SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079653456> ?p ?o ?g. }

Showing items 1 to 100 of ±109 with 100 items per page.

W2079653456 endingPage "7251" @default.
W2079653456 startingPage "7244" @default.
W2079653456 abstract "We tested the involvement of N-terminal six disulfide bonds (Cys-1 through Cys-12) of human apolipoprotein (apo) B in the assembly and secretion of lipoproteins using two C-terminal-truncated apoB variants, namely B50 and B18. In transfected rat hepatoma McA-RH7777 cells, B50 could assemble very low density lipoproteins (VLDL), and B18 was secreted as high density lipoproteins. When all 12 cysteine residues were substituted with alanines in B50, the mutant protein (B50C1–12) lost its ability to assemble lipid and was degraded intracellularly. However, mutation had no effect on B50C1–12 translation or translocation across the microsomal membrane. Post-translational degradation of B50C1–12 was partially inhibited by the proteasome inhibitor MG132. To determine which cysteines were critical in VLDL assembly and secretion, we prepared three additional mutant B50s, each containing four selected Cys-to-Ala substitutions in tandem (i.e. Cys-1 to Cys-4, Cys-5 to Cys-8, and Cys-9 to Cys-12). Expression of these mutants showed that disruption of disulfide bond formation within Cys-5 to Cys-8 diminished apoB secretion, whereas within Cys-1 to Cys-4 or Cys-9 to Cys-12 had lesser or no effect. In another two mutants in which only one disulfide bond (i.e. between Cys-5 and Cys-6 or between Cys-7 and Cys-8) was eliminated, only secretion of B50 with mutations at Cys-7 and Cys-8 was decreased. Thus, the disulfide bond involving Cys-7 and Cys-8 is most important for VLDL assembly and secretion. In addition, assembly and secretion of VLDL containing endogenous B100 or B48 were impaired in cells transfected with B50s containing Cys-7 and Cys-8 mutation. The Cys-to-Ala substitution abolished recognition of B50 by MB19, a conformational antibody with an epitope at the N terminus of human apoB. The Cys-to-Ala substitution also attenuated secretion of B18, but the effect of the mutation on B18 secretion was less evident than on B50. We tested the involvement of N-terminal six disulfide bonds (Cys-1 through Cys-12) of human apolipoprotein (apo) B in the assembly and secretion of lipoproteins using two C-terminal-truncated apoB variants, namely B50 and B18. In transfected rat hepatoma McA-RH7777 cells, B50 could assemble very low density lipoproteins (VLDL), and B18 was secreted as high density lipoproteins. When all 12 cysteine residues were substituted with alanines in B50, the mutant protein (B50C1–12) lost its ability to assemble lipid and was degraded intracellularly. However, mutation had no effect on B50C1–12 translation or translocation across the microsomal membrane. Post-translational degradation of B50C1–12 was partially inhibited by the proteasome inhibitor MG132. To determine which cysteines were critical in VLDL assembly and secretion, we prepared three additional mutant B50s, each containing four selected Cys-to-Ala substitutions in tandem (i.e. Cys-1 to Cys-4, Cys-5 to Cys-8, and Cys-9 to Cys-12). Expression of these mutants showed that disruption of disulfide bond formation within Cys-5 to Cys-8 diminished apoB secretion, whereas within Cys-1 to Cys-4 or Cys-9 to Cys-12 had lesser or no effect. In another two mutants in which only one disulfide bond (i.e. between Cys-5 and Cys-6 or between Cys-7 and Cys-8) was eliminated, only secretion of B50 with mutations at Cys-7 and Cys-8 was decreased. Thus, the disulfide bond involving Cys-7 and Cys-8 is most important for VLDL assembly and secretion. In addition, assembly and secretion of VLDL containing endogenous B100 or B48 were impaired in cells transfected with B50s containing Cys-7 and Cys-8 mutation. The Cys-to-Ala substitution abolished recognition of B50 by MB19, a conformational antibody with an epitope at the N terminus of human apoB. The Cys-to-Ala substitution also attenuated secretion of B18, but the effect of the mutation on B18 secretion was less evident than on B50. Apolipoprotein (apo) 1The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoproteins; ER, endoplasmic reticulum; MTP, microsomal triglyceride transfer protein; PAGE, polyacrylamide gel electrophoresis; RARE, RecA-assisted restriction endonuclease; kb, kilobase(s). 1The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoproteins; ER, endoplasmic reticulum; MTP, microsomal triglyceride transfer protein; PAGE, polyacrylamide gel electrophoresis; RARE, RecA-assisted restriction endonuclease; kb, kilobase(s).B100 is a large, hydrophobic polypeptide consisting of 4536 amino acids (1Chan L. J. Biol. Chem. 1992; 267: 25621-25624Abstract Full Text PDF PubMed Google Scholar, 2Segrest J.P. Jones M.K. Mishra V.K. Anantharamaiah G.M. Garber D.W. Arterioscler. Thromb. 1994; 14: 1674-1685Crossref PubMed Scopus (167) Google Scholar). In humans, apoB100 serves as a backbone for the assembly of triacylglycerol-enriched very low density lipoproteins (VLDL) (3Kane J.P. Annu. Rev. Physiol. 1983; 45: 637-650Crossref PubMed Scopus (212) Google Scholar). Assembly of VLDL requires coordinate biosynthesis of apoB100 and lipid components (4Yao Z McLeod R.S. Biochim. Biophys. Acta. 1994; 1212: 152-166Crossref PubMed Scopus (154) Google Scholar, 5Yao Z. Tran K. McLeod R.S. J. Lipid Res. 1997; 38: 1937-1953Abstract Full Text PDF PubMed Google Scholar). The association with lipid may occur during apoB100 translation and its translocation across the endoplasmic reticulum (ER), a process facilitated by the microsomal triglyceride transfer protein (MTP) (6Gordon D.A. Wetterau J.R. Gregg R.E. Trends Cell Biol. 1995; 5: 317-321Abstract Full Text PDF PubMed Scopus (118) Google Scholar). The sequence elements within apoB that are responsible for lipid recruitment have not been precisely defined. Mutational studies with C-terminal-truncated apoB variants have shown that the amount of core lipid in the resulting lipoprotein particles is positively correlated with the apoB polypeptide length (7Yao Z. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar, 8Graham D.L. Knott T.J. Jones T.C. Pease R.J. Pullinger C.R. Scott J. Biochemistry. 1991; 30: 5616-5621Crossref PubMed Scopus (54) Google Scholar, 9Borén J. Graham L Wettesten M. Scott J. White A. Olofsson S.-O. J. Biol. Chem. 1992; 267: 9858-9867Abstract Full Text PDF PubMed Google Scholar, 10Spring D.J. Chen-Liu L.W. Chatterton J.E. Elovson J. Schumaker V.N. J. Biol. Chem. 1992; 267: 14839-14845Abstract Full Text PDF PubMed Google Scholar, 11McLeod R.S. Zhao Y. Selby S.L. Westerlund J. Yao Z. J. Biol. Chem. 1994; 269: 2852-2862Abstract Full Text PDF PubMed Google Scholar), suggesting that the lipid recruiting ability is a function of the number of hydrophobic sequences. However, when abundant lipid is available, assembly of VLDL can be achieved by utilizing C-terminal-truncated apoB forms (e.g. apoB48) (12Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 13McLeod R.S. Wang Y. Wang S. Rusiñol A. Links P. Yao Z. J. Biol. Chem. 1996; 271: 18445-18455Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 14Wang Y. McLeod R.S. Yao Z. J. Biol. Chem. 1997; 272: 12272-12278Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Thus, both the length of apoB polypeptide and sufficient lipid supply are important determinants in the assembly and secretion of VLDL.Despite the absence of information concerning specific sequence elements involved in VLDL assembly, several experimental observations suggest that the N-terminal 17% of apoB100 (i.e. apoB17) is critical in lipoprotein formation. Transfection studies have shown that although the C-terminal-truncated apoB forms that contain the autologous N terminus of apoB100 are usually secretion-competent (7Yao Z. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar,11McLeod R.S. Zhao Y. Selby S.L. Westerlund J. Yao Z. J. Biol. Chem. 1994; 269: 2852-2862Abstract Full Text PDF PubMed Google Scholar, 13McLeod R.S. Wang Y. Wang S. Rusiñol A. Links P. Yao Z. J. Biol. Chem. 1996; 271: 18445-18455Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), recombinant apoB variants that lack the N-terminal sequences of apoB are often secreted poorly (13McLeod R.S. Wang Y. Wang S. Rusiñol A. Links P. Yao Z. J. Biol. Chem. 1996; 271: 18445-18455Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 15Gretch D.G. Sturley S.L. Wang L. Lipton B.A. Dunning A. Grunwald K.A.A. Wetterau J.R. Yao Z. Talmud P. Attie A.D. J. Biol. Chem. 1996; 271: 8682-8691Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Several studies have demonstrated that apoB17 has relatively low ability to associate with neutral lipids (7Yao Z. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar, 16Herscovitz H. Hadzopoulou-Cladaras M. Walsh M.T. Cladaras C. Zannis V.I. Small D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7313-7317Crossref PubMed Scopus (50) Google Scholar). Secondary structure analysis has predicted that apoB17 is composed mainly of amphipathic α-helices (2Segrest J.P. Jones M.K. Mishra V.K. Anantharamaiah G.M. Garber D.W. Arterioscler. Thromb. 1994; 14: 1674-1685Crossref PubMed Scopus (167) Google Scholar, 16Herscovitz H. Hadzopoulou-Cladaras M. Walsh M.T. Cladaras C. Zannis V.I. Small D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7313-7317Crossref PubMed Scopus (50) Google Scholar) and may form a globular structure owing to the existence of concentrated disulfide bonds (17Yang C.-Y. Kim T.W. Weng S.-A. Lee B. Yang M. Gotto Jr., A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5523-5527Crossref PubMed Scopus (84) Google Scholar). It is not clear why the region of apoB with low lipid binding ability is essential for efficient assembly of lipid into a lipoprotein.Of eight disulfide linkages within human plasma apoB, six are located within the N-terminal 500 amino acids (17Yang C.-Y. Kim T.W. Weng S.-A. Lee B. Yang M. Gotto Jr., A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5523-5527Crossref PubMed Scopus (84) Google Scholar). Recently, involvement of the concentrated disulfide bonds within the N terminus of apoB in lipoprotein assembly and secretion has been examined. Using dithiothreitol to disrupt disulfide bonding, Shelness and Thornburg (18Shelness G.S. Thornburg J.T. J. Lipid Res. 1996; 37: 408-419Abstract Full Text PDF PubMed Google Scholar) have observed that formation of the N-terminal disulfide bonds, which occurs within 1 min of translation, is required for the initiation of MTP-dependent lipid recruitment and the secretion of lipoproteins. It has been postulated that folding of the N-terminal region, presumably mediated through disulfide bonding, is essential for lipoprotein assembly (19Ingram M.F. Shelness G.S. J. Biol. Chem. 1997; 272: 10279-10286Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Considering the intrinsic nature of protein folding and the role of disulfide bonds in stabilizing native protein conformation, we hypothesized that the N-terminal six disulfide bonds play a crucial role in VLDL assembly and secretion.In the current studies, we have directly examined the function of six disulfide bonds using two truncated forms of apoB, apoB50 that has the ability to form VLDL and apoB18 that does not associate with neutral lipids. In these proteins, the cysteine residues were selectively