PRAP1 is a novel lipid-binding protein that promotes lipid absorption by facilitating MTTP-mediated lipid transport

Abstract

Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers lipids to lipid acceptors and whether there are other biomolecules involved in MTTP-mediated lipid transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially colocalized in the endoplasmic reticulum. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a 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 diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing lipid. Interestingly, 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. Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers lipids to lipid acceptors and whether there are other biomolecules involved in MTTP-mediated lipid transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially colocalized in the endoplasmic reticulum. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a 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 diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing lipid. Interestingly, 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. Lipoproteins are macromolecular complexes of various lipids and proteins, and their principal function is to transport lipids. One important class is apolipoprotein B (apoB)-containing lipoproteins that deliver lipids, mainly triglycerides (TGs) and cholesterol, from intestine and liver to other tissues. Two isoforms have been identified for apoB, apoB48 and apoB100, which are encoded by the same gene but generated via a unique mRNA editing process. ApoB100 is synthesized in the liver and is the major structural component of very low-density lipoprotein (VLDL) and its metabolic products. ApoB48, which contains the N-terminal 48% of apoB100, is synthesized in the intestine and is essential for the formation and secretion of chylomicrons (CMs) (1Hooper A.J. Burnett J.R. Watts G.F. Contemporary aspects of the biology and therapeutic regulation of the microsomal triglyceride transfer protein.Circ. Res. 2015; 116: 193-205Crossref PubMed Scopus (38) Google Scholar). Unlike human apoB48, the mouse apoB48 isoform is also found in mouse liver (2Greeve J. Altkemper I. Dieterich J.H. Greten H. Windler E. Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins.J. Lipid Res. 1993; 34: 1367-1383Abstract Full Text PDF PubMed Google Scholar). MTTP is an ER resident protein (3Wetterau J.R. Lin M.C. Jamil H. Microsomal triglyceride transfer protein.Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (276) Google Scholar, 4Wetterau J.R. Zilversmit D.B. Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues.Biochim. Biophys. Acta. 1986; 875: 610-617Crossref PubMed Scopus (92) Google Scholar). It principally transfers TG to facilitate optimal folding of nascent apoB and also shuttles other lipid classes such as cholesteryl esters, free cholesterol, phospholipids, ceramides, and sphingomyelin to further promote lipoprotein formation (5Iqbal J. Walsh M.T. Hammad S.M. Cuchel M. Tarugi P. Hegele R.A. Davidson N.O. Rader D.J. Klein R.L. Hussain M.M. Microsomal triglyceride transfer protein transfers and determines plasma concentrations of ceramide and sphingomyelin but not glycosylceramide.J. Biol. Chem. 2015; 290: 25863-25875Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 6Rava P. Hussain M.M. Acquisition of triacylglycerol transfer activity by microsomal triglyceride transfer protein during evolution.Biochemistry. 2007; 46: 12263-12274Crossref PubMed Scopus (31) Google Scholar, 7Wetterau J.R. Zilversmit D.B. A triglyceride and cholesteryl ester transfer protein associated with liver microsomes.J. Biol. Chem. 1984; 259: 10863-10866Abstract Full Text PDF PubMed Google Scholar). MTTP is predominantly expressed in hepatocytes and enterocytes and in several other cell types with relatively less abundance (8Hussain M.M. Rava P. Walsh M. Rana M. Iqbal J. Multiple functions of microsomal triglyceride transfer protein.Nutr. Metab. (Lond.). 2012; 9: 14Crossref PubMed Scopus (163) Google Scholar, 9Sirwi A. Hussain M.M. Lipid transfer proteins in the assembly of apoB-containing lipoproteins.J. Lipid Res. 2018; 59: 1094-1102Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). It forms a heterodimer with the ubiquitous ER chaperone protein disulfide isomerase (PDI). PDI itself lacks lipid transfer activity, but its noncovalent association with MTTP significantly promotes the solubility and the lipid transfer activity of MTTP (10Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity.Biochem. J. 1996; 315: 533-536Crossref PubMed Scopus (55) Google Scholar). Defective or missing MTTP function is linked to a human disease state, abetalipoproteinemia (11Hooper A.J. van Bockxmeer F.M. Burnett J.R. Monogenic hypocholesterolaemic lipid disorders and apolipoprotein B metabolism.Crit. Rev. Clin. Lab. Sci. 2005; 42: 515-545Crossref PubMed Scopus (78) Google Scholar, 12Zamel R. Khan R. R.L. Hegele R.A. and J. PubMed Scopus Google Scholar). mice with of the MTTP gene manifested a of lipids in the of the small intestine R.L. J. Davidson N.O. increase in hepatic in mice with Biol. Chem. Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Both the lipid transfer and of MTTP are involved in assembly (8Hussain M.M. Rava P. Walsh M. Rana M. Iqbal J. Multiple functions of microsomal triglyceride transfer protein.Nutr. Metab. (Lond.). 2012; 9: 14Crossref PubMed Scopus (163) Google Scholar, 9Sirwi A. Hussain M.M. Lipid transfer proteins in the assembly of apoB-containing lipoproteins.J. Lipid Res. 2018; 59: 1094-1102Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Although the structure of human complex been I. The structure of human microsomal triglyceride transfer Sci. A. 116: PubMed Scopus Google how exactly MTTP transfers lipids and whether there are other involved in MTTP-mediated lipid transport remain elusive. protein identified as a protein that expressed in the during the to of J. E. A acid is and expressed in the during J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). It found that PRAP1 is expressed in the of both and mice and is mainly in the small with a expression along the M. J. A.J. M. R.A. and in the mammalian intestine from of gene expression and study of the 2005; PubMed Scopus Google Scholar, J. and expression of the mouse protein gene and its rat in the intestine and Biophys. Acta. PubMed Scopus Google Scholar). of the human of PRAP1 cell in and liver cell J. H. A. The protein is and of cell Res. Google Scholar). It also that PRAP1 is involved in cell PRAP1 is a of in cell 2012; PubMed Scopus Google Scholar). the and the functions of PRAP1 remain We to in the function of the PRAP1 in for that were in we that PRAP1 is a protein that facilitates MTTP-mediated lipid transport. The mouse gene a of amino with a expression PRAP1 with or that both were the of the suggesting that PRAP1 is a protein the functions of we generated PRAP1 mice by a were with an and to in their a chow the body weight increase of the mice to that of the mice of the small from the mice with that the PRAP1 protein expressed in cell the the expression of PRAP1 of PRAP1 not significantly the or in the small as by the with the or for various cell for for and A for the of mice with also in the of and mice we that the length of the small intestine in mice significantly that in mice PRAP1 a a PRAP1 N-terminal and cell from of mice of proteins by that MTTP is the that with PRAP1. PRAP1 or that PRAP1 in from but not the E85V knock-in mutant mice with MTTP at the that PRAP1 and MTTP are partially colocalized at the ER as a in the to whether PRAP1 the lipid transfer activity of that from mice manifested significantly TG and transfer activity compared with from mice and MTTP protein in from both were Interestingly, PRAP1 the N-terminal to as both TG and transfer activity of MTTP in activity to a that very to that such we identified that the at position 85 is very important for PRAP1 to promote the lipid transfer function of MTTP, as mutation of such to (E85V) in activity to transfer both TG and in we that PDI to the of PRAP1 on MTTP-mediated assembly and secretion of apoB48 lipoprotein Jamil H. J. of apolipoprotein lipoproteins from is on expression of the microsomal triglyceride transfer protein and is by lipid Sci. A. PubMed Scopus Google Scholar). The that of PRAP1 MTTP-mediated secretion of apoB48 the with the in the E85V mutant of PRAP1 manifested a activity in apoB48 secretion in this to and of by A. Apolipoprotein 1996; PubMed Google in this expression apoB48 the when PRAP1 with apoB48 in the same we the that PRAP1 is a that as in the to TG in a and the to its to important for TG the of the in the protein as not further increase the lipid activity of and in Interestingly, the PRAP1 which activity in MTTP-mediated lipid manifested a as the the E85V mutant manifested a its to TG compared with the or the mutant lipids that significantly with TG for to PRAP1 suggesting that the of lipids to PRAP1 significantly with or for cholesterol, or cholesteryl in this The E85V mutant binds to TG with a the and in but to promote the lipid transfer activity of MTTP a that MTTP PRAP1-bound TG as a cargo and transfer TG to the lipid apoB and that the lipids are not as to MTTP as the lipids. such