The male seahorse synthesizes and secretes a novel C-type
lectin into the brood pouch during early pregnancy
Philippa Melamed, Yangkui Xue, Jia Fe David Poon, Qiang Wu, Huangming Xie, Julie Yeo,
Tet Wei John Foo and Hui Kheng Chua
Department of Biological Sciences, National University of Singapore, Singapore
The seahorse (Hippocampus) species, which are highly
sought after for both ornamental and traditional Chi-
nese medicine purposes, are in danger of extinction and
their culture presents unique problems in aquaculture,
particularly in rearing of the young. The seahorse
belongs to the Syngnathidae family of fish, which
includes also the pipefish, pipehorses and seadragons. In
all of these, the males incubate the young on or within
their bodies. In the seahorse, this incubation resembles a
true male pregnancy, as the female deposits her eggs into
an enclosed brood pouch on the ventral side of the
male’s abdomen. This brood pouch comprises epithelial
and stoma-like tissue which lines a thick muscular wall.
The epithelium thickens and becomes more vascularized
as the reproductive season approaches (Fig. 1). After
uptake and fertilization of the eggs, the pouch is sealed
and the developing embryos become embedded in the
epithelium. Each embryo becomes compartmentalized
as the epithelium forms a surrounding pit in which it
remains until after yolk absorption is complete [1]. The
embryos continue to develop and grow for several weeks
(depending on the species) until they are able to with-
stand the external environmental conditions independ-
ently, at which point the juveniles are released.
Although appearing to be a true male pregnancy, in
contrast to mammals but comparable to most other

shown, using Western analysis and 2D electrophoresis, that this protein is
secreted in significant quantities into the pouch fluid specifically during early
pregnancy. Preliminary functional studies indicate that this CTL causes
erythrocyte agglutination and may help to repress bacterial growth.
Abbreviations
AP, alkaline phosphatase; CTL, C-type lectin; CRD, carbohydrate recognition domain; 2DE, 2D gel electrophoresis; DIG, digoxygenin; hcCTL,
Hippocampus comes C-type lectin; HRP, horseradish peroxidase; IPG, immobilized pH gradient; LB, Luria–Bertani; MBP, mannose binding
protein; NBT ⁄ BCIP, Nitro Blue tetrazolium 5-bromo-4-chloroindol-2-yl-phosphate.
FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS 1221
teleost fish, these fry appear to obtain most of their
nutrition from the yolk sac [2]. Instead, the father’s role
seems to be related to providing a suitable osmotic envi-
ronment for the young, while also supplying oxygen and
calcium, and presumably removing waste products [3,4].
Histological studies have demonstrated the presence of
mitochondria-rich cells in the epithelia lining the pouch
which are postulated to act as ion transporters, as they
do in the gills; the number of these increases with dur-
ation of the incubation period, after which they undergo
apoptosis [4]. In the gills, these cells contain receptors to
prolactin which is one of the major piscine osmoregula-
tory hormones [4,5], and also has a central role in
governing parental behaviour in most animals. The
presence of prolactin receptors in the brood pouch,
however, has yet to be reported.
The aim of this project was to construct and charac-
terize a cDNA library made from the epithelium and
stroma-like tissue lining the incubation pouch, in order
to help understand the molecular mechanisms regula-
ting the development and function of this unique male

