Báo cáo khoa học: The male seahorse synthesizes and secretes a novel C-type lectin into the brood pouch during early pregnancy pdf

The aim of this project was to construct and characterize a cDNA library made from the tissue lining the pouch, in order to help understand the molecular mecha-nisms regulating its devel

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

Keywords

Hippocampus comes; C-type lectin; cDNA

library; male pregnancy

Correspondence

P Melamed, Department Biological

Sciences, National University of Singapore,

14 Science Drive 4, Singapore 117542

Fax: +65 6872 2013

Tel: +65 6874 1882

E-mail: dbsmp@nus.edu.sg

(Received 23 November 2004, revised 26

December 2004, accepted 6 January 2005)

doi:10.1111/j.1742-4658.2005.04556.x

The male seahorse incubates its young in a manner resembling that of a mammalian pregnancy After the female deposits her eggs into the male’s brood pouch they are fertilized and the embryos develop and grow for several weeks until they are able to withstand the external environmental conditions independently, at which point they are irreversibly released Although the precise function of the brood pouch is not clear, it is probably related to pro-viding a suitable protective and osmotic environment for the young The aim

of this project was to construct and characterize a cDNA library made from the tissue lining the pouch, in order to help understand the molecular mecha-nisms regulating its development and function The library profile indicates expression of genes encoding proteins involved in metabolism and transport,

as well as structural proteins, gene regulatory proteins, and other proteins whose function is unknown However, a large portion of the library con-tained genes encoding C-type lectins (CTLs), of which three full-length proteins were identified and found to contain a signal peptide and a single C-lectin domain, possessing all the conserved structural elements We have produced recombinant protein for one of these and raised antisera; we have 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

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 incubdur-ation 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

pregnancy.

Results

Identification of cDNA clones from the pouch

tissue

A cDNA library was constructed from the tissue

lin-ing the incubation pouch, and over 250 cloned

inserts were sequenced; of these 151 were found to

match sequences in the nucleotide and ⁄ or protein databases Another 80 inserts appeared to encode novel proteins for which matches could not be found As expected, the identified inserts contained genes for ubiquitous proteins such as actin, globin, keratin, ribosomal proteins and also for transferrins, and generally showed closest matches with homolog-ous sequences from other teleosts, where available All sequences have been entered to the NCBI Gen-Bank data base (Table 1).

Many of the cloned inserts encode metabolic enzymes, including those involved in oxidative phos-phorylation, fatty acid oxidation and reductive biosyn-thesis The presence of these enzymes presumably reflects the large number of mitochondria in this tissue Genes encoding putative regulatory proteins were also identified, including those for kinases, transcription factors and binding proteins, indicating that this tissue

is probably regulated by specific signalling pathways Genes encoding proteases and protease inhibitors were also present and a gene with high homology to the carp zinc endopeptidase, nephrosin, was identified This proteinase, which is stimulated by high concentra-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

Table 1 Identified cDNA clones from male seahorse brood pouch, based on gene and⁄ or protein comparisons.

YK1 Beta globin [Oryzias latipes] (4e-87) Adult beta-type globin [O latipes] (1e-54) CV863925

YK4 NADH ubiquinone oxidoreductase 49 kDa

subunit [Bos taurus] (3e-32)

NADH2 dehydrogenase 49 kDa subunit [B taurus] (8e-88) CV863928

YK7 Myosin regulatory light

chain 2 [Mus musculus] (9e-70)

Myosin regulatory light chain 2 [Gallus gallus] (7e-47) CV863930

YK14 40S Ribosomal protein S25

[Ictalurus punctatus] (1e-61)

Similar to ribosomal protein S25 [Rattus norvegicus] (2e-32) CV863934

YK16 ATPase subunit 8 (ATPase8)

and ATPase subunit 6 (ATPase6)

[Rhamdia sp.] (7e-30)

ATP synthase F0 subunit 6 [Emmelichthys struhsakeri] (4e-75) CV863935

YK20 Lysyl-tRNA synthetase

[Xenopus laevis] (1e-13)

Lysyl-tRNA synthetase [X laevis] (1e-60) CV863936

YK26 Farnesyl diphosphate farnesyl

transferase 1 [H sapiens] (3e-12)

Farnesyl diphosphate farnesyl transferase 1 [R norvegicus] (6e-50) CV863938

YK35 Clone MGC:55674 [Danio rerio] (1e-18) Makorin 3 [zinc finger protein 127] [M musculus] (2e-05) CV863940

