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

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

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)

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.

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.

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.

Although appearing to be a true male pregnancy, in contrast to mammals but comparable to most other

doi:10.1111/j.1742-4658.2005.04556.x

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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.

P. Melamed et al. C-type lectins in the male seahorse pregnancy

match sequences in the nucleotide and ⁄ or protein databases. Another 80 inserts appeared to encode novel proteins could not be for which matches 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).

the

Many of

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.

including those for kinases,

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.

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.

Results

encode metabolic cloned inserts 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, 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].

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

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.

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P. Melamed et al. C-type lectins in the male seahorse pregnancy

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

Clone Gene Protein Accession number

Beta globin [Oryzias latipes] (4e-87)

NADH ubiquinone oxidoreductase 49 kDa YK1 YK2 YK3 YK4 Adult beta-type globin [O. latipes] (1e-54) Serum lectin isoform 1 precursor [Salmo salar] (1e-23) C-type lectin [Anguilla japonica] (4e-15) NADH2 dehydrogenase 49 kDa subunit [B. taurus] (8e-88) CV863925 CV863926 CV863927 CV863928 subunit [Bos taurus] (3e-32)

Myosin regulatory light YK5 YK7 C-type lectin 2 [A. japonica] (4e-16) Myosin regulatory light chain 2 [Gallus gallus] (7e-47) CV863929 CV863930 chain 2 [Mus musculus] (9e-70)

40S Ribosomal protein S25 YK8 YK10 YK13 YK14 FC-epsilon RII [M. musculus] (7e-18) Zymogen granule protein 16 [Homo sapiens] (6e-16) Polyubiquitin [Arabidopsis lyrata] (4e-92) Similar to ribosomal protein S25 [Rattus norvegicus] (2e-32) CV863931 CV863932 CV863933 CV863934

[Ictalurus punctatus] (1e-61) ATPase subunit 8 (ATPase8) YK16 ATP synthase F0 subunit 6 [Emmelichthys struhsakeri] (4e-75) CV863935

and ATPase subunit 6 (ATPase6) [Rhamdia sp.] (7e-30) Lysyl-tRNA synthetase YK20 Lysyl-tRNA synthetase [X. laevis] (1e-60) CV863936 [Xenopus laevis] (1e-13)

Farnesyl diphosphate farnesyl YK23 YK26 Serotransferrin precursor [O. latipes] (3e-46) CV863937 Farnesyl diphosphate farnesyl transferase 1 [R. norvegicus] (6e-50) CV863938 transferase 1 [H. sapiens] (3e-12)

Clone MGC:55674 [Danio rerio] (1e-18)

Ribosomal L6 [Pargus major] (1e-111) Galectin-like protein YK29 YK35 YK37 YK39 YK40 Nephrosin precursor [Cyprinus carpio] (3e-35) Makorin 3 [zinc finger protein 127] [M. musculus] (2e-05) Brevican core protein [M. musculus] (5e-15) 60S ribosomal protein L6 [R. norvegicus] (6e-61) Galectin like protein [O. mykiss] (3e-56) CV863939 CV863940 CV863941 CV863942 CV863943 [Oncorhynchus mykiss] (2e-09) YK41 Adult beta type globin [O. latipes] (3e-55) CV863944

Adult beta type globin [O. latipes] (6e-79) Actin related protein 2 YK43 CV863945 homolog [X. laevis] (2e-13)

YK45 YK46 YK47 YK49 CV863946 CV863947 CV863948 CV863949 Transferrin [Melanogrammus aeglefinus] (1e-25) Mannose receptor precursor [G. gallus] (2e-05) Actin-like protein [G. gallus] (8e-93) Novel protein similar to vertebrate mitochondrial enoyl Coenzyme A hydratase 1 (ECHS1) [D. rerio] (2e-39)

DJ-1 [S. salar] (3e-31)