tions of potassium, is expressed specifically in immune
and hematopoietic tissue in carp and shares some
homology with other members of the astacin or fish
hatching enzyme family [6].
By far the most common inserts, however, were
cDNAs encoding proteins with homology to various
C-type lectins (CTL); these comprised inserts in over
15% of all of the clones sequenced.
Fig. 1. Morphology of the seahorse brood
pouch. (A) The brood pouch consists of a
muscular wall (#) which is lined with an
easily detachable layer of stroma (*) and
epithelium (e) which extends towards the
incubation cavity. (B) By the time the male
is ready to receive the female eggs, the epi-
thelium has thickened and is well vascular-
ized (arrow marks blood vessels). (C) With
uptake and fertilization of the eggs, the epi-
thelium becomes more extensive and enve-
lopes the developing embryos (Em). (D) By
the time the fully developed young seahors-
es are hatched and getting ready to leave
the pouch, this tissue has thinned consider-
ably.
C-type lectins in the male seahorse pregnancy P. Melamed et al.
1222 FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS
Table 1. Identified cDNA clones from male seahorse brood pouch, based on gene and ⁄ or protein comparisons.
Clone Gene Protein Accession number
YK1 Beta globin [Oryzias latipes] (4e-87) Adult beta-type globin [O. latipes] (1e-54) CV863925
YK2 Serum lectin isoform 1 precursor [Salmo salar] (1e-23) CV863926

[Oncorhynchus mykiss] (2e-09)
Galectin like protein [O. mykiss] (3e-56) CV863943
YK41 Adult beta type globin
[O. latipes] (6e-79)
Adult beta type globin [O. latipes] (3e-55) CV863944
YK43 Actin related protein 2
homolog [X. laevis] (2e-13)
CV863945
YK45 Transferrin [Melanogrammus aeglefinus] (1e-25) CV863946
YK46 Mannose receptor precursor [G. gallus] (2e-05) CV863947
YK47 Actin-like protein [G. gallus] (8e-93) CV863948
YK49 Novel protein similar to vertebrate mitochondrial enoyl
Coenzyme A hydratase 1 (ECHS1) [D. rerio] (2e-39)
CV863949
YK50 C-type lectin 2 [A. japonica] (4e-15) CV863950
YK51 Cytochrome c oxidase subunit II [Exocoetus volitans] (1e-110) CV863951
YK52 Serotransferrin precursor [O. latipes] (3e-67) CV863952
YK54 Brevican core protein [M. musculus] (2e-15) CV863953
YK55 Brevican core protein [M. musculus] (2e-15) CV863954
YK56 Neurocan core protein precursor [H. sapiens] (9e-11) CV863955
YK57 Transketolase [P. flesus] (1e-15) CV863956
YK59 Similar to ADP-ribosylation factor 2 [M. musculus] (2e-05) CV863957
YK61 DJ-1 [S. salar] (3e-31) Similar to DJ-1 protein [M. musculus] (5e-69) CV863958
YK62 FC-epsilon RII [H. sapiens] (5e-06) CV863959
YK63 Ribosomal protein L23
[Gillichthys mirabilis] (1e-24)
60S ribosomal protein L23 [H. sapiens] (5e-22) CV863960
YK64 Microsatellite marker
[Poecilia reticulata] (8e-54)
CV863961

clone XX-184L24
[D. rerio] (1e-12)
Novel protein [D. rerio] (1e-46) CV863979
YK92 Retinoic acid binding
protein 1-cellular
[H. sapiens] (1e-18)
Retinoic acid binding protein 1-cellular [T. rubripes] (4e-60) AY437393
YK95 Metalloproteinase inhibitor 4 precursor [R. norvegicus] (4e-13) CV863980
YK98 NIKs-related kinase
[H. sapiens] (8e-08)
Traf2 and NCK interacting kinase [H. sapiens] (2e-14) CV863981
YK99 Ferritin heavy subunit
[S. salar] (6e-16)
Selenocysteine methyltransferase [Astragalus bisulcatus] (5e-12) CV863982
YK102 Ribosomal protein L21
[I. punctatus] (5e-39)
Ribosomal protein L21 [I. punctatus] (1e-55) AY357070
YK103 EF1alpha [Drosophila
melanogaster] (1e-36)
CV863983
YK104 Ribosomal protein L35
[I. punctatus] (7e-29)
60S ribosomal protein L35 [Sus scrofa] (1e-42) AY357071
WQ4 Cytochrome c oxidase polypeptide subunit VIb
[H. sapiens] (4e-14)
CV863984
WQ5 Cytochrome c oxidase subunit I [Mugil cephalus] (1e-11) CV863985
WQ6 Programmed cell death 6
[M. musculus] (1e-06)
Programmed cell death protein 6 [M. musculus] (2e-37) CV863986

