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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10698-10702, November 1995 Immunology

Role of the integument in 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 and 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 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 . 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. . 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 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 . 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). Colloidal Gold Immunocytochemistry for Localization of 0.69 Pro-phenol Oxidase. All cuticle samples were excised in ice-cold 10 mM Tris-HCI buffer (pH 7.2) containing 664 ,uM APMSF and immediately fixed in 3% (vol/vol) glutaraldehyde and embedded in Lowicryl resin (8). Ultrathin sections were made using a diamond knife on several different body wall and tracheal integ- .?0.4' umental samples. Ultrathin sections were immunolabeled for pro-phenol oxidase using a polyclonal rabbit antisilkworm pro- C) Ce phenol oxidase IgG (affinity purified). A goat anti-rabbit IgG ' co conjugated to 10-nm colloidal gold was used to reveal the first c antibody (8). It was (H. I. Yamazaki and M.A., unpublished o 0.2- observation) shown that this antibody did not cross-react with 0 cuticular laccase (p-diphenol: 02 oxidoreductase, E.C. 1.10.3.2,), o0 which is involved in the sclerotization process (9). Control ex- periments were conducted in the same fashion except that anti-pro-phenol oxidase IgG was substituted by the nonimmune 0.0. rabbit IgG at the same concentration. Sections were viewed and photographed at 80 kV on a JEOL model 100C electron micro- 0 10 20 scope. Time, min SDS/PAGE and Immunoblotting of Pro-phenol Oxidase. FIG. 1. Activation of cuticular pro-phenol oxidase. The addition of SDS/PAGE (10) was carried out in a 14% acrylamide gel the CM-ToyoPearl flow-through fraction containing pro-phenol oxi- except that N,N'-methylenebisacrylamide was replaced by dase to the Ca2+-treated AC fraction resulted in the appearance of AcrylAide crosslinker according to the manufacturer's (FMC) phenol oxidase activity (a). If the AC fraction was not treated with instructions. Immunoblotting of pro-phenol oxidase onto an Ca2+ and mixed with the pro-phenol oxidase fraction, no activity Immobilon poly(vinylidene difluoride) membrane (Millipore) appeared (0). If, however, the AC fraction was mixed with the was carried out according to Ashida et al. (8). pro-phenol oxidase fraction and Ca2+ (final concentration 4 mM) was Tissue Collection for RNA Isolation. Epidermal cells were added at the same time, phenol oxidase activity gradually appeared collected and pooled as described (11) from larvae on the (o). For all three experiments the mixtures were held on ice. Aliquots of 30 ,ul of the mixtures were used to assay phenol oxidase activity, second day of fifth instar at which developmental stage which is expressed as AA520 per 30 Al per 5 min. (Inset) Appearance pro-phenol oxidase was found to be accumulating in body wall of PPAE activity in the AC fraction during its incubation with or cuticle. Hemocytes were collected as described (12). Tissues without 4 mM CaCl2 at 25°C. Prior to mixing the AC fraction with the were stored in liquid N2 until used. pro-phenol oxidase fraction (1:1, vol/vol), the AC fraction was incu- RNA Extraction and Northern Blot Hybridization. Total bated at 25°C in the presence or the absence of 4 mM CaC12 for up to cellular RNA was prepared from pooled frozen epidermal cells 30 min. At the indicated times, the AC fractions were added to the and hemocytes using a Stratagene RNA purification kit. The pro-phenol oxidase fractions and incubated on ice for 5 min after pro-phenol oxidase hybridization probe consisted of a 32p_ which phenol oxidase activity (30 ,ul of the mixture) was assayed. The observed phenol oxidase activity (AA520 per 30 Al per 5 min) corre- labeled random-primed 2.0-kb EcoRI fragment of hemocyte sponded to the PPAE activity generated during incubation with Ca2+ pro-phenol oxidase (pPO17) cDNA (13). A 32P-labeled ran- at 25°C. 0, PPAE activity in the AC fraction incubated in the presence dom-primed a-tubulin probe was used as a constitutively ex- of Ca2+; o, PPAE activity in the