substituted with alanine. Our results demonstrate that the fourth disulfide bond (between Cys-7 and Cys-8) is most important for apoB50 to assemble VLDL. However, the requirement for disulfide bonds is less manifest in apoB18. The present results suggest that an interaction between the disulfide-bonded domain and the downstream lipid binding sequences may play an important role in the post-translational stability of apoB and in VLDL assembly and secretion.DISCUSSIONProtein folding and conformational stabilization by disulfide bonds during translation and translocation into the ER is re-garded as a crucial step for the sorting of many secretory proteins. Using site-directed mutagenesis to selectively eliminate disulfide bond formation within B50, we have observed that not all, but only the fourth pair of cysteines (Cys-7 and Cys-8) is essential for the formation and secretion of B50-VLDL (Fig. 4 B) and for efficient B50 secretion (Fig. 5 B). Mutation at other disulfide bonds has less or no effect, whereas elimination of all six disulfide bonds (B50C1–12) almost abolishes B50 secretion as lipoproteins. The decreased secretion of mutant B50 is not caused by reduced protein expression nor is it attributable to impaired translocation across the ER membrane. Rather, the failure to secrete mutant B50 results from a defect at the post-translational level, leading to degradation of the secretion-incompetent proteins. It is most likely that the inefficiency of lipid recruitment impairs secretion of the mutant proteins. The specific requirement for the fourth disulfide bond in B50 is striking, which suggests that the failed assembly and secretion of mutant B50-VLDL may not be simply the consequence of misfolding of the N terminus of apoB. In fact, mutation at cysteine residues other than Cys-7 and Cys-8 equally impairs proper folding of apoB (as determined by the reduced affinity to MB19 shown in Fig. 8 D) but has less effect on B50 secretion. Therefore, it is possible that the fourth disulfide bond plays an important functional role in lipid assembly other than a structural role in folding. However, the possibility that mutation at Cys-7 and Cys-8 may affect the correct formation of other disulfide bonds within apoB has not been ruled out.Unexpectedly, mutation at Cys-7 and Cys-8 also impairs assembly and secretion of endogenous VLDL (Figs. 2 A, 3 C, and4 C). Intracellular degradation of endogenous B100 and B48 are markedly increased in cells transfected with mutant B50. Expression of misfolded proteins inside the ER has been shown to induce expression of some molecular chaperones that assist both the folding and degradation processes (33Kozutsumi Y. Segal M. Normington K. Gething M.-J. Sambrook J. Nature. 1988; 332: 462-464Crossref PubMed Scopus (965) Google Scholar, 34Knittler M.R. Dirks S. Haas I.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1764-1768Crossref PubMed Scopus (131) Google Scholar). Considering that lipid assembly is a general process for both endogenous and exogenous apoBs, expression of mutant B50 lacking the functional disulfide bond at Cys-7 and Cys-8 may trigger a stress response that typically affects lipid assembly. This would then generate unstable apoB molecules that are more susceptible to degradation. The nature of this stress response is not known. Identification of molecular chaperones or protease systems that are induced in cells transfected with mutant B50 may provide an explanation for the enhanced degradation of endogenous apoBs.Another important observation made in the current study is the oleate-induced covalent complex formation of apoB in the microsomal membrane (Fig. 8). Direct interaction of apoB with MTP and other ER resident proteins, including BiP/grp78 and calnexin, has been recently demonstrated (35Patel S.B. Grundy S.M. J. Biol. Chem. 1996; 271: 18686-18694Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 36Wu X. Zhou M. Huang L.-S. Wetterau J. Ginsberg H.N. J. Biol. Chem. 1996; 271: 10277-10281Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Our results are the first demonstration that complex formation is sensitive to mutations at the N-terminal cysteines of apoB. The concept of a high-order association of ER resident proteins forming a matrix or a gel inside the ER lumen led to a new paradigm that newly synthesized proteins form complexes of heterogeneous sizes with each other or with ER molecular chaperones to acquire more intrachain disulfide bonds until they are fully oxidized and released from the aggregates (37Tatu U. Helenius A. J. Cell Biol. 1997; 136: 555-565Crossref PubMed Scopus (186) Google Scholar, 38Kellokumpu S. Suokas M. Risteli L. Myllylä R. J. Biol. Chem. 1997; 272: 2770-2777Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Vesicular stomatitis virus G proteins and procollagen chains are a few of the examples demonstrating complex formation with protein folding catalysts such as BiP/grp78 and protein disulfide isomerase before their maturation (38Kellokumpu S. Suokas M. Risteli L. Myllylä R. J. Biol. Chem. 1997; 272: 2770-2777Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 39de Silva A. Braakman I. Helenius A. J. Cell Biol. 1993; 120: 647-655Crossref PubMed Scopus (90) Google Scholar). Similar biochemical events may also occur during apoB-lipoprotein assembly, involving transient protein-protein interactions until the apoB polypeptide achieves a certain degree of lipidation to become secretion-competent.The current studies have emphasized that interfering with folding of the N terminus of apoB in general has less effect on the secretion of B18 than on B50. Although secretion of apoB18 is abolished when the six pairs of cysteines are