Ribosomal protein L23 YK50 YK51 YK52 YK54 YK55 YK56 YK57 YK59 YK61 YK62 YK63 C-type lectin 2 [A. japonica] (4e-15) Cytochrome c oxidase subunit II [Exocoetus volitans] (1e-110) Serotransferrin precursor [O. latipes] (3e-67) Brevican core protein [M. musculus] (2e-15) Brevican core protein [M. musculus] (2e-15) Neurocan core protein precursor [H. sapiens] (9e-11) Transketolase [P. flesus] (1e-15) Similar to ADP-ribosylation factor 2 [M. musculus] (2e-05) Similar to DJ-1 protein [M. musculus] (5e-69) FC-epsilon RII [H. sapiens] (5e-06) 60S ribosomal protein L23 [H. sapiens] (5e-22) CV863950 CV863951 CV863952 CV863953 CV863954 CV863955 CV863956 CV863957 CV863958 CV863959 CV863960 [Gillichthys mirabilis] (1e-24) YK64 Microsatellite marker CV863961 [Poecilia reticulata] (8e-54)

Beta actin 1 YK66 YK67 C-type lectin 2 [A. japonica] (4e-15) Actin [Strongylocentrotus purpuratus] (2e-20) CV863962 CV863963 [Takifugu rubripes] (1e-101)

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YK68 YK69 YK70 C-type lectin 2 [A. japonica] (7e-15) Gluthionine S-transferase [H. sapiens] (2e-40) C-type lectin 2 [A. japonica] (1e-16) CV863964 CV863965 CV863966

P. Melamed et al. C-type lectins in the male seahorse pregnancy

Table 1. (Continued).

Clone Gene Protein Accession number

Cytochrome c sububit 1 [Trachipterus trachypterus] (4e-14) Serotransferrin II precursor [S. salar] (6e-23) C-type mannose-binding lectin [O. mykiss] (6e-08) Flavin reductase (NADPH) H. sapiens (1e-38) C-type lectin 2 [A. japonica] (7e-12) C-type lectin 2 [A. japonica] (4e-10) c-src family protein tyrosine kinase [T. rubripes] (3e-29) Transferrin [D. rerio] (7e-44) CV863967 CV863968 CV863969 CV863970 CV863971 CV863972 CV863973 CV863974 YK72 YK74 YK75 YK78 YK79 YK80 YK81 YK82

Ornithine decarboxylase antizyme [D. rerio] (7e-25) CV863975 YK84 Transferrin [Pagrus major] (8e-40) Ornithine decarboxylase antizyme [D. rerio] (2e-43)

Arachidonate 15-lipoxygenase type II [H. sapiens] (2e-08) Type II cytokeratin [D. rerio] (4e-62) CV863976 CV863977 YK85 YK86 Type II keratin [O. mykiss] (5e-74)

Transferrin [O. latipes] (3e-65) Novel protein [D. rerio] (1e-46) CV863978 CV863979 YK87 YK91

Retinoic acid binding protein 1-cellular [T. rubripes] (4e-60) AY437393 YK92

DNA sequence from clone XX-184L24 [D. rerio] (1e-12) Retinoic acid binding protein 1-cellular [H. sapiens] (1e-18)

Metalloproteinase inhibitor 4 precursor [R. norvegicus] (4e-13) Traf2 and NCK interacting kinase [H. sapiens] (2e-14) CV863980 CV863981 YK95 YK98

Selenocysteine methyltransferase [Astragalus bisulcatus] (5e-12) CV863982 YK99

Ribosomal protein L21 [I. punctatus] (1e-55) AY357070 YK102

CV863983 YK103

60S ribosomal protein L35 [Sus scrofa] (1e-42) AY357071 YK104 NIKs-related kinase [H. sapiens] (8e-08) Ferritin heavy subunit [S. salar] (6e-16) Ribosomal protein L21 [I. punctatus] (5e-39) EF1alpha [Drosophila melanogaster] (1e-36) Ribosomal protein L35 [I. punctatus] (7e-29) Cytochrome c oxidase polypeptide subunit VIb CV863984 WQ4 [H. sapiens] (4e-14)

Cytochrome c oxidase subunit I [Mugil cephalus] (1e-11) Programmed cell death protein 6 [M. musculus] (2e-37) CV863985 CV863986 WQ5 WQ6