+
transporting, mitochondrial F1 complex,
O subunit [B. taurus] (9e-52)
CV863996
WQ36 C-type lectin 2 [A. japonica] (4e-10) CV863997
WQ39 Ribosomal protein L38
[Branchiostoma belcheri] (2e-24)
Similar to ribosomal protein L38, cytosolic [R. norvegicus] (7e-14) CV863998
WQ40 Cytochrome c oxidase subunit II [E. volitans] (3e-70) CV863999
WQ42 Chromosome 20 ORF 42
(C20orf42) [H. sapiens] (1e-06)
Protein c20orf42 homolog [M. musculus] (1e-69) CV864000
WQ43 Transferrin [O. latipes] (0.59) Transferrin [O. latipes] (3e-13) CV864001
WQ44 C-type mannose-binding lectin [O. mykiss] (3e-09) CV864002
WQ51 FC-epsilon-RII (9e-07) CV864003
WQ52 Ferritin heavy subunit
[Oreochromis mossambicus] (2e-64)
Ferritin H [S. salar] (1e-67) CV864004
WQ56 Ribosomal protein L18
[Oreochromis niloticus] (5e-16)
Ribosomal protein L18 [S. salar] (2e-08) CV864005
WQ59 Ferritin heavy subunit
[S. salar] (2e-60)
Ferritin heavy subunit; ferritin H [S. salar] (4e-56) CV864006
WQ60 Villin 2 [ezrin] (VIL2)
[B. taurus] (6e-09)
Ezrin [G. gallus] (2e-23) CV864007
WQ62 40S ribosomal protein S28
[I. punctatus] (2e-6)
40S ribosomal protein S28 [I. punctatus] (7e-12) AY357067

WQ82 Hypothetical protein LOC51255
[D. rerio] (9e-11)
Zinc finger protein 364 [M. musculus] (7e-11) CV864060
WQ83 Transferrin [O. latipes] (0.52) Transferrin [Salvelinus namaycush] (5e-10) CV864061
WQ86 Metalloproteinase inhibitor 2 precursor (TIMP-2) [Canis familiaris] (1e-22) CV864062
WQ87 cAMP responsive element binding protein-like 2 [H. sapiens] (1e-21) CV864056
WQ89 Lectin C-type [C. elegans] (1e-06) CV864017
WQ90 Elongation factor 1-alpha
[Sparus aurata] (2e-22)
CV864018
WQ93 C-type lectin 2 [A. japonica] (5e-12) CV864019
WQ95 Transferrin Salmo trutta (4e-31) CV864020
WQ97 Lectin C-type domain containing protein [C. elegans] (3e-15) CV864021
WQ99 Serum lectin isoform 1 precursor [S. salar] (1e-15) CV864022
P. Melamed et al. C-type lectins in the male seahorse pregnancy
FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS 1225
Table 1. (Continued).
Clone Gene Protein Accession number
WQ100 Lectin C-type domain containing protein precursor family member
[C. elegans] (2e-15)
CV864023
WQ101 Similar to opioid receptor, sigma 1 [D. rerio] (1e-42) CV864024
WQ102 Cyclophilin A
[Canis familiaris] (2e-18)
Peptidylprolyl isomerase F (cyclophilin F) [H. sapiens] (6e-47) CV864025
WQ104 40S ribosomal protein S30
[I. punctatus] (1e-42)
40S ribosomal protein S30 [I. punctatus] (7e-51) AY357069
WQ105 C-type lectin 2 [A. japonica] (1e-11) CV864026
WQ106 ADP,ATP translocase

