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-139- Melanocyte(1) 9/5 Room-C
SMALL GTP BINDING PROTEIN RAB 8 IS NOT ASSOCIATED WITH MELANOSOME IN PIGMENT CELLS.
Keishi Araki, Tatsuya Horikawa, Masamitsu Ichihashi1.
1Department of Dermatology, Kobe University Medical School, Kobe, Japan.
Rab 8 is a small GTP-binding protein which may play an important role in intracellular vesicular transport. In this study,we investgated subcellular distribution of Rab8 protein in pigment cells. We found that Rab8 is associated with membrane fraction but not melanosome enriched fraction by Westernblot analysis. . Immunofluorescense microscopic analysis also revealed that Rab8 was not associated with melanosome. These result indicate that Rab8 is not involved in melanosome transport.
-140- Melanocyte(1) 9/5 Room-C
ANALYSIS OF THE ROLE OF COP I PROTEIN AND SMALL GTP-BINDING PROTEIN IN INTRACELLULAR COATED VESICLE TRANSPORT.
Youko Funasaka, Keishi Araki, Ashok K. Chakraborty, Akira Ito, Eri Nishioka, Tatsuya Horikawa, Masamitsu Ichihashi.
Department of Dermatology, Kobe University School of Medicine, Kobe, Japan.
The melanin biosynthetic pathway is critically regulated at the enzymatic level by tyrosinase, which is carried by coated vesicle to melanosome resulting in melanin synthesis in the latter compartment. Coated vesicle consists of assembly of ARF and coatmer units on the membrane, and conversion of GDP to GTP or vice versa, takes place during budding and fusing. To search the biochemical mechanism of coated vesicle in the pigment cells, we investigated the localization of coat proteins and small GTP binding protein. Coated vesicle enriched fraction was obtained from cultured B 16 mouse melanoma cells by sucrose density gradient method. B-COP, one constituent of the COP I coatmer complex was identified in the coated vesicle enriched fraction by Western blotting. GTP-binding assay showed the presence of small MW ( 25-28 kDa ) GTP-binding protein in the coated vesicle enriched fraction. These results indicate that COP I and small GTP-binding protein play a role in melanin biosynthesis in pigment cells.
-141- Melanocyte(1) 9/5 Room-C
ANALYSIS OF CHAPERONE FUNCTION OF CALNEXIN IN FOLDING OF MELANOSOMAL MEMBRANE PROTEINS.
Fumihiro Kato1, Ikuo Wada2, Kowichi Jimbow1.
1Department of Dermatology, 2Department of Biochemistry , Sapporo Medical University, Sapporo, Japan.
Calnexin is a unique chaperone residing in the endoplasmic reticulum (ER) where it binds to the monoglucose of glycoproteins. We have recentry shown that the chaperone function is required for the correct folding of tyrosinase. However, its mechanism remains unclear. In this study, we examined if this function is distinctively required for tyrosinase folding or could be replaced by other chaperon e.g. stress-inducible one. We have prepared the melanocytes with elevated levels of heat-shock proteins by the treatment of cyclopentenome prostaglandins which stimulate expression of BiP through the unfolded protein response element, and analyzed folding of tyrosinase and TRP-1 in the cells. Calnexin was not induced by the treatment. The examination using a glucosidase inhibitor, castanospermine, which abolished their binding to calnexin, showed that BiP did not substitute the calnexin function, although their folding kinetics were altered in the treated cells, suggesting that calnexin is functionally distinct from stress-induced chaperon
-142- Melanocyte(1) 9/5 Room-C
LOCALIZATION OF HEAT SHOCK PROTEIN 60 IN MELANOSOMES OF MELANOCYTE.
Takeshi Kobayashi1,2, Vincent J. Hearing2, Yoshinori Takema1, Genji Imokawa1.
1Biological Science Laboratories, Kao Corporation, Tochigi, Japan. 2LCB/NCI/NIH, MD USA.
In melanosomes of melanocytes, melanogenic enzymes catalyze several reactions initiated from tyrosine to form melanin biopolymer. Since most of them include the oxidization and reduction of substrates, there may be the stress of oxidization in melanosomes, to which melanosomal proteins are exposed. Therefore, it is possible that molecular chaperone may function in melanosomes as seen in mitochondria. To locate of heat shock proteins (HSPs), we prepared the melanosome-rich fraction from murine melanocytes using sucrose-gradient centrifugation and performed western immunoblotting. In melanosome-rich fraction, anti-HSP60 Abs, but not anti-HSP70 and anti-HSP90, identified the specific 60-kDa band on blottings. These findings suggest that HSP60 contribute to melanogenesis as molecular chaperone in melanosomes.
-143- Melanocyte(1) 9/5 Room-C
EXPRESSION AND ENZYME ACTIVITY OF TYROSINASE, TRP-1 AND TRP-2 AFTER UVB IRRADIATION.
Eri Nishioka, Yoko Funasaka, Ashock K.Chakraborty, Masamitsu Ichihashi.
Department of Dermatology, Kobe University School of Medicine, Kobe, Japan.
UVB activates melanin production by increasing tyrosinase activity. Tyrosinase-related proteins, TRP-1 and TRP-2, are involved in melanin formation, and also play a role in protecting from cell death. To elucidate the role of tyrosinase, TRP-1 and TRP-2 in UVB irradiated melanocytes, the expression and activity of these proteins were studied in relevance to melanin production and cell proliferation. Among two lines of B16 mouse melanoma, six human melanoma ( hMel ) , and two normal human melanocytes ( hMC ) two B16, one hMel, and two hMC showed enhanced melanin content with increment of tyrosinase activity after 2.5-30 mJ /cm2 of UVB iradiation, but two B16 and one hMel showed decreased TRP-2 activity and cell number. Three hMel with unchanged melanin content showed increment of TRP-2 activity and protein amount, and cell number. Taken together, TRP-2 might play a role in UVB induced cell proliferation but not in melanogenesis.
-144- Melanocyte(1) 9/5 Room-C
THE LINKAGE BETWEEN POLYMORPHIC GA REPEAT SEQUENCE AND PATHOLOGICAL MUTATIONS OF THE TYROSINASE GENE IN JAPANESE OCA1 PATIENTS.
Eriko Nakamura1, Jun Matsunaga1, Yoshinori Miyamura1, Muneo Tanita1, Yasuko Takisawa2, Hiroshi Shimizu2, Takeji Nishikawa2, Yasushi Tomita1.
1Department of Dermatology, Akita University School of Medicine, Akita, 2Department of Dermatology, Keio University School of Medicine, Tokyo,Japan.
The human tyrosinase gene has a complex dinucleotide (GA) sequence 5' upstream of the translational start site. The GA repeat sequence shows polymorphism that results from insertions or deletions of dinucleotides. To examine the linkage between this polymorphic site and mutations of the tyrosinase gene, we performed length analysis of the GA prepeat sequence in OCA1 patients and their relatives. We analyzed the product of polymerase chain reaction (PCR) using GeneScan. The predicted length of the amplified sequence was 349bp. As results, the GA repeat sequence is classified into 5 lengths (339bp, 349bp, 357bp, 359bp and 370bp). Although the 357bp fragment is linked to 3 different mutations, including +¦¤C310, and the normal tyrosinase gene, +¦¤C310 is primarily linked to the 357bp GA repeat sequence in 8 cases (13 chromosomes) without exception. Similarly, R77Q is linked to the 359bp GArepeat sequence in 4 cases (7 chromosomes). Therefore, we presume that the +¦¤C310 and R77Q mutations derived from a common origin, respectively.