Retinol binding protein and transthyretin are secreted as a complex formed in the endoplasmic reticulum in HepG2 human hepatocarcinoma cells.
Retinol binding protein (RBP), the retinol-specific carrier, circulates in blood as a 1:1 complex with the homotetrameric protein transthyretin (TTR). Both RBP and TTR are synthesized and secreted by the hepatocyte. In this work we have demonstrated, using HepG2 cells as a model system, that the association between the two proteins occurs inside the cell before secretion. The intracellular complex was detected only when metabolically labeled cells were lysed under mild detergent conditions (1.5% octylglucoside), followed by immunoprecipitation and SDS-PAGE. Alternatively, the immunoprecipitates from unlabeled cells lysed with the same buffer were analyzed by Western blotting. This finding was confirmed using the cross-linking agent dithiobis(succinimidyl) propionate before cell lysis. Moreover, we found that in cells treated with brefeldin A to block the exit of proteins from the endoplasmic reticulum (ER), the complex was present in the microsomal fraction. Thus, we can conclude that the RBP-TTR complex is formed inside the cell, more precisely within the ER. As RBP and TTR both lack an ER retention signal, we considered the possible involvement of chaperones in RBP and TTR retention in the ER and in complex formation. We found that calnexin, an ER integral membrane protein which functions as a chaperone, coprecipitates with RBP and TTR when cell lysis and immunoprecipitation are performed under mild conditions (1% Triton X-100). This result strongly suggests that calnexin may be involved in RBP and TTR retention in the ER, in TTR tetramer assembly, and possibly in complex formation.
Istituto Nazionale della Nutrizione, Rome, Italy.
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Bellovino
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Morimoto
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Tosetti
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Gaetani
D020126:Brefeldin A; D002135:Calcium-Binding Proteins / Q000032:analysis; D037281:Calnexin; D006528:Carcinoma, Hepatocellular; D003432:Cross-Linking Reagents; D003517:Cyclopentanes / Q000494:pharmacology; D003902:Detergents; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D005960:Glucosides; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008862:Microsomes, Liver / Q000737:chemistry; D018832:Molecular Chaperones / Q000032:analysis; D008970:Molecular Weight; D011228:Prealbumin / Q000032:analysis / Q000378:metabolism*; D011233:Precipitin Tests; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012177:Retinol-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D013388:Succinimides; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D003517:Cyclopentanes; D003902:Detergents; D005960:Glucosides; D018832:Molecular Chaperones; D011228:Prealbumin; D011500:Protein Synthesis Inhibitors; D012177:Retinol-Binding Proteins; D013388:Succinimides; D037281:Calnexin; D020126:Brefeldin A; C018619:octyl-beta-D-glucoside; C011240:dithiobis(succinimidylpropionate)