Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis

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Insect Biochemistry and Molecular Biology

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Re-examination of a a-chymotrypsin-solubilized laccase in the pupalcuticle of the silkworm, Bombyx mori: Insights into the regulationsystem for laccase activation during the ecdysis process

Tsunaki Asano a, *, Masato Taoka b, Yoshio Yamauchi b, R. Craig Everroad c, Yosuke Seto a,Toshiaki Isobe b, Masaharu Kamo d, Naoyuki Chosa d

a Department of Biological Sciences, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japanb Department of Chemistry, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japanc Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USAd Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, Yahaba, Iwate 028-3694, Japan

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Article history:Received 25 August 2014Received in revised form12 October 2014Accepted 14 October 2014Available online xxx

Keywords:LaccaseActivationPhylogenetic analysis

* Corresponding author. Tel.: þ81 42 677 2570; faxE-mail address: [email protected] (T. Asano

http://dx.doi.org/10.1016/j.ibmb.2014.10.0040965-1748/© 2014 Elsevier Ltd. All rights reserved.

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Please cite this article in press as: Asano, T., eBombyx mori: Insights into the regulation sy(2014), http://dx.doi.org/10.1016/j.ibmb.2014

a b s t r a c t

The laccase in the pupal cuticle of the silkworm, Bombyx mori, is thought to accumulate as an inactiveprecursor that can be activated stage-dependently. In this study we isolated an 81-kDa laccase fromcuticular extract of B. mori that was prepared by digestion of the pupal cuticles with a-chymotrypsin. Themass spectrometric analysis of the purified protein indicates that this 81-kDa laccase is a product of theBombyx laccase2 gene. The purified 81-kDa laccase (a-chymotrypsin-solubilized Bombyx laccase2: Bm-clac2) has an N-terminal sequence of RNPADS that corresponds to Arg146 to Ser151 of the deduced pro-tein sequence of Bmlaccase2 cDNA, indicating that Bm-clac2 lacks the N-terminal part upstream fromresidue Arg146. Bm-clac2 shows enzymatic activity, but its specific activity is increased around 17-foldafter treatment with trypsin, which involves cleavage of peptide bonds at the C-terminal region. Wealso found that the activity of Bm-clac2 is increased in the presence of isopropanol. In previous reports,proteolytic processing has been hypothesized as a system for laccase activation in vivo, but the presentresult implies that this type of processing is not the only way to convert Bm-clac2 to the high-activityenzyme.

© 2014 Elsevier Ltd. All rights reserved.

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1. Introduction

Laccase (EC 1.10.3.2) is a member of the three-domain multi-copper blue oxidase (3dMCO) protein group. Since the first dis-covery of laccase from the Japanese lacquer tree, Rhus vernicifera,over 130 years ago (Yoshida, 1883), a number of laccases andlaccase-like 3dMCOs from plant, fungi and bacteria have beenextensively studied, and their structures and molecular propertieshave been characterized in detail (Mayer and Staples, 2002;Nakamura and Go, 2005). However, the presence of laccase andlaccase-like 3dMCO genes was not known in metazoans until 10years ago when it was reported that cDNAs for a laccase-like oxi-dase were isolated from the tobacco hornworm,Manduca sexta andthe malaria vector mosquito, Anopheles gambiae (Dittmer et al.,

: þ81 42 677 2559.).

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t al., Re-examination of a a-cstem for laccase activation du.10.004

2004). In that study, genes for two types of proteins were found,differentiated by the structure of their regions on the N-terminalside of the catalytic domain, and they were designated as laccase1and laccase2. It has long been hypothesized that phenol-oxidizingenzymes, including laccase-like enzymes, are involved in cuticlesclerotization and pigmentation (Andersen, 2010). The expressionpatterns of these newly found genes suggested that laccase2participated in these processes. A subsequent study of the red flourbeetle, Tribolium castaneum, demonstrated that knockdown of notlaccase1, but laccase2 resulted in lethality associated with defectsin both cuticle hardening and pigmentation (Arakane et al., 2005).Since then, the expression or gene functions of laccase2 genes havebeen studied in insect species from multiple orders (Arakane et al.,2005; Yatsu and Asano, 2009; Dittmer et al., 2009; Gorman et al.,2008; Elias-Neto et al., 2010; Futahashi and Tanaka et al., 2011;Masuoka et al., 2013). In contrast, it was reported recently that alaccase1 ortholog in the fruitfly, Drosophila melanogaster, has fer-roxidase activity (Lang et al., 2012). It was shown that a strong

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hymotrypsin-solubilized laccase in the pupal cuticle of the silkworm,ring the ecdysis process, Insect Biochemistry and Molecular Biology

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knockdown of the gene for this protein resulted pupal lethality,indicating that the laccase1 gene is essential in this insect, possiblyvia controlling iron metabolism.

In 2009, the amino acid sequence of a laccase-like enzyme fromthe pupal cuticle of the silkworm, Bombyx mori, was analyzed. Theobtained sequence corresponded to that predicted from the cDNAofthe Bombyx ortholog of laccase2. Therefore it was confirmed at theprotein level that the translation product of laccase2 gene has lac-case activity. Then, a more-detailed analysis of the endogenous andrecombinant laccase2s of M. sexta was reported (Dittmer et al.,2009). More recently, the enzymatic properties of the two alterna-tively spliced isoforms (A-type and B-type) of laccase2 werecompared in two species,A. gambiaeand T. castaneum (Gormanet al.,2012). The purified and recombinant laccase2s oxidize endogenoussubstrates for both melanin synthesis (dopa and dopamine) andcuticle hardening (N-acetyl-dopamine and N-b-alanyl-dopamine)(Andersen, 2010; Dittmer et al., 2009; Gorman et al., 2012).

