Acanthoscurrin: a novel glycine-rich antimicrobial peptide
constitutively expressed in the hemocytes of the spider
Acanthoscurria gomesiana
Daniel M. Lorenzini
a
, Pedro I. da Silva Jr.
b
, Andre´a C. Fogac¸a
a
, Philippe Bulet
c,1
,
Sirlei Daffre
a,
*
a
Departamento de Parasitologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes,
1374, Sa˜o Paulo CEP 05508-900, Brazil
b
Laborato´rio de Artro´podes, Instituto Butantan. Av. Vital Brazil, 1500, CEP 05503-900, Sa˜o Paulo, Brazil
c
Institut de Biologie Mole´culaire et Cellulaire, UPR 9022, CNRS, ‘Re´ponse Immunitaire et De´veloppement chez les Insectes’,
15, rue Rene´ Descartes, 67084 Strasbourg, France
Received 25 November 2002; revised 4 March 2003; accepted 6 March 2003
Abstract
We report the isolation of a novel antimicrobial peptide, acanthoscurrin, from the hemocytes of unchallenged tarantula spider
Acanthoscurria gomesiana. A combination of Edman degradation, mass spectrometry and cDNA cloning revealed the presence
of two isoforms of acanthoscurrin, differing by two glycine residues. Both displayed cationic properties and a high percentage
of glycine residues. However, acanthoscurrins have no structural similarities with already known glycine-rich antimicrobial
peptides from animals and plants. As deduced from cDNA cloning and mass spectrometry, the amino acid sequence of
acanthoscurrin begins with a putative signal peptide of 23 amino acids followed by the mature peptide, which is post-
translationally modified by a C-terminal amidation. Acanthoscurrins are constitutively expressed in hemocytes and released to
plasma following an immune challenge.
q 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Antimicrobial peptide; Glycine-rich peptide; Hemocyte; Spider; cDNA; Cloning; Secreted
1. Introduction
Invertebrates have developed efficient mechanism
against microbial infection through the production of
potent antimicrobial peptides and polypeptides
(AMP). During the past two decades, several AMPs
have been isolated and characterized from inverte-
brates including insects, horseshoe crabs, mollusks,
shrimps, and arachnids [1]. Due to the extreme
diversity in their primary structure, AMPs can be
classified according to their amino acid composition
and 3D structure, into three main classes: (i) linear
peptides forming amphipathic and hydrophobic a-
helices, (ii) open-ended cyclic peptides with one or
0145-305X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0145-305X(03)00058-2
Developmental and Comparative Immunology 27 (2003) 781–791
www.elsevier.com/locate/devcompimm
1
Present address: Atheris Labs, Case Postale 314, CH-1233
Bernex, Geneva, Switzerland.
*
Corresponding author. Fax: þ55-11-30917417.
E-mail address: sidaffre@icb.usp.br (S. Daffre).

more cysteine bonds, which form b-sheet or a-helix/
b-sheet structures, and (iii) peptides with an over-
representation of particular amino acids such as
proline or/and glycine residues [2].
In invertebrates, AMPs can be produced differently
depending on the species. In holometabolous insects,
AMPs are produced predominantly in the fat body, a
functional equivalent of the mammalian liver, and
their synthesis is induced within few hours following
an infection and then released into the hemolymph. In
contrast, in hemimetabolous insects, such as the
fungus-growing termite Pseudacanthotermes spiniger
(Isoptera), AMPs were found to be constitutively
present in granular hemocytes [3]. A similar pattern
occurs in other invertebrates such as mussels, shrimps
and horseshoe crabs, where AMPs are stored in
hemocyte granules, and released into the hemolymph
[4,5] and/or fused with phagocytic vesicles following
an infection [6]. In arachnids, several AMPs were
characterized in the venom of scorpions and spiders.
Two peptides with antimicrobial properties were
isolated from scorpion venom, scorpine from Pandi-
nus imperator [7] and hadrurin from Hadrurus
aztecus [8]. Scorpine is a 75-amino acid polypeptide
with three disulfide bridges, no sequence similarity
with known molecules, and with antimalarial and
antibacterial properties. Hadrurin is a 41 amino-acid
peptide with no cysteine residues and adopting a two-
a-helical structure. This peptide has antimicrobial
properties at low micromolar concentration and also
shows some hemolytic activity. In addition, several
linear AMPs with no cysteine residues were isolated
from spider venom. Two peptide toxins, lycotoxins I
and II (25 and 27 amino acids, respectively), were
isolated from wolf spider Lycosa carolinensis [9].
Besides the antimicrobial activity, both peptides were
able to dissipate ion and voltage gradients across the
membrane of excitable cells and, therefore, contribute
to paralysis of the prey. From Cupiennius salei
venom, a family of peptides with 35 amino acids,
cupiennin 1, showed antimicrobial and insecticidal
activities [10]. Five AMPs isolated from wolf spider
Oxyopes kitabensis, the oxyopinins, exhibited both
hemolytic and insecticidal activity [11]. Oxyopinin 1
is a 48-amino acid peptide while oxyopinins 2a–2d
are composed of 37 amino acid residues. They showed
sequence similarity to the ant insecticidal peptide
ponericinL2 [12].
In contrast to venom AMPs, the presence of such
molecules has been poorly investigated in the
hemolymph of arachnids. Only few reports have
been published. Insect defensin-like molecules were
identified in the hemolymph of unchallenged scor-
pions of the species Leiurus quinquestriatus and
Androctonus australis [13,14]. The scorpion defen-
sins are mainly active against Gram-positive bacteria.
Besides defensins, two other cysteine-rich AMPs have
been characterized from A. australis: buthinin and
androctonin [14]. Buthinin is a 34-amino acid peptide
with three internal disulfide bridges with no sequence
similarity to known molecules, and is active against
Gram-positive and Gram-negative bacteria. In con-
trast, androctonin is a 25-residue peptide with two
disulfide bridges, showing sequence similarities to
tachyplesin and polyphemusin from horseshoe crabs
[15,16]. Androctonin was found to be active against
bacteria and fungi.
Recently, we have purified a small sized anti-
microbial peptide, named gomesin, from the hemo-
cytes of the unchallenged mygalomorph spider
Acanthoscurria gomesiana [17]. Gomesin, with its
18 amino acids and two disulfide bridges, adopts in
water a well-defined b-hairpin-like structure as
determined by 2-D NMR and molecular dynamics
studies [18]. This open-ended cyclic peptide shows
marked sequence and structural similarities to tachy-
plesin and polyphemusin from horseshoe crabs
[19–22] and to androctonin from the scorpion A.
australis [14,23]. Interestingly, gomesin shares also
structural similarities with protegrins, antimicrobial
peptides from porcine leucocytes [21,22,24].
In the present study, we report the isolation and
structural characterization of a novel family of AMPs,
acanthoscurrin, from the tarantula spider A. gomesi-
ana. Two isoforms of acanthoscurrin were found in
the hemocytes of unchallenged spiders. They are
cationic peptides containing a high percentage of
glycine residues. Mass spectrometry and cDNA
sequence analysis revealed that acanthoscurrin results
from the processing of a large precursor containing a
signal peptide immediately followed by the mature
acanthoscurrin. In addition, acanthoscurrins were
found to be C-terminally amidated. We also showed
that acanthoscurrins are constitutively synthesized in
the hemocytes and released into the plasma after an in
vivo immune challenge.
D.M. Lorenzini et al. / Developmental and Comparative Immunology 27 (2003) 781–791782