The microsomal triglyceride transfer protein (MTP), which catalyses the transport of triglyceride, cholesteryl ester and phospholipid between phospholipid surfaces, is a heterodimer composed of the multifunctional protein, protein disulphide isomerase, and a unique large subunit with an apparent M(r) of 88K (refs 1-3). It is isolated as a soluble protein from the lumen of the microsomal fraction of liver and intestine. The large subunit of MTP was not detectable in four unrelated subjects with abetalipoproteinaemia, a rare autosomal recessive disease characterized by a defect in the assembly or secretion of plasma lipoproteins that contain apolipoprotein B (ref. 6). We report here the isolation and sequencing of complementary DNA encoding the large subunit of MTP. A comparison of this sequence to corresponding genomic sequences from two abetalipoproteinaemic subjects revealed a homozygous frameshift mutation in one subject and a homozygous nonsense mutation in the other. The results indicate that a defect in the gene for the large subunit of MTP is the proximal cause of abetalipoproteinaemia in these two subjects, and that MTP is required for the secretion of plasma lipoproteins that contain apolipoprotein B.

To Recent studies showing that a defect in the MTP is the proximal cause of abetalipoproteinemia indicate that this protein is required for assembly and secretion of apoBcontaining lipoproteins (10, 11). Abetalipoproteinemic patients have only trace amounts of plasma apoB-containing lipoproteins, resulting in extremely low TG and cholesterol levels (12). The cause of this phenotype is a defect in the pathway responsible for assembly and secretion of apoBcontaining lipoproteins since the apoB gene (13, 14) and lipid synthesis (12) have been shown to be normal. Thus, it is clear that MTP is required for the efficient assembly and secretion of apoB-containing lipoprotein particles.Although studies of abetalipoproteinemic patients indicate that MTP is required for the production of plasma lipoproteins containing apoB, the role of MTP in this process remains unclear. Also, it is not known whether MTP is the only tissue-specific factor needed by hepatocytes and enterocytes to synthesize and secrete these particles. To address these issues, apoB-53 was expressed in either a nonlipoprotein-producing cell line (HeLa) or a derivative cell line stably expressing the large subunit ofMTP and MTP activity. These cells were evaluated for their ability to secrete apoBcontaining lipoproteins into the tissue culture medium. tTo whom reprint requests should be addressed. MATERIALS AND METHODS 7628The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the multifunctional enzyme, protein disulfide-isomerase, and a unique large, 97 kDa, subunit. It is found as a soluble protein within the lumen of the endoplasmic reticulum of liver and intestine and is required for the assembly of very low density lipoproteins and chylomicrons. Mutations in MTP which result in an absence of MTP function have been shown to cause abetalipoproteinemia. Here, the gene encoding the MTP 97-kDa subunit of an abetalipoproteinemic subject, which we have previously demonstrated lacks MTP activity and protein (Wetterau, J. R., Aggerbeck, L. P., Bouma, M.-E., Eisenberg, C., Munck, A., Hermier, M., Schmitz, J., Gay, G., Rader, D. J., and Gregg, R. E. (1992) Science 258, 999-1001), was isolated and sequenced. A nonsense mutation, which predicts the truncation of the protein by 30 amino acids, was identified. To investigate if this apparently subtle change in MTP could explain the observed absence of MTP, protein disulfide-isomerase was co-expressed with either the normal or mutant MTP 97-kDa subunit in Sf9 insect cells using a baculovirus expression system. Although there were high levels of expression of both the normal and mutant forms of the MTP 97-kDa subunit, only the normal subunit was able to form a stable, soluble complex with protein disulfide-isomerase. These results indicate that the carboxyl-terminal 30 amino acids of the MTP 97-kDa subunit plays an important role in its interaction with protein disulfide-isomerase.

Microsomal triglyceride transfer protein (MTP) is a heterodimer consisting of the multifunctional enzyme protein disulfide isomerase and a unique, large 97-kDa subunit. MTP is required for the assembly and secretion of very low density lipoproteins and chylomicrons by the liver and intestine, respectively. In vitro, MTP catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between phospholipid surfaces. We have characterized the gene encoding the large subunit of human MTP. It contains 18 exons and spans approximately 55-60 kb. Fluorescent in situ hybridization localized this gene to band 4q24 of chromosome 4. A (CA)n repeat polymorphic marker, which may be useful for investigating a link between the MTP gene and genetic defects in lipid metabolism, was identified in intron 10. Sequence analysis of the 5' flanking region of the gene revealed potential sites which may bind transcriptional factors and control MTP expression.

Equine (e) CG and LH beta-subunits have identical amino acid sequences, including an extended carboxyl-terminal peptide (CTP). This suggests that unlike the corresponding human genes, the beta-subunits of eCG and eLH may be encoded by a single gene and share a common proximal promotor region. To explore this, we isolated and characterized the eLH/CG beta gene(s). Data from Southern analyses suggest that the eCG beta and eLH beta subunits are products of the same single copy gene (eLH/CG beta). Overlapping fragments of the eLH/CG beta gene and cDNA were amplified from equine genomic DNA and pituitary gland mRNA by the polymerase chain reaction, cloned, and sequenced. The eLH/CG beta gene spans less than 1.2 kilobase-pairs and has three exons that translate a CTP-containing polypeptide identical in sequence to that previously reported for the mature equine protein. There is, however, little amino acid homology shown between the CTP of human or equine CG beta subunit. In addition, unlike the human genes, the same TATAA-like element appears to be involved in directing initiation of transcription of the eLH/CG beta gene in placenta and anterior pituitary. Based upon these differences, we suggest that the CG beta genes evolved independently in humans and equids and that different mechanisms are involved in their patterns of placenta-specific expression.