Ribosomal protein S19 (3e-62) CV878464 WQ7 Programmed cell death 6 [M. musculus] (1e-06) Ribosomal protein S19 [Gillichthys mirabilis] (8e-72)

eEF-1 beta [X. laevis] (6e-14) Similar to Eukaryotic initiation factor 4a [D. rerio] (8e-08) CV863987 CV863988 WQ18 WQ19 DEAD (Asp-Glu-Ala-Asp) box polypeptide (D. rerio) [1e-11]

Ependymin 2 [M. musculus] (7e-15) Hypothetical protein MGC63929 [D. rerio] (1e-29) Serotransferrin I precursor [S. salar] (2e-20) CV863989 CV863990 CV863991 WQ22 WQ25 WQ27 Transferrin [Gadus morhua] (1e-07)

WQ29 WQ30 WQ31 C-type lectin 2 [A. japonica] (6e-11) C-type lectin 2 [A. japonica] (2e-11) Similar to ATP synthase C, subunit C, isoform 3 [D. rerio] (1e-35) CV863992 CV863993 CV863994

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40S ribosomal protein S15A [P. olivaceus] (1e-56) AY319480 WQ32 Similar to ATP synthase H+ transporting, mitochondrial F0 complex, subunit c (subunit 9) isoform 3 [X. laevis] (5e-40) 40S ribosomal protein S15A [Paralichthys olivaceus] (1e-115) Similar to CG13623-PA [H. sapiens] (1e-28) CV863995 WQ33

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Table 1. (Continued).

Clone Gene Protein Accession number

WQ34 CV863996 ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit [B. taurus] (9e-52)

WQ36 WQ39 Ribosomal protein L38 C-type lectin 2 [A. japonica] (4e-10) Similar to ribosomal protein L38, cytosolic [R. norvegicus] (7e-14) CV863997 CV863998 [Branchiostoma belcheri] (2e-24)

WQ40 WQ42 Chromosome 20 ORF 42 Cytochrome c oxidase subunit II [E. volitans] (3e-70) Protein c20orf42 homolog [M. musculus] (1e-69) CV863999 CV864000 (C20orf42) [H. sapiens] (1e-06)

WQ43 Transferrin [O. latipes] (0.59) WQ44 WQ51 WQ52 Ferritin heavy subunit Transferrin [O. latipes] (3e-13) C-type mannose-binding lectin [O. mykiss] (3e-09) FC-epsilon-RII (9e-07) Ferritin H [S. salar] (1e-67) CV864001 CV864002 CV864003 CV864004 [Oreochromis mossambicus] (2e-64) WQ56 Ribosomal protein L18 Ribosomal protein L18 [S. salar] (2e-08) CV864005 [Oreochromis niloticus] (5e-16) WQ59 Ferritin heavy subunit Ferritin heavy subunit; ferritin H [S. salar] (4e-56) CV864006 [S. salar] (2e-60) Ezrin [G. gallus] (2e-23) CV864007 WQ60 Villin 2 [ezrin] (VIL2) [B. taurus] (6e-09) WQ62 40S ribosomal protein S28 40S ribosomal protein S28 [I. punctatus] (7e-12) AY357067 [I. punctatus] (2e-6) WQ63 40S ribosomal protein S29 40S ribosomal protein S29 [I. punctatus] (1e-21) AY357068 [I. punctatus] (2e-25)

WQ65 WQ69 WQ70 WQ71 WQ72 WQ73 Haplotype VIB.313 cytochrome b Similar to cystatin B (stefin B) [D. rerio] (7e-23) TANK-binding kinase 1 [M. musculus] (2e-10) Lithostathine 1 beta [H. sapiens] (2e-07) Hypothetical protein XP_148064 [M. musculus] (2e-19) C-type mannose-binding lectin [O. mykiss] (5e-07) Cytochrome b [Hippocampus comes] (1e-89) CV864008 CV864009 CV864010 CV864011 CV864012 AF192657 [Hippocampus comes] (0) WQ74 Lectin C-type domain containing protein CV864013 [Caenorhabditis elegans] (1e-08) WQ75 Type II keratin E3 Type II keratin E3 [O. mykiss] (5e-24) CV864014 [O. mykiss] (2e-58)

WQ76 WQ77 Similar to eIF3 subunit 9 Serotransferrin precursor [O. latipes] (3e-30) Eukaryotic translation initiation factor 3 subunit 9 [H. sapiens] (1e-11) CV864015 CV864016 [M. musculus] (6e-18)