AC fraction incubated in the absence pressed internal standard (14). of Ca2+. Downloaded by guest on September 27, 2021 10700 Immunology: Ashida and Brey Proc. Natl. Acad. Sci. USA 92 (1995) of two polypeptides with mobilities in SDS/PAGE corre- activated in the mixture of pro-phenol oxidase fraction and AC sponding to 71 kDa and 70 kDa (16). This antibody gave fraction when incubated on ice (Fig. 1). However, at 25°C in identical results with regard to cross-reactive polypeptides in the presence of Ca2+ the pro-PPAE in the AC fraction is the cuticular extract (i.e., 71 and 70 kDa; Fig. 2, lane c) but did rapidly converted to PPAE as shown by the rapid increase of not detect any polypeptides in the AC fraction, indicating the PPAE activity (Fig. 1 Inset). presence of pro-phenol oxidase only in the pro-phenol oxidase It is not known at present whether components other than fraction. It was confirmed by using homogeneous hemolymph pro-PPAE and Ca2+ are involved in the activation process of pro-phenol oxidase that components other than pro-phenol pro-PPAE in the AC fraction. oxidase in the cuticular pro-phenol oxidase fraction are not Immunoblotting with Anti-Hemolymph Pro-phenol Oxi- involved in the appearance of phenol oxidase activity (data not dase IgG. Among proteins extracted from cuticles with 5% shown). SDS, 3% 2-mercaptoethanol, and 5 M urea, no polypeptide Factor(s) Responsible for Pro-phenol Oxidase Activation in other than a polypeptide doublet of pro-phenol oxidase cross- the AC Fraction. One of the factors responsible for pro-phenol reacted with anti-hemolymph pro-phenol oxidase IgG (Fig. 2, oxidase activation in the AC fraction was shown to be a specific lane b). The pro-phenol oxidase polypeptides were calculated protease because pro-phenol oxidase was converted to a to be 71 kDa and 70 kDa, respectively, from their relative smaller polypeptide doublet in SDS/PAGE during incubation mobilities to the marker proteins. Native pro-phenol oxidase of pro-phenol oxidase fraction with Ca2+-treated AC fraction. appears to be a heterodimer of the polypeptides or a mixture We will refer to the protease as pro-phenol oxidase activating of two kinds of homodimers because the molecular mass of enzyme (PPAE). Furthermore, PPAE in the Ca2+-treated AC native pro-phenol oxidase was determined to be 128 kDa by fraction was completely inactivated by DFP. On the contrary, gel-permeation chromatography (M.A., unpublished data). DFP did not show any appreciable effect on the ability for the The mobilities of the pro-phenol oxidase polypeptides in the AC fraction to acquire PPAE activity when DFP had been pro-phenol oxidase fraction were similar to those in the incubated with the AC fraction and subsequently removed by SDS/urea cuticular extract (Fig. 2, lane c). If the pro-phenol dialysis. These results strongly suggest that PPAE is a serine oxidase fraction is incubated with the AC fraction in the proteinase that exists as a zymogen (pro-PPAE) in the AC absence of Ca2+, no difference in mobility could be detected fraction. In the presence of Ca2+, the pro-PPAE is slowly (Fig. 2, lane e). If however, the pro-phenol oxidase fraction was incubated with the Ca2+-treated AC fraction, a 4-kDa decrease in molecular mass of both polypeptides could be observed (Fig. 2, lane f). 92 Immunocytochemical Detection of Pro-phenol Oxidase in Integuments and . Ultra-thin sections of integument 66 2 and trachea were stained with polyclonal rabbit anti-pro- phenol oxidase IgG (affinity purified) and subsequently with a goat anti-rabbit IgG-10-nm colloidal gold conjugate. Elec- 45 tron micrographs of the stained sections are presented in Fig. 3. Pro-phenol oxidase was localized in the nonlamellate en- docuticle, lamellate endocuticle, cuticular assembly zone, and along the basal surface (hemocoel side) of the epidermal basement membrane of the tracheal and body wall integument. 