Since the first characterization of laccase-like activity in thepuparium cuticle of Drosophila virillis, there have been observationsthat the activity of phenol-oxidizing enzymes in the cuticle isactively changed during puarium or pupal cuticle formation(Yamazaki, 1969, 1972; Barrett and Anderson, 1988; Yatsu andAsano, 2009). The activity of the enzyme appeared or highlyincreased at the very time when puparium or pupal cuticle wassclerotizing. It is reasonable that the activity of the phenol-oxidizing enzymes is actively controlled during these processes.During puparium formation and ecdysis, the cuticle should be softor flexible enough for smooth transition of the shape from late larvato puparium, or for molting and the following expansion of the newcuticle. On the other hand, after the shape-transition or expansion,the cuticle should quickly harden to protect the insect from attackfrom predators or infection by pathogens. In addition, the cuticleshould be strong enough to support movements of insects such aswalking or flight for escaping fromdanger. To precisely facilitate thetime-dependent change in the mechanical properties of the cuticle,control systems for substrate supply (Davis et al., 2007) or laccaseactivity may have developed. As one possible model for regulationof laccase activity, it has been suggested that an inactive precursorof laccase in the newly formed pupal cuticle is activated after pupalecdysis (Yamazaki, 1972; Yatsu and Asano, 2009). In a key previousreport, a laccase precursor was purified from the pupal cuticles ofBombyx (Yamazaki, 1989). This laccase precursor was tightlyattached to the cuticle matrix. Consequently, a-chymotrypsin wasused to breakdown the cuticle structure that anchoring the pre-cursor. The molecular mass of the purified laccase precursor wasestimated to be 81 kDa in SDS-PAGE. In in-gel staining analysis ofthe laccase precursor after native-PAGE, a faint signal for enzymeactivity was observed after a long period of incubation of the gelwith substrate. In contrast, the protein was converted to anapparently active enzyme after artificial processing by trypsintreatment, suggesting that a proteolytic activation of the precursorwas needed. Unfortunately, the electrophoretical patterns of thepurified enzyme, or those of the mobility shifts after trypsintreatment had not been presented. In addition, no structural in-formation of this protein, either as amino acid sequence or as site ofprocessing by protease treatment, was presented. Based on theseresults from Yamazaki (1989), the aim of the present study is tobetter understand the regulation system for laccase activity inBombyx. In the present study, we re-isolate the 81-kDa laccase tohomogeneity in SDS-PAGE, and by structural analyses we show thatthe purified protein is a product of the Bmlac2 gene. We alsoperform molecular characterization of the purified protein withrespect to its activation mechanisms. The observations in this studyoffer new insight for the possible mechanisms of laccase activationduring the process of cuticle formation in vivo.

Please cite this article in press as: Asano, T., et al., Re-examination of a a-cBombyx mori: Insights into the regulation system for laccase activation d(2014), http://dx.doi.org/10.1016/j.ibmb.2014.10.004

2. Materials and methods

2.1. Animals

Silkworms, B. mori (Kinsyu� Showa) were reared on an artificialdiet (Shirukumeito 2M, Nihon Nosan Kougyo) at 25 �C under a 12-h photoperiod.

2.2. Native-PAGE and activity staining

Native-PAGE and in-gel activity staining assay was performed asdescribed previously (Yatsu and Asano, 2009).

2.3. Purification of laccase from cuticle proteins solubilized with a-chymotrypsin

Throughout the purification processes, for detection of laccaseprecursor in the collected fractions, the laccase was artificiallyactivated by trypsin digestion followed by in-gel analysis for laccaseactivity. To activate the laccase in the samples, 10e20 aliquots ofeach fraction were mixed with one tenth volume of 1 M TriseHClpH 7.5 and 1 ml of trypsin solution (25 mg per ml in 5 mM HCl) andincubated at 37 �C for 30min. The resulting mixturewas assayed byactivity staining analysis after native-PAGE.

As starting material, cuticles from newly ecdysed pupae werecollected and stored at �70 �C until use. The cuticles (17.2 g) werebroken to powder in mortar pre-cooled with liquid nitrogen. Theresulting cuticle powderwas suspended into 90ml of ice-cold 0.1MTriseHCl buffer, pH 7.5 with crystals of phenylthiourea (2e3 mg)(hereafter buffer I), containing 1 M urea. After incubation on ice for5 min, the suspension was centrifuged at 8000 � g for 10 min (inthis purification, centrifugation was performed at 4 �C). Theprecipitated cuticle was re-suspended into 90ml of ice-cold buffer Icontaining 1 M sodium chloride. After incubation on ice for 10 min,the suspension was centrifuged at 8000 � g for 15 min. Theprecipitated cuticle was re-suspended into 75 ml of ice-cold bufferI. To solubilize the laccase, 75 mg of a-chymotrypsin (Wako Pure-chemicals, Wako, Japan) that was pre-dissolved into 1 ml of 5 mMHCl was added to the suspension. The mixture was incubated at37 �C for 75 min, and then centrifuged at 8000 g � 15 min. To thesupernatant, 35 g of ammonium sulfate was dissolved (~60% satu-ration) and stirred over night. The solution was centrifuged at8000 g� 15 min, and then the precipitated proteins were dissolvedinto a minimum volume of 10 mM TriseHCl, pH 7.5, containing1 mM PMSF and dialyzed against 2 L of the same buffer (4 �C). Thedialyzed solution was centrifuged at 10,000 g � 15 min, and thenthe supernatant was filtered through a 0.22 mm filter (MILLEX GP,Millipore). The filtrate was applied to Hi-trap Q HP column (5 ml),pre-equilibrated with 10 mM TriseHCl, pH 7.5. After washing thecolumnwith the same buffer, the adsorbed proteins were eluted bya linear gradient of sodium chloride (0e500 mM/80 min at a flowrate of 0.5 ml per minute). The laccase was eluted at elution volumeof 16e26 ml. The fraction (10 ml in total) was diluted five fold with10 mM TriseHCl, pH 7.5, and subjected to re-chromatography withthe same Hitrap Q HP column. This time, a smaller size column(Hitrap Q HP (1 ml)) was used for obtaining sample with a smallervolume. After washing the column pre-equilibrated with the10 mM TriseHCl, pH 7.5, the proteins were eluted by a lineargradient of sodium chloride (0e350 mM/100 min at a flow rate of0.2 ml per minute). The laccase was eluted from the column at theelution volume of 10e12 ml. The fractions were combined and thesolution (2 ml in total) was concentrated to 0.5 ml by centrifugeevaporator. The solution was applied to Superdex-200 HR10/30column (GE healthcare) that was pre-equilibrated with 10 mMTriseHCl buffer, pH 7.5 containing 150 mM NaCl. Gel permeation

