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 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. 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 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. 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 YK3 C-type lectin [Anguilla japonica] (4e-15) CV863927 YK4 NADH ubiquinone oxidoreductase 49 kDa subunit [Bos taurus] (3e-32) NADH2 dehydrogenase 49 kDa subunit [B. taurus] (8e-88) CV863928 YK5 C-type lectin 2 [A. japonica] (4e-16) CV863929 YK7 Myosin regulatory light chain 2 [Mus musculus] (9e-70) Myosin regulatory light chain 2 [Gallus gallus] (7e-47) CV863930 YK8 FC-epsilon RII [M. musculus] (7e-18) CV863931 YK10 Zymogen granule protein 16 [Homo sapiens] (6e-16) CV863932 YK13 Polyubiquitin [Arabidopsis lyrata] (4e-92) CV863933 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 YK23 Serotransferrin precursor [O. latipes] (3e-46) CV863937 YK26 Farnesyl diphosphate farnesyl transferase 1 [H. sapiens] (3e-12) Farnesyl diphosphate farnesyl transferase 1 [R. norvegicus] (6e-50) CV863938 YK29 Nephrosin precursor [Cyprinus carpio] (3e-35) CV863939 YK35 Clone MGC:55674 [Danio rerio] (1e-18) Makorin 3 [zinc finger protein 127] [M. musculus] (2e-05) CV863940 YK37 Brevican core protein [M. musculus] (5e-15) CV863941 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 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 YK66 C-type lectin 2 [A. japonica] (4e-15) CV863962 YK67 Beta actin 1 [Takifugu rubripes] (1e-101) Actin [Strongylocentrotus purpuratus] (2e-20) CV863963 YK68 C-type lectin 2 [A. japonica] (7e-15) CV863964 YK69 Gluthionine S-transferase [H. sapiens] (2e-40) CV863965 YK70 C-type lectin 2 [A. japonica] (1e-16) CV863966 P. Melamed et al. C-type lectins in the male seahorse pregnancy FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS 1223 Table 1. (Continued). Clone Gene Protein Accession number YK72 Cytochrome c sububit 1 [Trachipterus trachypterus] (4e-14) CV863967 YK74 Serotransferrin II precursor [S. salar] (6e-23) CV863968 YK75 C-type mannose-binding lectin [O. mykiss] (6e-08) CV863969 YK78 Flavin reductase (NADPH) H. sapiens (1e-38) CV863970 YK79 C-type lectin 2 [A. japonica] (7e-12) CV863971 YK80 C-type lectin 2 [A. japonica] (4e-10) CV863972 YK81 c-src family protein tyrosine kinase [T. rubripes] (3e-29) CV863973 YK82 Transferrin [Pagrus major] (8e-40) Transferrin [D. rerio] (7e-44) CV863974 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 YK87 Transferrin [O. latipes] (3e-65) CV863978 YK91 DNA sequence from 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 WQ7 Ribosomal protein S19 [Gillichthys mirabilis] (8e-72) Ribosomal protein S19 (3e-62) CV878464 WQ18 eEF-1 beta [X. laevis] (6e-14) CV863987 WQ19 DEAD (Asp-Glu-Ala-Asp) box polypeptide (D. rerio) [1e-11] Similar to Eukaryotic initiation factor 4a [D. rerio] (8e-08) CV863988 WQ22 Ependymin 2 [M. musculus] (7e-15) CV863989 WQ25 Hypothetical protein MGC63929 [D. rerio] (1e-29) CV863990 WQ27 Transferrin [Gadus morhua] (1e-07) Serotransferrin I precursor [S. salar] (2e-20) CV863991 WQ29 C-type lectin 2 [A. japonica] (6e-11) CV863992 WQ30 C-type lectin 2 [A. japonica] (2e-11) CV863993 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 WQ33 Similar to CG13623-PA [H. sapiens] (1e-28) CV863995 C-type lectins in the male seahorse pregnancy P. Melamed et al. 1224 FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS Table 1. (Continued). Clone Gene Protein Accession number WQ34 ATP synthase, H + 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 WQ63 40S ribosomal protein S29 [I. punctatus] (2e-25) 40S ribosomal protein S29 [I. punctatus] (1e-21) AY357068 WQ65 Similar to cystatin B (stefin B) [D. rerio] (7e-23) CV864008 WQ69 TANK-binding kinase 1 [M. musculus] (2e-10) CV864009 WQ70 Lithostathine 1 beta [H. sapiens] (2e-07) CV864010 WQ71 Hypothetical protein XP_148064 [M. musculus] (2e-19) CV864011 WQ72 C-type mannose-binding lectin [O. mykiss] (5e-07) CV864012 WQ73 Haplotype VIB.313 cytochrome b [Hippocampus comes] (0) Cytochrome b [Hippocampus comes] (1e-89) AF192657 WQ74 Lectin C-type domain containing protein [Caenorhabditis elegans] (1e-08) CV864013 WQ75 Type II keratin E3 [O. mykiss] (2e-58) Type II keratin E3 [O. mykiss] (5e-24) CV864014 WQ76 Serotransferrin precursor [O. latipes] (3e-30) CV864015 WQ77 Similar to eIF3 subunit 9 [M. musculus] (6e-18) Eukaryotic translation initiation factor 3 subunit 9 [H. sapiens] (1e-11) CV864016 WQ78 C-type lectin 2 [A. japonica] (2e-11) CV864057 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 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 [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 WQ113 Chitinase3 [P. olivaceus] (4e-13) CV864030 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 WQ118 C-type lectin 2 [A. japonica] (4e-16) CV864034 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 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) 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 C-type lectins in the male seahorse pregnancy P. Melamed et al. 1226 FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS 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). Clone Gene Protein Accession number 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 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 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. C-type lectins in the male seahorse pregnancy P. Melamed et al. 1228 FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS 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 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- 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. P. Melamed et al. C-type lectins in the male seahorse pregnancy FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS 1229 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 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 by the hcCTL III, the same assay was repeated using 4.5 l M hcCTL III with the addition of fructose, sucrose, maltose, glucose, galactose or mannose at 12.5–100 m M. 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. C-type lectins in the male seahorse pregnancy P. Melamed et al. 1230 FEBS Journal 272 (2005) 1221–1235 ª 2005 FEBS [...]... 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,... 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 MMLVreverse transcriptase (Stratagene, La Jolla, CA), 2.8 lg linker primer and 6 lg of the poly (A) + RNA at 37 °C for 1 h After synthesis using DNA polymerase I, the. .. pipefishes and seahorse (Syngnathidae) Am Zool 31, A8 3 A8 3 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 (1997) Morphological organization of the male brood pouch epithelium of Syngnathus abaster Risso (Teleostea, Syngnathidae) before, during, and after egg incubation Tissue Cell 29, 21–30 4 Watanabe S, Kaneke... which 10 lL was electrophoresed on a 5% nondenaturing acrylamide gel and silver stained: the fractions containing larger cDNAs were combined and purified 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 °C for 2 days and then packaged into Gigapack III Gold Packaging Extract (Stratagene) by incubating at room temperature for... Expression of recombinant lectin type III and raising of antisera 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... mm 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 supernatant 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. .. shares some common elements with the hcCTLs, perhaps 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 functional studies indicate that this CTL causes cell agglutination and may act to help repress bacterial... 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 functional 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... 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 epithelium Although the Japanese eel CTLs appear to be galactose specific, it is possible that their regulation... PAGE, and the protein content determined using a Bradford assay (Bio-Rad, Hercules, CA) 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 antibody titres monitored Western analysis . 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. 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