215 Very few gold particles were detected in the cuticulin layer, in the electron dense granules in the non-lamellate endocuticle, or in the underlying epidermal cells (Fig. 3 A and B). A 14.4 conspicuous feature of the labeling pattern in the lamellate e...... g." .. endocuticle was that the gold particles were seen in an orderly a b c d e f g array along the basal border of helicoidal lamellae; other portions of the lamellae were not labeled or very sparsely B labeled (Fig. 3C). Contrary to this orderly labeling pattern in - 92 lamellate endocuticle, a random labeling pattern was observed ag in nonlamellate endocuticle (Fig. 3A). In trachea, the taenidial - 66.2 cushion was densely labeled for pro-phenol oxidase, but the distribution pattern appeared random (Fig. 3D). Sparse label- ing of pro-phenol oxidase also was observed in the taenidial a b c d e f g 45 cuticle of the tracheae, whereas the endocuticle and cuticulin were not labeled (Fig. 3D). Labeling in control sections stained FIG. 2. Detection of pro-phenol oxidase and phenol oxidase by with nonimmune rabbit IgG was insignificant (data not SDS/PAGE and immunoblotting. (A) The SDS/PAGE pattern of shown). cuticular protein samples stained by Coomassie brilliant blue. Samples Northern Blot Analysis of Pro-phenol Oxidase mRNA in applied to each lane were as follows: lane a, marker proteins (phos- phorylase b, 92 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 Epidermal Cells. As shown in Fig. 4, abundant pro-phenol kDa; carbonic anhydrase, 31 kDa; trypsin inhibitor, 21.5 kDa; and oxidase mRNA was detected in total hemocyte RNA by a lysozyme, 14.4 kDa); lane b, SDS/urea extract of cuticle; lane c, 32P-labeled random-primed EcoRI fragment of the pro-phenol pro-phenol oxidase fraction; lane d, AC fraction; lane e, a mixture (1:1, oxidase cDNA probe. However, pro-phenol oxidase mRNA vol/vol) of the pro-phenol oxidase fraction and the AC fraction could not be detected in total RNA extracted from epidermal incubated on ice for 30 min; lane f, mixture (1:1, vol/vol) of the cells. One might point out that the pro-phenol oxidase probe pro-phenol oxidase fraction and the Ca2+-treated AC fraction incu- used here was produced from cDNA originating from hemo- bated for 30 min on ice; lane g, marker proteins. The amount of protein cyte pro-phenol oxidase mRNA and thus argue that cuticular applied to lanes c-f was 2.2 j,g, 1.3 j,g, 3.5 ,.g, and 3.5 j±g, respectively. pro-phenol oxidase and hemolymph pro-phenol oxidase are To lane b, 2.5 ,ul of SDS/urea extract of cuticle was applied. (B) Immunoblotting of polypeptides cross-reactive to anti-(hemolymph) nonhomologous. However, we have determined amino acid pro-phenol oxidase IgG after SDS/PAGE. Samples were identical to sequences of several peptide fragments obtained from peptide those in A. The positions and sizes (in kDa) of marker proteins are mapping of purified cuticular pro-phenol oxidase and found indicated at right. them to be identical to amino acid sequences deduced from the Downloaded by guest on September 27, 2021 Immunology: Ashida and Brey Proc. Natl. Acad. Sci. USA 92 (1995) 10701

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w; * E *i i. C *D.; . . . *.. . f (;...... X . * Fs*. ._~t - * . . .; .

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...... : :*.....: ...... :: ...... :...... : :...... ::: ...... w ...... : FIG. 3. Immunocytochemical localization of pro-phenol oxidase in fifth-instar (day 5) larval body wall cuticle and tracheal cuticle. (A) Outer surface of the body wall integument. (B) Cuticular epidermal cell and assembly zone. (C) Lamellate endocuticle. (D) Tracheal integument. Diameter of colloidal gold particles = 10 nm. c, Cuticulin; edg, electron dense granules; nlc, nonlamellate endocuticle; 1, lamina of lamellate endocuticle; pc, pore canal; ec, epidermal cell; az, assembly zone; t, taenidium; tc, taenidial cushion; bm, basement membrane. (A and C, bar = 500 nm; B and D, bar = 200 nm.) hemolymph phenol oxidase cDNA (M.A., T. Ozawa, M. Ochiai, Another common injury or defense response is cuticular and Y. Yasuhara, unpublished data). Thus, the present result melanization. This response can be elicited by a mechanical shows the absence of pro-phenol oxidase transcripts in the scratch or by microbial invasion. This defense response is epidermal cells at the time when pro-phenol oxidase is being known to play a role in the sequestration of fungal pathogens accumulated in cuticle. This leads us to postulate that hemolymph as they attempt to penetrate the cuticle (18). It is well known and cuticular pro-phenol oxidase are the same gene product that melanin is synthesized from phenolic substances