mutated, its secretion is relatively resistant to one- or two-pair cysteine mutations. Thus, the stringent requirement for the disulfide bonding appears to be by and large dependent on MTP-mediated lipid assembly. On the other hand, gross disruption of disulfide bonding affects both lipid recruitment and protein secretion. On the basis of these observations, we postulate that correct folding and lipid association may represent two interdependent biochemical events that, in concert with the ER quality control system, regulate the assembly and secretion of VLDL. (a) Proper folding of a critical region involving the fourth disulfide bond might signal a lipid recruitment sensor to initiate VLDL assembly. This forward-signaling event (that is, appropriate folding of the N terminus precedes lipid assembly by acting as an acceptor for MTP) has been suggested by studies with dithiothreitol-treated HepG2 cells (19Ingram M.F. Shelness G.S. J. Biol. Chem. 1997; 272: 10279-10286Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). (b) The downstream lipid binding sequences, after acquiring adequate lipid load might in turn signal the ER quality control to release the secretion-competent particle. This reverse-signaling event has been suggested by studies with oleate-treated Sf-21 cells transfected with apoB, MTP, and protein disulfide isomerase (40Wang L. Fast D.G. Attie A.D. J. Biol. Chem. 1997; 272: 27644-27651Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Apolipoprotein (apo) 1The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoproteins; ER, endoplasmic reticulum; MTP, microsomal triglyceride transfer protein; PAGE, polyacrylamide gel electrophoresis; RARE, RecA-assisted restriction endonuclease; kb, kilobase(s). 1The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoproteins; ER, endoplasmic reticulum; MTP, microsomal triglyceride transfer protein; PAGE, polyacrylamide gel electrophoresis; RARE, RecA-assisted restriction endonuclease; kb, kilobase(s).B100 is a large, hydrophobic polypeptide consisting of 4536 amino acids (1Chan L. J. Biol. Chem. 1992; 267: 25621-25624Abstract Full Text PDF PubMed Google Scholar, 2Segrest J.P. Jones M.K. Mishra V.K. Anantharamaiah G.M. Garber D.W. Arterioscler. Thromb. 1994; 14: 1674-1685Crossref PubMed Scopus (167) Google Scholar). In humans, apoB100 serves as a backbone for the assembly of triacylglycerol-enriched very low density lipoproteins (VLDL) (3Kane J.P. Annu. Rev. Physiol. 1983; 45: 637-650Crossref PubMed Scopus (212) Google Scholar). Assembly of VLDL requires coordinate biosynthesis of apoB100 and lipid components (4Yao Z McLeod R.S. Biochim. Biophys. Acta. 1994; 1212: 152-166Crossref PubMed Scopus (154) Google Scholar, 5Yao Z. Tran K. McLeod R.S. J. Lipid Res. 1997; 38: 1937-1953Abstract Full Text PDF PubMed Google Scholar). The association with lipid may occur during apoB100 translation and its translocation across the endoplasmic reticulum (ER), a process facilitated by the microsomal triglyceride transfer protein (MTP) (6Gordon D.A. Wetterau J.R. Gregg R.E. Trends Cell Biol. 1995; 5: 317-321Abstract Full Text PDF PubMed Scopus (118) Google Scholar). The sequence elements within apoB that are responsible for lipid recruitment have not been precisely defined. Mutational studies with C-terminal-truncated apoB variants have shown that the amount of core lipid in the resulting lipoprotein particles is positively correlated with the apoB polypeptide length (7Yao Z. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar, 8Graham D.L. Knott T.J. Jones T.C. Pease R.J. Pullinger C.R. Scott J. Biochemistry. 1991; 30: 5616-5621Crossref PubMed Scopus (54) Google Scholar, 9Borén J. Graham L Wettesten M. Scott J. White A. Olofsson S.-O. J. Biol. Chem. 1992; 267: 9858-9867Abstract Full Text PDF PubMed Google Scholar, 10Spring D.J. Chen-Liu L.W. Chatterton J.E. Elovson J. Schumaker V.N. J. Biol. Chem. 1992; 267: 14839-14845Abstract Full Text PDF PubMed Google Scholar, 11McLeod R.S. Zhao Y. Selby S.L. Westerlund J. Yao Z. J. Biol. Chem. 1994; 269: 2852-2862Abstract Full Text PDF PubMed Google Scholar), suggesting that the lipid recruiting ability is a function of the number of hydrophobic sequences. However, when abundant lipid is available, assembly of VLDL can be achieved by utilizing C-terminal-truncated apoB forms (e.g. apoB48) (12Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 13McLeod R.S. Wang Y. Wang S. Rusiñol A. Links P. Yao Z. J. Biol. Chem. 1996; 271: 18445-18455Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 14Wang Y. McLeod R.S. Yao Z. J. Biol. Chem. 1997; 272: 12272-12278Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Thus, both the length of apoB polypeptide and sufficient lipid supply are important determinants in the assembly and secretion of VLDL. Despite the absence of information concerning specific sequence elements involved in VLDL assembly, several experimental observations suggest that the N-terminal 17% of apoB100 (i.e. apoB17) is critical in lipoprotein formation. Transfection studies have shown that although the C-terminal-truncated apoB forms that contain the autologous N terminus of apoB100 are usually secretion-competent (7Yao Z. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar,11McLeod R.S. Zhao Y. Selby S.L. Westerlund J. Yao Z. J. Biol. Chem. 1994; 269: 2852-2862Abstract Full Text PDF PubMed Google Scholar, 13McLeod R.S. Wang Y. Wang S. Rusiñol A. Links P. Yao Z. J. Biol. Chem. 1996; 271: 18445-18455Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), recombinant apoB variants that lack the N-terminal sequences of apoB are often secreted poorly (13McLeod R.S. Wang Y. Wang S. Rusiñol A. Links P. Yao Z. J. Biol. Chem. 1996; 271: 18445-18455Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 15Gretch D.G. Sturley S.L. Wang L. Lipton B.A. Dunning A. Grunwald K.A.A. Wetterau J.R. Yao Z. Talmud P. Attie A.D. J. Biol. Chem. 1996; 271: 8682-8691Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Several studies have demonstrated that apoB17 has relatively low ability to associate with neutral lipids (7Yao Z. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar, 16Herscovitz H. Hadzopoulou-Cladaras M. Walsh M.T. Cladaras C. Zannis V.I. Small D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7313-7317Crossref PubMed Scopus (50) Google Scholar). Secondary structure analysis has predicted that apoB17 is composed mainly of amphipathic α-helices (2Segrest J.P. Jones M.K. Mishra V.K. Anantharamaiah G.M. Garber D.W. Arterioscler. Thromb. 1994; 14: 1674-1685Crossref PubMed Scopus (167) Google Scholar, 16Herscovitz H. Hadzopoulou-Cladaras M. Walsh M.T. Cladaras C. Zannis V.I. Small D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7313-7317Crossref PubMed Scopus (50) Google Scholar) and may form a globular structure owing to the existence of concentrated disulfide bonds (17Yang C.-Y. Kim T.W. Weng S.-A. Lee B. Yang M. Gotto Jr., A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5523-5527Crossref PubMed Scopus (84) Google Scholar). It is not clear why the region of apoB with low lipid binding ability is essential for efficient assembly of lipid into a lipoprotein. Of eight disulfide linkages within human plasma apoB, six are located within the N-terminal 500 amino acids (17Yang C.-Y. Kim T.W. Weng S.-A. Lee B. Yang M. Gotto Jr., A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5523-5527Crossref PubMed Scopus (84) Google Scholar). Recently, involvement of the concentrated disulfide bonds within the N terminus of apoB in lipoprotein assembly and secretion has been examined. Using dithiothreitol to disrupt disulfide bonding, Shelness and Thornburg (18Shelness G.S. Thornburg J.T. J. Lipid Res. 1996; 37: 408-419Abstract Full Text PDF PubMed Google Scholar) have observed that formation of the N-terminal disulfide bonds, which occurs within 1 min of translation, is required for the initiation of MTP-dependent lipid recruitment and the secretion of lipoproteins. It has been postulated that folding of the N-terminal region, presumably mediated through disulfide bonding, is essential for lipoprotein assembly (19Ingram M.F. Shelness G.S. J. Biol. Chem. 1997; 272: 10279-10286Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Considering the intrinsic nature of protein folding and the role of disulfide bonds in stabilizing native protein conformation, we hypothesized that the N-terminal six disulfide bonds play a crucial role in VLDL assembly and secretion. In the current studies, we have directly examined the function of six disulfide bonds using two truncated forms of apoB, apoB50 that has the ability to form VLDL and apoB18 that does not associate with neutral lipids. In these proteins, the cysteine residues were selectively substituted with alanine. Our results demonstrate that the fourth disulfide bond (between Cys-7 and Cys-8) is most important for apoB50 to assemble VLDL. However, the requirement for disulfide bonds is less manifest in apoB18. The present results suggest that an interaction between the disulfide-bonded domain and the downstream lipid binding sequences may play an important role in the post-translational stability of apoB and in VLDL assembly and secretion. DISCUSSIONProtein folding and conformational stabilization by disulfide bonds during translation and translocation into the ER is re-garded as a crucial step for the sorting of many secretory proteins. Using site-directed mutagenesis to selectively eliminate disulfide bond formation within B50, we have observed that not all, but only the fourth pair of cysteines (Cys-7 and Cys-8) is essential for the formation and secretion of B50-VLDL (Fig. 4 B) and for efficient B50 secretion (Fig. 5 B). Mutation at other disulfide bonds has less or no effect, whereas elimination of all six disulfide bonds (B50C1–12) almost abolishes B50 secretion as lipoproteins. The decreased secretion of mutant B50 is not caused by reduced protein expression nor is it attributable to impaired translocation across the ER membrane. Rather, the failure to secrete mutant B50 results from a defect at the post-translational level, leading to degradation of the secretion-incompetent proteins. It is most likely that the inefficiency of lipid recruitment impairs secretion of the mutant proteins. The specific requirement for the fourth disulfide bond in B50 is striking, which suggests that the failed assembly and secretion of mutant B50-VLDL may not be simply the consequence of misfolding of the N terminus of apoB. In fact, mutation at cysteine residues other than Cys-7 and Cys-8 equally impairs proper folding of apoB (as determined by the reduced affinity to MB19 shown in Fig. 8 D) but has less effect on B50 secretion. Therefore, it is possible that the fourth disulfide bond plays an important functional role in lipid assembly other than a structural role in folding. However, the possibility that mutation at Cys-7 and Cys-8 may affect the correct formation of other disulfide bonds within apoB has not been ruled out.Unexpectedly, mutation at Cys-7 and Cys-8 also impairs assembly and secretion of endogenous VLDL (Figs. 2 A, 3 C, and4 C). Intracellular degradation of endogenous B100 and B48 are markedly increased in cells transfected with mutant B50. Expression of misfolded proteins inside the ER has been shown to induce expression of some molecular chaperones that assist both the folding and degradation processes (33Kozutsumi Y. Segal M. Normington K. Gething M.