YK39 Ribosomal L6 [Pargus major] (1e-111) 60S ribosomal protein L6 [R norvegicus] (6e-61) CV863942 YK40 Galectin-like protein

[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

YK49 Novel protein similar to vertebrate mitochondrial enoyl

Coenzyme A hydratase 1 (ECHS1) [D rerio] (2e-39)

CV863949

YK51 Cytochrome c oxidase subunit II [Exocoetus volitans] (1e-110) CV863951

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

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

YK67 Beta actin 1

[Takifugu rubripes] (1e-101)

Actin [Strongylocentrotus purpuratus] (2e-20) CV863963

Table 1 (Continued).

YK72 Cytochrome c sububit 1 [Trachipterus trachypterus] (4e-14) CV863967

YK81 c-src family protein tyrosine kinase [T rubripes] (3e-29) CV863973 YK82 Transferrin

[Pagrus major] (8e-40)

YK84 Ornithine decarboxylase

antizyme [D rerio] (2e-43)

Ornithine decarboxylase antizyme [D rerio] (7e-25) CV863975

YK85 Arachidonate 15-lipoxygenase type II [H sapiens] (2e-08) CV863976 YK86 Type II keratin

[O mykiss] (5e-74)

Type II cytokeratin [D rerio] (4e-62) CV863977

YK91 DNA sequence from

clone XX-184L24

[D rerio] (1e-12)

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

[H sapiens] (4e-14)

CV863984

WQ6 Programmed cell death 6

[M musculus] (1e-06)

Programmed cell death protein 6 [M musculus] (2e-37) CV863986

WQ7 Ribosomal protein S19

[Gillichthys mirabilis] (8e-72)

WQ19 DEAD (Asp-Glu-Ala-Asp) box

polypeptide (D rerio) [1e-11]

Similar to Eukaryotic initiation factor 4a [D rerio] (8e-08) CV863988

WQ27 Transferrin

[Gadus morhua] (1e-07)

Serotransferrin I precursor [S salar] (2e-20) CV863991

WQ31 Similar to ATP synthase H+

transporting, mitochondrial

F0 complex, subunit c

(subunit 9) isoform 3

[X laevis] (5e-40)

Similar to ATP synthase C, subunit C, isoform 3 [D rerio] (1e-35) CV863994

WQ32 40S ribosomal protein S15A

[Paralichthys olivaceus] (1e-115)

40S ribosomal protein S15A [P olivaceus] (1e-56) AY319480

Table 1 (Continued).

WQ34 ATP synthase, H+transporting, mitochondrial F1 complex,

O subunit [B taurus] (9e-52)

CV863996

WQ39 Ribosomal protein L38

[Branchiostoma belcheri] (2e-24)

Similar to ribosomal protein L38, cytosolic [R norvegicus] (7e-14) CV863998

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

WQ52 Ferritin heavy subunit

[Oreochromis mossambicus] (2e-64)

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)

WQ62 40S ribosomal protein S28

[I punctatus] (2e-6)

40S ribosomal protein S28 [I punctatus] (7e-12) AY357067

WQ63 40S ribosomal protein S29

[I punctatus] (2e-25)

40S ribosomal protein S29 [I punctatus] (1e-21) AY357068

WQ73 Haplotype VIB.313 cytochrome b

[Hippocampus comes] (0)

Cytochrome b [Hippocampus comes] (1e-89) AF192657

[Caenorhabditis elegans] (1e-08)

CV864013

WQ75 Type II keratin E3

[O mykiss] (2e-58)

Type II keratin E3 [O mykiss] (5e-24) CV864014

WQ77 Similar to eIF3 subunit 9

[M musculus] (6e-18)

Eukaryotic translation initiation factor 3 subunit 9 [H sapiens] (1e-11) CV864016

WQ79 (i) Kinesin light chain

[G gallus] (7e-56);

(ii) 40S ribosomal protein S2

[R norvegicus] (1e-51)

40S ribosomal protein S2 [I punctatus] (2e-37) CV864058

WQ81 Similar to lysyl-tRNA synthetase

[M musculus] (3e-17)

Lysyl-tRNA synthetase [X laevis] (3e-49) CV864059

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

WQ90 Elongation factor 1-alpha

[Sparus aurata] (2e-22)

CV864018

WQ97 Lectin C-type domain containing protein [C elegans] (3e-15) CV864021

Table 1 (Continued).

