|Budget Amount *help
¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 2000 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1999 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1998 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Apolipoprotein B (apoB) is the huge glycoprotein that is essential for the assembly of very low density lipoproteins (VLDL). In this study, we investigated the role of N-linked glycosylation of apoB in the assembly and secretion of VLDL.Treatment of rat hepatocytes with tunicamycin (TM) decreased synthesis of apoB100 and secretion of apoB100 and apoB48. Secretion of recombinant human apoB variants (i.e. apoB17, B29, B34, B42, and B48 representing the amino-terminal 17%, 29% and so on of apoB) from transfected McA-RH7777 cells was also decreased by TM to different degrees. Since the amino-terminal 17% of apoB is essential for VLDL assembly, we tested the function of the singly glycosylation site (Asn^<158>) within B17. Asn^<158>-to-Gln substitution decreased secretion of apoB17 but had little effect on apoB37 and apoB48 secretion. In contrast, mutagenesis of all (five) glycosylation sites decreased the secretion of apoB50 at levels similar to that of their wild type counterparts treated with TM.Furthermore, the mutagenesis markedly diminished the secretion of mutated apoB50 as VLDL, although it had relatively little effect on apoB50 secretion as dense lipoproteins. Similarly, treatment of primary rat hepatocytes with TM decreased secretion of apoB48 as VLDL.In these cells, TM decreased the movement of apoB from the microsomal membranes to the lumen and thereby diminished the appearance of VLDL in the lumen. Thus, we conclude that N-linked glycosylation of apoB plays an important role in the assembly and secretion of VLDL.