WQ78 WQ79 (i) Kinesin light chain C-type lectin 2 [A. japonica] (2e-11) 40S ribosomal protein S2 [I. punctatus] (2e-37) CV864057 CV864058 [G. gallus] (7e-56); (ii) 40S ribosomal protein S2

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

[R. norvegicus] (1e-51) WQ81 Similar to lysyl-tRNA synthetase [M. musculus] (3e-17) WQ82 Hypothetical protein LOC51255 Zinc finger protein 364 [M. musculus] (7e-11) CV864060

Transferrin [Salvelinus namaycush] (5e-10) CV864061 Metalloproteinase inhibitor 2 precursor (TIMP-2) [Canis familiaris] (1e-22) CV864062 CV864056 cAMP responsive element binding protein-like 2 [H. sapiens] (1e-21) CV864017 Lectin C-type [C. elegans] (1e-06) CV864018 [D. rerio] (9e-11) WQ83 Transferrin [O. latipes] (0.52) WQ86 WQ87 WQ89 WQ90 Elongation factor 1-alpha [Sparus aurata] (2e-22)

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WQ93 WQ95 WQ97 WQ99 C-type lectin 2 [A. japonica] (5e-12) Transferrin Salmo trutta (4e-31) Lectin C-type domain containing protein [C. elegans] (3e-15) Serum lectin isoform 1 precursor [S. salar] (1e-15) CV864019 CV864020 CV864021 CV864022

P. Melamed et al. C-type lectins in the male seahorse pregnancy

Table 1. (Continued).

Protein Accession number Clone Gene

Lectin C-type domain containing protein precursor family member CV864023 WQ100 [C. elegans] (2e-15)

Similar to opioid receptor, sigma 1 [D. rerio] (1e-42) Peptidylprolyl isomerase F (cyclophilin F) [H. sapiens] (6e-47) WQ101 WQ102 Cyclophilin A CV864024 CV864025

40S ribosomal protein S30 [I. punctatus] (7e-51) WQ104 [Canis familiaris] (2e-18) 40S ribosomal protein S30 AY357069 [I. punctatus] (1e-42)

C-type lectin 2 [A. japonica] (1e-11) ADP,ATP translocase [P. flesus] (1e-14) WQ105 WQ106 ADP,ATP translocase CV864026 CV864027 [P. flesus] (8e-19) WQ107 Similar to ribosomal Similar to ribosomal protein L27[H. sapiens] (3e-53) AY437394 protein L27 [D. rerio] (5e-89) WQ110 Similar to retinoid-inducible Similar to retinoid-inducible serine caroboxypetidase [D. rerio] (4e-55) CV864028

serine caroboxypetidase [D. rerio] (8e-08) WQ111 Heat shock protein 90 beta Heat shock protein 90 beta [P. flesus] (2e-09) CV864029 [P. flesus] (3e-13)

Chitinase3 [P. olivaceus] (4e-13) ATP synthase F0 subunit 6 [P. olivaceus] (4e-25) CV864030 CV864031 WQ113 WQ114 ATPase subunit 8 (ATPase8) and

ATPase subunit 6 (ATPase6) – mito- chondrial [Rhamdia laticauda] (4e-17) WQ115 Microsatellite marker Pret-15 CV864032 [Poecilia reticulata] (1e-37)

WQ116 WQ118 WQ119 Leucine-rich repeat-containing Lectin C-type domain containing protein [C. elegans] (5e-07) C-type lectin 2 [A. japonica] (4e-16) Leucine-rich repeat-containing protein 8 [M. musculus] (9e-79) CV864033 CV864034 CV864035 protein 8 [R. norvegicus] (5e-63)

WQ124 WQ127 Fructose-1, 6-bisphosphate Lectin C-type domain containing protein [C. elegans] (2e-08) Fructose-1, 6-bisphosphate aldolase [S. aurata] (2e-67) CV864036 CV864037 aldolase [Sparus aurata] (6e-55) WQ130 Ribosomal protein L19 mRNA Ribosomal protein L19 [I. punctatus] (2e-64) CV864038 [I. punctatus] (4e-99) WQ131 Ribosomal protein L31 mRNA 60S ribosomal protein L31 [P. olivaceus] (2e-46) CV864039

