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Role of the Integument in Insect Defense: Pro-Phenol Oxidase Cascade in the Cuticular Matrix MASAAKI ASHIDA* and PAUL T

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Role of the Integument in Insect Defense: Pro-Phenol Oxidase Cascade in the Cuticular Matrix MASAAKI ASHIDA* and PAUL T Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10698-10702, November 1995 Immunology Role of the integument in insect defense: Pro-phenol oxidase cascade in the cuticular matrix MASAAKI ASHIDA* AND PAUL T. BREYt The Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan Communicated by John H. Law, The University ofArizona, Tucson, AZ, July 31, 1995 (received for review June 9, 1995) ABSTRACT The cuticle of the silkworm Bombyx mori was nearly three decades (6). In this study we demonstrate the demonstrated to contain pro-phenol oxidase [zymogen of presence of the pro-phenol oxidase cascade in the cuticle of the phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, silkworm. EC 1.14.18.1)] and its activating cascade. The activating cascade contained at least one serine proteinase zymogen (latent form of pro-phenol oxidase activating enzyme). When the extracted MATERIALS AND METHODS cascade components were incubated with Ca2+, the latent form Organisms. Silkworms, Bombyx mori, were reared on the ofpro-phenol oxidase activating enzyme was itselfactivated and, artificial diet Silkmate 2M (Kyodo Shiryo, Tokyo), at 24°C in turn, converted through a limited proteolysis of pro-phenol under a 12-hr photoperiod. Experiments used fifth-instar oxidase to phenol oxidase. Immuno-gold localization of pro- larvae reared to day 5 for pro-phenol oxidase cascade and phenol oxidase in the cuticle using a cross-reactive hemolymph localization studies. Fifth-instar larvae reared to day 2 were anti-pro-phenol oxidase antibody revealed a random distribu- used for Northern blot analysis. tion ofthis enzyme in the nonlamellate endocuticle and a specific Extraction of the Pro-phenol Oxidase Cascade from the orderly arrayed pattern along the basal border of the laminae in Cuticle. Twenty silkworm larvae were dissected in ice-cold the lamellate endocuticle of the body wall. Furthermore, pro- acetate buffer 1 (50 mM acetate buffer at pH 5.2 containing 5 phenol oxidase was randomly distributed in the taenidial cush- mM EDTA). The integuments were then meticulously sepa- ion of the tracheal cuticle. At the time of pro-phenol oxidase rated with a spatula to remove all tissues adhering to the accumulation in the body wall cuticle, no pro-phenol oxidase cuticle. The tissue-free cuticles were subsequently rinsed twice mRNA could be detected in the epidermal tissue, whereas in fresh acetate buffer I and then blotted with absorbent paper free-circulating hemocytes contained numerous transcripts of to remove excess liquid. Each cuticle was individually extracted pro-phenol oxidase. Our results suggest that the pro-phenol with 1 ml of acetate buffer II [same as acetate buffer I except oxidase is synthesized in the hemocytes and actively transported containing 0.33 mM amidinophenylmethanesulfonyl fluoride into the cuticle via the epidermis. (APMSF)] for 1-1.5 hr on ice. The extract was centrifuged at 7000 x g for 10 min at 4°C. Combined supernatants (total The chitinous exoskeleton of the insect is a nonliving matrix of volume of 18.5 ml) were then subjected to ion-exchange carbohydrate and protein secreted from a monolayer of epi- chromatography. dermal cells, which cover the entire surface of the insect, Column Chromatography of Cuticular Extract on CM- including respiratory tracheae, the anterior and posterior Toyopearl. To separate pro-phenol oxidase from its activating portion of the digestive tract, and reproductive ducts (1). The cascade, cuticular extract was applied to a CM-Toyopearl cuticle serves as a protective barrier between the internal (Tosoh, Tokyo) column (1.5 x 7 cm) that had been equili- tissues and the external environment. Injury causes the cuticle brated with acetate buffer I, and the column was eluted to darken or melanize around the damaged zone. Pasteur (2), sequentially with 60 ml of acetate buffer I and 50 ml of Tris while studying silkworm diseases, noted that the cuticles of buffer I (10 mM Tris HCl containing 0.5 M KCI and 5 mM healthy silkworms were often topically scratched, and as a EDTA at pH 7.5). During the chromatography, the flow rate result the cuticle darkened around the injured area. Further- was maintained at 2 ml/min, and effluent was monitored at more, he observed that silkworms infected with the micro- 280 nm using a UV-1 monitor (Pharmacia LKB). The flow- sporidian Nosema bombysis often manifested a cuticular mel- through fraction and the eluant corresponding to the first peak anization reaction. Cuticular melanization is thought to be an after application of the Tris buffer I were saved. After their pH important defense reaction of insects because it is involved in was adjusted to 7.5 with 1 M Tris solution, both fractions were