-J. Sambrook J. Nature. 1988; 332: 462-464Crossref PubMed Scopus (965) Google Scholar, 34Knittler M.R. Dirks S. Haas I.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1764-1768Crossref PubMed Scopus (131) Google Scholar). Considering that lipid assembly is a general process for both endogenous and exogenous apoBs, expression of mutant B50 lacking the functional disulfide bond at Cys-7 and Cys-8 may trigger a stress response that typically affects lipid assembly. This would then generate unstable apoB molecules that are more susceptible to degradation. The nature of this stress response is not known. Identification of molecular chaperones or protease systems that are induced in cells transfected with mutant B50 may provide an explanation for the enhanced degradation of endogenous apoBs.Another important observation made in the current study is the oleate-induced covalent complex formation of apoB in the microsomal membrane (Fig. 8). Direct interaction of apoB with MTP and other ER resident proteins, including BiP/grp78 and calnexin, has been recently demonstrated (35Patel S.B. Grundy S.M. J. Biol. Chem. 1996; 271: 18686-18694Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 36Wu X. Zhou M. Huang L.-S. Wetterau J. Ginsberg H.N. J. Biol. Chem. 1996; 271: 10277-10281Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Our results are the first demonstration that complex formation is sensitive to mutations at the N-terminal cysteines of apoB. The concept of a high-order association of ER resident proteins forming a matrix or a gel inside the ER lumen led to a new paradigm that newly synthesized proteins form complexes of heterogeneous sizes with each other or with ER molecular chaperones to acquire more intrachain disulfide bonds until they are fully oxidized and released from the aggregates (37Tatu U. Helenius A. J. Cell Biol. 1997; 136: 555-565Crossref PubMed Scopus (186) Google Scholar, 38Kellokumpu S. Suokas M. Risteli L. Myllylä R. J. Biol. Chem. 1997; 272: 2770-2777Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Vesicular stomatitis virus G proteins and procollagen chains are a few of the examples demonstrating complex formation with protein folding catalysts such as BiP/grp78 and protein disulfide isomerase before their maturation (38Kellokumpu S. Suokas M. Risteli L. Myllylä R. J. Biol. Chem. 1997; 272: 2770-2777Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 39de Silva A. Braakman I. Helenius A. J. Cell Biol. 1993; 120: 647-655Crossref PubMed Scopus (90) Google Scholar). Similar biochemical events may also occur during apoB-lipoprotein assembly, involving transient protein-protein interactions until the apoB polypeptide achieves a certain degree of lipidation to become secretion-competent.The current studies have emphasized that interfering with folding of the N terminus of apoB in general has less effect on the secretion of B18 than on B50. Although secretion of apoB18 is abolished when the six pairs of cysteines are mutated, its secretion is relatively resistant to one- or two-pair cysteine mutations. Thus, the stringent requirement for the disulfide bonding appears to be by and large dependent on MTP-mediated lipid assembly. On the other hand, gross disruption of disulfide bonding affects both lipid recruitment and protein secretion. On the basis of these observations, we postulate that correct folding and lipid association may represent two interdependent biochemical events that, in concert with the ER quality control system, regulate the assembly and secretion of VLDL. (a) Proper folding of a critical region involving the fourth disulfide bond might signal a lipid recruitment sensor to initiate VLDL assembly. This forward-signaling event (that is, appropriate folding of the N terminus precedes lipid assembly by acting as an acceptor for MTP) has been suggested by studies with dithiothreitol-treated HepG2 cells (19Ingram M.F. Shelness G.S. J. Biol. Chem. 1997; 272: 10279-10286Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). (b) The downstream lipid binding sequences, after acquiring adequate lipid load might in turn signal the ER quality control to release the secretion-competent particle. This reverse-signaling event has been suggested by studies with oleate-treated Sf-21 cells transfected with apoB, MTP, and protein disulfide isomerase (40Wang L. Fast D.G. Attie A.D. J. Biol. Chem. 1997; 272: 27644-27651Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Protein folding and conformational stabilization by disulfide bonds during translation and translocation into the ER is re-garded as a crucial step for the sorting of many secretory proteins. Using site-directed mutagenesis to selectively eliminate disulfide bond formation within B50, we have observed that not all, but only the fourth pair of cysteines (Cys-7 and Cys-8) is essential for the formation and secretion of B50-VLDL (Fig. 4 B) and for efficient B50 secretion (Fig. 5 B). Mutation at other disulfide bonds has less or no effect, whereas elimination of all six disulfide bonds (B50C1–12) almost abolishes B50 secretion as lipoproteins. The decreased secretion of mutant B50 is not caused by reduced protein expression nor is it attributable to impaired translocation across the ER membrane. Rather, the failure to secrete mutant B50 results from a defect at the post-translational level, leading to degradation of the secretion-incompetent proteins. It is most likely that the inefficiency of lipid recruitment impairs secretion of the mutant proteins. The specific requirement for the fourth disulfide bond in B50 is striking, which suggests that the failed