Transepithelially transported pro-phenoloxidase in the cuticle of the silkworm, Bombyx mori. Identification of its methionyl residues oxidized to methionine sulfoxides

Pro-phenoloxidase (proPO) in insects is activated through the action of a protease cascade triggered by minute amounts of microbial cell wall components. It is an important molecule for the defense against invading microorganisms and for the repair of wounds. In the accompanying paper (Asano, T., and Ashida, M. (2001) J. Biol. Chem. 276, 11100-11112), a proPO isoform, proPO-HS, in the hemolymph of the silkworm, Bombyx mori, is reported to be transported to the cuticle. The transported proPO isoform was recovered from the cuticle and named proPO-CS. The elution profiles of proPO-CS and proPO-HS in reversed-phase high performance liquid chromatography (HPLC) were found to be different, giving a basis to the inference that proPO-CS is a modified form of proPO-HS. In the present study, we investigated the nature of the modifications occurring in proPO-CS, in which proteolytically and chemically cleaved fragments originating from the subunits of proPO-CS and proPO-HS were analyzed by reversed-phase HPLC, amino acid sequencing, and mass spectrometry. A subunit of the heterodimeric proPO-CS was found to contain five or six methionine sulfoxides, and another subunit was found to contain one methionine residue oxidized to the sulfoxide. All of the oxidized methionyl residues were identified. Other than oxidation of the methionyl residues, no additional modification of proPO-CS was found. In the model structure of each subunit of proPO-CS constructed by protein modeling with the known structures of the horseshoe crab, Limulus polyphemus, hemocyanin type II subunit as templates, the methionine residues identified as methionine sulfoxide had high degrees of accessibility to the solvent. The implication of the oxidation at the methionine residues is discussed in relation to the mechanism of transepithelial transport of proPO from the hemolymph to the cuticle.     

A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia larvae

The prophenoloxidase (proPO) activation pathway, like the vertebrate complement system, consists of a protease cascade and functions as a non-self-recognition system in these animals. Determining the molecular mechanism by which pattern recognition molecules differentiate non-self from self and transduce signals that stimulate defense responses is a key for understanding the ways in which innate immune systems are regulated. However, the proPO system is poorly defined at the molecular level. The proPO-activating system of the insect Holotrichia diomphalia comprises several components, some of which have been cloned and characterized, such as the novel 27-kDa proPO-activating factor-III (PPAF-III) from the plasma of H. diomphalia larvae and two prophenoloxidases. The PPAF-III gene encodes an easter-type serine protease zymogen consisting of 351 amino acid residues with a mass of 40 kDa. The purified 27-kDa PPAF-III specifically cleaved a 55-kDa proPPAF-II to generate a 45-kDa PPAF-II with or without Ca2+ present. Furthermore, two Holotrichia prophenoloxidases (proPO-I and -II) have been characterized, and their structural changes during activation were examined by in vitro reconstitution experiments. When the proPOs were incubated with PPAF-I, the 79-kDa proPOs were converted to 76-kDa proPOs, which did not exhibit any phenoloxidase (PO) activity. However, when the proPOs were incubated simultaneously with PPAF-I, proPPAF-II, and PPAF-III in the presence of Ca2+, a 60-kDa protein (PO-1) with PO activity was detected in addition to the 76-kDa proPO-II protein. These results indicate that the conversion of Holotrichia proPOs to enzymatically active phenoloxidase is accomplished by PPAF-I, PAF-II, and PPAF-III through a two-step limited proteolysis in the presence of Ca2+.     

Purification and characterization of Manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating proteinase-3

The proteolytic activation of prophenoloxidase (proPO) is a critical defense mechanism in insects and crustaceans. We have isolated three prophenoloxidase-activating proteinases (PAPs) from cuticular extracts or hemolymph of Manduca sexta pharate pupae, which are negatively regulated by serpin-1J and serpin-3. To test if other serpins may also inhibit the PAPs, we fractionated the induced hemolymph by ammonium sulfate precipitation, gel filtration, and lectin affinity chromatography. A 47 kDa protein, designated M. sexta serpin-6, was identified in concanavalin A-bound fractions, which formed an SDS-stable complex with PAP-3. This inhibitor, not recognized by the serpin-1 or serpin-3 antibodies, was further purified on HPLC anion exchange and hydroxylapatite columns. The molecular mass and isoelectric point of serpin-6 were found to be 46,710 +/- 10 Da and 5.4. While its amino terminus was blocked, we obtained five internal peptide sequences, one of which is highly similar to M. sexta serpins-1, -2, and -3. Serpin-6 strongly inhibited PAP-3 but not PAP-1 or PAP-2, suggesting that the proPO activation by PAPs is differentially regulated by multiple serpins. When included in the reaction mixture containing proPO, PAP-3, and its cofactor, serpin-6 efficiently blocked the cleavage activation of proPO.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

In vitro translation of nematode cuticular collagens.