hymotrypsin-solubilized laccase in the pupal cuticle of the silkworm,uring the ecdysis process, Insect Biochemistry and Molecular Biology

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chromatographywas performed at a flow-rate of 0.2ml per minute.The laccase was eluted at the elution volume of 13e15 ml. Thelaccase-containing fractions (2 ml) were combined and diluted fivefold with 10 mM TriseHCl pH 7.5, and then applied to a Mono QHR5/50 column (GE healthcare) pre-equilibrated with the samebuffer. The proteins were eluted with a linear gradient of NaCl(75e300mM/200min) at a flow rate of 0.1 ml per min. Laccase waseluted at the elution volume from 10 to 11 ml. The fractions werestored and used as the purified sample of the 81-kDa laccase or a-chymotrypsin-solubilized Bombyx laccase2.

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2.4. N-terminal sequence determination and MS analysis of the81 kDa laccase and its proteolytic fragments

Mass spectrometry and Edman degradation were performed asdescribed in previous reports (Taoka et al., 2009; Yatsu and Asano,2009; Asano et al., 2013). For high-resolution electrophoresis ofsmall peptides, TriseTricine SDS-PAGE was performed (Sch€aggerand von Jagow, 1987).

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2.5. Enzyme assay of the purified laccase

The unit of the enzyme activity in the purified laccase wasdetermined using catechol as described by Yamazaki (1972).However, the sample was treated with trypsin before the assay. Thepurified laccase solution (Mono Q fraction; 0.25 mg per ml in 10 mMTriseHCl, pH 7.5, containing approximately 200 mM sodium chlo-ride) was mixed with one tenth volume of 1 M TriseHCl pH 7.5 andthenmixed with one tenth volume of 0.25 mg per ml of trypsin. After1-h incubation at 37 �C, the solution was used as enzyme solutionwith activated laccase. Determination of specific activity against anendogenous substrate, dopamine (DA), was performed in 150 ml of0.1 M potassium phosphate buffer pH 6.5, containing 2.5 mMsubstrate and 0.25 mg of laccase. The reaction mixture was incu-bated at 25 �C for 5 min and the increase of absorbance at 475 nmwasmeasured. Protein concentrationwas determined using ProteinAssay (BioRad) using a Bovine serum albumin as standard.

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2.6. Molecular phylogenetic analysis

To collect homologous amino acid sequences of 3dMCOs, aBLAST search was conducted at the NCBI website using the aminoacid sequence of Bmlac2A as a query against the non-redundantprotein database. For the alignment, we selected 33 sequences,and extracted only the catalytic domains from each of the selectedsequences. Here, the boundary between the N-terminal region andthe catalytic region was defined as a DGV/M/L-like sequence (andthe corresponding sequences) located at around 40e50 residuesfrom the N-terminally located HxH copper-binding motif. Usingthese extracted sequences, an alignment was constructed using theMUSCLE program (Edgar, 2004) implemented in MEGA5 software(Tamura et al., 2011) with the default setting. The resulting align-ment was used to construct the phylogenetic tree using theMaximum Likelihood (ML) method with the WAG model withproportion of invariable site, discrete gamma distribution of ratevariation among sites (5 categories) and observed amino acid fre-quencies (WAG þ I þ G þ F) [1] using MEGA5. Initial trees for theheuristic search were obtained automatically by applyingNeighbor-Joining and BioNJ algorithms to a matrix of pairwisedistances estimated using a JTT model, and then the ML tree searchwas conducted by Nearest-Neighbor-Interchange (NNI) method.Statistical support of the topology was assessed by 100 bootstrapreplicates.

Please cite this article in press as: Asano, T., et al., Re-examination of a a-cBombyx mori: Insights into the regulation system for laccase activation du(2014), http://dx.doi.org/10.1016/j.ibmb.2014.10.004

3. Results

3.1. Purification and characterization of a-chymotrypsin-solubilizedlaccase

Extraction of laccase was performed according to the method ofYamazaki with minor modifications (1989). To confirm successfulextraction, the extract was subjected to in-gel activity staininganalysis (Fig. 1A). In the lanewhere the extract was directly applied,little signal for laccase activity was detected (lane 1). In contrast inthe lane where the trypsin-treated sample was applied (lane 2), anapparent activity band was detected (open arrowhead). It seemsthat the laccase precursor in the extract is activated through pro-teolytic processing by trypsin. To study further the molecularproperties, we purified the laccase precursor from the extract. Afterammonium sulfate fractionation, chromatography was performedwith four different columns. Fig. 1B, panel a shows the elutionprofile of anion exchange chromatography with Mono Q HR5/50column, the final procedure of the purification. There was a majorpeak of UV absorbance at 10e11 ml of the elution volume. In SDS-PAGE of the fraction eluted with the peak (horizontal bar), a single81-kDa band was found (panel b, lane 1). The fractions that wereeluted in this area were mixed and used as the purified protein inlater analyses. The specific activity of the 81-kDa laccase aftertrypsin treatment was 2106 units per mg protein, which is similarto that of trypsin-solubilized 70-kDa laccase (2280 units per mgprotein) that was purified in the previous study (Yatsu and Asano,2009).