assembly and secretion of mutant B50-VLDL may not be simply the consequence of misfolding of the N terminus of apoB. In fact, mutation at cysteine residues other than Cys-7 and Cys-8 equally impairs proper folding of apoB (as determined by the reduced affinity to MB19 shown in Fig. 8 D) but has less effect on B50 secretion. Therefore, it is possible that the fourth disulfide bond plays an important functional role in lipid assembly other than a structural role in folding. However, the possibility that mutation at Cys-7 and Cys-8 may affect the correct formation of other disulfide bonds within apoB has not been ruled out. Unexpectedly, mutation at Cys-7 and Cys-8 also impairs assembly and secretion of endogenous VLDL (Figs. 2 A, 3 C, and4 C). Intracellular degradation of endogenous B100 and B48 are markedly increased in cells transfected with mutant B50. Expression of misfolded proteins inside the ER has been shown to induce expression of some molecular chaperones that assist both the folding and degradation processes (33Kozutsumi Y. Segal M. Normington K. Gething M.-J. Sambrook J. Nature. 1988; 332: 462-464Crossref PubMed Scopus (965) Google Scholar, 34Knittler M.R. Dirks S. Haas I.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1764-1768Crossref PubMed Scopus (131) Google Scholar). Considering that lipid assembly is a general process for both endogenous and exogenous apoBs, expression of mutant B50 lacking the functional disulfide bond at Cys-7 and Cys-8 may trigger a stress response that typically affects lipid assembly. This would then generate unstable apoB molecules that are more susceptible to degradation. The nature of this stress response is not known. Identification of molecular chaperones or protease systems that are induced in cells transfected with mutant B50 may provide an explanation for the enhanced degradation of endogenous apoBs. Another important observation made in the current study is the oleate-induced covalent complex formation of apoB in the microsomal membrane (Fig. 8). Direct interaction of apoB with MTP and other ER resident proteins, including BiP/grp78 and calnexin, has been recently demonstrated (35Patel S.B. Grundy S.M. J. Biol. Chem. 1996; 271: 18686-18694Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 36Wu X. Zhou M. Huang L.-S. Wetterau J. Ginsberg H.N. J. Biol. Chem. 1996; 271: 10277-10281Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Our results are the first demonstration that complex formation is sensitive to mutations at the N-terminal cysteines of apoB. The concept of a high-order association of ER resident proteins forming a matrix or a gel inside the ER lumen led to a new paradigm that newly synthesized proteins form complexes of heterogeneous sizes with each other or with ER molecular chaperones to acquire more intrachain disulfide bonds until they are fully oxidized and released from the aggregates (37Tatu U. Helenius A. J. Cell Biol. 1997; 136: 555-565Crossref PubMed Scopus (186) Google Scholar, 38Kellokumpu S. Suokas M. Risteli L. Myllylä R. J. Biol. Chem. 1997; 272: 2770-2777Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Vesicular stomatitis virus G proteins and procollagen chains are a few of the examples demonstrating complex formation with protein folding catalysts such as BiP/grp78 and protein disulfide isomerase before their maturation (38Kellokumpu S. Suokas M. Risteli L. Myllylä R. J. Biol. Chem. 1997; 272: 2770-2777Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 39de Silva A. Braakman I. Helenius A. J. Cell Biol. 1993; 120: 647-655Crossref PubMed Scopus (90) Google Scholar). Similar biochemical events may also occur during apoB-lipoprotein assembly, involving transient protein-protein interactions until the apoB polypeptide achieves a certain degree of lipidation to become secretion-competent. The current studies have emphasized that interfering with folding of the N terminus of apoB in general has less effect on the secretion of B18 than on B50. Although secretion of apoB18 is abolished when the six pairs of cysteines are mutated, its secretion is relatively resistant to one- or two-pair cysteine mutations. Thus, the stringent requirement for the disulfide bonding appears to be by and large dependent on MTP-mediated lipid assembly. On the other hand, gross disruption of disulfide bonding affects both lipid recruitment and protein secretion. On the basis of these observations, we postulate that correct folding and lipid association may represent two interdependent biochemical events that, in concert with the ER quality control system, regulate the assembly and secretion of VLDL. (a) Proper folding of a critical region involving the fourth disulfide bond might signal a lipid recruitment sensor to initiate VLDL assembly. This forward-signaling event (that is, appropriate folding of the N terminus precedes lipid assembly by acting as an acceptor for MTP) has been suggested by studies with dithiothreitol-treated HepG2 cells (19Ingram M.F. Shelness G.S. J. Biol. Chem. 1997; 272: 10279-10286Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). (b) The downstream lipid binding sequences, after acquiring adequate lipid load might in turn signal the ER quality control to release the secretion-competent particle. This reverse-signaling event has been suggested by studies with oleate-treated Sf-21 cells transfected with apoB, MTP, and protein disulfide isomerase (40Wang L. Fast D.G. Attie A.D. J. Biol. Chem. 1997; 272: 27644-27651Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). We thank L. Curtiss, Y. Marcel, M. Michalak, R. Milne, L. Wong, and S. Young for providing antibodies against apoB or protein disulfide isomerase." @default.