by the synthesized in the hemocytes, released into the hemolymph, and action of phenol oxidase (19). Lai-Fook (5) and Barrett (20) then transported into cuticle. suggested that cuticular melanization resulted from injury by the activation of a pro-phenol oxidase by a proteinaceous activator. The presence of pro-phenol oxidase, however, has DISCUSSION not been unambiguously demonstrated, and no information One of the distinguishing features of is their about its activation has been elucidated since. According to exoskeleton, which provides them with a protective armor Barrett (6), the isolation and characterization of cuticular against the aggressions of the external environment. It has phenol oxidase as well as the relationships between hemo- been considered for the most part that the integument is only lymph and cuticular phenol oxidases remain important unre- an inert physical barrier (1). Recently, however, we demon- solved questions in cuticular biochemistry. strated that the integument can actively respond to minor Our present results clearly demonstrate that cuticular phe- injury in the presence of microbial cell wall components nol oxidase is truly in a zymogen form, which is activated (lipopolysaccharide and peptidoglycan) by de novo synthesis of through a limited proteolysis by the serine proteinase PPAE, cecropin antibacterial peptides and relaying them to the zone which is also in a zymogen form. Hence, two distinct steps have of cuticular aggression (11, 17). been elucidated in the activation of pro-phenol oxidase. Ca2+ Downloaded by guest on September 27, 2021 10702 Immunology: Ashida and Brey Proc. Natl. Acad. Sci. USA 92 (1995) matrix. This could explain our observation of pro-phenol oxidase localization along the basal surface (hemocoel side) of the epidermal basement membrane of the tracheal and body wall integument. The mechanism with which particular hemo-

A;:..:; lyph protein is destined to be transported to cuticle remains 2.7 kb .- unknown. 1.7 kb-- Our present results along with those of previous studies (8, 12) demonstrate the ubiquity of pro-phenol oxidase through- out the insect body (i.e., hemolymph and cuticle). The insect

..; -i u., tissues are literally bathed in or surrounded by this enzyme :-.-' zymogen and its activating cascade. Defense functions have already been attributed to the pro-phenol oxidase cascade, but we are now questioning whether pro-phenol oxidase could fulfill other functions, such as an oxygen transport like hemo- E H H H H cyanin or because silkworm pro-phenol oxidase 1 1 1 1 was shown to be a protein homologous to hemocy- 40 20 10 1 anin (13). One could speculate that pro-phenol oxidase picks FIG. 4. Northern blot analysis of pro-phenol oxidase mRNA in up oxygen from both the vast body surface and tracheal system cuticular epidermal cells and hemocytes. Total RNA from pooled and transports it through the hemolymph to the tissues. epidermal cells and hemocytes was analyzed by Northern hybridiza- Further studies are necessary to validate or invalidate such tion. The blot was probed simultaneously with the 32P-labeled random- speculation. primed 2.0-kb EcoRI fragment of hemocyte pro-phenol oxidase (pPO17) cDNA and a tubulin cDNA. Lanes E and H received 17.9 ijg We sincerely thank Professor Fotis Kafatos for his encouragement of epidermal cell total RNA and hemocyte total RNA, respectively. To and valuable discussions. We are grateful to Miss Yoshiko Koizumi the lanes labeled 1/10, 1/20, and 1/40, reduced amounts of hemocyte and Mr. Takashi Ozawa (Institute of Low Temperature Science, RNA were applied (1/1Oth, 1/20th, and 1/40th of the amount applied Hokkaido University, Sapporo) and Won-Jae Lee (Institut Pasteur, to lane H, respectively). From separate experiments, pro-phenol Paris) for their technical support. This work was supported by research mRNA mRNA were to to the oxidase and tubulin proved migrate and travel grants from the Japan Society for the Promotion of Science, positions corresponding to 2.7 kb and 1.7 kb, respectively. Institut Pasteur, Japan Ministry of Education, Science and Culture of and Fish- is necessary for the conversion of pro-PPAE to PPAE; how- (Grant 02454019), Japan Ministry Agriculture, Forestry eries (BMP 95-III-2-1-4), and La direction du Developpement et de la ever, we do not know if is involved in this reaction Ca2+ specific Cooperation Scientifique Technique et Educative du Ministere des or if it is involved in cascade events prior to this step. The Affaires Etrangeres Francaises. mechanism by which injury or pathogen invasion set the cuticular cascade into motion remains unknown. 