After trypsin treatment, the band shifted to a position at 70 kDa(lane 2 in panel b of Fig.1B). In native-PAGE analysis, a similar band-shift was observed (Fig. 2A, panel a). After trypsin treatment, theband for the 81-kDa laccase in CBB staining gel (indicated withopen arrowhead (left lane)) also shifted to a higher mobility area(indicated with closed arrowhead (right lane)). In in-gel activitystaining assay using an endogenous substrate (dopamine: DA),little signal for laccase activity was observed in the lane where thesample without trypsin treatment was applied (left lane). The bandfor laccase activity was seen only in the trypsin-treated sample(panel b, right lane). In spectrophotometric analysis (Fig. 2B),enzyme activity was detected in the sample that was untreatedwith trypsin (�try), but the specific activity was much lower (~6%total activity) than that measured after the trypsin treatment(þtry).

In previous reports, it has been shown that the inactive pre-cursor of phenoloxidase (proPO), another copper enzyme in insectswith activity to oxidize catechols, can be converted to activeenzyme in buffered iso-propanol (Asada et al., 1993; Asano andAshida, 2001). In this study, we used buffered isopropanol (30%)for in-gel activity staining assay as described in a previous report(Yatsu and Asano, 2009). In this condition, a new activity bandappeared at the position corresponding to that of CBB band (Fig. 2A,panel c, left lane), in addition to the band of the trypsin-treatedsample (right lane). This result indicates that in the presence ofisopropanol, the activity of 81-kDa laccase is highly increased.

3.2. N-terminal sequencing and MS analysis of the 81 kDa laccase

To obtain information about the amino-acid sequence of the 81-kDa laccase, the purified protein was subjected to Edman degra-dation analysis. The determined N-terminal sequence was RNPALS,which corresponds to Arg146 to Ser151 of Bombyx laccase2 (Bmlac2)(Fig. 3A) (hereafter the 81-kDa laccase is referred to as a-chymo-trypsin-solubilized Bmlaccase2: Bm-clac2). The sequence startsfrom Arg146, therefore it is assumed that Bm-clac2 lacks N-terminalpart of laccase 2 from Arg145. This assumption is supported by the


Fig. 1. Extraction and purification of 81-kDa laccase. A. In-gel activity staining analysis of cuticle extract that was prepared by a-chymotryptic digestion of the pupal cuticles. Theextraction and trypsin treatment were performed as described in Materials and Methods. 10 ml of aliquot was applied to each lane after mixing with the same volume of 2� native-PAGE sample buffer. In lane 1, sample was directly applied. In lane 2, sample was applied after trypsin treatment (try). B. Purification of the 81-kDa laccase. The last purificationprocedure was anion exchange chromatography with Mono Q column (panel a). The horizontal bar indicates the elution volume of the absorbance peak where the purified samplewas eluted. The purified protein was subjected to SDS-PAGE of 10% acryl-amide gel, followed by CBB staining (panel b). In each lane 0.5 mg of the purified protein was applied, butthe sample in lane 2 was pre-treated with trypsin.


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result in LC-MS/MS analysis of the peptide fragments derived fromBm-clac2. The SDS-PAGE band for Bm-clac2 was subjected to in-geldigestion with trypsin, followed by mass spectrometric analysis.From the digest, many peptides corresponding to those predictedfrom Bmlac2 cDNA were identified. However, we could not detectany of the fragments from the region of the N-terminal 145 residues(Fig. 3B and Table 1). In laccase2 of other insect species, the ex-pressions of alternatively spliced variants with variable C-terminalregions of ~250 residues have been shown (Arakane et al., 2005;Dittmer and Kanost, 2010). In our preparation only the peptidefragments from A-type isoform were detected. This is consistentwith the two previous reports of laccase2 purification from B. moriand M. sexta (Yatsu and Asano, 2009; Dittmer et al., 2009).

Next we checked the site of proteolytic processing after trypsintreatment. The trypsin-treated sample was subjected to SDS-PAGE

Fig. 2. Activation of the purified 81-kDa laccase. A. The purified 81-kDa laccase was subjec(panels, b and c). In each lane, 2 mg of the purified laccase was applied. B. Specific activity


and the proteins in the gel were transferred to a PVDF membrane.The N-terminal sequence of the protein contained in the 70-kDa-band (Fig. 1C) was RNPALS; identical to that of the sample beforetrypsin treatment. As second major sequence, NPALSA was alsoread. Except for the absence of Arg146, this sequence matches theabove motif, indicating that a portion of the N-terminally locatedArg146 in Bm-clac2 was released by the trypsin treatment. Thisresult indicated that the band shift was not due to cleavage at theN-terminus. Thus, to obtain the putative peptide fragment that isderived by the trypsin cleavage, TriseTricine SDS-PAGE was per-formed for higher resolution in the low molecular weight regioncompared to Triseglycine SDS-PAGE (Fig. 4A). In the lane where thetrypsin treated sample was applied (middle lane), a band wasnewly detected at around the position of 7.0 kDa as indicated withclosed arrowhead. This band is not observed in the control lane

ted to native-PAGE, followed by CBB staining (panel a) or in-gel activity staining assayof the 81-kDa laccase. In both A and B, DA indicates dopamine.

