Abstract 232: A Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of the N-Terminal ß-Sheet in the Lipid Transfer and ApoB Secretion Activities of Microsomal Triglyceride Transfer Protein

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Meghan T Walsh ◽  
Enza Di Leo ◽  
Eda Ozaydin ◽  
Patrizia Tarugi ◽  
Mahmood Hussain
Keyword(s):  
Missense Mutation ◽  
Structural Integrity ◽  
Microsomal Triglyceride Transfer Protein ◽  
Salt Bridge ◽  
Missense Mutations ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Transfer Activity ◽  
Apob Secretion ◽  
Β Sheet

Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. Mutations in the MTTP gene cause abetalipoproteinemia (ABL). Missense mutations in ABL have revealed that the central α-helical and C-terminal β-sheet domains are important for the lipid transfer activity of MTP and for the assembly and secretion of apoB-containing lipoproteins. The N-terminal domain, on the other hand, has mainly been implicated in apoB- and membrane-binding. Here, we describe a novel ABL missense mutation (D169V) in the N-terminal β-sheet of MTP. Although this mutant MTP (MTPD169V) is expressed and localized to the endoplasmic reticulum, it is unable to transfer triglycerides and phospholipids. Further, MTPD169V does not support the assembly and secretion of apoB-containing lipoproteins. Computational molecular modeling suggests that D169 could form an internal salt bridge with K187 and K189. Indeed, mutagenesis of these lysine residues to leucine abolishes triglyceride transfer and apoB secretion activities of MTP. Furthermore, conservative mutagenesis that preserves charges on these residues partially restores triglyceride transfer and apoB secretion activities of MTP. Therefore, D169 is probably involved in an internal salt bridge with K187 and K189. Disruption of this internal salt bridge in the N-terminal region affects the lipid transfer activity present in the C-terminal end of the MTP molecule. We speculate that this salt bridge, although away from the speculated lipid transfer site, might be important in providing structural integrity necessary for the lipid transfer activity of MTP.

Abstract 192: Characterization of Microsomal Triglyceride Transfer Protein Missense Mutations Found in Abetalipoproteinemia and Hybobetalipoproteinemia Subjects

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Meghan T Walsh ◽  
Enza Di Leo ◽  
Patrizia Tarugi ◽  
M. Mahmood Hussain
Keyword(s):  
Plasma Lipid ◽  
Microsomal Triglyceride Transfer Protein ◽  
The Other ◽  
Plasma Lipoproteins ◽  
Missense Mutations ◽  
Lipid Transfer ◽  
Novel Proteins ◽  
Transfer Protein ◽  
Apob Secretion ◽  
Familial Hypobetalipoproteinemia

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels and lipid malabsorption with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their effects on function. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. Similarly, H297Q, D384A, and G661A mutants supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP’s three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, there probably exist novel mutations in other genes that cause severe hypolipidemia and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.

scholarly journals MTP regulated by an alternate promoter is essential for NKT cell development

2007 ◽  
Vol 204 (3) ◽  
pp. 533-545 ◽  
Author(s):  
Stephanie K. Dougan ◽  
Paul Rava ◽  
M. Mahmood Hussain ◽  
Richard S. Blumberg
Keyword(s):  
Lipid Transfer Protein ◽  
Microsomal Triglyceride Transfer Protein ◽  
Cell Development ◽  
Northern Analysis ◽  
Lipid Transfer ◽  
Nkt Cell ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Apob Secretion

Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum lipid transfer protein critical for apolipoprotein B (apoB) secretion, regulates CD1d antigen presentation. We identified MTP variant 1 (MTPv1), a novel splice variant of mouse MTP, by polymerase chain reaction and Northern analysis in non–apoB-secreting tissues, including thymocytes and antigen-presenting cells (APCs). Edman degradation of MTPv1 isolated from transfected cells revealed three unique residues; however, recombinant MTP and MTPv1 had an equivalent protein disulfide isomerase association, subcellular localization, triglyceride transfer, phospholipid transfer, response to inhibitors, and ability to support apoB secretion. MTP and MTPv1 efficiently transferred phosphatidylethanolamine to CD1d in vitro. NKT cells fail to develop in fetal thymic organ culture (FTOC) treated with MTP antagonists. MTP-inhibited FTOCs produced negligible numbers of CD1d tetramer–positive cells and exhibited marked defects in IL-4 production upon stimulation with anti-CD3 or α-galactosylceramide–pulsed APCs. CD1d expression on CD4+CD8+ FTOC cells was unaffected by MTP inhibition. Thus, our results demonstrate that MTPv1 in thymocytes is critical to NKT cell development. We hypothesize that, when MTP is inactive, CD1d traffics to the cell surface and presents no lipid or a lipid that is incapable of mediating NKT cell selection and/or is refractory to lysosomal editing.