WQ100 Lectin C-type domain containing protein precursor family member

[C elegans] (2e-15)

CV864023

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

WQ106 ADP,ATP translocase

[P flesus] (8e-19)

ADP,ATP translocase [P flesus] (1e-14) CV864027

WQ107 Similar to ribosomal

protein L27 [D rerio] (5e-89)

Similar to ribosomal protein L27[H sapiens] (3e-53) AY437394

WQ110 Similar to retinoid-inducible

serine caroboxypetidase

[D rerio] (8e-08)

Similar to retinoid-inducible serine caroboxypetidase [D rerio] (4e-55) CV864028

WQ111 Heat shock protein 90 beta

[P flesus] (3e-13)

Heat shock protein 90 beta [P flesus] (2e-09) CV864029

WQ114 ATPase subunit 8 (ATPase8) and

ATPase subunit 6 (ATPase6) –

mito-chondrial [Rhamdia laticauda] (4e-17)

ATP synthase F0 subunit 6 [P olivaceus] (4e-25) CV864031

WQ115 Microsatellite marker Pret-15

[Poecilia reticulata] (1e-37)

CV864032

WQ116 Lectin C-type domain containing protein [C elegans] (5e-07) CV864033

WQ119 Leucine-rich repeat-containing

protein 8 [R norvegicus] (5e-63)

Leucine-rich repeat-containing protein 8 [M musculus] (9e-79) CV864035

WQ124 Lectin C-type domain containing protein [C elegans] (2e-08) CV864036 WQ127 Fructose-1, 6-bisphosphate

aldolase [Sparus aurata] (6e-55)

Fructose-1, 6-bisphosphate aldolase [S aurata] (2e-67) CV864037

WQ130 Ribosomal protein L19 mRNA

[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 Length¼ 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)

RAB37, member of RAS oncogene family; GTPase Rab37 [M musculus] (1e-22)

CV864046

WQ142 Translocon-associated protein

alpha mRNA [D rerio] (3e-05)

Translocon-associated protein alpha [D rerio] (2e-17) CV864047

WQ147 Cytokeratin mRNA

Stizostedion vitreum vitreum] (4e-15)

Type I cytokeratin, enveloping layer;

type I cytokeratin [D rerio] (1e-38)

CV864048

Three different CTLs are expressed

in the incubation pouch

The inserts encoding CTL-like proteins were aligned

and found to comprise three different sequences For

each of these, a full-length sequence was found in the

library, and the deduced proteins were aligned Two

of the Hippocampus comes CTLs (hcCTLs), types I

and III are highly similar, while a third, type II differs.

Alignment with the C-type lectins found in whole body

extracts of H kuda and in the gills of the Japanese eel

[7,8], reveals similarity with the hcCTL II, but less so

to the other two hcCTLs (Fig 2A) All three novel

hcCTLs contain a signal peptide and a single C-type

lectin domain without other associated domains

(Fig 2A), defining them as group VII lectins They

contain many of the 37 residues of the C-type

carbohy-drate recognition domain (CRD), as defined by Weis

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 the cDNA library This protein spot was cut and tryp-sin-digested for peptide fingerprinting using MALDI

MS Comparison of the peptide masses with the deduced peptides for the three hcCTLs revealed pep-tides that matched the predicted sizes for the novel hcCTL III and covered 28% of the mature protein.

Analysis of the levels of lectin proteins in the pouch fluid during pregnancy

The cDNA encoding the hcCTL III was expressed in Escherichia coli and the recombinant protein (shown in

Table 1 (Continued)

WQ149 Chromosome 20 open reading

frame 52 (C20orf52), mRNA

[H sapiens] (2e-28)

Chromosome 20 open reading frame 52; homolog of mouse RIKEN 2010100O12 gene [H sapiens] (2e-21)

CV864049

WQ150 40S ribosomal protein S15A mRNA,

complete [P olivaceus]

(1e-119)

40S ribosomal protein S15A [P olivaceus] (3e-61) AY319480

WQ154 mRNA for embryonic alpha-type

globin [O latipes] (9e-29)

Embryonic alpha-type globin [O latipes] (1e-52) CV864050

WQ156 Type I cytokeratin (cki),

mRNA [D rerio] (3e-06)

Type I cytokeratin, enveloping layer; type I cytokeratin [D rerio] (3e-18)

CV864051

WQ158 Lectin C-type domain containing protein precursor family

member [C elegans] (1e-09)

CV864052

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

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 usingPSIPREDat http://bioinf.cs.ucl.ac.uk/psipred/) and the three disulphide bridges (joined by lines and labelled with boxed numbers) are shown The five residues comprising the part of the CRD that determines mannose binding (according to Drickamer [12]) are noted in italics above the sequence

the preimmune rabbit sera was used (data not shown).