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Fig. 3. Structure of the purified 81-kDa laccase (Bm-clac2). A. In panel a, the above is the structure of the full-length Bmlac2 that was predicted from the cDNA for Bmlac2A(AB379590), and the below is the structure of the purified 81-kDa laccase. The sequence that was determined by Edman degradation is shown next to the arrow. Panel b is thecomparison between the sequences around the N-termini of Bm-clac2 (upper) and Ms-clac2 (lower). The cleavage sites are indicated with arrowheads. The underlines indicate thesequences determined by Edman degradation. B. The predicted amino acid sequence of the full-length Bmlac2 is shown. Signal sequence is indicated with open box. N-terminalregion that is thought to be absent in the purified Bm-clac2 is indicated with dotted-lined gray box. Peptide fragments that were identified in LC-MS analysis are indicated withclosed box and white letters. In both A and B, characteristic motifs and amino acid residues (the sites of modifications or copper binding sites) are indicated as depicted below. Thepositions of the possible sites for glycosylation and signal sequence were predicted in the servers, NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/), NetOGlyc (Steentoft et al.,2005 Q4) and SignalP 3.0 (Peterson et al., 2011).


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where only the trypsin (right lane) or untreated Bm-clac2 (left lane)was applied. The N-terminal sequence of this small peptide wasQGDLP, corresponding to Gln678 to Pro681, indicating that the pep-tide bond at Lys677eGln678 was cut by trypsin. In addition to thissequence, HLKQG was read as second major sequence, suggestingthat peptide bond at Arg674eHis675 was also a cleavage site. Thecleavage at Lys677eGln678 or Arg674eHis675 is consistent with atrypsin-cleaved peptide bond at the C-terminal side of Lys or Arg.The predicted sizes of the peptides with the N-terminal residues ofGln678 and His675 are 8403.9 and 8782.3, respectively. This is alsoconsistent with the observation that the estimated size of thepeptide newly detected in TriseTricine gel was 7.0 kDa. In thealignments of laccase2s from 7 insect species, the sequencesaround these cleavage sites are not highly conserved (Fig. 4B). The


N-terminally located cleavage site (Arg674) is found in all laccase2s,but the C-terminally located site (Lys677) is found only in laccase2sof lepidopteran species.

3.3. Comparison of insect laccase2 with 3dMCOs from otheranimals

In previous studies of insect laccases, molecular phylogenetictrees for laccase-like proteins have been constructed to discuss theevolution of 3dMCOs in insect species. Recently ESTs or genomicdatabases have become available for a variety of animals, especiallyin non-insect arthropods like chelicerata, crustacea and myliapoda(Grbic et al., 2011; Colbourne et al., 2011; i5k consortium, 2013(https://www.hgsc.bcm.edu/arthropods/geophilimorph-


https://www.hgsc.bcm.edu/arthropods/geophilimorph-centipede-genome-project

http://www.cbs.dtu.dk/services/NetNGlyc/

Table 1Peptide fragments identified by MS analysis.

Experimental mass Theoretical mass Observed mass Position Sequence

1469.824248 1469.704468 735.9194 (2þ) 146e158 RNPALSAPDECAR1313.705048 1313.603363 657.8598 (2þ) 147e158 NPALSAPDECAR1616.777648 1616.753815 809.3961 (2þ) 218e231 MLPGPSIQVCENDK2366.264472 2366.109344 789.7621 (3þ) 279e298 YQWQGNAGTHFWHAHTGLQK1190.773448 1190.665924 596.394 (2þ) 299e309 LDGLYGSIVVR3471.783296 3471.567734 868.9531 (4þ) 315e345 DPNSHLYDYDLTTHVMLISDWLHEDAAER1753.893448 1753.921021 877.954 (2þ) 348e364 LAVNTGQDPESVLINGK2323.237448 2323.115479 1162.626 (2þ) 369e389 DPNTGFMTNTPLEVFTITPGR1053.730848 1053.640686 527.8727 (2þ) 467e475 RAQQLGILR897.471648 897.539581 449.7431 (2þ) 468e475 AQQLGILR2957.623272 2957.45932 986.8817 (3þ) 479e506 GPYQPSSLAPTYDVGIPQGVVMNPLDAR1160.602448 1160.585938 581.3085 (2þ) 512e521 NDAICVSQLK1919.958448 1920.006454 960.9865 (2þ) 522e538 NAQNIDPAILQERPDVK2220.289872 2221.077866 741.1039 (3þ) 543e559 FFVYRPEMLFQPNTYNR1543.815448 1543.835831 772.915 (2þ) 695e708 DTIAVPNNGYVILR


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100101


centipede-genome-project)). This enables us to discuss more aboutthe detail on the origin or evolution of insect 3dMCPs. To under-stand the position of insect laccase2s including Bmlac2 in theevolutionary history of laccase-like 3dMCOs in animals, we decidedto perform a phylogenetic analysis of animal 3dMCOs. Using BLASTwith our Bmlac2 sequence as the query sequence, we found puta-tive genes for 3dMCOs in a wide diversity of animals across severalphyla, including hydra (3), water flea (1), centipede (1), nematode(2), oyster (1), sea urchin (4), ascidian (1), acorn worm (1) andlancelet (4) (numbers in parentheses mean the total numbers of theputative proteins found in each animal). Fig. 5 shows schematicstructures (A) and the phylogenetic tree (B) of 3dMCOs from ani-mals (in the phylogenetic tree, five insects (B. mori, M. sexta,D. melanogaster, A. gambiae and T. custaenum) were selected as therepresentative species). Unlike the already-characterized laccasesfrom plants or fungi, animal 3dMCOs have additional extensions atthe N-terminal side of the catalytic domain. Previously, a cysteine-rich region, which locates next to the catalytic domain, had been