[I. punctatus] (4e-99)
Ribosomal protein L19 [I. punctatus] (2e-64) CV864038
WQ131 Ribosomal protein L31 mRNA
[P. olivaceus] (1e-126)
60S ribosomal protein L31 [P. olivaceus] (2e-46) CV864039
WQ133 Cisplatin resistance related protein
mRNA Length ¼ 2058
[M. musculus] (3e-60)
CRR9p (Crr9-pending), Crr9-pending protein
[M. musculus] (2e-64)
AY437395
WQ134 Machado-Joseph disease protein 1 (Ataxin-3) [M. musculus] (6e-63) CV864040
WQ135 Cytochrome c oxidase subunit VIII
liver form (COX8L) mRNA
[Trachypithecus cristatus] (0.054)
Cytochrome c oxidase subunit VIII liver form [Eulemur fulvus] (9e-08) CV864041
WQ136 Mannose receptor, C type 2; novel lectin [M. musculus] (4e-07) CV864042
WQ137 Eukaryotic translation initiation
factor gamma 2, subunit 3
[D. rerio] (3e-33)
Eukaryotic translation initiation factor 2G; eukaryotic translation
initiation factor 2, subunit 3 (gamma, 52 kDa) [H. sapiens] (1e-99)
CV864043
WQ138 Fatty acyl-CoA hydrolase precursor, medium chain
(thioesterase B) [Anas platyrhynchos] (4e-48)
CV864044
WQ139 Transferrin [O. latipes] (0.85) Transferrin [Oncorhynchus nerka] (4e-31) CV864045
WQ140 RAB26, member RAS
oncogene family (Rab26),
mRNA [R. norvegicus] (1e-07)

et al. [9], as well as six conserved cysteines (Fig. 2A).
The secondary structure of hcCTL III is predicted to
form two helices at the N-terminal end, eight strands
and three disulphide bridges (Fig. 2B). The five residues
crucial in determining mannose binding specificity [10]
are absent in all of the hcCTLs (Fig. 2B), although the
hcCTL II and most of the other aligned CTLs contain
the QPD motif endowing galactose specificity (Fig. 2A).
However, the highly conserved proline contained within
QPD is found in all the CTLs shown (Figs 2A and B).
In situ hybridization confirmed the specific expres-
sion of the hcCTL III in the tissue lining the brood
pouch. Using a digoxygenin (DIG)-labelled 300-bp
fragment of the cDNA, a particularly strong signal
was seen in the stroma-like pouch lining which exten-
ded in the cavity along the epithelial protrusions that
surround the developing embryos. The negative control
completely lacked this signal (Figs 3A and B).
2D gel electrophoresis reveals that hcCTL III
is secreted into the brood pouch
To verify that the hcCTLs are indeed secreted into the
pouch cavity, and to examine other proteins present in
the fluid surrounding the embryos, the proteome of the
pouch fluid of a single incubating male was examined
using 2D gel electrophoresis (2DE) over a pI range of
3–10. After silver staining, several proteins were vis-
ible, the most prominent of which had a low pI and
an apparent relative molecular mass just over 15 kDa
(Fig. 4); this matches the predicted relative molecular
mass (16 kDa) and pI (4) of the hcCTLs identified in