CRR9p (Crr9-pending), Crr9-pending protein AY437395 [M. musculus] (2e-64) [P. olivaceus] (1e-126) WQ133 Cisplatin resistance related protein mRNA Length ¼ 2058 [M. musculus] (3e-60)

Machado-Joseph disease protein 1 (Ataxin-3) [M. musculus] (6e-63) CV864040 Cytochrome c oxidase subunit VIII liver form [Eulemur fulvus] (9e-08) CV864041 WQ134 WQ135 Cytochrome c oxidase subunit VIII

liver form (COX8L) mRNA [Trachypithecus cristatus] (0.054)

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WQ136 WQ137 Eukaryotic translation initiation Mannose receptor, C type 2; novel lectin [M. musculus] (4e-07) Eukaryotic translation initiation factor 2G; eukaryotic translation CV864042 CV864043 initiation factor 2, subunit 3 (gamma, 52 kDa) [H. sapiens] (1e-99) factor gamma 2, subunit 3 [D. rerio] (3e-33) WQ138 Fatty acyl-CoA hydrolase precursor, medium chain CV864044 (thioesterase B) [Anas platyrhynchos] (4e-48) Transferrin [O. latipes] (0.85) WQ139 WQ140 RAB26, member RAS Transferrin [Oncorhynchus nerka] (4e-31) RAB37, member of RAS oncogene family; GTPase Rab37 CV864045 CV864046 [M. musculus] (1e-22) oncogene family (Rab26), mRNA [R. norvegicus] (1e-07) WQ142 Translocon-associated protein alpha [D. rerio] (2e-17) CV864047 Translocon-associated protein alpha mRNA [D. rerio] (3e-05) WQ147 Cytokeratin mRNA CV864048 Stizostedion vitreum vitreum] (4e-15) Type I cytokeratin, enveloping layer; type I cytokeratin [D. rerio] (1e-38)

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Table 1. (Continued).

Clone Gene Protein Accession number

WQ149 Chromosome 20 open reading frame 52; homolog of mouse CV864049 RIKEN 2010100O12 gene [H. sapiens] (2e-21) Chromosome 20 open reading frame 52 (C20orf52), mRNA [H. sapiens] (2e-28) WQ150 40S ribosomal protein S15A mRNA, 40S ribosomal protein S15A [P. olivaceus] (3e-61) AY319480

complete [P. olivaceus] (1e-119) WQ154 mRNA for embryonic alpha-type Embryonic alpha-type globin [O. latipes] (1e-52) CV864050 globin [O. latipes] (9e-29) WQ156 Type I cytokeratin (cki), Type I cytokeratin, enveloping layer; type I cytokeratin CV864051 mRNA [D. rerio] (3e-06) [D. rerio] (3e-18) WQ158 Lectin C-type domain containing protein precursor family CV864052 member [C. elegans] (1e-09)

Three different CTLs are expressed in the incubation pouch

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

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).

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.

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).

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

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

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

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WQ159 WQ162 C-type lectin 2 [A. japonica] (4e-15) Tubulin alpha chain [Notophthalmus viridescens] (3e-64) Alpha tubulin mRNA CV864053 CV864054 [Notothenia coriiceps] (1e-134) WQ166 Ribosomal protein L28 mRNA 60S ribosomal protein L28 [H. sapiens] (7e-55) AY437397 [I. punctatus] (1e-49) WQ168 S6 ribosomal protein mRNA 40S S6 ribosomal protein [O. mykiss] (8e-63) CV864055 [O. mykiss] (1e-123)

P. Melamed et al. C-type lectins in the male seahorse pregnancy

A

B

Fig. 5A, lane 3 after elution from Ni-NTA affinity col- umn) was used to raise antisera in rabbits. The anti- the rabbits was highly specific, sera from one of

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

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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 PSIPRED at 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.

P. Melamed et al. C-type lectins in the male seahorse pregnancy

the brood pouch fluid proteome reveals that Fig. 4. 2DE of 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 ((cid:1) 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.

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

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).

