Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase-dependent cross-linking Manabu Iijima*, Tomonori Hashimoto*, Yasuyuki Matsuda, Taku Nagai, Yuichiro Yamano, Tomohiko Ichi, Tsukasa Osaki and Shun-ichiro Kawabata Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan The arthropod cuticle functions principally as an exo- skeleton covering the total body surface, and is a highly organized structure produced by extracellular secretion from the epidermis. It is constructed as a composite consisting of chitin filaments (a homopoly- mer of N-acetyl glucosamines conjugated by b-1,4 linkages), structural proteins, lipids, catecholamine derivatives, and minerals. Its structural properties, however, vary among species and also according to surface location and developmental stage in individuals [1–3]. The mechanical properties of the cuticle depend on the content of chitin, the microarchitecture of chitin filaments, and the interaction between the chitin- filament system and cuticular proteins. Furthermore, the cuticle can be modified by sclerotization, namely the oxidative incorporation of o-diphenols into cuticular Keywords chitin-binding proteins; exoskeleton; horseshoe crab; innate immunity; transglutaminase Correspondence Shun-ichiro Kawabata, Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812–8581, Japan Tel & Fax: +81 92 6422632 E-mail: skawascb@mbox.nc.kyushu-u.ac.jp Note nucleotide sequence data are available in the DDBJ databases under the accession numbers AB201765, AB201766, AB201767, AB201768, AB201769, AB201770, AB201771, AB201772, AB201773, AB201774, AB201775, AB201776, AB201777, AB201778 and AB201779. *These authors contributed equally to this work. (Received 14 June 2005, revised 25 July 2005, accepted 29 July 2005) doi:10.1111/j.1742-4658.2005.04891.x Arthropod cuticles play an important role as the first barrier against inva- ding pathogens. We extensively determined the sequences of horseshoe crab cuticular proteins. Proteins extracted from a part of the ventral side of the cuticle were purified by chitin-affinity chromatography, and separated by two-dimensional SDS ⁄ PAGE. Proteins appearing on the gel were designa- ted high molecular mass chitin-binding proteins, and these proteins were then grouped into classes based on their approximate isoelectric points and predominant amino acid compositions. Members of groups designated basic G, basic Y, and acidic S groups contained a so-called Rebers and Riddiford consensus found in arthropod cuticular proteins. Proteins desig- nated acidic DE25 and DE29 each contained a Cys-rich domain with sequences similar to those of insect peritrophic matrix proteins and chitin- ases. In contrast, basic QH4 and QH10 contained no consensus sequences found in known chitin-binding proteins. Alternatively, a low molecular mass chitin-binding fraction was prepared by size exclusion chromatogra- phy, and 15 low molecular mass chitin-binding proteins, named P1 through P15, were isolated. With the exception of P9 and P15, all were found to be identical to known antimicrobial peptides. P9 consisted of a Kunitz-type chymotrypsin inhibitor sequence, and P15 contained a Cys-rich motif found in insulin-like growth factor-binding proteins. Interestingly, we observed transglutaminase-dependent polymerization of nearly all high molecular mass chitin-binding proteins, a finding suggests that transgluta- minase-dependent cross-linking plays an important role in host defense in the arthropod cuticle, analogous to that observed in the epidermal cornified cell envelope in mammals. Abbreviations DCA, monodansylcadaverine; IGFBP, insulin-like growth factor-binding protein; HMM, high molecular mass; LMM, low molecular mass; R & R, Rebers and Riddiford; RACE, rapid amplification of cDNA ends; TGase, transglutaminase. 4774 FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS matrix [4–7]. For insect cuticular proteins sufficient sequence information is available to allow recognition of consensus sequences. The motif first identified is the so-called Rebers and Riddiford (R & R) consen- sus: -G-X(8)-G-X(6)-Y-X-A-X-G-X-G-Y-X(7)-P-X(2)-P-, where X represents any amino acid, and the values in parentheses indicate the number of intervening residues [8–10]. A slightly modified R & R consensus has also been reported, -G-X(7)-(D, E, or N)-G-X(6)-(F or Y)- X-A-(D, G, or N)-X(2 or 3)-G-(F or Y)-X-(A or P)- X(6)-P [3]. The region flanking the N-terminus of the R & R consensus is enriched in hydrophilic amino acid residues [11,12], and a conserved stretch of approxi- mately 68 amino acid residues is referred as the exten- ded R & R consensus [13]. The extended R & R consensus has no sequence similarity to other known cysteine-containing chitin-binding domains [14], such as plant chitin-binding proteins and horseshoe crab antimicrobial peptides