is to a ternary complex MTTP and the but the E85V mutation significantly the formation of such ternary this MTTP from to to or the E85V mutant of PRAP1 or with of The mutant of which protein in various and A and in this that significantly TG with MTTP in a or in a significantly or with MTTP in the of but not in the of lipids with this MTTP but not the E85V mutant of PRAP1 in Together, a that PRAP1 with which TG to to MTTP during its lipid transfer We whether PRAP1 is involved in lipid in of a of significantly lipid by or by of in the small of the mice compared with that in the the of detectable in the less in mice in the mice with this the plasma from mice lipid not as as that from mice suggesting that PRAP1 lipid absorption. MTTP is essential for the of apoB-containing lipoproteins J.R. C. A. M. J. Rader D.J. of microsomal triglyceride transfer protein in with PubMed Scopus Google we whether PRAP1 assembly and secretion from major cell and that the and secretion of synthesized apoB48 significantly from from or the E85V mutant mice compared with that from the mice PRAP1 or the E85V mutation not significantly apoB mRNA expression were in hepatocytes significantly less apoB48 and were synthesized and from both and the E85V mutant hepatocytes compared with we the and of apoB in plasma of mice that been for were with lipoprotein and an of lipid and of plasma from mice for lipoprotein Lipid Res. Full Text Full Text PDF PubMed Google that significantly less apoB48 and and TG were to the of plasma compared with the same from mouse plasma of plasma from mice by that PRAP1 apoB and secretion hepatic suggesting that a very low of PRAP1 in the liver with that in on MTTP-mediated apoB lipoprotein assembly and plasma from mice that the E85V mutant mice manifested very as the significantly less apoB48 and and TG were the of and E85V mutant plasma compared with of plasma and assembly and secretion the low-density or mice were for and an lipid the plasma from mice of the same by by of the protein or of triglyceride in The apoB protein in and as a of apoB from from plasma E85V mutation assembly and secretion the low-density with the were for and an lipid of plasma from mice of the same by by of the proteins by the apoB protein in in and as a of apoB from from triglyceride mass or in in is from also for apoB and TG of mouse plasma in in the in and PRAP1 detectable in the plasma of A at the plasma for the Interestingly, the E85V mutant protein in the by less or with a secretion compared with the protein PRAP1 as and The of to Although on chow diet the body weight increase of and mice were on high-fat diet from mice gained significantly less body weight compared with mice 12 on a the fat mass of the mice significantly compared with that of the mice with this both were significantly in in mice compared with that in the in the mice and were significantly the that both and mice very on a we compared their and lipid Both of mice manifested very as by the of mice lipids of mice also that were in in suggesting that the of mice on a mainly to a in lipid absorption. 12 on a a in the to and plasma of TG and in mice or free acid in the plasma from both of mice to very mice lipid and less from the lipid of and mice fed with a chow were from to mice and lipid and mice at of were fed with a high-fat diet for from were and the lipid a of from mice in and to in of and are are from with to or mice from the same Interestingly, the E85V mutant mice manifested very as the mice in their small intestine that of littermate controls fed on a chow lipids were in the small of the E85V compared with mice of lipids on for 12 compared with the E85V mutant mice gained significantly less body weight and manifested and and plasma TG we the of PRAP1 or the E85V mutation on lipid in the liver this mice were fed a for 12 or their were and for and lipid The that lipid by hepatocytes with A and and hepatic TG B and were significantly in and the E85V mutant compared with suggesting that PRAP1 or the E85V mutation or the E85V mutation high-fat diet hepatosteatosis. A and and mice were fed with a for 12 of from mice are in and their weight and liver triglyceride are in mice with the were fed with a for of the and liver from mice are as of lipid in by the liver TG of mice fed with a for an and the we evidence that PRAP1 is a in MTTP-mediated transfer of lipids TG and that the ability to form a ternary complex with TG and MTTP with the ability of PRAP1 to promote MTTP-mediated lipid transfer. that MTTP binds and shuttles lipid and such for lipid transport that a complex is an in the transfer (3Wetterau J.R. Lin M.C. Jamil H. Microsomal triglyceride transfer protein.Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (276) Google Scholar, A. J.R. of microsomal triglyceride transfer protein lipid 1993; PubMed Scopus Google Scholar, A. J.R. of classes of lipid on the microsomal triglyceride transfer PubMed Scopus Google Scholar). that PRAP1 the that promotes the formation of the complex in this study the that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with PRAP1 to the lipid acceptors apoB, PRAP1 to component of that PRAP1 is a protein and is partially colocalized with MTTP at we a during its secretion PRAP1 TG in the ER and a to TG and to required to this this study identified an mutant of PRAP1 E85V that promote the activity of The E85V mutant binds to TG but to form a ternary complex with TG and It is but is less or with a mutation a that is very to that by PRAP1 in of apoB secretion and lipid absorption. on we that lipids not as as the lipids and are less to MTTP but not E85V lipids, to in the of structural required to this PRAP1 lipid absorption. We that apoB in mice but is significantly less in and in the of The length of the small intestine of mice compared with mice on the same chow diet that this is a for the to lipids. MTTP mice during the mice with of MTTP gene expression are with secretion of apoB and plasma apoB M. M.M. R.L. of the abetalipoproteinemia gene in lipoprotein secretion in and in Sci. A. PubMed Scopus Google Scholar). The of mice partially mice are MTTP is mainly expressed in hepatocytes and of MTTP in hepatocytes hepatic M. M.M. J. R.L. of the role of microsomal triglyceride transfer protein in the liver of Clin. PubMed Scopus Google Scholar). hepatic TG significantly in MTTP which is with lipid R.L. J. Davidson N.O. increase in hepatic in mice with Biol. Chem. Full Text Full Text PDF PubMed Scopus (92) Google Scholar). PRAP1 suggesting that PRAP1 its role in MTTP activity in in at in with this PRAP1 expression is found to in mouse in that the assembly of is likely a process. The is the transfer of a small of lipid to nascent apoB during its the ER to form a apoB The been to the of the apoB with ER lipid that not apoB A. Hussain M.M. Lipid transfer proteins in the assembly of apoB-containing lipoproteins.J. Lipid Res. 2018; 59: 1094-1102Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, Davidson N.O. of the in lipid Rev. 2012; PubMed Scopus Google Scholar, evidence for a assembly of lipoproteins in the of the endoplasmic reticulum of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. C. T. J. of very low a process of apolipoprotein B PubMed Google Scholar). MTTP is required for the formation of apoB and ER lipid R.L. J. Davidson N.O. increase in hepatic in mice with Biol. Chem. Full Text Full Text PDF PubMed Scopus (92) Google Scholar, M. M.M. J. R.L. of the role of microsomal triglyceride transfer protein in the liver of Clin. PubMed Scopus Google Scholar, A. Microsomal triacylglycerol transfer protein is required for of triacylglycerol not associated with as well as for Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, The activity of microsomal triglyceride transfer protein is essential for of triglyceride in A for the assembly of very low lipoproteins.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the formation of the complex is involved in both of assembly to the other during the of we that PRAP1 is also detectable in fractions, The and function of PRAP1 in remain to of low-density lipoprotein cholesterol, lipoprotein cholesterol, and TG are the important for Two association have significantly associated with lipid M. A. M. and of for PubMed Scopus Google Scholar, A. J. A. and of associated with lipid PubMed Scopus Google Scholar). of from as well as several other T. A.J. a for the and of association J. PubMed Scopus Google J. J. T. P. H. A. P. T. H. The a of Res. 42: PubMed Scopus Google M. association study with J. PubMed Scopus Google T. D.B. P. R. of expression and Res. 2005; PubMed Scopus Google in in as an in J. PubMed Scopus Google and P. R. M. E. A. M. T. P. a for therapeutic and Res. PubMed Scopus Google association single on PRAP1 or PRAP1 gene expression and does not the that other to identified on PRAP1 associated with plasma lipid there to that in PRAP1 which the study for PRAP1. PRAP1 or the E85V mutation weight and of mice on a One for liver is liver which is characterized in by of TG in study that PRAP1 as a therapeutic for lipid PRAP1 the were from a mouse and to the by in such a a linked to a of the and to that at the Two and the mutant were to The mice were with to mice to for were to and mice for this by with from the mouse for