Fig. 4. Analysis of tryptic cleavage site on Bm-clac2. A. TriseTricine SDS-PAGE of Bm-clac2.amount of Bm-clac2 was applied after the treatment with 0.5 mg of trypsin. In the right lane,(middle lane) that is absent in trypsin solution (right lane) and the purified sample itself (left70-kDa and 7-kDa were derived (panel a). Their N-terminal sequences were RNPALS (NPALmajor sequences). The sequence alignments around the cleavage sites were constructed wiet al., 2007). On the left, the abbreviations of each species names are shown. The accession nAAN17507.1 (M. sexta), NP_001034487 (T. castaneum), XP_006562317.1 (Apis mellifera), NP_0(Acyrthosiphon pisum). In B, light and deep gray arrowheads indicate the cleavage sites.


identified in the study of the first cDNA cloning of insect 3dMCOs(Dittmer et al., 2004). Other than insects, proteins with the Cys-richregion are found in the water flea (Daphnia pulex), centipede(Strigamia maritima), sea urchin (Strongylocentrotus purpuratus)and ascidian (Ciona intestinalis) (the Cys-rich region is indicatedwith “C” in Fig. 5A). Therefore, the Cys-rich region may have a rolethat is not related to the insect specific physiology, but to moregeneral biological phenomenon. The 3dMCO proteins from hydra(Hydra vulgaris), nematode (Caenorhabditis elegans and Caeno-rhabditis briggsae), oyster (Crassostrea gigas), acorn worm (Sacco-glossus kowalevskii) and lancelet (Branchiostoma floridae) lack thecysteine-rich region. Instead the proteins from hyrda and nema-tode have their own specific short sequences at their N-terminus(see also Supplemental Fig. S1).

In the phylogenetic tree of the catalytic domains (Fig. 5B),3dMCOs from the animal species are divided into two majorgroups, one with the Cys-rich region and the other lacking the Cys-rich region (tentatively named a-type and b-type, respectively). All

In the left lane, the purified Bm-clac2 (10 mg) was applied. In the next lane, the same5 mg of trypsin was applied. The closed arrowhead indicates the newly detected peptidelane). B. By trypsin treatment Bm-clac2 is cleaved at C-terminal region, and peptides of

SD) and QGDLPP (HLKQGD), respectively (sequences in the parentheses are the secondth laccase2 sequences of 7 insect species (panel b), by using ClustalW program (Larkinumbers of laccase2 sequences used for making the alignments are AB379590.1 (B. mori),01137606.1 (Drosophila melanogaster), AAY29698.1 (Aedes aegypti) and XP_001950788.1

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https://www.hgsc.bcm.edu/arthropods/geophilimorph-centipede-genome-project

Fig. 5. Comparison of 3dMCOs from animals. A. Schematic structures of animal laccase-like proteins are shown. Characteristic motifs and regions are indicated as depicted underthe structures. Proteins with and without cysteine-rich regions are categorized as a-type and b-type, respectively. The accession numbers of the proteins are as follows; AB379590.1(Bombyx lac2), NP_609287.1 (Drosophila lac1), EFX81873.1 (Daphnia MCO), SMAR014928 (Strigamia MCO), XP_786321.3 (Strongylocentrotus MCO1), XP_002159531.1 (Hydra MCO1),XP_002598432.1 (Branchiostoma MCO1), NP_001255321.1 (Caenorhabditis MCO (C. elegans MCO)), AAL07440.1 (Trametes pubescens lac) and BAB63411.2 (Rhus vernicifera lac). B.Maximum Likelihood phylogenetic tree of 3dMCOs in animals. a-type and b-type groups are indicated with vertical double bar (grey color). In a-type, cluster I and II, that containlaccase1s (MCO1s) and laccase2s, respectively, are indicated with deep and light grey bars, respectively (they are also indicated with deep and light grey background, respectively).The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The numbers along the branches are the bootstrap values. The accession numbersof the sequences are AAX49501 (AgLac2), ABQ95972 (AgMCO3), ABY84643 (AgMCO4), ABY84644 (AgLac5), DAA06286 (BmLac1), AAN17505 (AgLac1), AY135186 (Mslac2),AAN17506.1 (Mslac1), NP_001034514.1 (TcMCO1), XP_002121236.1 (Ciona MCO), XP_789921.1 (Strongylocentrotus MCO2), XP_798506.3 (StrongylocentrotusMCO9), XP_001199445.2(Strongylocentrotus MCO4), NP_651441.1 (DmCG5959), NP_573249.1 (DmCG32557), XP_002159531.2 (Hydra MCO2), XP_002159531.2 (Hydra MCO4), XP_002164125.2 (HydraMCO11), EKC25244.1 (Crassostrea MCO), XP_002731346 (Saccoglossus MCO), XP_002599414.1 (Branchiostoma MCO2), XP_002598431.1 (Branchiostoma MCO3), XP_002604762.1(Branchiostoma MCO4), CAP34412.2 (Caenorhabditis briggsae MCO) (the accession numbers that were mentioned in Fig. 4B or Fig. 5A are omitted here).


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of the insect proteins are included in the group of a-type proteinswith the Cys-rich region. Inside this group, there are two majorclusters, one of which (cluster I) includes insect laccase1s (insectLac1s) and the other (cluster II) includes insect laccase2s (insectLac2s). Putative MCOs in cluster I include proteins from not onlyinsects, but also from the water flea, a centipede, an ascidian and asea urchin. In contrast, cluster II is composed of only insect proteins.Inside cluster II, two subclasses can be seen as has been shownpreviously (Gorman et al., 2008); one that includes laccase2s fromall five species used here and the other that includes only Anopheles(mosquito) specific proteins (AgMCO3, 4 and 5). The insect lac-case2s are characterized by the presence of FFSSTHGLVQTHPS likemotif at N-terminal region as indicated in Supplemental Fig. S2, butthe Anopheles specific proteins do not possess this motif. As can beseen by the short distance after branching of the cluster, the se-quences of insect laccase2s show high sequence identity. Thelepidopteran proteins (Bmlac2 andMslac2) show very high identityof 96% in their catalytic domains, and even the lowest value is 85%found between Mslac2 and Tclac2A. Since the emergence of lac-case2 in the ancestors of insects, amino acid replacements haveoccurred infrequently. This indicates that even a small alteration inthe amino acid sequence of insect laccase2 can affect greatly theirmolecular functions.

