The resulting MTPlck mice had >95% deletion of mttp at the genomic level (unpublished data) but showed no decrease in mttp transcription. Therefore, in an attempt to parse out the role of MTP in thymic selection of NKT cells, we intercrossed the MTP-floxed mouse to an Lck-Cre mouse that has Cre expression driven by the proximal Lck promoter. The Mx1 promoter displays low activity in the thymus ( 37). ER and endosomal lipids differ in their potential to select NKT cells a tail-deleted form of CD1d, which remains at the cell surface and fails to traffic to endosomes, can activate NKT hybridomas with diverse, but not invariant, TCRs and exclusively supports development of diverse NKT cells ( 30, 31). Loss of AP-3, prosaposin, or cathepsins L or S decreased production of iGb3 caused by β-hexosaminidase B deficiency or altered lipid storage caused by loss of Niemann-Pick type C1 all result in impaired NKT cell development ( 23– 29).
#Alternote mobile Activator#
Lysosomal saposins, generated by cathepsin L–mediated cleavage of prosaposin, and GM2 activator protein can exchange ER-derived lipids bound by CD1d for lysosomal lipids such as iGb3. The vesicular trafficking protein AP-3 transports CD1d from the cell surface to the endosomal/lysosomal compartment. Phosphatidylinostitol and phosphatidylcholine, presumably of ER origin, have been found associated with CD1d, and at least one phosphatidylinositol-reactive NKT cell hybridoma has been characterized ( 20– 22). Humans homozygous for mutations in MTP develop abetalipoproteinemia, a disease characterized by malabsorption of lipids and lipophilic vitamins ( 11).Įndogenous lipids, derived from both the ER and the endosomal compartment, likely play a role in NKT cell selection. MTP deletion is embryonic lethal in mice, presumably because of insufficient lipid nutrient transfer from the placenta ( 9, 10). MTP is highly abundant in hepatocytes and IECs and has been found, along with modest apoB secretion, in cardiac myocytes, retina, and placenta ( 5– 8). This finding demonstrates that the phospholipid transfer function of MTP is sufficient for apoB secretion. Although apoB-containing lipoproteins are largely composed of triglycerides, drosophila MTP, which cannot transfer triglycerides but can transfer phospholipids, can support apoB secretion ( 4). Kinetic studies of MTP-mediated transfer of various lipid classes revealed that MTP has at least two lipid transfer sites: a fast site that transfers triglycerides and cholesterol esters and a slow site that transfers phospholipids ( 2, 3). MTP transfers phospholipids, triglycerides, and cholesterol to nascent apoB during its translocation into the ER. Microsomal triglyceride transfer protein (MTP) is a mainly ER-resident 97-kD lipid transfer protein that, when complexed with protein disulfide isomerase (PDI), transfers lipids to apolipoprotein B (apoB) for the production of very low density lipoprotein (VLDL) from hepatocytes or chylomicrons from intestinal epithelial cells (IECs) ( 1– 3). 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.
Thus, our results demonstrate that MTPv1 in thymocytes is critical to NKT cell development. CD1d expression on CD4 +CD8 + FTOC cells was unaffected by MTP inhibition. 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. NKT cells fail to develop in fetal thymic organ culture (FTOC) treated with MTP antagonists. MTP and MTPv1 efficiently transferred phosphatidylethanolamine to CD1d in vitro. 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. 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). Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum lipid transfer protein critical for apolipoprotein B (apoB) secretion, regulates CD1d antigen presentation.
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