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

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

[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

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

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

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

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

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-

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

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.

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

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

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

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

[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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