The N-linked oligosaccharides at the amino terminus of human apoB are important for the assembly and secretion of VLDL

Abstract

We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N 158 ) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N 158 , N 956 , N 1341 , N 1350 , and N 1496 ), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by Ͼ 50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d Ͻ 1.02 and d Ͼ 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments. These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB. The asparagine (N)-linked oligosaccharides of proteins are an important component of the quality control mechanisms of eucaryotic cells. Multiple roles have been assigned to N-linked oligosaccharides, including folding of nascent polypeptides, protection from proteolytic degradation, intracellular trafficking, secretion, cell surface expression, maintenance of protein conformation, and enzymatic activity (1-3). Human apolipoprotein (apo) B-100, a major structural protein of VLDL synthesized in the liver, is a 4,536 amino acid glycoprotein. There are 20 potential N-linked glycosylation sites within apoB-100, of which 16 Asn residues are conjugated with oligosaccharides on plasma LDL. Each mole of apoB-100 contains 5-6 mol of high-mannose type, and 8-10 mol of complex type oligosaccharides (4). As a member of the vitellogenin family of lipid transport and storage proteins (5-7), apoB-100 possesses numerous amphipathic ␣ -helices and ␤ -strands that constitute the major structural framework for the assembly and integrity of triglyceride-rich lipoproteins (8, 9). The precise amino acid sequences within apoB-100 that are involved ...

Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins

“…It is possible that the impaired glycosylation induced by glucosamine causes a conformational change in the apoB-100 molecule, resulting in increased availability of apoB-100 for ubiquitin-proteasomal degradation (3). This observation is well supported by Vukmirica et al (26), who found that the N-glycans at the N terminus of several apoB constructs are required for secretion. In addition, improperly folded apoB-100 molecules could stimulate ER-stress chaperones such as Grp78 (27).…”

Disruption of N-linked glycosylation promotes proteasomal degradation of the human ATP-binding cassette transporter ABCA3

“…Second, the immunoblot bands of single and double mutants of N124Q and N140Q supports the notion that the prominent changes in molecular weights (compared with WT ABCA3) are likely due to the absence of large sugar moiety and not due to the replacement of a single amino acid. Third, because of asparagine's close resemblance to glutamine in both size and structure, the reliability of glutamine for asparagine substitution mutation in the study of N-linked glycosylation is frequently demonstrated in previous studies of glycoproteins (1,39,53,57,63) including other ABC transporters such as the multidrug resistance protein (MRP) (28), ABCC7 (CFTR) (21), and ABCG2 (16,45).…”

Missense Mutations in APOB within the βα1 Domain of Human APOB-100 Result in Impaired Secretion of ApoB and ApoB-containing Lipoproteins in Familial Hypobetalipoproteinemia

“…Preparation of Expression Plasmids-The expression plasmids encoding human apoB-100 (39), B-48 (40), and B-17 (41) cDNA, respectively, were prepared as described previously. Numbers following apoB represent the percent of full-length apoB (apoB-100).…”