wound healing and sequestration of pathogens (3). More individually dialyzed against 1 liter of Tris HCl buffer II (10 recently it has been speculated that intermediate compounds mM Tris HCI containing 0.1 mM EDTA at pH 7.5 at 23°C) for (quinones) in the melanin synthesis from mono- or diphenols 24 hr with one change of the buffer at 4°C. The dialyzed are highly toxic to living cells, including infectious microor- flow-through fraction (40 ml) and the first peak fraction (9.4 ganisms (4). ml) were referred to as the pro-phenol oxidase fraction and Lai-Fook (5) studied the mechanism of the cuticular mel- activating cascade (AC) fraction, respectively. Protein concen- anization reaction with regard to injury. She speculated that trations of the pro-phenol oxidase and AC fractions were phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, about 370 ,ug/ml and 220 ,ug/ml, respectively. EC 1.14.18.1) was responsible for melanization and was Preparation of Pro-phenol Oxidase Activating Enzyme present in a latent form, which in turn was activated by a (PPAE) by Ca2+ Treatment of the AC Fraction and Its Inacti- proteinaceous activator upon injury. The regulation mecha- vation by Treatment with Diisopropylfluorophosphate (DFP). nism of the activation of pro-phenol oxidase (a zymogen of Eighty millimolar CaC12 was added to the AC fraction to make phenol oxidase) and the site of its synthesis have remained important unresolved questions in cuticular biochemistry for Abbreviations: APMSF, amindinophenylmethanesulfonyl fluoride; DFP, diisopropylfluorophosphate; AC, activating cascade; PPAE, pro- phenol oxidase activating enzyme. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in tPresent address: Unite d'Ecologie des Systemes Vectoriels, Institut accordance with 18 U.S.C. §1734 solely to indicate this fact. Pasteur, 25 rue du Dr. Roux 75724 Paris, Cedex 15, France. 10698 Downloaded by guest on September 27, 2021 Immunology: Ashida and Brey Proc. Natl. Acad. Sci. USA 92 (1995) 10699 its final concentration 4 mM and subsequently incubated on ice Assay of Phenol Oxidase Activity. Phenol oxidase activity overnight. The incubated mixture was used as PPAE. Two was assayed spectrophotometrically according to Ashida et al. milliliters of PPAE was added to 100 ,ul of 1 M Tris HCl buffer (15). Under the assay conditions, activated homogeneous at pH 8.6 to circumvent the drop in pH upon addition of DFP cuticular pro-phenol oxidase gives a specific activity (AA520 per (final concentration of20 mM). The mixture was incubated for 10 5 min per mg of protein) of 3500-4000 (M.A., unpublished hr and subsequently dialyzed against 1 liter of Tris buffer II data). overnight at 4°C. In addition, the AC fraction was also treated Assay of PPAE. PPAE activity was measured by the con- with DFP prior to Ca2+ treatment. version of its substrate, pro-phenol oxidase, to phenol oxidase Extraction of Cuticle with SDS and Urea. Cuticles were and is expressed in terms of the increase of phenol oxidase obtained as described above and immediately frozen in liquid activity over time. nitrogen. The tissue-free cuticles were ground to a fine powder Protein Quantification. The protein concentration was mea- in liquid nitrogen and immediately lyophilized without thaw- sured by using the Micro BCA protein assay reagent (Pierce) ing. The powder was homogenized in a glass homogenizer with using bovine serum albumin as a standard. a glass pestle (20 mg of powder per ml of 20 mM Tris HCl containing 5 M urea, 5% SDS, and 3% 2-mercaptoethanol) followed by heating in boiling water for 3 min and then held RESULTS at 20°C for 12 hr. The homogenate was centrifuged at 15,000 Appearance of Phenol Oxidase Activity in the Incubation x g for 10 min. The supernatant was dialyzed against 20 mM Mixture Consisting of the Pro-phenol Oxidase Fraction and Tris HCl buffer (pH 7.2) containing 1% SDS and 1% 2-mer- the AC Fraction in the Presence of Ca2 . Phenol oxidase captoethanol overnight. The dialyzed supernatant was used as activity increased with time when the pro-phenol oxidase an extract containing total extractable cuticular proteins. fraction was incubated with the AC fraction that had been Pro-phenol Oxidase and Anti-Pro-phenol Oxidase IgG. previously treated with Ca2 . It was demonstrated that Ca2+ Pro-phenol oxidase was purified from larval hemolymph of B. was necessary for the AC fraction to acquire the activity to mori as reported (7). The pro-phenol oxidase was dissolved in activate pro-phenol oxidase in the mixture of both fractions Tris buffer II at a protein concentration of 100 ,ug/ml. Rabbit (Fig. 1). polyclonal antibody was raised against homogeneous pro- Presence of Pro-phenol Oxidase in the Pro-phenol Oxidase phenol oxidase obtained from hemolymph of larval silkworm Fraction. Anti-hemolymph pro-phenol oxidase IgG was mono- hemolymph, and anti-pro-phenol oxidase IgG was purified specific to hemolymph pro-phenol oxidase, which is composed according to the method described before (8).
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