scholarly journals Regulation of microsomal triglyceride transfer protein by apolipoprotein A-IV in newborn swine intestinal epithelial cells

2011 ◽  
Vol 300 (2) ◽  
pp. G357-G363 ◽  
Author(s):  
Ying Yao ◽  
Song Lu ◽  
Yue Huang ◽  
Casey C. Beeman-Black ◽  
Rena Lu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Microsomal Triglyceride Transfer Protein ◽  
Lipid Absorption ◽  
Intestinal Epithelial ◽  
Lipid Transfer ◽  
Protein Levels ◽  
Transfer Protein ◽  
Transfer Activity ◽  
The Impact

Apolipoprotein (apo) A-IV overexpression enhances chylomicron (CM) assembly and secretion in newborn swine intestinal epithelial cells by producing larger particles (Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW, Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg RB, Black DD. J Biol Chem 281: 3473–3483, 2006). To determine the impact of apo A-IV on microsomal triglyceride transfer protein (MTTP), IPEC-1 cell lines containing a tetracycline-regulatable expression system were used to overexpress native swine apo A-IV and “piglike” human apo A-IV, a mutant human apo A-IV with deletion of the EQQQ-rich COOH-terminus, previously shown to upregulate basolateral triglyceride (TG) secretion 5-fold and 25-fold, respectively. Cells were incubated 24 h with and without doxycycline and oleic acid (OA, 0.8 mM). Overexpression of the native swine apo A-IV and piglike human apo A-IV increased MTTP lipid transfer activity by 39.7% ( P = 0.006) and 53.6% ( P = 0.0001), respectively, compared with controls. Changes in mRNA and protein levels generally paralleled changes in activity. Interestingly, native swine apo A-IV overexpression also increased MTTP large subunit mRNA, protein levels, and lipid transfer activity in the absence of OA, suggesting a mechanism not mediated by lipid absorption. Overexpression of piglike human apo A-IV significantly increased partitioning of radiolabeled OA from endoplasmic reticulum (ER) membrane to lumen, suggesting increased net transfer of membrane TG to luminal particles. These results suggest that the increased packaging of TG into nascent CMs in the ER lumen, induced by apo A-IV, is associated with upregulation of MTTP activity at the pretranslational level. Thus MTTP is regulated by apo A-IV in a manner to promote increased packaging of TG into the CM core, which may be important in neonatal fat absorption.

scholarly journals A missense mutation dissociates triglyceride and phospholipid transfer activities in zebrafish and human microsomal triglyceride transfer protein

2019 ◽  
Author(s):  
Meredith H. Wilson ◽  
Sujith Rajan ◽  
Aidan Danoff ◽  
Richard J. White ◽  
Monica R. Hensley ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Apolipoprotein B ◽  
Lipid Droplets ◽  
Transfer Functions ◽  
Microsomal Triglyceride Transfer Protein ◽  
Normal Growth ◽  
Selective Inhibition ◽  
Transfer Protein ◽  
Transfer Activity ◽  
Phospholipid Transfer

SUMMARYMicrosomal triglyceride transfer protein (MTP) transfers triglycerides and phospholipids and is essential for the assembly of Apolipoprotein B (ApoB)-containing lipoproteins in the endoplasmic reticulum. We have discovered a zebrafish mutant (mttpc655) expressing a C-terminal missense mutation (G863V) in Mttp, one of the two subunits of MTP, that is defective at transferring triglycerides, but retains phospholipid transfer activity. Mutagenesis of the conserved glycine in the human MTTP protein (G865V) also eliminates triglyceride but not phospholipid transfer activity. The G863V mutation reduces the production and size of ApoB-containing lipoproteins in zebrafish embryos and results in the accumulation of cytoplasmic lipid droplets in the yolk syncytial layer. However, mttpc655 mutants exhibit only mild intestinal lipid malabsorption and normal growth as adults. In contrast, zebrafish mutants bearing the previously identified mttpstl mutation (L475P) are deficient in transferring both triglycerides and phospholipids and exhibit gross intestinal lipid accumulation and defective growth. Thus, the G863V point mutation provides the first evidence that the triglyceride and phospholipid transfer functions of a vertebrate MTP protein can be separated, arguing that selective inhibition of the triglyceride transfer activity of MTP may be a feasible therapeutic approach for dyslipidemia.