W2079653456 created "2016-06-24" @default.
W2079653456 creator A5025258103 @default.
W2079653456 creator A5026085971 @default.
W2079653456 creator A5027914982 @default.
W2079653456 creator A5030295153 @default.
W2079653456 creator A5034366374 @default.
W2079653456 creator A5046743882 @default.
W2079653456 creator A5051320270 @default.
W2079653456 creator A5052821016 @default.
W2079653456 date "1998-03-01" @default.
W2079653456 modified "2023-10-02" @default.
W2079653456 title "Functional Analysis of Disulfide Linkages Clustered within the Amino Terminus of Human Apolipoprotein B" @default.
W2079653456 cites W11746928 @default.
W2079653456 cites W1524184984 @default.
W2079653456 cites W1524608424 @default.
W2079653456 cites W1541084827 @default.
W2079653456 cites W1551374056 @default.
W2079653456 cites W1604410629 @default.
W2079653456 cites W1626408496 @default.
W2079653456 cites W1652993674 @default.
W2079653456 cites W1775749144 @default.
W2079653456 cites W1820484722 @default.
W2079653456 cites W1874360846 @default.
W2079653456 cites W1964375961 @default.
W2079653456 cites W1968506143 @default.
W2079653456 cites W1974591888 @default.
W2079653456 cites W1975931718 @default.
W2079653456 cites W1977820944 @default.
W2079653456 cites W1997826161 @default.
W2079653456 cites W1999233154 @default.
W2079653456 cites W2014589819 @default.
W2079653456 cites W2015804255 @default.
W2079653456 cites W2019585786 @default.
W2079653456 cites W2020437934 @default.
W2079653456 cites W2021892283 @default.
W2079653456 cites W2026058880 @default.
W2079653456 cites W2038799520 @default.
W2079653456 cites W2040666005 @default.
W2079653456 cites W2041920247 @default.
W2079653456 cites W2061645854 @default.
W2079653456 cites W2062661754 @default.
W2079653456 cites W2069903573 @default.
W2079653456 cites W2073630907 @default.
W2079653456 cites W2074407729 @default.
W2079653456 cites W2086183522 @default.
W2079653456 cites W2113120828 @default.
W2079653456 cites W2114627734 @default.
W2079653456 cites W2122865563 @default.
W2079653456 cites W2166851392 @default.
W2079653456 cites W2177551494 @default.
W2079653456 cites W2187455213 @default.
W2079653456 cites W2342431794 @default.
W2079653456 doi "https://doi.org/10.1074/jbc.273.13.7244" @default.
W2079653456 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9516417" @default.
W2079653456 hasPublicationYear "1998" @default.
W2079653456 type Work @default.
W2079653456 sameAs 2079653456 @default.
W2079653456 citedByCount "46" @default.
W2079653456 countsByYear W20796534562012 @default.
W2079653456 countsByYear W20796534562015 @default.
W2079653456 countsByYear W20796534562018 @default.
W2079653456 countsByYear W20796534562019 @default.
W2079653456 countsByYear W20796534562023 @default.
W2079653456 crossrefType "journal-article" @default.
W2079653456 hasAuthorship W2079653456A5025258103 @default.
W2079653456 hasAuthorship W2079653456A5026085971 @default.
W2079653456 hasAuthorship W2079653456A5027914982 @default.
W2079653456 hasAuthorship W2079653456A5030295153 @default.
W2079653456 hasAuthorship W2079653456A5034366374 @default.
W2079653456 hasAuthorship W2079653456A5046743882 @default.
W2079653456 hasAuthorship W2079653456A5051320270 @default.
W2079653456 hasAuthorship W2079653456A5052821016 @default.
W2079653456 hasBestOaLocation W20796534561 @default.
W2079653456 hasConcept C185592680 @default.
W2079653456 hasConcept C27256138 @default.
W2079653456 hasConcept C2778163477 @default.
W2079653456 hasConcept C55493867 @default.
W2079653456 hasConcept C62746215 @default.
W2079653456 hasConcept C70721500 @default.
W2079653456 hasConcept C86803240 @default.
W2079653456 hasConceptScore W2079653456C185592680 @default.
W2079653456 hasConceptScore W2079653456C27256138 @default.
W2079653456 hasConceptScore W2079653456C2778163477 @default.
W2079653456 hasConceptScore W2079653456C55493867 @default.
W2079653456 hasConceptScore W2079653456C62746215 @default.
W2079653456 hasConceptScore W2079653456C70721500 @default.
W2079653456 hasConceptScore W2079653456C86803240 @default.
W2079653456 hasIssue "13" @default.
W2079653456 hasLocation W20796534561 @default.
W2079653456 hasOpenAccess W2079653456 @default.
W2079653456 hasPrimaryLocation W20796534561 @default.
W2079653456 hasRelatedWork W1982843405 @default.
W2079653456 hasRelatedWork W2045355728 @default.
W2079653456 hasRelatedWork W2060268288 @default.
W2079653456 hasRelatedWork W2329655268 @default.
W2079653456 hasRelatedWork W2333726361 @default.
W2079653456 hasRelatedWork W2341499342 @default.