Phenanthroline treatment of growing cultures of the free-living nematode Panagrellus silusiae was used to lower the degree of hydroxylation of nascent collagen chains at the polysomal level. Under these conditions, the bound pentasome-hexasome fraction provided substrate for prolyl hydroxylase. When this polysomal fraction was subsequently tested in a cell-free wheat germ system, collagenase-susceptible translation products were observed after sodium dodecyl sulfate-acrylamide gel electrophoresis. The electrophoretic mobilities of each of these four major collagen products were similar to four collagens that are isolated from intact cuticles. In addition, purified polysomal RNA that adhered to unmodified cellulose directed the synthesis of four pepsin-resistant polypeptides that had molecular weights that coincided with four pepsin-resistant collagens that can be purified from the cuticle of this species. Thus, the polysomal site of the messenger RNAs for the cuticular collagens of P. silusiae was located. Although precursor forms of the cuticular collagens were not produced in the cell-free system, the question whether additional amino acid segments occur on the primary translational products of the cuticular collagens in vivo remains open.     

Distribution of serpins in the tissues of the tobacco hornworm (Manduca sexta) larvae. Existence of new serpins possibly encoded by a gene distinct from the serpin-1 gene

Polyclonal antibodies were raised against the isolated hemolymph serine proteinase inhibitors (serpins) of Manduca sexta larvae. Two of these antibodies, MsH49a and MsH49b, displayed characteristic differences in labelling patterns of hemocytes, fat body, integumental epidermis and cuticle on immunoblots, and in light- and electronmicroscopic sections. The serpin composition of the latter three tissue homogenates was determined by native immunoblots and inhibitor binding assays. The results were compared to the hemolymph samples containing all the known inhibitors encoded by the well-characterized serpin-1 gene. The enzyme specificity of the MsH49b-labelled cuticular serpin was similar to serpin-1J, although its electrophoretic mobility on native PAGE was not identical with any of the known proteinase inhibitors encoded by the serpin-1 gene. Based on these data, we suggest that the cuticle and hemolymph may contain novel serpin(s) encoded by a gene other than the serpin-1 gene. Since the serpin-1J proved to be involved in the activation pathway of the prophenoloxidase system in the hemolymph, the in vivo function of cuticular MsH49b serpin was investigated by prophenoloxidase tests in native cuticular homogenates. Our results demonstrated that the cuticular serpin(s) that are labelled by the MsH49b antibody may play a determinant role in the regulation of the prophenoloxidase system of the integumental cuticle.     

Review of sorption and diffusion of lipophilic molecules in cuticular waxes and the effects of accelerators on solute mobilities

Many agrochemicals are applied to the leaf surfaces of crop plants. Systemic chemicals have to penetrate through the cuticle, which forms an effective transport barrier. The barrier properties of cuticles are mainly due to the cuticular waxes deposited as partially crystalline aggregates on the outer surfaces of leaves. Substances increasing the mobilities of agrochemicals in cuticular waxes are called accelerators and it is shown that they act as plasticizers when absorbed by cuticular waxes. They decrease the barrier properties of the waxes and thus increase the mobilities of the agrochemicals through them. In order to analyse the efficiency of different accelerators, the sorption and mobility of both agrochemicals and accelerators within cuticular waxes was measured. Such information was used to establish correlations between the internal concentrations of accelerators and their mobility-enhancing effects on agrochemicals in the cuticle. This, in turn, allowed the determination and comparison of the intrinsic effects of different accelerators and to rationalize the effect of accelerators on the cuticular permeability of agrochemicals. Results describing the sorption (partition coefficients) and mobility (diffusion coefficients) of lipophilic organic molecules in reconstituted cuticular waxes from different plant species, and the effect of two different classes of accelerators (alcohol ethoxylates and n-alkyl esters), on the mobility of organic molecules are presented and discussed.     

Prophenoloxidase (proPO) activation in Manduca sexta: an analysis of molecular interactions among proPO, proPO-activating proteinase-3, and a cofactor