the were and and the for the mutant were and the E85V knock-in mutant the H. H. M.M. R. of mice in by Full Text Full Text PDF PubMed Scopus Google Scholar). the E85V mutation the gene editing of a mutant are and mRNA the mouse and of mice and and and mice were found to or and were with mice to mice were to and as E85V for this The E85V mice from both and manifested very and were from in for the were and and the for the mutant were and were in a with a on at with to and We to mice for were in with the by and is a expression that the of PRAP1 the N-terminal to the expression in the various of PRAP1 were by as a and are expression that the of PRAP1 amino acid of PRAP1 as in their as in the and various other proteins were with with to the by of by to a of PRAP1 or with or in the to proteins the N-terminal The of the small intestine of mice and in for at to The from mice were and in for at with were with and in a and with and or to were to of for at were with the and proteins on the were by and with protein in by but not by were for mass to their as The isoform of mouse is a protein with PubMed Scopus Google Scholar). generated both in and were by to from the small intestine of or mutant (E85V) mice were in the and and were with of PRAP1 in or MTTP The protein complexes were with the by and to the and the proteins were by with PRAP1 in or MTTP were in the and by The complex on the with the with A and by with or mutant PRAP1 in of A to of for the complex with A and with of The with of of the to for and the to lipids by of proteins with to a J. E. A. association study a that to liver 46: PubMed Scopus Google Scholar). The proteins were in a and by and of MTTP and or or or MTTP were the MTTP activity to the from or mice in and were on in a Two of with of and of in with the the the of triglyceride or in the by A not cell as a for this of were from mice to by and the of phospholipids, or free were from or to were with during to for the at mice were with body weight of by an of of of and of as a in that by the small T. T. M. A. T. M. of an of microsomal triglyceride transfer PubMed Scopus Google Scholar). One to the of the small intestine the in the cell were and the of and were at to lipid and the of in the the in the small intestine or the the were by the as in the to the of the of to or mutant PRAP1 proteins as C. of triglyceride to fat proteins and is important for lipid Sci. A. PubMed Scopus Google with of or proteins or to with A and and in of A the of at were with of A by and in of and and for a as J. Davidson N.O. lipoprotein assembly in mice in triglyceride secretion with a to J. PubMed Scopus Google with from the were for with and for to in modified with and A lipid concentrations to both and the both cell and were with apoB and the complexes by and The were by the of metabolic of mouse we the as M. M.M. J. R.L. of the role of microsomal triglyceride transfer protein in the liver of Clin. PubMed Scopus Google with hepatocytes were of to at that this is both cell and were with apoB and the complexes by and The were by the of from of with the and The mRNA expression of of were by on a to the of the in this are in in were with ApoB48, MTTP along with or by the and with modified of for were to and and for at to cell lipoproteins in the were by as J. Hussain M.M. of transport the Biol. Chem. Full Text Full Text PDF PubMed Scopus Google with of the to with with of of and of in a at for at the of the as The were with of at for at the of the as The were with of and at for at lipoproteins were from the in were to lipids by of proteins with to a J. E. A. association study a that to liver 46: PubMed Scopus Google Scholar). The proteins were in a and by and and plasma from mice of the by a single as for lipoprotein Lipid Res. Full Text Full Text PDF PubMed Google Scholar). in and and for 12 to were for lipid and protein plasma from to mice of the were by as A. Apolipoprotein 1996; PubMed Google Scholar). of plasma to with and with of in a A of of of and of on of the at for at the of the as The with of and at for and The as The with and The as The of the from mice were at for and to in of weight to were from mice that been fed a for to a Lipid from mouse 2015; PubMed Google Scholar). with for or of for are the and associated The that have of with the of this We E. of for on this for in and and of for their with the to the by of for in the and of mice and the of the for and the of of for mutant mice for this H. C. and the H. and M. M. H. and H. J. M. M. H. and H. P. and the study in by an from and by and from the of and of to by a from

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