WQ158 Lectin C-type domain containing protein precursor family
member [C. elegans] (1e-09)
CV864052
WQ159 C-type lectin 2 [A. japonica] (4e-15) CV864053
WQ162 Alpha tubulin mRNA
[Notothenia coriiceps] (1e-134)
Tubulin alpha chain [Notophthalmus viridescens] (3e-64) CV864054
WQ166 Ribosomal protein L28 mRNA
[I. punctatus] (1e-49)
60S ribosomal protein L28 [H. sapiens] (7e-55) AY437397
WQ168 S6 ribosomal protein mRNA
[O. mykiss] (1e-123)
40S S6 ribosomal protein [O. mykiss] (8e-63) CV864055
P. Melamed et al. C-type lectins in the male seahorse pregnancy
FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS 1227
Fig. 5A, lane 3 after elution from Ni-NTA affinity col-
umn) was used to raise antisera in rabbits. The anti-
sera from one of the rabbits was highly specific,
reacting with only a single sized protein in the pouch
fluid of a pregnant but not a nonpregnant seahorse
(Fig. 5B), this reactive protein was not apparent when
A
B
Fig. 2. Three novel H. comes brood pouch C-lectins are homologous with similar proteins from other species and show conserved structural
constaints. (A) The three CTLs identified from screening of the pouch cDNA library (HcI, HcII and HcIII) are aligned with five CTL protein
sequences found in whole body extracts of H. kuda (H00011, H00359, H00385, H00386, H00395 [8]) and two isolated from the gills of the
Japanese eel (Eel1, Eel2 [7]). All of the H. comes and eel CTLs and one H. kuda CTL (H00386) contain a signal peptide (underlined). Con-
served residues of CTLs, as defined by Weis et al. [9] are shown in bold; the six cysteines are marked with asterisks, and the QPD motif
determining galactose binding, where present, is boxed. (B) The predicted structure of hcCTL III, comprising two helices at the N terminus
(marked in bold), eight strands (S1–S8; underlined: both predicted using

tination assay. Concentrations of 2.25–18 lm of the
hcCTL III were able to agglutinate mouse red blood
cells after 1–1.5 h of incubation (Fig. 7A). In an
attempt to identify the sugars bound by the lectin, the
same assay was repeated after addition of various
mono-, di- and complex carbohydrates, including
mannose, galactose, glucose, maltose, sucrose, fructose,
raffinose, N-acetyl glucosamine and N-acetyl galactosa-
mine, using hcCTL III at a final concentration of
4.5 lm. However none of these was able to inhibit the
agglutination, even at a concentration of 100 mm
(Fig. 7B and not shown).
Discussion
We have created and partially characterized a cDNA
library comprising genes expressed in the epithelium
and stroma-like tissue lining the male seahorse brood
pouch. The profile indicates a high level of expression
of genes encoding proteins involved in metabolism and
transport, as well as structural proteins, gene regula-
tory proteins, and other proteins whose function is
Fig. 3. Confirmation of expression of hcCTL III in the pouch tissue
by in situ hybridization. (A) H. comes pouch tissue was formalin-
fixed and paraffin-embedded before sectioning at 6–8 l
M. The
cDNA for the novel hcCTL III was labelled with DIG and detected
using AP-conjugated antisera and NBT ⁄ BCIP, to give a dark purple
reaction product (*). (B) The negative control, which lacks the same
intense staining, is also shown.
Fig. 4. 2DE of the brood pouch fluid proteome reveals that
hcCTL III is secreted. The incubation fluid that surrounds the sea-

the end of the CRD, the second is shorter and located
at the C-terminal end of the CRD, and the third is
found towards the N-terminal end and spans the first
strand; the latter is lacking in the short (i.e. 115 resi-
due) form. All of the cysteines forming these bridges
are found in the conserved locations in the novel
hcCTLs, as are the positions of the two a helices.
Fig. 6. The novel hcCTL III inhibits growth of E. coli. E. coli cells
(1 mL at an D
595
of 0.1) were incubated with recombinant hcCTL III
at 0.7 l
M, or vehicle alone, for up to 2 h, and D
595
readings taken
every 30 min to assess the rate of bacterial growth. The D values
were calculated relative to the initial readings in the same samples.
An asterisk denotes mean values statistically different (Welch two-
sample t-test, P < 0.05) in hcCTL-treated and control samples
(mean ± SEM, n ¼ 4).
A
B
Fig. 7. The hcCTL III causes erythrocyte agglutination which is not
inhibited by common sugars. (A) A haemagglutination assay was
carried out to test the ability of the hcCTL III to cause erythrocyte
agglutination. After 1 h incubation of mouse erythrocytes with
hcCTL III at 2.25–18 l
M, plaque formation resulted indicating ability
of the hcCTL III to cause agglutination which was absent in the
control samples. (B) In order to verify the carbohydrates recognized

