Promotion of Tyrosinase Folding in Cos 7 Cells by Calnexin
To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immun...
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Published in | Journal of biochemistry (Tokyo) Vol. 125; no. 1; pp. 82 - 89 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
01.01.1999
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Subjects | |
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Abstract | To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced ˜2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When α-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immuno-precipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER. |
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AbstractList | To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER. To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced ˜2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When α-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immuno-precipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER. |
Author | Hori, Yoshiaki Park, Jong Sung Toyofuku, Kazutomo Hirosaki, Kuninori Jimbow, Kowichi Wada, Ikuo |
Author_xml | – sequence: 1 givenname: Kazutomo surname: Toyofuku fullname: Toyofuku, Kazutomo organization: Division of Dermatology & Cutaneous Sciences, Faculty of Medicine, University of Alberta Canada T6G 2S2 – sequence: 2 givenname: Ikuo surname: Wada fullname: Wada, Ikuo organization: Departments of Biochemistry Sapporo 060–8543 – sequence: 3 givenname: Kuninori surname: Hirosaki fullname: Hirosaki, Kuninori organization: Dermatology, Sapporo Medical University School of Medicine Sapporo 060–8543 – sequence: 4 givenname: Jong Sung surname: Park fullname: Park, Jong Sung organization: Division of Dermatology & Cutaneous Sciences, Faculty of Medicine, University of Alberta Canada T6G 2S2 – sequence: 5 givenname: Yoshiaki surname: Hori fullname: Hori, Yoshiaki organization: Department of Dermatology, Faculty of Medicine, Kyushu University Fukuoka 812–8582 – sequence: 6 givenname: Kowichi surname: Jimbow fullname: Jimbow, Kowichi email: jimbow@sapmed.ac.jp, 2To whom correspondence should be address at: Department of Dermatology, Sapporo Medical University School of Medicine, S–l, W–16, Chuo-ku, Sapporo 060–8543. jimbow@sapmed.ac.jp organization: Division of Dermatology & Cutaneous Sciences, Faculty of Medicine, University of Alberta Canada T6G 2S2 |
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Notes | ArticleID:125.1.82 ark:/67375/HXZ-7MXZFJNH-D istex:E1D0EF713D23BE2CCB7E178DE0817391C975DEBE 1This study was supported by grants from the Medical Research Council of Canada (#MT-12866) and the Ministry of Education, Science, Sports and Culture of Japan (#08407022). K.T. was from the Department of Dermatology, Faculty of Medicine, Kyushu University, Japan, as part of a graduate student program training. Technical help was obtained by Mr. Hua Chen. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
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SubjectTerms | Animals Calcium-Binding Proteins - genetics Calcium-Binding Proteins - metabolism Calnexin Carbohydrate Sequence COS Cells - metabolism DNA, Complementary - metabolism Endoplasmic Reticulum - chemistry Glycosylation Humans melanin Melanins - analysis Melanins - biosynthesis melanocyte melanogenesis Microscopy, Confocal Molecular Sequence Data Monophenol Monooxygenase - chemistry Monophenol Monooxygenase - genetics Monophenol Monooxygenase - metabolism Oligosaccharides - metabolism Precipitin Tests Protein Folding Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Transfection tyrosinase |
Title | Promotion of Tyrosinase Folding in Cos 7 Cells by Calnexin |
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