Proteolytic activation of prophenoloxidase (proPO) is an integral part of the insect immune system against pathogen and parasite infection. This reaction is mediated by a proPO-activating proteinase (PAP) and its cofactor in the tobacco hornworm, Manduca sexta (Proc. Natl. Acad. Sci. USA 95 (1998) 12220; J. Biol. Chem. 278 (2003) 3552; Insect Biochem. Mol. Biol. 33 (2003) 1049). The cofactor consists of two serine proteinase homologs (SPHs), which associate with immulectin-2, a calcium-dependent lectin that binds to lipopolysaccharide (Insect Biochem. Mol. Biol. 33 (2003) 197). In order to understand the auxiliary effect of SPH-1 and SPH-2 in proPO activation, we started to investigate the molecular interactions among proPO, PAP-3, and the proteinase-like proteins. M. sexta SPH-1 and SPH-2 were purified from hemolymph of prepupae by hydroxylapatite, gel filtration, lectin-affinity, and ion exchange chromatography. They existed as non-covalent oligomers with an average molecular mass of about 790 kDa. MALDI-TOF mass fingerprint analysis revealed a new cleavage site in SPH-1 before Asp85. The PAP cofactor did not significantly alter Michaelis constant (KM) or kcat of PAP-3 towards a synthetic substrate, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide, but greatly enhanced proPO activation by PAP-3. The apparent KM for proPO was determined to be about 9.4 microg/ml, close to its estimated concentration in larval hemolymph. In the presence of excess proPO and a set amount of PAP-3, increasing levels of phenoloxidase (PO) activity were detected as more SPHs were added. Half of the maximum proPO activation occurred when the molar ratio of PAP-3 to SPH was 1:1.4. Gel filtration experiments suggested that proPO, PAP-3, and the cofactor formed a ternary complex.     

Identification of cuticular hydrocarbons and the alkene precursor to the pheromone in hemolymph of the female gypsy moth, Lymantria dispar

Hydrocarbons were extracted from the surface of the cuticle and from the hemolymph of adult female gypsy moths. GC and GC/MS analysis indicated that the cuticular hydrocarbons with chain lengths >21 carbons were the same as those found in the hemolymph. These consisted of mostly saturated straight chain hydrocarbons with heptacosane the major component. Methyl branched hydrocarbons were also identified including a series of tetramethylalkanes with chain lengths of 30, 32, and 34 carbons. In addition to those found on the cuticle surface, the hemolymph contained the alkene pheromone precursor, 2-methyl-Z7-octadecene and two saturated analogues, 2-methyl-octadecane and 2-methyl-hexadecane. No evidence was obtained for the presence of the pheromone 2-methyl-7, 8-epoxy-octadecane in the hemolymph. Pheromone gland extracts indicated that small amounts (<1 ng) of the alkene precursor were also present in the gland. Relatively larger amounts of the alkene precursor were found in the hemolymph at the time when pheromone titers were higher on the gland. The presence of the hydrocarbon pheromone precursor in the hemolymph is discussed in relation to possible biosynthetic pathways for producing the gypsy moth pheromone.     

Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization

We investigated the enzymatic properties and immunohistochemical localization of cuticular hemocyanin, a known oxygen transporter in the prawn Penaeus japonicus. The molecular weight of hemocyanin purified from the cuticle was estimated to be 67-77 k using SDS-PAGE, and the purified protein was effectively converted into a phenoloxidase-like enzyme by an SDS-treatment. The activated enzyme catalyzed the o-hydroxylation of monophenols and the oxidation of o-diphenols and was inhibited by typical inhibitors of phenoloxidase. These characteristics were nearly identical to the enzymatic properties of hemolymph hemocyanin. Immunological detection showed a diffuse distribution of hemocyanin over the exocuticle and endocuticle, and a higher signal level was observed in the latter. Based on these results, roles of hemocyanin in various physiological processes such as immune response and sclerotization of the cuticle were discussed.     

Rhodnius prolixus infected with Trypanosoma rangeli: In vivo and in vitro experiments

Studies were carried out on the activation of the prophenoloxidase (proPO) in adults of Rhodnius prolixus infected by short and long epimastigote forms of Trypanosoma rangeli. The in vitro activation of the proPO cascade using l-DOPA as substrate was very low in the absence of fat body extract, hemolymph, and parasites. On the other hand, a higher PO activity was observed when short, but not long, epimastigotes of T. rangeli were incubated with fresh hemolymph, fat body extract, and l-DOPA. Supernatant from lysed long epimastigotes increased the PO activity at levels identical to those observed with supernatants from lysed short epimastigotes. Similarly, the PO activity of hemolymph obtained from inoculated insects with long epimastigotes of T. rangeli showed a very low activity when incubated with l-DOPA compared to the PO activity of hemolymph taken from insects inoculated with short epimastigotes of T. rangeli. Control insects inoculated with sterile PBS showed no PO activity. These data indicate the presence of (a) factor(s) in the hemolymph as well as in the fat body extract that may be released (or induced) by the presence of short epimastigotes of T. rangeli and which results in the activation of the R. prolixus proPO system. The implications of these findings are discussed in relation to the development of T. rangeli and its ability to overcome the proPO system, survive, and successfully colonize the hemolymph of R. prolixus.     

