Characterization of Cln3p, the gene product responsible for juvenile neuronal ceroid lipofuscinosis, as a lysosomal integral membrane glycoprotein

被引:62
作者
Ezaki, J
Takeda-Ezaki, M
Koike, M
Ohsawa, Y
Taka, H
Mineki, R
Murayama, K
Uchiyama, Y
Ueno, T
Kominami, E
机构
[1] Juntendo Univ, Sch Med, Dept Biochem, Bunkyo Ku, Tokyo 113, Japan
[2] Juntendo Univ, Sch Med, Div Biochem Anal, Cent Lab Med Sci, Tokyo 113, Japan
[3] Osaka Univ, Grad Sch Med, Dept Cell Biol & Neurosci, Osaka, Japan
关键词
CLN3; LC-MS; lectin; lysosomes; MS/MS; neuronal ceroid lipofuscinosis;
D O I
10.1046/j.1471-4159.2003.02132.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessively inherited lysosomal storage disease involving a mutation in the CLN3 gene. The sequence of CLN3 was determined in 1995; however, the localization of the CLN3 gene product (Cln3p) was not confirmed. In this study, we investigated endogenous Cln3p using two peptide antibodies raised against two distinct epitopes of murine Cln3p. Identification of the liver 60 kDa protein as Cln3p was ascertained by amino acid sequence analysis using tandem mass spectrometry. Liver Cln3p was predominantly localized in the lysosomal membranes, not in endoplasmic reticulum (ER) or Golgi apparatus. As the tissue concentration of brain Cln3p was much lower than that of liver Cln3p, it could be detected only after purification from brain extract using anti-Cln3p IgG Sepharose. The apparent molecular masses of liver Cln3p and brain Cln3p were determined to be about 60 kDa and 55 kDa, respectively. Both brain and liver Cln3p were deglycosylated by PNGase F treatment to form polypeptides with almost the same molecular mass (45 kDa). However, they were not affected by Endo h treatment. In addition, it was also elucidated that the amino terminal region of Cln3p faces the cytosol.
引用
收藏
页码:1296 / 1308
页数:13
相关论文
共 54 条
  • [1] Normal lysosomal morphology and function in LAMP-1-deficient mice
    Andrejewski, N
    Punnonen, EL
    Guhde, G
    Tanaka, Y
    Lüllmann-Rauch, R
    Hartmann, D
    von Figura, K
    Saftig, P
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (18) : 12692 - 12701
  • [2] Ausubel FM, 1995, SHORT PROTOCOLS MOL
  • [3] BARRETT AJ, 1981, METHOD ENZYMOL, V22, P130
  • [4] Cln3Δex7/8 knock-in mice with the common JNCL mutation exhibit progressive neurologic disease that begins before birth
    Cotman, SL
    Vrbanac, V
    Lebel, LA
    Lee, RL
    Johnson, KA
    Donahue, LR
    Teed, AM
    Antonellis, K
    Bronson, RT
    Lerner, TJ
    MacDonald, ME
    [J]. HUMAN MOLECULAR GENETICS, 2002, 11 (22) : 2709 - 2721
  • [5] EZAKI J, 1995, J NEUROCHEM, V64, P733
  • [6] Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis
    Ezaki, J
    Takeda-Ezaki, M
    Oda, K
    Kominami, E
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2000, 268 (03) : 904 - 908
  • [7] Specific delay in the degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis is derived from cellular proteolytic dysfunction rather than structural alteration of subunit c
    Ezaki, J
    Wolfe, LS
    Kominami, E
    [J]. JOURNAL OF NEUROCHEMISTRY, 1996, 67 (04) : 1677 - 1687
  • [8] THE SEQUENCE OF THE MAJOR PROTEIN STORED IN OVINE CEROID LIPOFUSCINOSIS IS IDENTICAL WITH THAT OF THE DICYCLOHEXYLCARBODIIMIDE-REACTIVE PROTEOLIPID OF MITOCHONDRIAL ATP SYNTHASE
    FEARNLEY, IM
    WALKER, JE
    MARTINUS, RD
    JOLLY, RD
    KIRKLAND, KB
    SHAW, GJ
    PALMER, DN
    [J]. BIOCHEMICAL JOURNAL, 1990, 268 (03) : 751 - 758
  • [9] Mutations in a novel CLN6-encoded transmembrane protein cause variant neuronal ceroid lipofuscinosis in man and mouse
    Gao, HL
    Boustany, RMN
    Espinola, JA
    Cotman, SL
    Srinidhi, L
    Antonellis, KA
    Gillis, T
    Qin, XB
    Liu, SM
    Donahue, LR
    Bronson, RT
    Faust, JR
    Stout, D
    Haines, JL
    Lerner, TJ
    MacDonald, ME
    [J]. AMERICAN JOURNAL OF HUMAN GENETICS, 2002, 70 (02) : 324 - 335
  • [10] CLN3 protein regulates lysosomal pH and alters intracellular processing of Alzheimer's amyloid-β protein precursor and cathepsin D in human cells
    Golabek, AA
    Kida, E
    Walus, M
    Kaczmarski, W
    Michalewski, M
    Wisniewski, KE
    [J]. MOLECULAR GENETICS AND METABOLISM, 2000, 70 (03) : 203 - 213