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 incubation (Fig. 7A). In an cells after 1–1.5 h of 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).

Functional analysis of hcCTL III

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.

Discussion

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 the bacteria was 2 h, during which the growth of assessed by O.D. readings every 30 min. Under these conditions, hcCTL III at a final concentration of

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

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P. Melamed et al. C-type lectins in the male seahorse pregnancy

A

A

B

B

C

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.

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.

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.

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.

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Fig. 6. The novel hcCTL III inhibits growth of E. coli. E. coli cells (1 mL at an D595 of 0.1) were incubated with recombinant hcCTL III at 0.7 lM, or vehicle alone, for up to 2 h, and D595 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).

the likelihood that

supports

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 Ca2+ binding. The presence of these structural constraints the novel hcCTLs are indeed functional lectins.

these residues have probably been selected as a group [11]. The proline of the EPN motif is however, extre- mely well conserved also in CTLs that do not bind 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].

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 [13,14]). This innate response for destruction (e.g. 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- cated specifically in the inhibition of bacterial growth (e.g. [7,16–21]).

recognition

specific

than just distinguishing self

The presence of a developed innate defence mech- anism in the male seahorse brood pouch organ could be vital as a large number of fry are contained in an enclosed environment, requiring efficient mecha- nisms for preventing bacterial colonization. The lec- tin-mediated pathway could provide a more adept mechanism than the classical antibody pathway in of microorganisms, allowing rather from nonself which would leave the embryos, obviously immuno- logically distinct from the father, also at risk. This may, however, rely on quite complex recognition of pathogen-associated molecular patterns on the sur- face of the microorganisms [14].

Notably, of the group VII fish CTLs reported to date, nearly all were found to recognize mannose or galactose, and their respective related sugars [15]. The Ca2+ binding sites of the MBP have been well charac- terized; one of them requires six key residues including the EPN motif between loops 3 and 4 [10]. Most of these residues are not present in the novel hcCTLs, confirming our own findings that the hcCTL III does not bind mannose, and also previous observations that

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 which is comprised of around 20 conserved residues and is distinct from other characterized CRDs [24,25] and bears no resemblance to that found in the novel hcCTLS reported here, which appear more related functionally and structurally to the skin mucous CTLs. Interestingly, a study was published recently descri- bing a cDNA library made from the Chinese seahorse H. kuda [8], in which a large number of clones enco- ding CTL proteins was detected. However of the H. kuda CTL sequences published, only five contain partial similarity with those found in our study, and notably more so with the hcCTL II lectin than with the other two. Given that the library was obtained from entire animals, it is difficult to conclude much about the possible expression patterns of these proteins as localization studies were not carried out, nor were any functional studies performed; furthermore the repro- ductive state of the fish used for that study was not reported. However the authors did attribute a likely role of the H. kuda CTLs in innate immunity and

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possibly also some of the beneficial effects associated with this animal in traditional Chinese medicine.

was created using Pfu DNA polymerase. The purified blunted cDNA was ligated with EcoRI adapters and the EcoRI ends phosphorylated with T4 polynucleotide kinase. The product was digested with XhoI. The DNA was precipitated and re- suspended in STE buffer before passing through a sepharose CL-2B gel filtration column. Twenty 100-lL fractions were collected, from each of which 10 lL was electrophoresed on a 5% nondenaturing acrylamide gel and silver stained: the fractions containing larger cDNAs were combined and puri- fied before re-suspension in 5 lL sterile water.

The cDNA was ligated into the Uni-ZAP XR vector (Stratagene) using T4 DNA ligase at 4 (cid:1)C for 2 days and then packaged into Gigapack III Gold Packaging Extract (Stratagene) by incubating at room temperature for 2 h, before addition of SM buffer (500 lL) and chloroform (20 lL). After centrifugation, the phage were stored at 4 (cid:1)C while serial dilutions of primary cDNA library were incuba- ted with bacteria at 37 (cid:1)C for 15 min before plating of the mixtures onto Luria–Bertani (LB) top agar plates.

The secretory profile of the hcCTL III in the pouch fluid indicates that it is expressed at highest levels after uptake of the eggs, but that its levels drop off after the larvae are hatched and as the juvenile seahorses are preparing to leave the pouch. This suggests the passage of a signal between the young and the father which 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.