Given the similarity of protein sequences between the

three novel seahorse lectins and their close sizes, this

reactive band could, however, represent more than just

the hcCTL III Samples of the pouch fluid from

sea-horses at various stages of incubation were collected

and run on SDS gels for Western analysis to

com-pare the levels of the immunoreactive (ir)-hcCTL III

proteins The ir-hcCTL III protein was detected only

during incubation of early embryos, but not the

devel-oped seahorses, and was also undetectable both before

uptake of the eggs and after hatching and release of

the juveniles (Fig 5C).

Functional analysis of hcCTL III

In order to verify a possible antibacterial role for the

novel hcCTL III, bacteriostatic tests were performed.

These involved incubation of E coli cells with or

with-out addition of the recombinant hcCTL III for up to

2 h, during which the growth of the bacteria was

assessed by O.D readings every 30 min Under these

conditions, hcCTL III at a final concentration of

0.7 lm started to inhibit E coli growth after 1.5 h, and reached a 25% reduction after 2 h (Fig 6).

The ability of the novel hcCTL III to recognize cell-surface glycoproteins was assessed using a haemagglu-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 lM 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-horse embryos was extracted from the pouch of a pregnant male

H comes comes for analysis of the proteome The proteins were separated using 2DE (over the pI range 3–10), and a prominent pro-tein spot (circled) corresponding to the approximate mass and pI of the novel hcCTL proteins (
16 kDa, pI 4) was cut and digested with trypsin, for peptide fingerprinting using MALDI MS Of the peptides obtained, three matched the predicted sizes for the novel hcCTL III, covering 28% of the mature protein

unknown However, an unusually large portion of the library contained genes encoding CTLs Three full-length CTLs were identified, which share some similar-ity to CTLs expressed H kuda and to a lesser degree, those in the gills of the Japanese eel [7,8] The localiza-tion of hcCTL III mRNA transcripts specifically in the stroma-like tissue and epithelium of the pouch tissue was confirmed by in situ hybridization, while 2DE and Western analysis revealed that it is secreted into the incubation fluid that surrounds the embryos during early pregnancy.

CTLs are found universally in eukaryotes and pro-karyotes and have diverse functions [10] Although often containing several domains, they are character-ized by their ability to bind carbohydrates in a cal-cium-dependent manner, through a CRD The CRD contains two a helices and several strands separated by loops [11] At least three disulphide bridges are com-mon in the long form (approximately 130 residues), one of which spans from the end of the first helix to 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 D595of 0.1) were incubated with recombinant hcCTL III

at 0.7 lM, or vehicle alone, for up to 2 h, and D595readings 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 two-samples

(mean ± SEM, n¼ 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 lM, 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

by the hcCTL III, the same assay was repeated using 4.5 lM

hcCTL III with the addition of fructose, sucrose, maltose, glucose, galactose or mannose at 12.5–100 mM However, no inhibition of agglutination was apparent with addition of any of the sugars +C, Positive control to which no sugars were added; -C, negative con-trol in which hcCTL III was lacking

A

C

B

Fig 5 The amounts of ir-hcCTL III in the pouch fluid vary with

pro-gression of pregnancy (A) Recombinant hcCTL III was raised and

purified on a Ni–NTA affinity column; the cell lysate (lane 1), column

flow-through (lane 2) and eluted protein (lane 3) are shown on an

SDS⁄ PAGE gel (12%) stained with Coomassie blue (B) The eluted

recombinant hcCTL III was used to raise antisera, which recognized

just a single sized-protein in the pouch fluid of a pregnant male

(third lane); shown also are the rainbow marker (first lane) and fluid

from a nonpregnant male (second lane) The proteins were

resolved on an SDS⁄ PAGE gel (12%); primary antisera was used at

1 : 1000 dilution, and a goat antirabbit IgG–HRP-conjugated

secon-dary antibody (at 1 : 1000 dilution) was used for detection by

chemiluminescence (C) This antisera was then used in the same

manner to compare levels of ir-hcCTL III in the same volume of

pouch fluid for individuals at various stages of pregnancy: before

uptake of the eggs, during incubation of the developing embryos or

seahorses, or after their release