Moreover, significantly more GST-WT or GST-E118-119V was coprecipitated with FLAG-tagged MTTP than GST-E85V in the presence (40C80 pmole of 3H-TG under our assay conditions) but not in the absence of lipids (Fig.?2and 0.05. ternary complex with TG and MTTP, as well as impairs its ability to facilitate MTTP-mediated apoB-containing lipoprotein assembly and secretion, suggesting that the ternary complex formation is required for PRAP1 to facilitate MTTP-mediated lipid transport. PRAP1 is detectable in chylomicron/VLDL-rich plasma fractions, suggesting that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with?PRAP1 to lipid acceptors. Both PRAP1-deficient and E85V knock-in mutant mice fed a chow Sstr1 diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing lipid. Interestingly, GPDA both genetically modified mice gained significantly less body weight and fat mass when on high-fat diets compared with littermate controls and were prevented from hepatosteatosis. Together, this study provides evidence that PRAP1 plays an important role in MTTP-mediated lipid transport and lipid absorption. gene, because it was pulled out in our screen for genes that were upregulated in uterus shortly after embryo implantation. Here, we demonstrate that PRAP1 is actually a lipid-binding protein that facilitates MTTP-mediated lipid transport. Results PRAP1-deficient mice manifest increased length of the small intestine The mouse gene encodes a proline-rich, acidic polypeptide of 149 amino acids (aa) with a putative 20-aa signal peptide. Transient transfection assays using expression vectors encoding PRAP1 C-terminally tagged with HA or FLAG revealed that both tagged molecules were secreted into the culture medium of the transfected cells, suggesting that PRAP1 is a secreted protein (Fig.?S1showed that IEC from PRAP1?/? mice manifested significantly reduced TG and phospholipids transfer activity compared with cells from control mice (compare first and second bars), albeit MTTP protein levels in intestinal cells from GPDA both genotypes were quite similar (Fig.?1(Fig.?1, assay, the E85V mutant of PRAP1 manifested a weaker activity in promoting apoB48 secretion in this cell-based assay (Fig.?S4V). Further fractionation of cultured medium by density gradient ultracentrifugation (21) revealed that, in this transient expression system, apoB48 was?secreted into the HDL-sized fraction when PRAP1 was coexpressed with apoB48 in the same cells (Fig.?S4point to examples of colocalized regions. shows the Coomassie blue staining of the GPDA recombinant protein used in that assay. 0.05. and and C), was included in this assay. Figure?2shows that significantly more 3H-labeled TG was coprecipitated with FLAG-tagged MTTP in a binding reaction containing GST-WT or GST-E118-119V than in a reaction containing GST-E85V. Moreover, significantly more GST-WT or GST-E118-119V was coprecipitated with FLAG-tagged MTTP than GST-E85V in the presence (40C80 pmole of 3H-TG under our assay conditions) but not in the absence of lipids (Fig.?2and 0.05. Note, except PRAP1-FL (and 0.05. Open in a separate window Figure?4 PRAP1 deficiency reduces apoB-lipoprotein assembly and secretion into the chylomicron/very low-density lipoproteinCrich fractions. Control (+/+) or PRAP1?/? mice were fasted for 16 h before tyloxapol injection and receiving an oral lipid bolus. Four hours later, the plasma pooled from three mice of the same genotype was fractionated by fast phase liquid chromatography, followed by immunoblotting analysis of the indicated protein (access to food and water) plasma level of TG and phospholipids was observed in PRAP1?/? mice (Fig.?6 0.05. Open in a separate window Figure?7 PRAP1?/?mice had increased GPDA fecal lipid content GPDA material and absorbed less calories from the diet.test. Interestingly, the E85V mutant mice manifested very similar phenotypes as the knockout mice in many aspects. For good examples, their small intestine was consistently longer than that of littermate settings (Fig.?8and and 0.05. Open in a separate window Number?9 PRAP1 deficiency or the E85V mutation diminishes high-fat diet (HFD)-induced hepatosteatosis.and genomic locus were first isolated from a 129/Svj mouse genomic library and used to construct the targeting vector. This focusing on vector was constructed by PCR-assisted cloning in such a way that, after homologous recombination, a LacZ reporter linked to a floxed Neo marker would be put into exon 3 of the locus. This focusing on vector was then electroporated into.