Proteomic profiling of the prechylomicron transport vesicle involved in the assembly and secretion of apoB‐48‐containing chylomicrons in the intestinal enterocytes

Abstract

Intracellular assembly of chylomicrons (CM) occurs in intestinal enterocytes through a series of complex vesicular interactions. CM are transported from the ER to the Golgi using a specialized vesicular compartment called the prechylomicron transport vesicle (PCTV). In this study, PCTVs were isolated from the enteric ER of the Syrian Golden hamster, and characterized using 2-DE and MS. Proteomic profiles of PCTV-associated proteins were developed with the intention of identifying proteins involved in the formation, transport, lipidation, and assembly of CM particles. Positively identified proteins included those involved in lipoprotein assembly, namely microsomal triglyceride transfer protein and apolipoprotein B-48, as well as proteins involved in vesicular transport, such as Sar1 and vesicle-associated membrane protein 7. Other groups of proteins found were chaperones, intracellular vesicular trafficking proteins, fatty acid-binding proteins, and lipid-related proteins. These findings have increased our understanding of the transport vesicle involved in the intracellular assembly and transport of CM and can provide insight into potential cellular factors responsible for dysregulation of intestinal CM production.

Proteomic Analysis of the Very Low Density Lipoprotein (VLDL) transport vesicles

“…Interestingly, we identified apoB100, an essential VLDL structural protein, which is known to be present in VTVs but could not be recognized by 2D-gel and MALDI-TOF approach. We could not identify apoB48 using both approaches, which is consistent with previous published reports from Adeli’s group [41,42], however, the presence of both apoB100 and apoB48 in VTVs was confirmed by immunoblotting data (Fig. 2A).…”

“…These data suggest that MTP and PDI are present in VTV but they are not the preferential cargo for the VTV. The inability to identify the PDI using mass spectrometric approach raises two possibilities: (i) since immunoblotting data indicate no enrichment of PDI in the VTV; therefore it is possible that detection may be less sensitive; (ii) secondly, since there are several isoforms of PDI, it is very likely that information of a particular isoform of PDI (which is present in VTVs) is not available in the database, which is consistent with other published reports in which the identification of apoB48, VAMP7 Sar1 could not be accomplished using LC-MS/MS but these proteins were detected by immunoblotting [41,42]. Identification of rab1 and Sec31A by 2D-gel and MALDI-TOF approach was interesting because these two proteins play an important role in ER-to-Golgi transport of nascent proteins.…”

Cargo loading at the ER

“…Further proteomic analyses of isolated PCTVs might shed light on this apparent contraction and help to identify new candidates to define function and elucidate the mechanism. Such experiments have already revealed cytoskeletal proteins and a large number of enzymes and chaperones to be associated with PCTVs (Wong et al 2009). In addition, clear and definitive morphological data, such as immunogold labelling and electron tomography would resolve the issue of whether PCTVs are encapsulated by COPII.…”

The identification of the SNARE complex required for the fusion of VLDL-transport vesicle with hepatic cis-Golgi

“…We have shown previously that a unique composition of SNARE complex is required for the fusion of PCTVs (pre-chylomicron transport vesicles) with intestinal cis -Golgi [30,31]. The PCTV is the largest ER-derived vesicle [32,33] that uniquely utilizes VAMP7 (vesicle-associated membrane protein 7) as a v-SNARE [31,34]. Although both VTVs and PCTVs are the large ER-derived vesicles engaged in ER–Golgi transport of TAG-rich lipoproteins, VLDL and chylomicrons respectively, both vesicles appeared to be fundamentally different in their budding and fusion mechanisms.…”