4. Discussion

Here, according to the method of Yamazaki (1989), we suc-ceeded in isolating the 81-kDa laccase. By sequence analysis of thepurified protein, we could confirm that the 81-kDa laccase is theproduct of Bmlac2 gene. It seems that the 81-kDa laccase (Bm-clac2) lacks the N-terminal 145 residues as depicted in Fig. 3. Thepredicted molecular mass without the N-terminal region is69,220 Da, which is smaller than the size estimated from themobility in SDS-PAGE. The difference between the observed massand the calculated mass may be explainable by posttranslational


modifications like addition of glyco-chains as mentioned in thestudies of laccase2s from other species (Dittmer et al., 2009;Gorman et al., 2012). As shown in Fig. 3A, there are three possibleN-glycosilation sites in the predicted sequence for Bm-clac2 (in ourMS analysis, we could not detect peptide fragments harboringthese sites.), and one O-glycosilation site (Ser95) in the N-terminalpart that lacks in the purified Bm-clac2. It is possible that the N-terminal part may be involved in anchoring laccase2 protein to thecuticle matrix. As has been assumed in the study of Manduca lac-case2 (Dittmer et al., 2009), the N-terminal part of Bmlac2 in thecuticle might be degraded during extraction with a-chymotrypsin.It had been postulated that the N-terminal part of insect laccase2 isa regulatory domain for suppression of enzyme activity. However,the specific activities of the recombinant full-length Mslac2 and itsN-terminally truncated form are almost the same (Dittmer et al.,2009). Also in the present study, as shown in Fig. 4, the cleavageat the C-terminal region is responsible for the in vitro activation ofBm-clac2. The presence or absence of the N-terminal part of theprotein does not seem to affect strongly the activity of laccase2.

The N-terminal sequence of Bm-clac2 was RNPALS (Fig. 3A,panel b, upper sequence), which can be aligned to the RRNPALSAPsequence determined for the a-chymotrypsin-solubilized Manducalaccase2 (Ms-clac2) (Fig. 3A, panel b, lower sequence). This resultindicates that almost the same positions were cleaved in thesepolypeptides during extraction with a-chymotrypsin. The se-quences around the cleavage sites (AELRRNPALS) are exactly thesame between the two proteins. In Mslac2, the peptide bond atLue118eArg119 (AEL-RRNP) is the cleavage site. This result isconsistent with the rule that a-chymotrypsin cleaves peptide bondsat the C-terminal side of hydrophobic residues. In contrast, Bm-clac2 seems to be derived by the cleavage between the two argi-nines (AELR-RNP), though in both Manduca and Bombyx, solub-izations were performedwith a-chymotrypsin. It is assumed that inBmlac2 the peptide bond at Lue144eArg145 (Fig. 3A, panel b, greyarrowhead), corresponding to Lue118eArg119 of Mslac2, might be



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cleaved first. Then, the N-terminal Arg145 in the resulting sequencemight be secondly liberated by endogenous proteases or minuteimpurity in a-chymotrypsin used in this study.

In a previous study (Yatsu and Asano, 2009), a protein mixturewas extracted from the newly ecdysed pupae of B. mori using a ureasolution, and the laccase activity in the mixture was checked by in-gel activity staining assay and spectrophotometric methods. In bothanalyses, little laccase activity was observed, but after trypsintreatment of the extract, an apparent enzyme activity was detected.The data suggested that the urea-solubilized extract contained aninactive laccase precursor (pro-laccase), and that the pro-laccasewas proteolytically activated by trypsin. These observations arevery similar to those for Bm-clac2 in this study. In in-gel activitystaining assay of Bm-clac2 (Fig. 1A or panel b of Fig. 2A), the bandfor the enzyme activity was negative or trace-like (left lanes), butafter trypsin treatment the signal became very clear (right lanes).Also, in the quantitative analysis, the specific activity of the un-treated Bm-clac2 is much lower than that of the trypsin-treatedBm-clac2 (Fig. 2B). Bm-clac2 seems to preserve the characters ofthe hypothetical pro-laccase, with respect to the low active stateand the potential to become a highly active enzyme. Previously a70-kDa Bmlac2 was purified from the protein extract that wasprepared by tryptic digestion of the cuticles from newly ecdysedpupae (Yatsu and Asano, 2009). The purified protein (trypsin-sol-ubilized Bmlac2: Bm-tlac2) is apparently active enzyme with aspecific activity comparable to that of Bm-clac2 pre-treated withtrypsin, as described in the RESULT section. It is thought that thepro-laccase in the cuticle might be proteolytically processed intoBm-tlac2 during the extraction with trypsin.

Until now, there have been studies on two a-chymotrypsin-solubilized laccase2s (Ms-clac2 from M. sexta (Dittmer et al., 2009)and Bm-clac2 from B. mori (this study)) and the recombinant lac-case2s from three species (M. sexta (Dittmer et al., 2009), A. gambiaeand T. castaneum (Gorman et al., 2012)). In all cases, laccase2s arepositive for enzyme activity, but Bm-clac2 seems to be a low-activeprecursor rather than fully-active molecule (Fig. 2). It is not knownif Bm-clac2 is equivalent to laccase2s from other species that havebeen characterized, since the in vitro activation of enzyme activityhas been reported only in Bm-clac2. During the preparation of thismanuscript, we have observed that the activity of the purified Ms-clac2 is increased (~13 fold) after trypsin-treatment (Asano et al., inpreparation). This result is similar to that of Bm-clac2 in the presentstudy. This activatable nature of laccase2 might be generalized toother insects, though more comparative studies are needed tounderstand this phenomenon.

