Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine

Kwang Moon Lee, Kum Young Lee, Hye Won Choi, Mi Young Cho, Tae Hyuk Kwon, Shun-Ichiro Kawabata, Bok Luel Lee

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxidase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis induced by activated phenoloxidase. In this study, to isolate and characterize proteins involved in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution), specifically showing phenoloxidase activity in the presence-of Ca2+ and β-1,3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 column. When G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca2+ and β-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, pheholoxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160- kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase induced melanin synthesis. Furthermore, when the purified 160-kDa melanization-engaging protein was added to a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conclusion that the 160-kDa vitellogenin-like protein is involved in arthropod melanin synthesis.

Original languageEnglish
Pages (from-to)3695-3703
Number of pages9
JournalEuropean Journal of Biochemistry
Volume267
Issue number12
DOIs
Publication statusPublished - Jul 12 2000

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Tenebrio
Vitellogenins
Monophenol Monooxygenase
Melanins
Larva
Dopamine
Proteins
Arthropods
Glucans
Phenylthiourea
Hemolymph
Caenorhabditis elegans
Polyacrylamide Gel Electrophoresis
Complementary DNA
Western Blotting

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine. / Lee, Kwang Moon; Lee, Kum Young; Choi, Hye Won; Cho, Mi Young; Kwon, Tae Hyuk; Kawabata, Shun-Ichiro; Lee, Bok Luel.

In: European Journal of Biochemistry, Vol. 267, No. 12, 12.07.2000, p. 3695-3703.

Research output: Contribution to journalArticle

Lee, Kwang Moon ; Lee, Kum Young ; Choi, Hye Won ; Cho, Mi Young ; Kwon, Tae Hyuk ; Kawabata, Shun-Ichiro ; Lee, Bok Luel. / Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine. In: European Journal of Biochemistry. 2000 ; Vol. 267, No. 12. pp. 3695-3703.
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abstract = "One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxidase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis induced by activated phenoloxidase. In this study, to isolate and characterize proteins involved in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution), specifically showing phenoloxidase activity in the presence-of Ca2+ and β-1,3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 column. When G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca2+ and β-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, pheholoxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160- kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7{\%}). Western blot analysis showed that it disappeared when active phenoloxidase induced melanin synthesis. Furthermore, when the purified 160-kDa melanization-engaging protein was added to a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conclusion that the 160-kDa vitellogenin-like protein is involved in arthropod melanin synthesis.",
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AU - Kawabata, Shun-Ichiro

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