showed t hat 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 t o complex with protein disulfide isomerase and form the active MTP hol oprotein, These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive a ssociation with protein disulfide isomerase, Of the 13 mutant MTP 97-k Da subunit alleles described to date, this is the first encoding a mis sense mutation.