Furthermore, a highly conserved proline positioned to
induce a turn just before the second disulphide bridge
[11] is also conserved. In the mannose binding protein
(MBP) this is thought to position the flanking side
chains so allowing the correct fold of the CRD to faci-
litate Ca
2+
binding. The presence of these structural
constraints supports the likelihood that the novel
hcCTLs are indeed functional lectins.
Lectins are classified into seven groups, according to
their structural arrangement, including the number and
position of the CRD in relationship to the other func-
tional domains [12]. The CTLs revealed in the current
study belong to group VII, as they contain just a single
CRD and a signal peptide, and are clearly secreted
from the cell. Many of these CTLs, as well as those in
group III (i.e. the collectins, including the MBP), have
been implicated in innate immunity which provides a
rapid first line of defence to help prevent pathogen
penetration. An essential part of this response is the
lectin’s recognition of the pathogen’s cell-surface car-
bohydrates as nonself, in order to target the pathogens
for destruction (e.g. [13,14]). This innate response
appears particularly important in lower vertebrates in
which the acquired immune response may be less well
developed than in mammals [15]. Indeed, lectins of this
type have been found in abundance in mucous of the
skin, gills and intestine, as well as in the blood of sev-
eral species of fish, and some of these have been impli-

mannose, and a change of the motif to QPD alters the
binding preference to galactose. Although the latter
motif was apparent in the novel hcCTL II, the other
two novel hcCTLs conform to neither. This is in agree-
ment with our finding that hcCTL III-mediated eryth-
rocyte agglutination was not inhibited by galactose or
mannose, or any of the other sugars that we tested,
and suggests that it may be a novel type of CTL pos-
sibly possessing highly specific carbohydrate recogni-
tion. The fact that these hcCTLs in the brood pouch
lack significant similarity with the other CTL reported
in related teleosts further supports the likelihood that
they contain distinct functions, which may be specific
to their role in this distinctive organ. A general lack of
conservation amongst CTLs across species has previ-
ously been noted, as their evolution is thought to have
paralleled that of complex oligosaccharides on cell sur-
face glycoproteins and glycolipids [10].
A number of publications have reported the expres-
sion of high levels of CTLs in fish gonads. These gen-
erally show binding specificity for rhamnose, but also
recognize related sugars containing hydroxyl groups at
C2 and C4 [22–26]. Some of these have been implica-
ted in innate immunity as they inhibit growth of cer-
tain bacteria, however, they also share homology with
the vitellogenic receptor ligand biding domain and
interact with yolk proteins [27]. These are thus thought
to comprise a distinct family of lectins related to the
low density lipoprotein receptor superfamily. They
generally comprise two or three repeats of the CRD

regulates the levels of synthesis and secretion of these
proteins. To date there is little, if any, information on
the ways in which such communication occurs during
this unique pregnancy. Notable however, is that the
drop in hcCTL III levels in the pouch parallels an
increase in osmolality of the pouch fluid, which reaches
that of the seawater just prior to release of the young.
This correlation was also seen in the levels of Japanese
eel CTLs in the gills which are markedly elevated in
fish held in freshwater conditions [7]. That study also
attributed a likely function of the CTLs to a role in
innate immunity, as microorganisms may be more
abundant in freshwater than in seawater conditions,
and it was suggested their presence in the gill mucous
forms a protective layer between the water and the epi-
thelium. Although the Japanese eel CTLs appear to be
galactose specific, it is possible that their regulation
shares some common elements with the hcCTLs, per-
haps as a direct result of the increased salinity.
Our study has thus revealed a novel CTL that is
produced and secreted in significant quantities into the
male H. comes brood pouch in a regulated manner
during specific stages of pregnancy. Preliminary func-
tional studies indicate that this CTL causes cell agglu-
tination and may act to help repress bacterial growth,
but that this is not via the common lectin-binding
sugars. Further studies will be required to elucidate its
precise mechanisms of action and the ways in which its
expression is regulated.
Experimental procedures

while serial dilutions of primary cDNA library were incuba-
ted with bacteria at 37 °C for 15 min before plating of the
mixtures onto Luria–Bertani (LB) top agar plates.
After 10
7
pfu of the phage, 10
8
XL1-Blue MRF¢ cells and
10
9
pfu of ExAssist helper phage were incubated at 37 °C for
15 min, 20 mL of LB broth was added and the mixture incu-
bated for 2.5 h at 37 °C with shaking. The mixture was hea-
ted at 65 °C for 20 min, spun (1000 g for 10 min) and 1 lLof
the supernatant was added to 200 lL SOLR cells. After
15 min at 37 °C, 100 lL of the cell mixture was plated onto
LB–ampicillin agar plates. Plasmid DNA was isolated from
individual colonies and inserts larger than  500 bp (con-
firmed by colony PCR using T3 and T7 primers, and selected
in order to provide a reasonable chance for accurate identifi-
cation) were sequenced from the plasmid with T3 or T7 prim-
ers using the ABI prism Dye Terminator Cycle Sequencing
Ready Reaction Kit and an ABI PRISM 3100 DNA Se-
quencer (Perkin Elmer Applied Biosystems, Foster City, CA).
Nucleotide sequences were used to search the GenBank
using blastn, and they were also checked in all reading
frames against PDB and ⁄ or SwissProt protein databases
using blastx and blastp (all at NCBI: http://www.
ncbi.nlm.nih.gov/blast/). Insert sequences were also transla-
ted into possible peptides, using specific reading frames

The sections were incubated with 300 lL prehybridization
solution [formamide (25%), sodium citrate (NaCl ⁄ Cit · 4),
Denhardt’s solution, calf thymus DNA (0.5 mgÆmL
)1
), yeast
tRNA (0.25 mgÆmL
)1
), dextran sulfate (10%) and dithio-
threitol (0.01 m)] for 1 h at 37 ° C, followed by washing in
2· NaCl ⁄ Cit. Probes were denatured for 5 min at 95 °C and
immediately cooled on ice, and diluted in the prehybridiza-
tion solution to 200 ngÆmL
)1
. Hybridization was carried out
for 16 h at 42 °C.
The sections were washed in 2· NaCl ⁄ Cit buffer (1 h at
room temperature), 1· NaCl ⁄ Cit buffer (1 h at room tem-
perature) and then 0.5· NaCl ⁄ Cit buffer (30 min at 37 °C,
twice), and subsequently with Buffer 1 (100 mm Tris ⁄ HCl,
150 mm NaCl, pH 7.5) before incubation in fresh Blocking
solution (Buffer 1 containing 2% normal sheep serum,
0.3% Tritron X-100) for 30 min. The Anti-DIG-alkaline
phosphatase (AP) conjugate was diluted · 5000 with Buffer
1 and applied to the sections for 4 h. After washing in Buf-
fer 2 (100 mm Tris ⁄ HCl, 100 mm NaCl, 60 mm MgCl
2
,
pH 9.5), colour solution (500 lL 200 lm Nitro Blue tetra-
zolium 5-bromo-4-chloroindol-2-yl-phosphate (NBT ⁄ BCIP)
stock solution in 10 mL Detection Buffer) was applied and