Granular phenoloxidase involved in cuticular melanization in the tobacco hornworm: regulation of its synthesis in the epidermis by juvenile hormone

The granular phenoloxidase (PO) that is responsible for cuticular melanization in Manduca sexta larva was purified and an antibody was prepared. This granular PO was found to consist of four isozymes of 90 kDa with isoelectric points ranging from 5.7 to 5.85. The enzyme was immunologically and electrophoretically distinct from the cuticular wound PO, a second cuticular PO common to all larval cuticle, and the hemolymph PO. Both [14C]mannose and [14C]sialic acid were incorporated into the granular PO, showing that this granular PO was a glycoprotein whose sugar moiety was a complex oligosaccharide. When no juvenile hormone (JH) was present at the head capsule slippage (HCS) stage, the epidermis began synthesizing PO 6 hr later. This epidermal synthesis was maximal 12 hr after HCS at which time the PO appeared in the cuticle, and then synthesis declined. When synthesis ceased about 23 hr after HCS, no further incorporation into the cuticle was observed. As melanization proceeded, immunologically detectable cuticular PO decreased. Application of 0.1 microgram JH I at the time of HCS inhibited synthesis of PO by the epidermis and thus prevented melanization. JH application after PO synthesis had begun (8 hr after HCS) prevented its subsequent synthesis, causing partial melanization. Thus, the absence of JH is necessary during the period of epidermal synthesis of the granular PO to allow complete melanization.     

Immune suppression of Galleria mellonella (Insecta,Lepidoptera) humoral defenses induced by Steinernema feltiae (Nematoda,Rhabditida): involvement of the parasite cuticle

Immune depression of Galleria mellonella larvae was evaluated a short time after infection with the entomopathogenic nematode Steinernema feltiae. In the host the activity of the enzymatic cascade known as the proPO system was significantly reduced by the presence of either live or dead parasites. The presence of parasites decreased the LPS-elicited proPO system activity. In addition, this process seems to be related to a decrease in the activity of hemolymph proteases, more than to phenoloxidase damage. proPO inhibition was also achieved by injected isolated cuticle fragments, suggesting that the parasite body surface plays an important role in the early parasitation phase.     

黄粉虫幼虫体壁硬化过程中酚氧化酶活性的变化

为研究酚氧化酶(PO)在昆虫蜕皮过程中的功能和作用, 采用微量测定法研究了黄粉虫Tenebrio molitor体壁硬化过程中血淋巴和表皮中的PO活性变化。结果表明：初蜕皮幼虫血淋巴中PO活性较高, 但随着体壁的不断黑化与硬化, 其活性呈现下降趋势, 在3～4 h内达到最低点, 而后PO活性逐渐上升, 7 h左右活性上升至最高, 并接近于正常幼虫的水平;在刚蜕完皮后的1 h内, 体壁中 PO活性基本无变化, 但随后即开始下降, 3 h左右降到最低点, 然后开始回升, 6~7 h左右恢复到正常水平, 并趋于稳定;以L-DOPA为底物, 通过双倒数曲线作图法求得黄粉虫血淋巴PO的Km＝1.176 mmol/L, 体壁PO的Km＝0.881 mmol/L, 表明体壁PO与底物L-DOPA的亲和力要高于血淋巴PO。研究表明两种来源的酚氧化酶均参与了黄粉虫幼虫的体壁硬化过程, 但在作用方式及与底物的亲和力方面存在差异。     

The effects of exposure to critical thermal maxima on the physiological,metabolic, and immunological responses in adult white shrimp Litopenaeus vannamei (Boone)

Exposure to thermal stress was shown to have a significant effect on the osmotic pressure of the hemolymph, glucose levels, total count of hemocyte (TCH), and proPO activity in adult white shrimp Litopenaeus vannamei. Exposure of the shrimp to CTMax significantly increased the osmotic pressure of the hemolymph relative to the control group. In organisms reaching CTMax, temperature elicited a secondary stress response that included an increase in hemolymph glucose of 31?mg?mL^?1. Metabolites in hemolymph such as cholesterol, acylglycerides, and total protein were not significantly affected by exposure to CTMax. CTMax exposure affected several immunological parameters causing decreases in TCH and proPO activity. We suggested that biomarkers such as osmolality, glucose levels, TCH, and proPO activity could be used as sensitive predictors of exposure to CTMax in white shrimp.     

Penetration of cuticle and proliferation in hemolymph by Paecilomyces fumosoroseus isolates that differ in virulence against lepidopteran larvae.

Frequencies of cuticular penetration and speed of proliferation in hemolymph were demonstrated for two isolates of Paecilomyces fumosoroseus that differ in virulence against diamondback moth, Plutella xylostella. Penetrant hyphae of virulent isolate 4461 were visible in larval cuticle cross-sections of diamondback moth and fall armyworm, Spodoptera frugiperda, within 22 h after inoculation. Virtually no penetration was observed for isolate 1576 for up to 52 h after inoculation. Isolate 4461 also proliferated more quickly than isolate 1576 in the hemolymph of fall armyworm when the isolates were injected as blastospores.