Abstract 15800: Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein

Abstract

Microsomal triglyceride transfer protein (MTP) is heterodimeric protein complex consisting of a unique 97 kDa protein with lipid transfer activity and the ubiquitous 58 kDa protein disulfide isomerase. The complex is essential for assembly of triglyceride-rich apolipoprotein (apo) B-containing lipoproteins. In tissues that do not synthesize apoB nor assemble lipoproteins, it is presumed to serve specialized needs in lipid trafficking, such as in the lipidation of CD1d in antigen presenting cells, including adipocytes. Previous studies in our laboratory identified a novel isoform of MTP in mice that we named MTP-B. MTP-B has an alternative first exon (Ex1B) located 2.7 kbp upstream of the first exon (Ex1A) of MTP-A, the canonical isoform of MTP. The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study, we have identified another splice variant, MTP-C, that contains both exons Ex1B and Ex1A. PCR analysis revealed that MTP-C mRNA is expressed in a number of tissues including liver, ovary, heart, kidney, brain and testis. In CHO and HEK 293 cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’ untranslated region (UTR) of MTP-C revealed six ATGs (uATGs) upstream of the translation initiation site for MTP-A, which is the only viable start site in frame with the main open reading frame (ORF), and three potential upstream ORFs (uORFs). Since uATGs and uORFs are generally associated with repressed translational efficiency, we inserted the 5’ UTR of MTP-C into a pGL3 vector between the SV40 promoter and luciferase reporter gene and transfected it into CHO cells. Luciferase activity was significantly reduced (45.5 ± 6.4%) compared to pGL3-Control vector transfected cells. This suggests the reduced protein expression observed in MTP-C transfected cells resulted in part from decreased translation efficiency. We conclude that uORFs in the 5’ UTR of MTP-C repress translation, providing a mechanism to fine tune MTP levels to meet the needs of the cell. uORFs in human MTP-C could represent novel therapeutic targets for regulation of MTP expression and plasma lipid levels.