placing them onto 12% SDS ⁄ PAGE gels and sealing with
molten low-melting agarose (1% in electrode running buf-
fer). Electrophoresis was at 10 mA constant per gel for 1 h
followed by 24 mA constant for about 5 h, at room tem-
perature. The protein spots were visualized by silver-stain-
ing according to Blumb et al. [27].
Protein spots were excised from the gel and minced, before
washing and dehydrating three times with 50% acetonitrile
containing 50 mm ammonium bicarbonate, and acento-
nitrile, before drying. The gel pieces were re-swollen in
15–50 lL of digestion solution [12.5 ngÆlL
)1
trypsin (Prome-
ga, Sequencing grade modified trypsin) in 50 mm ammonium
bicarbonate] and incubated at 4 °C for 30 min. The excess
trypsin solution was removed and 15–50 lLof50mm
ammonium bicarbonate was added, for 15 h at 37 °C.
The supernatants were collected and the gel pieces were
treated with 20 mm ammonium bicarbonate and the super-
natant saved. The gel pieces were then treated with 15–
50 lL of 5% formic acid in 50% acetonitrile for 10 min
and centrifuged at 3800 g. The extracts were saved and the
extraction repeated twice. Lastly, all three supernatants
were combined and dried.
The digests were redissolved in 5 lL of 0.5% formic acid
in 50% acetonitrile and 1 lL of the peptide solution was
applied onto the MALDI plate with a solution of a-cyano-4-
hydroxycinnamic acid (10 mgÆmL
)1
in 50% acetonitri-

agen) were transformed by heat-shock and a single colony
was inoculated in 10 mL LB–ampicillin medium at 37 °C,
250 r.p.m., for 16 h. Subsequently, 3 mL of the culture was
used to inoculate 1500 mL medium, and incubated until the
D
600
was 0.4–0.6. Protein expression was induced by IPTG
(0.1 mm) and cells cultivated for 5 h (37 °C, 250 r.p.m),
before harvesting (3840 g, 15 min) and lysis in 40 mL lysis
buffer (50 mm NaH
2
PO
4
, 300 mm NaCl, 10 mm imidazole
pH 8.0, 1 mgÆmL
)1
lysozyme) with sonication (20 · 10-s
bursts at 200 W, with a 10-s cooling intervals). The lysate
was cleared by centrifugation (33 700 g, 40 min at 4 °C)
and the supernatant collected.
The lectin was purified by addition of 2 mL Ni–NTA
agarose (Qiagen, Valencia, CA) overnight at 4 °C before
loading onto a column and washing with modified wash
buffer (50 mm NaH
2
PO
4
, 300 mm NaCl, 50 mm imidazole
pH 8.0). The protein was eluted in fractions with modified
elution buffer (50 m m NaH

washing, the immunoreactive protein was detected using
the Super Signal Pico West chemiluminescent system
(Pierce Chemical Co., Rockford, IL), followed by exposure
to Cl-XPosure film (Pierce Chemical Co.) for 1–5 min.
Bacteriostatic assays
Bacteriostatic assays were carried out essentially as in
Biswas et al. [28]. LB media (1 mL) was inoculated with
DH5a E. coli and incubated at 37 °C, at 250 r.p.m. for
3–4 h, until D
595
reached 0.1. The recombinant hcCTL III
protein dissolved in NaCl ⁄ P
i
(20 lL) was added to half of
the tubes to a final concentration of 0.7 lm, while the vehi-
cle alone was added to the controls. The OD reading was
subsequently taken every 30 min in four samples for each
group at each time point.
Haemagglutination assays
Heparinized mouse blood was washed three times in
NaCl ⁄ P
i
(at 420 g, 10 min). The mouse erythrocytes [50 lL,
2% (v ⁄ v)] in NaCl ⁄ P
i
were added to the recombinant
hcCTL III (50 lL to final concentration of 0.5–18 lm) and
incubated at 25 °C for 60 min in a V-shaped 96-well plate.
For inhibition assays, the same volume of erythrocytes was
added to the hcCTL III protein (25 lL to final concentra-

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