with chitin-binding affinity [15–17]. Rebers and Willis reported that the extended R & R consensus is a chitin-binding domain and pos- tulated that the conserved aromatic residues in this domain may play an important role in chitin binding [18]. Short consensus repeats with the sequences -A-A- P-(A or V)- and -G-Y-G-G-L- have also been identi- fied in insect cuticular proteins [4]. Although cuticular proteins from other arthropods have not yet been characterized to the same extent as those from insects, a sequence motif similar to R & R consensus has been identified in proteins from an arachnid, the spider Araneus diadematus [19], as well as in those from a crustacean, the lobster Homarus americanus [20,21]. Several cuticular proteins from H. americanus and the crab Cancer pagurus contain a repeating consensus sequence that is 18-residues in length and contains Gly residues at positions 4, 7 and 13: -X-u-u-G-P-S-G-u-u-X-X-(D ⁄ N)-G-X-X-X-Q-u- (where u represents a hydrophobic residue). Gly resi- dues within this motif may play an important role in maintaining a conformation required for the essential interaction with calcium ions in the exoskeletal matrix [20–22]. Reports in the literature regarding the amino acid composition and sequence of horseshoe crab pro- teins have been sparse, and have primarily dealt with the American horseshoe crab Limulus polyphemus [23– 25]. A cuticular extract from the silkworm Bombyx mori has been shown to contain prophenoloxidase [26,27], an enzyme that typifies arthropod innate immune response and which is activated during the related processes of sclerotization, exoskeletal wound healing, and host defense response to microorganisms [28,29]. In order to identify novel cuticular chitin-bind- ing proteins and cuticular proteins involved in innate immunity, we undertook an extensive examination of cuticle proteins from the horseshoe crab Tachypleus tridentatus. This investigation involved fractionation by chitin-affinity chromatography, two-dimensional SDS ⁄ PAGE (2D SDS ⁄ PAGE) and reverse-phase HPLC, and culminated in the determination of numer- ous cuticle protein sequences. Results and Discussion Separation of cuticular chitin-binding proteins The cuticular proteins of T. tridentatus were extracted with acetic acid, and the resulting extract was subjec- ted to chitin-affinity column chromatography to obtain chitin-binding proteins. Proteins bound to chitin were eluted with acetic acid and lyophilized. Based on an extinction coefficient of 10 at 280 nm for a 1% protein solution, we estimate that approximately 20 mg of chi- tin-binding proteins were reproducibly obtained from 5 g of carapace fragments. The cuticular chitin-binding proteins were separated by 2D SDS ⁄ PAGE over a range of isoelectric points from 3 to 10, and were dis- tributed into two (acidic and basic) clusters on the gel (Fig. 1A). A similar pattern on 2D SDS ⁄ PAGE was observed for the cuticular chitin-binding proteins extracted with 8 m urea (data not shown). Proteins extracted with 10% acetic acid were used in all subse- quent experiments. The majority spots corresponded to proteins with apparent molecular masses of 16, 20 and 25 kDa, with most of these spots clustered in the basic region. In contrast, spots with higher molecular masses (ranging from 67 to 94 kDa) were observed in the acidic region of the gel. In the neutral region, only two major spots (32 and 38 kDa) were present. Thirty- seven spots on the gel were subjected for amino acid composition and sequence analyses. Cuticular proteins extracted from cuticle of the American horseshoe crab L. polyphemus with 6 m urea containing 0.1% trifluoroacetic acid have been separ- ated by 2D SDS ⁄ PAGE over a rage of isoelectric points from 3.5 to 10 [25]. Their isoelectric points range from 6.5 to 9.2, and the 2D gel shows no protein spots in the acidic range from 3.5 to 5.5, corresponding to the members of acidic S and acidic DE in T. tridentatus, as described below. This discrepancy in protein spots on the 2D gels between L. polyphemus and T. tridenta- tus may be due to a discrepancy in age of the samples, juvenile (L. polyphemus) and adult (T. tridentatus). Using a complementary approach, we also analyzed low molecular mass (LMM) chitin-binding proteins. The eluate from the chitin affinity column was fractionated by gel filtration on a Sephadex G-50 column, and M. Iijima et al. Cuticular proteins in horseshoe crabs FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS 4775 fractions containing proteins determined by SDS ⁄ PAGE analysis to be less than 10 kDa were further fractionated by reverse-phase HPLC to obtain LMM chitin-binding proteins (Fig. 1B). A single fraction from the gel filtration step contained a protein that appeared as a single band on SDS ⁄ PAGE with an apparent molecular weight of 10 kDa. This protein, which we designated P1, was identified as big defensin [30] by amino acid sequence analysis. Proteins isolated by reverse-phase HPLC were designated P2 through P15. Previous reports have demonstrated that arthropod cuticular proteins may be resistant to extraction. In the beetle Agrianome spinnicollis, for example, more than 50% of total cuticular protein is retained following extraction [31]. It is therefore possible that the proteins obtained here may not be representative of all cuticu- lar chitin-binding proteins in T. tridentatus. Amino acid compositions of high molecular mass (HMM) chitin-binding proteins HMM chitin-binding proteins resolved by 2D SDS ⁄ PAGE were categorized into seven groups based on amino acid composition: basic G, basic Y, basic QH, neutral, acidic S, acidic DE, and others (Table 1). The proteins in the basic G group had a disproportion- ately high content of Gly (19–35%). Those in the basic Y group were characterized by a high content of Tyr (10–15%), Gly (16–19%), and Asp (10–12%). Proteins in the basic QH group were abundant in Glu (13–16%) and His (7%) as determined by amino acid analysis, but their partial amino acid sequences indicated a high content of Gln rather than Glu. Proteins in the neutral group were abundant in Ala (15%), Pro (12%) and Gly (11–12%), and proteins in the acidic S group, while similar otherwise to those in the neutral group, were additionally characterized by an abundance of Ser (9– 12%). The members of acidic DE group had a high content of Asp (14%) and Glu (10–14%). N-Terminal amino acid sequence analysis of HMM chitin-binding proteins Eleven of 37 spots observed on 2D SDS ⁄ PAGE were resistant to sequence analysis by Edman degradation, presumably due to N-terminal blocking, whereas the remaining spots yielded sequences with lengths between five and 78 residues (Table 1). The N-terminal sequence of neutral 2 was identical to that of acidic S37 despite a significant difference in their apparent B A Fig. 1. (A) 2D SDS ⁄ PAGE of chitin-binding proteins. For experimental details see text. The members of the basic Y, basic G, basic QH, neutral, acidic S and acidic DE groups are bounded, and numerical designations used in the text are indicated. (B) Reverse- phase HPLC chromatograph of LMM chitin-binding proteins. LMM cuticular chitin- binding proteins were resolved by reverse- phase HPLC using an acetonitrile gradient. Cuticular proteins in horseshoe crabs M. Iijima et al. 4776 FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS Table 1. N-Terminal amino acid sequences of cuticular chitin-binding proteins. ND, not detectable. Number Group N-Terminal sequence 1 Neutral VFVPAPAPAP GPAPAPGL 18 2 Neutral TGFPPGGAPI FLHLVPHAKA KAAPPVVVPP VAA 33 3 Other SYVAPAIGGA SARQESGDGY GSVSGSYQLS DADGRQRNVQ YTA 43 4 Basic QH EVFPFNVPEG KHDPAFLQNL QQEAL 25 5 Basic QH EVFPFNVPEG KHDPA 15 6 Basic Y GYFYHPAYYY GAGGSTQYKT QDNIGNYNFG XNE 33 7 Basic Y GVLYNPYFYH PYYYHGLGAS VRHHAQDNLG NYNFGYNEE 39 8 Basic Y GYFYHPAYYY GAG 13 9 Basic Y GVFYNPYFAH PYDPH 15 10 Basic QH GIFPYNVPAG QHDPAYLQAL QQQALHYINL QQVPDLQLQK ARELEVIA 48 11 Basic QH GIFPY 5 12 Other GFLGAGGGGG 10 13 Basic G GFIGAGVGGA GLGGAGLGGA GRFITGGGLG RFVGGGARGL AGTGLVAAGG YFHGGHAGAF AGGVGGGLAR GYYGQQPV 78 14 Basic G GFIGAGVGGA GLGGAGLGGA GRFITGGGLG 30 15 Basic G GFIGAGVGGA GLGGAGLGGA GRFITGGGLG RFVGGGARGL AGTGL 45 16 Basic G GFIGAGVGGA GLGGA 15 17 Basic G GIFPYNVPAG QHDPAYL 17 18 Other ND 19 Basic G GYIGAGGGGT GGLYGGGGGG 20 20 Basic G SYAAPALGGF SARQE 15 21 Other ND 22 Other ND 23 Other ND 24 Other ND 25 Acidic DE EAYDLPDGVQ LLVGNLKHSF VXXSDGYYAA 30 26 Acidic DE EAYDLPDGVQ LLVGNLKHSF 20 27 Acidic DE EAYDLPDGVQ LLVGNLKH 18 28 Acidic DE ND 29 Acidic DE AAFELPDGAQ VLVK 14 30 Acidic S ND 31 Acidic S ND 32 Acidic S ND 33 Acidic S TGIPGDGAVI FHLVPHGYKG 20 34 Acidic S ND 35 Acidic S ND 36 Acidic S TGIPGDGAVI FHLVPHGYK 19 37 Acidic S TGFPPGGAPI FLHLVPHAKA PAAAPPV 27 P1 NPLIPAIYIG ATVG 14 P2 GPPKXATYGQ K 11 P3 XFRVXYRGIX YRKXR 15 P4 XFRVXYRGIX YRRX 14 P5 XFRVXYRGIX YRRXR 15 P6 KWXFRVXYRG IXYRR 15 P7 KWXFRVXYRG IXTRK 15 P8 XFRVXYRGIX YRRXR 15 P9 FDXWSKPDPG PXYAYAFTRY YYDPASH 27 P10 YVLFRGARXR VYSGR 15 P11 YVSXL 5 P12 YITXL 5 P13 SRXQLQGFNX VVRSYGL 17 P14 YSRXQLQGFN XVVRSYGLPT I21 P15 WPPFPIGXGN XAATFXPYVP PSSXPGGKTT RD 32 M. Iijima et al. Cuticular proteins in horseshoe crabs FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS 4777 isoelectric points and molecular masses (33 and 70 kDa, respectively), indicating that neutral 2 may be a proteolytic product corresponding to the N-terminus of acidic S37. Alternatively, neutral 2 and acidic S37 may be isoforms of one another, or result from differ- ential mRNA splicing. Certain members of the basic G group, such as G13, G14, G15 and G16 had similar apparent molecular mass of about 25 kDa, and were characterized by an abundance of Gly near the amino terminus. The N-terminal 78 residues of basic G13, for example, contained 34 Gly residues. The four proteins of this group were identical within at least the first 15 residues, whereas their isoelectric points differed, sug- gesting that they are either isoforms of one another or are differentially post-translationally modified. Simi- larly, within the acidic DE group, acidic DE25, DE26 and DE27 were identical throughout the first 18 resi- dues and had similar apparent molecular masses (20 kDa), though they differed in apparent isoelectric points. In contrast, while the N-terminal sequences of acidic S33 and acidic S36 were identical, these proteins differed from one another both in isoelectric point and in apparent molecular mass (25 and 40 kDa, respect- ively), suggesting that acidic S33 may be a proteolytic fragment of acidic S36. In addition, the N-terminal sequence of acidic S37 was highly similar to those of acidic S33 and S36, indicating that the acidic S may contain several protein isoforms. Finally, Tyr residues occurred repeatedly within the N-terminal sequences of all basic Y proteins (basic Y6 through Y9), and repeats of the di- and tri-peptide sequences QQ and QQQ were observed in the amino terminal sequences of basic QH proteins 4 and 10. Nucleotide sequences of HMM chitin-binding proteins cDNA fragments of HMM chitin-binding proteins were amplified by PCR using degenerate oligonucleo- tide primers based on amino acid sequences derived from intact proteins or from proteolytic fragments thereof. Sense and antisense primers based on the resulting cDNA sequences were selected and used to amplify full-length cDNAs by RACE PCR, resulting in 11 full-length and two partial cDNA clones for HMM chitin-binding proteins. The cDNA of basic G13 encoded a 206 residue pro- tein with a 16-residue signal peptide. Three types of cDNA clones, designated basic G13A (accession num- ber AB201771), basic G13B (AB201772) and basic G13C (AB201773), were identified. Gly8 in G13A was replaced by Glu in G3B, and Leu7 in G13A was replaced by Val in G13C. The cDNA of basic G19 encoded a 161-residue protein with a 16-residue signal peptide (AB201774). A homologous cDNA, designated G19 h (AB201775) encoded a 141-residue protein with a 16-residue signal peptide. The basic G13 variants, G19 and G19 h all contained an R & R consensus sequence, and they exhibited significant sequence simi- larity (G19 and G13, 58% identity; G19 h and G13, 48%; G19 and G19 h, 64%). A partial basic Y6 cDNA lacked the 5¢-region, but overlapped with the region determined by Edman degra- dation of the intact protein, thereby allowing deduction of the sequence of a mature protein consisting of 143 residues (AB201768). The basic Y7 cDNA encoded a mature protein of 131 residues (AB201769). At the pro- tein level, basic Y6 and Y7 showed 50% sequence iden- tity overall, and both possessed an R & R consensus sequence. A blast homology search using basic Y6 and Y7 revealed significant sequence similarity between these proteins and Ad-ACP15.7, a cuticular protein from the spider A. diadematus [19] (Y6 vs. Ad-ACP15.7, 33% identity; Y7 vs. Ad-ACP15.7, 26% identity) as well as between these proteins and LpCP14b, a cuticular pro- tein from L. polyphemus [25] (Y6 vs. LpCP14b, 78% identity; Y7 vs. LpCP14b, 50% identity). The acidic S37 cDNA encoded a 608-residue protein with a 16-amino acid signal peptide (AB201765). The deduced protein sequence contained four tandem repeats of a 68-residue extended R & R consensus sequence, with sequence identity among the four repeats ranging from 66 to 94%. In addition the cDNA encoded seven copies of the pentapeptide sequence -A-A-P-A ⁄ V-, a short consensus sequence found in insect cuticular proteins [4]. A blast homol- ogy search revealed no other regions of similarity between acidic S37 and other known proteins. The members of basic Y and G, and acidic S37 all contain the extended R & R motif commonly found in insect cuticular proteins (Fig. 2). The motif found in the horseshoe crab proteins shows the highest sequence similarity to RR-2, one of the three variants of the consensus [9,10]. A recombinant protein containing the extended R & R consensus of a putative cuticular pro- tein from the mosquito Anopheles gambiae has been shown to be necessary and sufficient for chitin binding [18]. A secondary structure prediction and homology modeling of the extended R & R consensus suggest an antiparallel b-sheet structure [13,32]. Interestingly, aci- dic S37 contained four tandem extended R & R con- sensus repeats. This tandem R & R repeat structure has not previously been identified in arthropod cuticu- lar proteins, and suggests that acidic S37 may interact polyvalently with chitin fibers to form a stable three- dimensional network. In addition to the R & R motif, Cuticular proteins in horseshoe crabs M. Iijima et al. 4778 FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS basic Y6 and Y7 contain an 18-residue motif found in the cuticular proteins isolated from calcified regions of crustacean exoskeletons [20–22], a finding suggests that basic Y6 and Y7 may play a role in the deposition of calcium ions required to maintain the mechanical properties of cuticles (Fig. 3). Basic QH4 cDNAs were identified and shown to encode a 135-residue protein with a 16-residue acid sig- nal peptide. Two cDNA variants were isolated and designated basic QH4A (Pro36 and His83) and QH4B (Leu36 and Tyr8) (AB201766 and AB201767). The cDNA of basic QH10 encoded a 110-residue protein with a 16-amino acid signal peptide (AB201770). Basic QH4 and QH10 showed significant sequence similarity to one another (53% identity), and neither contained the R & R consensus. As basic QH4 and QH10 do not contain the R & R consensus, they must have an unknown a chitin-binding motif. A homology search revealed high sequence similarity (84% identity) between basic QH 10 and the cuticular protein LpCP13 from L. polyphemus [25], a degree of similarity that is particularly notable given that the clottable pro- tein coagulogen shows 70% identity between the two species [33,34]. The sequences of basic QH4 and QH10 can be divided into two regions, the Gln-rich N-ter- minal half and the C-terminal half in which Tyr and His are abundant. It has been proposed that cross- linking between proteins and chitin fibers in insect cuticles is mediated by His-catechol-chitin linkages, the formation of which involves the oxidation of catechol- amines to quinonoid sclerotizing agents with subse- quent nucleophilic addition to certain His residues within cuticular proteins [35,36]. The abundance of His residues in the basic QH proteins therefore raises the possibility that these proteins play an important role in maintenance of the integrity of the exoskeleton. A cDNA of acidic DE25 encoded a 137-amino acid protein and a 22-amino acid signal peptide (AB201776). A partial cDNA of acidic DE29 lacked the 5¢-region and its N-terminal sequence determined by Edman degradation overlapped to the deduced sequence to obtain the sequence of a mature protein of 120 residues (AB201777). Acidic DE25 and DE29 had an overall sequence identity of 46%, and showed no sequence similarity to other known cuticular proteins. Acidic DE25 and DE29 also lack an R & R consen- sus. Rather, they contain six Cys residues within their central region in positions similar those of a Cys-rich motif found in insect chitinases and peritrophic mem- brane proteins [14,37–40] (Fig. 4). Peritrophin-44, a major peritrophic membrane protein identified in the larvae of the fly Lucilia cuprina, contains five tandem repeats of the Cys-rich motif as well as several conserved aromatic residues within the proposed domain bound- ary. The peritrophic membrane is a semipermeable chi- tinous matrix lining the gut of most insects and is thought to play an important role in the maintenance of insect gut structure, the facilitation of digestion, and the protection from invasion by microorganisms and para- sites [37]. The C-terminal three Cys residues of the Cys- rich motif in acidic DE25 and DE29 can be aligned with the C-terminal domain of tachycitin (Cys40 to Cys61), a horseshoe crab chitin-binding protein [15], as well as with the chitin-binding domain of hevein (Cys12 to Cys33), a plant chitin-binding protein [41,42], as shown in Fig. 4. This segment of tachycitin forms an antiparal- lel b-sheet and aligns with the known chitin-binding region of hevein [17]. It is therefore likely that the cor- responding region of the Cys-rich motif in acidic DE25 Fig. 2. Alignment of the extended R & R consensus regions of cuticular proteins of T. tridentatus showing the R & R domains of basic Y6, basic Y7, basic G13, basic G19, basic G19 h, and the four contiguous domains from acidic S37. Highly conserved residues are designated with black boxes. Numbers on the right indicate amino acid residue numbers. Fig. 3. Alignment of basic Y6, basic Y7 and an 18-residue motif identified in Cancer pagurus (motif) [22]. Three conserved glycine residues are indicated by an asterisk. u indicates a hydrophobic resi- due. Numbers on the right indicate amino acid residue numbers. M. Iijima et al. Cuticular proteins in horseshoe crabs FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS 4779 and DE29 is involved in chitin binding. In insects, cuticular proteins containing cysteine residues have not been reported, but analyses of total cuticles following performic acid oxidation have demonstrated the pres- ence of minor amounts of cysteic acid, suggesting the presence of disulfide bond-containing proteins in insect cuticles [43,44]. Nucleotide sequences of LMM chitin-binding proteins All of the LMM chitin-binding proteins identified, with the exception of P9 and P15, were determined to be known antimicrobial peptides, such as tachy- plesin, tachystatin, and their isoforms, or proteolytic fragments thereof (Table 2). In vertebrates, antimi- crobial peptides are expressed on epithelial cell surfa- ces and have been proposed to play a role in innate immunity by acting as ‘natural antibiotics’ [45–47]. In horseshoe crabs, antimicrobial peptides have been shown to be able to induce the intrinsic phenoloxi- dase activity of hemocyanin [48]. The localization of antimicrobial peptides in the cuticle therefore sug- gests that these peptides may facilitate wound heal- ing in the exoskeleton in addition to acting as antimicrobial substances. Fig. 4. Alignment of cysteine-rich domains of acidic DE25, acidic DE29, peritrophic membrane protein, chitinase, and antimicrobial peptides. The cysteine-rich domains of acidic DE25, acidic DE29, the first of five domains found in peritrophin-44 from the fly L. cuprina (Peritrophin) [37], chitinase from the parasitic nematode Brugia malayi (Chitinase) [38], tachycitin from the horseshoe crab T. tridentatus (Tachycitin) [15] and hevein from rubber tree (Hevein) [41,42] were aligned. The conserved Cys residues designated with black boxes, and the conserved aro- matic amino acids are indicated with asterisks. Numbers on the right indicate amino acid residue numbers. Table 2. Features of cuticular chitin-binding proteins. Number Protein Name Residue number Chitin-binding motif DCA incorporation D. melanogaster homolog A. gambiae homolog 4 Basic QH4 135 Unknown + 6 Basic Y6 143 R & R + GC2555, GC1327, GC2342 AACO5656-5668 7 Basic Y7 131 R & R + GC1919, GC1327 10 Basic QH10 110 Unknown + 13 Basic G13 196 R & R + GC2341, GC1252, GC2360 19 Basic G19 161 R & R + Basic G19 h 141 R & R 25 Acidic DE25 137 Peritrophin-like + GC32036, GC14959 29 Acidic DE29 120 Peritrophin-like + GC14959, GC14301 37 Acidic S37 608 R & R GC16963 P1 Big defensin 79 Antibacterial P2 Tachystatin C 41 Antibacterial P3 Tachyplesin II or III fragment Antibacterial P4 Tachyplesin I fragment Antibacterial P5 Tachyplesin I fragment Antibacterial P6 Tachyplesin I 17 Antibacterial P7 Tachyplesin III 17 Antibacterial P8 Tachyplesin I fragment Antibacterial P9 Kunitz-type inhibitor 56 Unknown P10 Tachystatin B2 fragments Antibacterial P11 Tachystatin B1 42 Antibacterial P12 Tachystatin B2 42 Antibacterial P13 Tachystatin A 44 Antibacterial P14 Tachystatin A fragment Antibacterial P15 IGFBP-like protein 47 Unknown Cuticular proteins in horseshoe crabs M. Iijima et al. 4780 FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS The cDNA for P9 encoded a 56-residue protein with a 17-residue signal peptide (AB201778). The cDNA for P15 encoded a 47-residue protein with a 29-residue signal peptide (AB201779). P9 shows signi- ficant sequence similarity (52% identity) to the Kunitz-type protease inhibitor from T. tridentatus hemocytes [49]. Based on sequence homology, the reactive site of P9 can be predicted to be at the Tyr18–Ala19 bond, suggesting that it is a Kunitz-type inhibitor of chymotrypsin-like activity (Fig. 5). P15 contains eight Cys residues in positions similar to those observed in an insulin-like growth factor bind- ing motif (IGFBP motif) [50] (Fig. 6). In mammals, insulin-like growth factor-binding proteins, which con- tain the IGFBP motif, modulate the actions of the insulin-like growth factors in endocrine, paracrine, and autocrine systems [51]. Insulin-like growth factors are essential for growth and development [52], and the presence of the IGFBP motif in P15 raises the possibility that it might play an analogous role in the exoskeleton. Tissue-specific expression of HMM and LMM chitin-binding proteins Basic G13 and G19, acidic DE25 and DE29 and S37 were shown by RT-PCR to be expressed predominantly in epidermis (Fig. 7). In contrast, basic Y6, Y7 and QH4 were broadly expressed, and basic QH10 was highly expressed predominantly in muscle, heart and exoskeleton. Tachyplesin and big defensin were highly expressed in all tissues, and P9 and P15 were expressed in all tissues except for the intestine. In plants, many protease inhibitors perform a protective function against insect infestation through the inhibition of insect pro- teases [53]. In a similar way the Kunitz-type chymotryp- sin inhibitor bikunin is expressed on the keratinocyte cell membrane in human skin, and has been suggested to play a regulatory role [54]. The presence of a Kunitz- type chymotrypsin inhibitor sequence in P9 suggests that it may regulate endogenous proteases within the exoskeleton or inactivate those of invading pathogens. TGase-dependent cross-linking of HMM chitin- binding proteins TGases catalyze the formation of isopeptide bonds between Gln and Lys residues and play an important role during the final stage of blood coagulation in mammals and crustaceans [55,56]. In T. tridentatus, TGase promotes the cross-linking of coagulin with hemocyte surface antigens called proxins and may faci- litate the formation of physiological barrier to invading pathogens [57]. In mammals, TGase-catalyzed forma- tion of e-(c-glutamyl)-lysine bonds is involved in the formation of the cornified cell envelope of the skin, which serves as a frontline barrier against invading pathogens [58,59]. Horseshoe crab TGase was expressed predominantly in hemocytes, and expression in epider- mis was not significant (Fig. 7). Horseshoe crab TGase Fig. 5. Alignment of LMM-P9 and horseshoe crab kunitz-type tryp- sin inhibitor (Trp inh) [49]. Conserved cysteine residues are designa- ted with black boxes. Numbers on the right indicate amino acid residue numbers. The arrow indicates the predicted reactive site. Fig. 6. Comparison of LMM-P15 and the N-terminal regions of IGFBP family members. Amino acid sequences of LMM-P15, mac25, IGFBP-1, -3, -4, -5, and -7 were aligned [50]. Conserved cysteine residues are designated with black boxes. The characteris- tic IGFBP motif (GCGCCXXC) is boxed by a solid line. Fig. 7. Expression patterns of cuticular chitin-binding proteins and TGase. Relative mRNA levels were investigated by RT-PCR as des- cribed in ‘Experimental procedures’. Lane 1, hemocytes; lane 2, heart; lane 3, stomach; lane 4, intestine; lane 5, hepatopancreas; lane 6, epidermis; lane 7, skeletal muscle. M. Iijima et al. Cuticular proteins in horseshoe crabs FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS 4781 is released from hemocytes into the extracellular fluid in response to external stimuli, such as bacterial lipopoly- saccharides [57]. Recently, an epidermal barrier wound repair pathway has been shown to be evolutionally con- served between Drosophila and mice. In Drosophila, the transcription factor grainy head regulates production of the enzymes dopa decarboxylase and tyrosine hydroxy- lase, which are required for covalent cross-linking of cuticular structural components [60]. Mice lacking a homologue of Drosophila grainy head display defective skin barrier function and deficient wound repair, accompanied by reduced expression of TGase 1, the key enzyme involved in protein cross-linking in main- tenance of the stratum corneum [61]. To determine whether potential TGase substrates are present in the arthropod exoskeleton, the cuticular chitin-binding proteins of T. tridentatus were incubated with TGase, and subjected to SDS ⁄ PAGE. TGase induced the formation of SDS-insoluble aggregates of HMM chitin-binding proteins, which were incapable of migrating into the gel. Upon TGase treatment, the major HMM chitin-binding proteins (16, 20 and 25 kDa) were no longer visible on SDS ⁄ PAGE, indica- ting that these proteins were cross-linked to form higher molecular weight polymers (Fig. 8, lane 2). Monodansylcadaverine (DCA), a synthetic fluorescent substrate for TGase, competitively inhibited TGase- dependent polymerization (Fig. 8, lane 3), and was incorporated into the major HMM chitin-binding proteins (Fig. 8, lane 5). When analyzed by 2D SDS ⁄ PAGE, DCA was incorporated into nearly all groups of HMM chitin-binding proteins including basic G, basic Y, basic QH, and acidic DE (Fig. 9), and the identity of most DCA-labeled proteins were confirmed by amino acid sequence analysis (numbered spots in figure). These finding indicate that cuticular proteins in arthropods are capable of acting as sub- strates for TGase and may be involved in a TGase- dependent cross-linking system analogous to that observed in the epidermal cornified cell envelope in mammals. Experimental procedures Protein extraction Cuticles were obtained from the horseshoe crab T. tridenta- tus, which had died of natural causes while in captivity, and stored at )80 °C until use. A part of cuticle from the ventral side of a single specimen, called the doublure, was excised, and epidermal cells were stripped from the cuticle with a sterilized spatula and used subsequently for the pre- paration of mRNA. The remaining cuticle fragments were washed with distilled water, cut into small pieces with steril- ized scissors and homogenized in ice-cold homogenization buffer (50 mm Tris ⁄ HCl, pH 7.5, 0.1 m NaCl) using a Poly- tron (Central Scientific Commerce Inc., Tokyo, Japan) at 15 000 r.p.m. for 1 min. The insoluble material was recov- ered by centrifugation at 3200 g for 30 min at 4 °C, and subjected to a second round of homogenization and clarifi- cation. The resulting precipitate was mixed with 10% acetic acid or 8 m urea for 16 h at 4 °C with gentle agitation, and centrifuged at 4500 g) for 20 min at 4 °C. The resulting supernatant constituted the cuticular extract. Purification of chitin-binding proteins The lyophilized extract was dissolved in a buffer consisting of 50 mm Tris ⁄ HCl, pH 7.5, 0.1 m NaCl, and applied to a chitin (Seikagaku Corp., Tokyo, Japan) affinity column (2.7 · 17.5 cm) equilibrated with the same buffer. After washing with the equilibration buffer, chitin-binding proteins were eluted with 10% (v ⁄ v) acetic acid or 8 m urea. For isola- tion of low molecular mass chitin-binding proteins, the cuticular proteins fractionated by chitin-affinity chromatog- raphy, and the resulting eluate was lyophilized, dissolved in 10% (v ⁄ v) acetic acid, and applied to a Sephadex G-50 (Pharmacia Fine Chemicals, Uppsala, Sweden) column (2.7 · 105 cm) equilibrated with 10% (v ⁄ v) acetic acid. Fig. 8. TGase-dependent protein cross-linking of cuticular chitin- binding proteins. Lane 1, nontreated cuticular proteins; lane 2, cuticular proteins + TGase; lane 3, cuticular proteins + TGase + DCA; lane 4, cuticular proteins + TGase + EDTA; lane 5, otherwise identical to lane 3 but illuminated by UV light. Cuticular proteins in horseshoe crabs M. Iijima et al. 4782 FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS Eluted fractions were analyzed by 15% SDS ⁄ PAGE, and the fractions containing proteins with molecular masses of less than 10 kDa were lyophilized, and subjected to reverse-phase HPLC. 2D SDS/PAGE The chitin-binding proteins purified by chitin-affinity chro- matography were reduced, S-alkylated with iodoacetamide, and an aliquot was precipitated with trichloroacetic acid. The precipitates were dissolved in 350 lL of 2% IPG buffer (pH 3–10) (Amersham Pharmacia Biotech, Uppsala, Swe- den) containing 8 m urea, 2% Chaps, 65 mm dithiothreitol, and a trace of bromophenol blue, and then applied to the IPG strip (18 cm, pH 3–10NL). The strip was covered with silicone oil and rehydrated overnight. The proteins were focused at 20 °C, according to the following voltage gradi- ent program: 500 V, 2 h; 700 V, 1 h; 1000 V, 1 h; 1500 V, 1 h; 2000 V, 1 h; 2500 V, 1 h; 3000 V, 1 h; 3500 V, 10 h, using a Multi Drive XL electrophoresis power supply. The strip was then equilibrated for 15 min in 50 mm Tris ⁄ HCl, pH 6.8, 6 m urea, 30% (v ⁄ v) glycerol, 2% (w ⁄ v) SDS, and 10 mgÆmL )1 dithiothreitol, then treated with 25 mgÆmL )1 iodoacetamide for 15 min to alkylate free cysteine residues. Resolution of proteins in the second dimension was per- formed by SDS ⁄ PAGE (8–18%), according to the manu- facturer’s instructions. Proteolytic digestion and reverse-phase HPLC High molecular mass (HMM) chitin-binding proteins separ- ated by 2D SDS ⁄ PAGE were transferred to polyvinylidene difluoride membranes overnight at 20 V using an electro- blotting apparatus (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was stained with Coomassie Brilliant Blue R-250, and major spots were excised. Proteins were digested on the membrane with TPCK-trypsin (Worthing- ton Biochemical Corporation, Freehold, NJ, USA) in a buffer consisting of 100 mm NH 4 HCO 3 , pH 7.8, 10 mm CaCl 2 and 10% acetonitrile at 25 °C for 16 h. Peptides in digested samples and LMM chitin-binding proteins were resolved by reverse-phase HPLC, using a Cosmosil 5C 18 - MS column (2.0 · 150 mm, Nacalai Tesque Inc., Kyoto). Peptides were eluted from the column with a linear gradient of 0–72% acetonitrile in 0.1% trifluoroacetic acid for 120 min at a flow rate of 0.2 mLÆmin )1 with effluent monit- oring at 210 nm. Amino acid composition and sequence analyses Amino acid analysis was analyzed using the AccQ-Tag system (Waters Corp., Milford, MA, USA). Amino acid sequence analysis was performed using an Applied Biosys- tems 491 protein sequencer. Purification of mRNA and cDNA synthesis Purification of mRNA derived from cuticular epidermal cells was performed using a QuickPrep mRNA Purification Kit (Amersham Pharmacia Biotech). The synthesis of double- stranded cDNA was performed using a SuperScript TM III RNase H – reverse transcriptase kit (Invitrogen Corp., Carls- bad, CA, USA), according to the manufacturer’s instruc- tions. Amplification of cDNA fragments of the chitin- binding proteins The sequences of degenerate oligonucleotide primers used for RT-PCR were based on amino acid sequences identified Fig. 9. TGase-dependent incorporation of DCA into chitin-binding proteins on 2D-SDS gel. DCA incorporation was examined in the presence of 10 m M CaCl 2 and 10 mM dithiothreitol. Samples were subjected to 2D SDS ⁄ PAGE after incubation at 37 °C for 1 h, and illuminated by UV light. M. Iijima et al. 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Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase-dependent cross-linking Manabu. protein cross-linking of cuticular chitin- binding proteins. Lane 1, nontreated cuticular proteins; lane 2, cuticular proteins + TGase; lane 3, cuticular proteins