Activation of prophenoloxidase A1 by an activating enzyme in Drosophila melanogaster

Chosa, Naoyuki; Fukumitsu, Takashi; Fujimoto, Kengo; Ohnishi, Eiji

doi:10.1016/s0965-1748(96)00070-7

Cited by 72 publications

(46 citation statements)

References 20 publications

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“…After separation of a cuticular extract by ammonium sulfate precipitation, DEAE cellulose chromatography, and Sephacryl S-300 gel permeation chromatography, the fractions containing PAP activity were pooled (8 ml) and loaded onto a dextran sulfate column. After washing (flow-through fractions [1][2][3][4][5][6][7][8][9][10][11][12][13][14], bound proteins were eluted by a linear NaCl gradient, beginning at fraction 15. (-) Absorbance at 280 nm; (E) amidase activity in 20 l of the fractions assayed by using IEAR-p-nitroanilide as a substrate.…”

Section: Methodsmentioning

confidence: 99%

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Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Jiang

Wang

Kanost

1998

Proc. Natl. Acad. Sci. U.S.A.

246

206

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Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the aminoterminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal ''clip'' domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3 H-diisopropyl f luorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.

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Section: Methodsmentioning

confidence: 99%

“…ProPO activating enzymes have been isolated from cuticle of Bombyx mori (7,8), a crayfish hemocyte lysate (9), and Drosophila melanogaster pupae (10), and from plasma of Holotrichia diomphalia (11). All of these enzymes are serine proteinases with masses of Ϸ30 kDa with specificity for cleavage after arginine residues.…”

mentioning

confidence: 99%

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Jiang

Wang

Kanost

1998

Proc. Natl. Acad. Sci. U.S.A.

246

206

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“…PO is present in almost all organisms, functioning as an initiator of melanin synthesis (4)(5)(6)(7). Several potential activators of PO activity in vivo have been identified such as fatty acids, phospholipids, detergents, small antimicrobial peptides, and alcohols (3,(8)(9)(10). SDS, an artificial activator, seems to mimic the natural activation of PO; however, the mode of this activation is not known.…”

Section: Introductionmentioning

confidence: 99%

Hemocyanin conformational changes associated with SDS-induced phenol oxidase activation

Baird

Kelly

Price

et al. 2007

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site.

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“…Finally, a septic wound triggers immediate reactions that depend on proteolytic cascades, including the deposition of melanin at the wound site, which is thought to constitute another arm of host defense. Several steps of the melanization reactions are catalyzed by phenoloxidase (PO), which becomes active after the cleavage of its prodomain after the activation of dedicated proteolytic cascades (26)(27)(28)(29). Of note, independently of its Tolldependent function, the GNBP3 PRR is also required for the activation of PO after a fungal challenge and is required for the agglutination of C. albicans cells in the hemolymph (30).…”

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confidence: 99%

The Drosophila Toll Pathway Controls but Does Not Clear Candida glabrata Infections

Quintin

Asmar

Matskevich

et al. 2013

The Journal of Immunology

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The pathogenicity of Candida glabrata to patients remains poorly understood for lack of convenient animal models to screen large numbers of mutants for altered virulence. In this study, we explore the minihost model Drosophila melanogaster from the dual perspective of host and pathogen. As in vertebrates, wild-type flies contain C. glabrata systemic infections yet are unable to kill the injected yeasts. As for other fungal infections in Drosophila, the Toll pathway restrains C. glabrata proliferation. Persistent C. glabrata yeasts in wild-type flies do not appear to be able to take shelter in hemocytes from the action of the Toll pathway, the effectors of which remain to be identified. Toll pathway mutant flies succumb to injected C. glabrata. In this immunosuppressed background, cellular defenses provide a residual level of protection. Although both the Gram-negative binding protein 3 pattern recognition receptor and the Persephone protease-dependent detection pathway are required for Toll pathway activation by C. glabrata, only GNBP3, and not psh mutants, are susceptible to the infection. Both Candida albicans and C. glabrata are restrained by the Toll pathway, yet the comparative study of phenoloxidase activation reveals a differential activity of the Toll pathway against these two fungal pathogens. Finally, we establish that the high-osmolarity glycerol pathway and yapsins are required for virulence of C. glabrata in this model. Unexpectedly, yapsins do not appear to be required to counteract the cellular immune response but are needed for the colonization of the wild-type host.

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Activation of prophenoloxidase A1 by an activating enzyme in Drosophila melanogaster

Cited by 72 publications

References 20 publications

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Hemocyanin conformational changes associated with SDS-induced phenol oxidase activation

The Drosophila Toll Pathway Controls but Does Not Clear Candida glabrata Infections

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