scholarly journals The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity

1996 ◽  
Vol 315 (2) ◽  
pp. 533-536 ◽  
Author(s):  
Arja LAMBERG ◽  
Matti JAUHIAINEN ◽  
Jari METSO ◽  
Christian EHNHOLM ◽  
Carol SHOULDERS ◽  
...  
Keyword(s):  
Β Subunit ◽  
Lipid Transfer ◽  
Α Subunit ◽  
Protein Disulphide Isomerase ◽  
Isomerase Activity ◽  
Transfer Protein ◽  
Transfer Activity ◽  
Disulphide Bond Formation ◽  
Catalytically Active

The microsomal triacylglycerol transfer protein (MTP), an αβ dimer, is obligatory for the assembly of apoB-containing lipoproteins in liver and intestinal cells. The β subunit is identical with protein disulphide isomerase, a 58 kDa endoplasmic reticulum luminal protein involved in ensuring correct disulphide bond formation of newly synthesized proteins. We report here the expression of the human MTP subunits in Spodoptera frugiperda cells. When the α subunit was expressed alone, the polypeptide formed insoluble aggregates that were devoid of triacylglycerol transfer activity. In contrast, when the α and β subunits were co-expressed, soluble αβ dimers were formed with significant triacylglycerol transfer activity. Expression of the α subunit with a mutant protein disulphide isomerase polypeptide in which both -CGHC- catalytic sites had been inactivated also yielded αβ dimers that had comparable levels of lipid transfer activity relative to wild-type dimers. The results indicate that the role of the β subunit in MTP seems to be to keep the α subunit in a catalytically active, non-aggregated conformation and that disulphide isomerase activity of the β subunit is not required for this function.

Lipoprotein assembly and function in an evolutionary perspective

2010 ◽  
Vol 1 (2) ◽  
pp. 165-183 ◽  
Author(s):  
Dick J. Van der Horst ◽  
Kees W. Rodenburg
Keyword(s):  
Lipid Transfer Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Microsomal Triglyceride Transfer Protein ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Density Lipoprotein Receptor ◽  
Exchangeable Basis ◽  
And Function

AbstractCirculatory fat transport in animals relies on members of the large lipid transfer protein (LLTP) superfamily, including mammalian apolipoprotein B (apoB) and insect apolipophorin II/I (apoLp-II/I). ApoB and apoLp-II/I, constituting the structural (non-exchangeable) basis for the assembly of various lipoproteins, acquire lipids through microsomal triglyceride-transfer protein, another LLTP family member, and bind them by means of amphipathic α-helical and β-sheet structural motifs. Comparative research reveals that LLTPs evolved from the earliest animals and highlights the structural adaptations in these lipid-binding proteins. Thus, in contrast to apoB, apoLp-II/I is cleaved post-translationally by a furin, resulting in the appearance of two non-exchangeable apolipoproteins in the single circulatory lipoprotein in insects, high-density lipophorin (HDLp). The remarkable structural similarities between mammalian and insect lipoproteins notwithstanding important functional differences relate to the mechanism of lipid delivery. Whereas in mammals, partial delipidation of apoB-containing lipoproteins eventually results in endocytic uptake of their remnants, mediated by members of the low-density lipoprotein receptor (LDLR) family, and degradation in lysosomes, insect HDLp functions as a reusable lipid shuttle capable of alternate unloading and reloading of lipid. Also, during muscular efforts (flight activity), an HDLp-based lipoprotein shuttle provides for the transport of lipid for energy generation. Although a lipophorin receptor – a homolog of LDLR – was identified that mediates endocytic uptake of HDLp during specific developmental periods, the endocytosed lipoprotein appears to be recycled in a transferrin-like manner. These data highlight that the functional adaptations in the lipoprotein lipid carriers in mammals and insects also emerge with regard to the functioning of their cognate receptors.

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