ASPARAGINE-LINKED OLIGOSACCHARIDES FACILITATE HUMAN CHORIONIC-GONADOTROPIN BETA-SUBUNIT FOLDING BUT NOT ASSEMBLY OF PREFOLDED BETA WITH ALPHA

被引:21
作者
FENG, WJ
HUTH, JR
NORTON, SE
RUDDON, RW
机构
[1] UNIV NEBRASKA, MED CTR, EPPLEY INST RES CANC & ALLIED DIS, OMAHA, NE 68198 USA
[2] UNIV NEBRASKA, MED CTR, DEPT PHARMACOL, OMAHA, NE 68198 USA
[3] UNIV NEBRASKA, MED CTR, DEPT BIOCHEM & MOLEC BIOL, OMAHA, NE 68198 USA
关键词
D O I
10.1210/en.136.1.52
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
To determine the role of asparagine (N)-linked oligosaccharide chains in protein folding and assembly, the well established hCG-beta in vitro folding and assembly assays were used to analyze how the human CG (hCG) beta-subunit devoid of one or two N-linked glycans folds and assembles under different conditions. Two approaches were used: 1) site-specific mutagenesis of hCG-beta synthesized in Chinese hamster ovary cells transfected with beta-mutants lacking the asparagine glycosylation sites; and 2) enzymatic deglycosylation of hCG-beta synthesized in JAR cells with peptide N-glycosidase F or endoglycosidase H. In both cases, [S-35]cysteine-labeled beta-subunits were used as substrates to measure the conversion of the hCG-beta folding intermediate p beta 1 into p beta 2 and assembly of p beta 2 with urinary alpha. Using the mutated substrates from Chinese hamster ovary cells, it was found that 60% of wild-type p beta 1 (two N-linked glycans), 60% of p beta 1 missing the Asn(13)-linked glycan, 40% of p beta 1 missing the Asn(30)-linked glycan, and 10% of p beta 1 missing two N-linked glycans were converted to the corresponding p beta 2, respectively. With the enzymatically deglycosylated substrate from JAR cells, 90% of p beta 1 (two N-linked glycans), 70% of p beta 1((1)) (one N-linked glycan), and 10% of p beta 1((0)) (without N-linked glycan) folded into p beta 2 under cysteamine and cystamine redox conditions with or without protein disulfide isomerase. These data demonstrate that at least one N-linked glycan is required for efficient folding of hCG-beta and that the Asn(30)-linked glycan is more important than Asn(13)-linked glycan for hCG-beta folding. It also was shown that the composition of N-linked glycans of hCG-p beta 1 did not change protein folding, since hCG-beta substrates with high mannose oligosacharides folded as efficiently as beta-substrates containing sialylated complex oligosaccharides. Moreover, assembly of the already folded, assembly-competent folding intermediate, p beta 2, was not affected by removal of one or both of the N-linked glycans of the beta-subunit. These data thus show that N-linked glycans play their most important role in the folding component-of the folding and assembly pathway for hCG-beta.
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页码:52 / 61
页数:10
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共 42 条
  • [21] KORNFELD S, 1978, J BIOL CHEM, V253, P7771
  • [22] CRYSTAL-STRUCTURE OF HUMAN CHORIONIC-GONADOTROPIN
    LAPTHORN, AJ
    HARRIS, DC
    LITTLEJOHN, A
    LUSTBADER, JW
    CANFIELD, RE
    MACHIN, KJ
    MORGAN, FJ
    ISAACS, NW
    [J]. NATURE, 1994, 369 (6480) : 455 - 461
  • [23] MACHAMER CE, 1988, J BIOL CHEM, V263, P5955
  • [24] THE ROLE OF THE ASPARAGINE-LINKED OLIGOSACCHARIDES OF THE ALPHA-SUBUNIT IN THE SECRETION AND ASSEMBLY OF HUMAN CHORIONIC-GONADOTROPIN
    MATZUK, MM
    BOIME, I
    [J]. JOURNAL OF CELL BIOLOGY, 1988, 106 (04) : 1049 - 1059
  • [25] MATZUK MM, 1988, J BIOL CHEM, V263, P17106
  • [26] MOYLE WR, 1975, J BIOL CHEM, V250, P9163
  • [27] DIRECT DEMONSTRATION OF THE ESSENTIAL ROLE OF THE INTRAMOLECULAR HIGH-MANNOSE OLIGOSACCHARIDE CHAINS IN THE FOLDING AND ASSEMBLY OF SOYBEAN (GLYCINE-MAX) LECTIN POLYPEPTIDES
    NAGAI, K
    YAMAGUCHI, H
    [J]. JOURNAL OF BIOCHEMISTRY, 1993, 113 (02) : 123 - 125
  • [28] PATTILLO RA, 1971, IN VITRO CELL DEV B, V6, P398
  • [29] PETERS BP, 1984, J BIOL CHEM, V259, P5123
  • [30] GLYCOPROTEIN HORMONES - STRUCTURE AND FUNCTION
    PIERCE, JG
    PARSONS, TF
    [J]. ANNUAL REVIEW OF BIOCHEMISTRY, 1981, 50 : 465 - 495