In B. mori, the changes in the strength of laccase activity in thepupal cuticle were examined by measuring oxygen consumption(Yamazaki, 1972) and by in situ cuticle staining (Yatsu and Asano,2009), in the presence of substrate. Little or only a very weaksignal for laccase activity was observed in newly ecdysed pupa,though the gene for BmLac2 is expressed mainly before pupalecdysis (Yatsu and Asano, 2009). After ecdysis, the activityincreased rapidly to reach the maximum in several hours(Yamazaki, 1972), indicating that the laccase that had accumulatedbefore pupal ecdysis was being activated. In contrast, the activity oflaccase in the pupal cuticle of Manduca is almost the same beforeand after pupal ecdysis (Dittmer et al., 2009). InManduca, the onsetof cuticle tanning is prior to pupal ecdysis, especially in theabdominal part. Therefore, it is possible that the activity of laccasereaches optimal levels at the prepupal stage. As a possible mech-anism for the laccase activation in Bombyx, participation of pro-teolytic enzyme has been discussed since the first report of cuticlelaccase isolation from Bombyx (Yamazaki, 1972; Ashida andYamazaki, 1990; Andersen, 2010). In in vitro activation of Bm-clac2 by trypsin treatment, peptide bonds at Lys676eGln677 or


Arg673eHis674 are the cleavage sites (Fig. 4). As described above, thesequences around these cleavage sites are not highly conservedbetween species (Fig. 4B). Since the Arg of the N-terminally locatedcleavage site (Arg673eHis674) is conserved, it would be possible thatthis cleavage occurs in in vivo activation. However, proteolysis is notthe only way to induce enzyme activity from the precursor. Asshown in panel c of Fig. 2A, by incubating the gel in buffered iso-propanol, a clear band for enzyme activity became detectable (leftlane) at the position corresponding to that of CBB staining signal(panel a, left lane). The band intensity is comparable to that oftrypsin-treated sample applied in the next lane. Some structuralchangesmight occur in the presence of isopropanol, and theymightinduce strong enzyme activity from Bm-clac2. As one of thepossible mechanisms by which some endogenous factors couldlead to a structure change without proteolysis, we hypothesize thepresence of a protein or peptide that can bind to the proenzyme foractivation. For instance, hemocyanin of the horseshoe crab, Tachy-pleus tridentatus, normally functions as an oxygen carrier, but isconverted to a melanin-producing enzyme after association withthe chitin-binding antibacterial peptide, tachyplesin (Nagai et al.,2001). We expect that Bm-clac2 can be used for assays and isola-tion of endogenous factor(s) for laccase2 activation.

As described above, genes for 3dMCOs are found in variety ofanimals. Considering the close relationship between crustacea andinsecta (Regier et al., 2010), it is surprising that D. pulex has only thegene for laccase1-like protein, but does not have the laccase2 gene.Furthermore, we could not find any genes for 3dMCOs in thegenome of two cheliceratan species, the spider mite, Tetranychusurticae, and the bear tick, Ixodes scapularis (our observation). Sincethe cuticle can be regarded to be the body structure that is indis-pensable and the most fundamental in all the arthropods, theremay be some common systems for the stabilization of cuticlestructure in this phylum. However, considering the assumption thatlaccase2 is an insect-specific protein (Fig. 5B), utilization of laccase2for cuticle construction (and probably the system for laccase2activation) seems to be insect specific (Fig. 5B). The acquisition ofthe laccase2 system might be one of the determinants that differ-entiated the last common ancestor of all insects from other ar-thropods during arthropod evolution. For instance, the hardeningof cuticle with the laccase system does not require active calcifi-cation that is important in cructacean species (Luquet and Marin,2004; Nagasawa, 2012). This low requirement for calcium ionswould be an advantageous adaptation to terrestrial and freshwaterenvironments where availability of calcium is restricted comparedto seawater. It is interesting to think that the ability of insects to flymay be in part attributable to the laccase2 system, as the incor-poration of a mineral phase would otherwise make the cuticle tooheavy to fly (Fernandez and Ingber, 2013). In addition, oxidationreactions catalyzed by laccase consume molecular oxygen as ac-ceptors of electrons from the substrates. The utilization of molec-ular oxygen for cuticle hardening would also be an adaptation tothe terrestrial environment, inwhich the partial pressure of oxygenis much higher than that in aquatic environment. Future analyseson laccase2 and its regulation system may provide valuable infor-mation towards understanding the possible contribution of lac-case2 system to the emergence, evolution and success of insects.

Acknowledgment

We thank Dr. Hiroko Yamazaki for her valuable experience andadvice on laccase experiments, Dr. Masaaki Ashida for encourage-ment and research suggestions, Dr. Masanori Ochiai for N-terminalsequencing, Mr.Kazuhiro Kusunoki for the help of experiments inearly stage of this study, Dr. Toshiro Aigaki for advice, Dr. MichaelKanost, Dr. Maureen Gorman and Dr. Neal Dittmer for valuable



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discussion about MCOs including the laccase2 of insects. This workwas supported by grant (16780038) from the Japanese Ministry ofEducation, Culture, Sports and Technology.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ibmb.2014.10.004.

Uncited references

Arakane et al., 2009; Barrett and Andersen, 1981; Futahashiet al., 2011; Gianfreda et al., 2006; Hsia et al., 2013; Riva, 2006;Steentoft et al., 2013.

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Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis