Báo cáo khoa học: Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase-dependent cross-linking potx

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:
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. Cuticular proteins in horseshoe crabs
FEBS Journal 272 (2005) 4774–4786 ª 2005 FEBS 4783
by N-terminal sequencing of intact proteins as well as on
those identified by sequencing of peptides derived from
TPCK-trypsin digestion. Sense and antisense oligonucleo-
tides were synthesized with an EcoRI site at the 5¢ end.
PCR was performed according to standard procedures with
a Takara PCR thermal cycler using an amount of cDNA
template corresponding to 0.1 lg of poly(A)
+
RNA, and
20 pmol of each primer. PCR products were treated with
EcoRI, resolved on agarose gels and extracted using stand-
ard techniques. DNA fragments were then ligated into the
plasmid Bluescript II SK
+
(Stratagene, La Jolla, CA, USA)
and subjected to DNA sequence analysis [62].
Rapid amplification of cDNA ends (RACE)
Analysis by 5¢- and 3¢-RACE was performed using a
SMART
TM
RACE cDNA amplification kit (Clontech
Laboratories, Palo Alto, CA, USA). The 5¢- and 3¢-ends of
each chitin-binding protein-encoding cDNA were amplified

using universal primers provided by the manufacturer and
the Advantage 2 PCR kit (Clontech Laboratories), accord-
ing to the manufacturer’s instructions [63].
Homology search
A computer-assisted homology search was performed using
the blast search algorithm at the National Center for Bio-
technology Information (NCBI) [64].
Extraction of RNA from various tissues and
RT-PCR
Total RNA was extracted from various tissues of T. trident-
atus, according to the method of Chomczynski and Sacchi
[65]. First-strand cDNA synthesis from 0.1 lg of poly(A)
+
RNA was performed using SuperScript III RNase H
–
reverse transcriptase (Invitrogen) and random primers. One
two-hundredth of the resulting first strand cDNA and
10 pmol of each primer were subjected to PCR (40 cycles)
with denaturation at 94 °C for 5 s, annealing at 50 °C for
10 s, and. extension at 72 °C for 10 s. PCR products were
analyzed on a 3% agarose gel and visualized by ethidium
bromide staining.
Analysis of TGase-dependent DCA incorporation
by 2D SDS/PAGE
Cuticular chitin-binding proteins were incubated with horse-
shoe crab TGase in a buffer consisting of 50 mm Tris ⁄ acet-
ate, pH 7.5, 10 mm CaCl
2
,10mm dithiothreitol, and 0.5 mm
DCA at 37 °C for 1 h according to the method of Osaki et al.

[57]. Following incubation, aliquots were subjected to 2D
SDS ⁄ PAGE and the fluorescence-labeled proteins were visu-
alized by a transilluminator (Atto Corp., Tokyo, Japan).
Tricine-SDS ⁄ PAGE
Tricine ⁄ SDS ⁄ PAGE was performed according to the
method of Scha
¨
gger and von Jagow [66], and gels were
stained with Coomassie Brilliant Blue R-250.
Acknowledgements
We thank M. Kawabata and N. Ichinomiya-Sato for
expert technical assistance. We also thank Dr John
Kulman (University of Washington, Seattle, WA,
USA) and Dr Takumi Koshiba (Kyushu University)
for helpful discussions and suggestion on this manu-
script. This work was supported by a Grant-in-Aid
for Scientific Research on Priority Area 839 from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan (to SK).
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