1. Wigglesworth, V. B. (1972) The Principles of In addition, we were able to specifically localize the pro- (Chapman and Hall, London), 7th Ed. phenol oxidase in both the body wall and tracheal cuticle. We 2. Pasteur, L. (1870) Etudes sur la Maladies des Vers a Soie (Gau- contend that pro-phenol oxidase is being localized instead of thier-Villars, Paris). phenol oxidase, since integuments were dissected in high 3. Vey, A. & Gotz, P. (1986) in Hemocytic and Humoral Immunity concentrations (664 ,uM) of the trypsin-type serine proteinase in Arthropods, ed. Gupta, A. P. (Wiley, New York), pp. 89-115. inhibitor APMSF. Immunolocalization of cuticular proteins 4. Nappi, A. J. & Vass, E. (1993) Pigment Cell Res. 6, 117-126. and peptides is common (21-24). However, to our knowledge, 5. Lai-Fook, J. (1966) J. Insect Physiol. 12, 195-226. the conspicuous orderly arrayed localization pattern of pro- 6. Barrett, F. M. (1991) in Physiology of the Insect Epidermis, eds. phenol oxidase we observed along the basal border of the Ratnakaran, R. & Binnington, K. (CSIRO, Melbourne), pp. laminae in the lamellate endocuticle of the body wall is 195-212. seemingly novel. The physiological significance of this pattern 7. Ashida, M. (1971) Arch. Biochem. Biophys. 144, 749-762. remains unknown. 8. Ashida, M., Ochiai, M. & Niki, T. (1988) Tissue Cell 20,599-610. 9. Yamazaki, H. I. (1972) Insect Biochem. 2, 431-444. With regard to the site of synthesis of cuticular pro-phenol 10. Laemmli, U. K. (1970) Nature (London) 227, 680-685. oxidase, our present results strongly suggest that the cuticular 11. Brey, P. T., Lee, W.-J., Yamakawa, M., Koizumi, Y., Perrot, S., pro-phenol oxidase is originally synthesized in hemocytes. The Madeleine, F. & Ashida, M. (1993) Proc. Natl. Acad. Sci. USA 90, identity of amino acid sequences between peptide fragments 6275-6279. from cuticular pro-phenol oxidase and the deduced amino acid 12. Iwama, R. & Ashida, M. (1986) Insect Biochem. 16, 547-555. sequence of hemolymph pro-phenol oxidase cDNA corrobo- 13. Kawabata, T., Yasuhara, Y., Ochiai, M., Matsuura, S. & Ashida, rated our contention and allowed us to use the hemocyte M. (1995) Proc. Natl. Acad. Sci. USA 92, 7774-7778. pro-phenol oxidase cDNA as a molecular probe. Furthermore, 14. Kalfayan, L. & Wensink, P. C. (1981) Cell 24, 97-106. we examined pro-phenol oxidase transcription at the time 15. Ashida, M., Kinoshita, K. & Brey, P. T. (1990) Eur. J. Biochem. when pro-phenol oxidase is being accumulated in the cuticle, 188, 507-515. so if the epidermal cells were involved, we would have expected 16. Yasuhara, Y., Koizumi, Y., Katagiri, C. & Ashida, M. (1995) both pro-phenol oxidase mRNA and its gene product to be Arch. Biochem. Biophys. 320, 14-23. present in the epidermal cells. In previous studies (8, 12), 17. Lee, W.-J. & Brey, P. T. (1994) Anal. Biochem. 217, 231-235. B. pro-phenol oxidase was localized in oenocytoid and plasmato- 18. Golkar, L., LeBrun, R. A., Ohayon, H., Gounon, P., Papierok, Other tissues examined the & Brey, P. T. (1993) J. Invertebr. Pathol. 62, 1-8. cyte-type hemocytes. by incorpo- 19. Mason, H. S. (1955) Adv. Enzymol. 16, 105-184. ration of [35S]methionine into anti-pro-phenol oxidase IgG 20. Barrett, F. M. (1984) Arch. Insect. Biochem. Physiol. 1, 213-223. immunoprecipitates-i.e., and epidermal cells-did 21. Wolfgang, W. J., Fristrom, D. & Fristrom, J.W. (1986)J. CellBiol. not synthesize detectable quantities of pro-phenol oxidase 102, 306-311. (12). 22. Sass, M., Kiss, A. & Locke, M. (1994) J. Insect Physiol. 40, Very recently, Sass and colleagues (22) showed not only that 407-421. cuticular peptides are synthesized by the epidermal cells but 23. Sass, M., Kiss, A. & Locke, M. (1994) J. Insect. Physiol. 40, also that they can also originate from hemocytes and be 561-575. transported across the epidermis and enter into the cuticular 24. Locke, M., Kiss, A. & Sass, M. (1994) Tissue Cell 26, 707-734. Downloaded by guest on September 27, 2021