A novel 43-kDa protein as a negative regulatory component of phenoloxidase-induced melanin synthesis.
The melanization reaction induced by activated phenoloxidase in arthropods is important in the multiple host defense innate immune reactions, leading to the sequestration and killing of invading microorganisms. This reaction ought to be tightly controlled because excessive formation of quinones and systemic hypermelanization are deleterious to the hosts, suggesting that a negative regulator(s) of melanin synthesis may exist in hemolymph. Here, we report the purification and cloning of a cDNA of a novel 43-kDa protein, from the meal-worm Tenebrio molitor, which functions as a melanization-inhibiting protein (MIP). The deduced amino acid sequence of 352 residues has no homology to known sequences in protein data bases. When the concentration of the 43-kDa protein was examined by Western blot analysis in a melanin-induced hemolymph prepared by injection of Candida albicans into T. molitor larvae, the 43-kDa protein specifically decreased in the melanin-induced hemolymph compared with control hemolymph. Recombinant MIP expressed in a baculovirus system had an inhibitory effect on melanin synthesis in vitro. RNA interference using a synthetic 445-mer double-stranded RNA of MIP injected into Tenebrio larvae showed that melanin synthesis was markedly induced. These results suggest that this 43-kDa MIP inhibits the formation of melanin and thus is a modulator of the melanization reaction to prevent the insect from excessive melanin synthesis in places where it should be inappropriate.
Zhao
Mingyi
M
National Research Laboratory of Defense Proteins, College of Pharmacy, Pusan National University, Jangjeon Dong, Kumjeong Ku, Busan 609-735, Korea.
Söderhäll
Irene
I
Park
Ji Won
JW
Ma
Young Gerl
YG
Osaki
Tsukusa
T
Ha
Nam-Chul
NC
Wu
Chun Fu
CF
Söderhäll
Kenneth
K
Lee
Bok Luel
BL
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002176:Candida albicans / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006458:Hemolymph / Q000737:chemistry / Q000378:metabolism; D015151:Immunoblotting; D019476:Insect Proteins / Q000737:chemistry / Q000502:physiology; D007313:Insecta; D008543:Melanins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011809:Quinones / Q000737:chemistry; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013712:Tenebrio; D014819:Vitellogenins / Q000737:chemistry* / Q000502:physiology*
D018076:DNA, Complementary; D019476:Insect Proteins; D008543:Melanins; D011809:Quinones; D011994:Recombinant Proteins; D014819:Vitellogenins; D049452:Green Fluorescent Proteins; D004247:DNA; D014442:Monophenol Monooxygenase; D000581:Amidohydrolases