P. Melamed et al. C-type lectins in the male seahorse pregnancy

Experimental procedures

After 107 pfu of the phage, 108 XL1-Blue MRF¢ cells and 109 pfu of ExAssist helper phage were incubated at 37 (cid:1)C for 15 min, 20 mL of LB broth was added and the mixture incu- bated for 2.5 h at 37 (cid:1)C with shaking. The mixture was hea- ted at 65 (cid:1)C for 20 min, spun (1000 g for 10 min) and 1 lL of the supernatant was added to 200 lL SOLR cells. After 15 min at 37 (cid:1)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 (cid:1) 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 suggested by the results of blastx. Lastly, the translated peptide sequences were used for pattern searching at Scan- Prosit (http://us.expasy.org/tools/scanprosite/) for identifi- cation of conserved protein domains.

Construction of cDNA library and identification of clones

In situ hybridization and histology

Probes were prepared using a 300-bp PCR amplified frag- from the hcCTL III protein cDNA which was ment labelled with digoxigenin-dUTP, using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics, Basel, Switzerland).

The pouch tissues were fixed in formalin and embedded in paraffin before sectioning at 6–8 lm. The paraffin was

RNA was extracted from the pouches of five male seahorses (Hippocampus comes: purchased from Pacific Marine Aqua- ria, Singapore, after import from Indonesia) at various stages of pregnancy. After removal of all embryos, the inner tissue lining the pouch was pulled away from the muscle wall, and RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). The cDNA library was constructed using MMLV- reverse transcriptase (Stratagene, La Jolla, CA), 2.8 lg linker primer and 6 lg of the poly(A)+ RNA at 37 (cid:1)C for 1 h. After synthesis using DNA polymerase I, the second-strand cDNA

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removed and they were rehydrated before additional fix- for ing in 0.4% (v ⁄ v) paraformaldehyde in NaCl ⁄ Pi 20 min, after which they were washed in NaCl ⁄ Pi before treatment with 0.3% Triton-X for 15 min. The sections were permeablized with 20 lgÆmL)1 proteinase K in TE buffer at 37 (cid:1)C for 30 min, and then acetylated with 0.5% (v ⁄ v) acetic anhydride in 100 mm Tris ⁄ HCl pH 8.0 for 5 min before washing with NaCl ⁄ Pi.

The 17-cm, pH 3–10 IPG strips (Bio-Rad) were rehyd- rated actively for 12 h. Isoelectric focusing was performed (Protean IEF Cell, Bio-Rad): 250 V (1 h), 500 V (for 1 h), 1000 V (for 1 h, 10 000 V (gradient over 3 h), and 10 000 V (for 90 000 Vh) at 20 (cid:1)C. The IPG strips were equilibrated for 20 min in equilibration buffer (6 m urea, 2% SDS, 50 mm Tris ⁄ HCl pH 8.8, 30% glycerol) before 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].

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 (cid:1)C, followed by washing in 2· NaCl ⁄ Cit. Probes were denatured for 5 min at 95 (cid:1)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 (cid:1)C.

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 (cid:1)C for 30 min. The excess trypsin solution was removed and 15–50 lL of 50 mm ammonium bicarbonate was added, for 15 h at 37 (cid:1)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 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 (cid:1)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 MgCl2, 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 incubated in the dark until a desired intensity was acquired. The colour reaction was stopped using TE buffer and the sections were rinsed with water and air dried before mount- ing using DPX resinous mount.

Histological staining was carried out on sections from the same serials as above, after rehydration and washing with running water. They were stained using Mayer’s haem- atoxylin and eosin. Sections were dehydrated and cleared in xylene before mounting using DPX resinous mount.

Proteomic analysis of incubation fluid

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- le ⁄ 0.3% trifluoroacetic acid) and left to air dry. MS was car- ried out using a Voyager Biospectrometry Workstation STR with Delayed Extraction Technology MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, MA), at The Protein and Proteomic Centre, Department Biological Sciences, National University of Singapore. The MALDI spectra were acquired in the delayed extraction, reflector mode. The mass scale was calibrated internally with the tryp- sin autodigestion products of known amino acid sequence, [M + H]+ ¼ 842.509 and 2211.104. Typically, 180 scans were averaged to produce the final spectrum.

