Structure-Function Analysis of Microsomal Triglyceride Transfer Protein using Human Mutations.

Abstract

Microsomal triglyceride transfer protein (MTP) is an evolutionarily conserved endoplasmic reticulum (ER) resident protein that is required for apolipoprotein B (apoB)-containing lipoprotein assembly and secretion. MTP physically interacts with apolipoprotein B and transfers lipids (triglycerides, phospholipids, choelsteryl esters, sphingomyelin, and ceramide) to nascent apoB. MTP is a heterodimer composed of the catalytically active M subunit and protein disulfide isomerase (PDI). The M subunit is divided into 3 domains: N-terminal -barrel, middle -helical, and C-terminal. Computational, biochemical, and mutagenesis studies of missense mutations in all three domains of the M subunit identified critical amino acids for MTP function and lent insight into MTP’s structure-function. Loss of function mutations in MTP lead to Abetalipoproteinemia (ABL), which is characterized by the absence of plasma apoB-containing lipoproteins. We identified three ABL patients, who each had a novel homozygous missense mutation in MTP: R46G, D169V, or D361Y. To determine if any of these missense mutations were detrimental to MTP’s function, we created these mutations via site-directed mutagenesis, expressed them in Cos-7 cells, and investigated their ability to support apoB secretion. R46G, a polymorphism, had no effect on MTP’s ability to support apoB secretion or transfer triglycerides, suggesting that it is not the basis for the ABL phenotype in this patient. D169V and D361Y, on the other hand, were both located in the ER and able to bind apoB17, but were not able to bind PDI, transfer lipids, or support apoB secretion, suggesting that they were the cause of the ABL phenotype. D361E, which has a similar helix forming potential and charge as D361, is able to restore PDI binding, lipid transfer, and apoB secretion. Therefore, we postulate that D361Y may disrupt the tightly packed middle -helical domain, and consequently alter the PDI binding site in this domain. Computational modeling of D169 revealed that it may be part of a crucial internal salt bridge in the N-terminal -barrel. Computational modeling suggested that D169 could form an internal salt bridge with K187 and K189. Mutagenesis of these lysines to leucines abolished PDI heterodimerization, lipid transfer, and apoB secretion without affecting apoB binding or subcellular location. Further, mutants with preserved charges (D169E, K187R, K189R) rescued these activities, suggesting that these three residues form a salt bridge and a PDI binding site in the N-terminal region. This binding site is independent of the apoB binding site, suggesting that the N-terminal domain is actually divided into two microdomains. Next, we investigated whether MTP missense mutations (H297Q, D384A, G661A) reported in a previously identified patient with hypobetalipoproteinemia (HBL), which is characterized by plasma apoB in the bottom 5th percentile. We hypothesized that missense mutations from this patient would modulate, but not abolish, MTP’s ability to support apoB secretion. We created these mutants individually and in combination and investigated their ability to transfer lipids and support apoB secretion. However, none of these mutations affected MTP’s ability to support apoB secretion or transfer triglycerides, suggesting that these mutants function normally and may be polymorphisms. Therefore, the HBL in this patient may be caused by confounding factors. In summary, these studies characterized 12 missense mutations in MTP and identified 4 critical amino acids for MTP’s function. D361Y destroys the PDI binding site in the middle -helical region. The other three residues, D169, K187, and K189 form a salt bridge in the N-terminal domain, which stabilizes the PDI binding site in this region. They were also the first detrimental missense mutations in the Nterminal domain, demonstrating that this domain is also important for MTP activity. Further mutagenesis studies may help refine the tertiary structure of MTP.