Expression of recombinant lectin type III and raising of antisera

The pouch fluid was obtained by making an incision along the abdominal fissure of the pouch and draining into a syringe. Aliquots of 50 lL were treated with 4 mm tributyl phosphine (for 1 h) followed by 15 mm iodoacet- amide (for 1.5 h in the dark) to reduce and alkylate the proteins. The fluid was diluted 10-fold with rehydration buffer [6 m urea, 2 m thiourea, 4% CHAPS, 0.2% Bio- loytes 3-10 (Bio-Rad), and Bromophenol blue], centri- fuged for 20 min before the supernatant was subjected to ultrafiltration twice with Microcon YM-3 at 12 400 g. The final retentate was reconstituted to 300 lL with rehy- dration buffer for in-gel rehydration of immobilized pH gradient (IPG) strip.

The coding sequence of hcCTL III, without the signal peptide, was amplified by PCR to incorporate BamHI and XhoI sites at either end, using the following primers: forward, 5¢-CGCGGATCCTGGTCTTTCCAAAATATTC AGGCCA-3¢ and reverse, 5¢-GTCCTCGAGGTACATCA CATCTCTGAT-3¢. After digestion, the PCR-amplified fragment was cloned into a modified BamHI ⁄ XhoI digested

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pET-32a(+) plasmid (Novagen, San Diego, CA) to produce a His6-tagged protein. The construct was verified by sequen- cing.

DH5a E. coli and incubated at 37 (cid:1)C, at 250 r.p.m. for 3–4 h, until D595 reached 0.1. The recombinant hcCTL III protein dissolved in NaCl ⁄ Pi (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

BL21(DE3)pLysS Singles E. coli competent cells (Nov- agen) were transformed by heat-shock and a single colony was inoculated in 10 mL LB–ampicillin medium at 37 (cid:1)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 D600 was 0.4–0.6. Protein expression was induced by IPTG (0.1 mm) and cells cultivated for 5 h (37 (cid:1)C, 250 r.p.m), before harvesting (3840 g, 15 min) and lysis in 40 mL lysis buffer (50 mm NaH2PO4, 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 (cid:1)C) and the supernatant collected.

Heparinized mouse blood was washed three times in NaCl ⁄ Pi (at 420 g, 10 min). The mouse erythrocytes [50 lL, in NaCl ⁄ Pi were added to the recombinant 2% (v ⁄ v)] hcCTL III (50 lL to final concentration of 0.5–18 lm) and incubated at 25 (cid:1)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- tion of 4.5 lm) and of carbohydrate (25 lL) and incubated at 25 (cid:1)C for 60 min.

P. Melamed et al. C-type lectins in the male seahorse pregnancy

Acknowledgements

research was

supported by The Academic

This Research Fund, National University of Singapore.

The lectin was purified by addition of 2 mL Ni–NTA agarose (Qiagen, Valencia, CA) overnight at 4 (cid:1)C before loading onto a column and washing with modified wash buffer (50 mm NaH2PO4, 300 mm NaCl, 50 mm imidazole pH 8.0). The protein was eluted in fractions with modified elution buffer (50 mm NaH2PO4, 2 m NaCl, 250 mm imi- dazole pH 8.0). The purity was checked by SDS ⁄ PAGE, and the protein content determined using a Bradford assay (Bio-Rad, Hercules, CA).

References

1 Wetzel J & Wourms JP (1991) Paternal-embryonic rela- tionships in pipefishes and seahorse (Syngnathidae). Am Zool 31, A83–A83.

For raising of antisera, proteins were dialysed against NaCl ⁄ Pi overnight and adjusted to 500 lL (0.6 mgÆmL)1). An equivalent volume of Freund’s adjuvant was added and mixed before injection into rabbits four times at 3-week intervals, during which blood was extracted and the anti- body titres monitored.

2 Azzarello MY (1991) Some questions concerning the syngnathidae brood pouch. B Mar Sci 49, 741–747. 3 Carcupino M, Baldacci A, Mazzini M & Franzoi P

Western analysis

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