Purification, characterization and cDNA cloning of a lectin from the brittle star Ophioplocus japonicus.

Lectins are carbohydrate-binding proteins that possess specific sugar-binding properties and are involved in various biological activities in different organisms. In this study, purification, characterization, and cDNA cloning of a brittle star lectin, designated as Ophioplocus japonicus agglutinin (OJA), were conducted. OJA was isolated from the brittle star O. japonicus by affinity chromatography on a Sephadex G-25 column, followed by ion-exchange chromatography on a Resource Q column. This lectin yielded distinct bands at approximately 176 or 17 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing or reducing conditions, respectively. It also exhibited Ca2+-dependent hemagglutination activity, which, however, was not affected by other metal cations, such as Ba2+, Co2+, Cu2+, Zn2+, Fe2+, Mg2+, and Mn2+. The OJA activity was strongly inhibited by glucose and xylose among the monosaccharides tested, and by bovine thyroglobulin among the glycoproteins tested. Cloning of the OJA cDNA revealed that its primary structure contained the C-type lectin domain (CTLD). The results of this study showed that OJA is an echinoderm-derived glucose/xylose-specific lectin that belongs to the C-type lectin superfamily.

The Taurine Content of Japanese Seaweed.

Japanese and South Koreans have a dietary habit of eating seaweed. Although it is known that some seaweed contains taurine, there have been few detailed analyses on the taurine content of seaweed other than the major types of edible seaweed. In the present study, we determined the content of free amino acids, including taurine, in seaweed obtained along the Sea of Japan coast. The taurine content in the seaweed varied according to the species. Among the 29 different types of seaweed that were studied, red algae contained relatively high concentrations of taurine. In contrast, the taurine content was low or undetectable in brown and green algae. The algal alanine level was relatively higher in brown sea algae, which was in sharp contrast to its taurine level. No clear trends were observed with regards to the distribution of the other free amino acids, including aspartic acid, glutamic acid, and phenylalanine. Considering the physiological role of taurine in cellular homeostasis, the algal taurine content may be associated with the growing environment. Taurine-rich red edible algae such as mafunori (Gloiopeltis tenax)/fukurofunori (Gloiopeltis furcata), kabanori (Gracilaria textorii), and ogonori (Gracilaria vermiculophylla) may be used to create functional foods that are rich in naturally occurring taurine.

Cloning and expression of cDNA encoding lysyl hydroxylase 1, 2 and 3 in tiger puffer Takifugu rubripes.

Lysyl hydroxylase (LH) catalyzes the hydroxylation of lysine residues in collagens, and contributes to the formation of more stable collagen cross-links. However, in teleost, there is little information about collagen modification enzymes including lysyl oxidase (LOX) family members. Here, we cloned cDNAs encoding LH1, 2 and 3 from tiger puffer Takifugu rubripes. To determine the mRNA expressions of LH family members in a tiger puffer, we performed a northern blot analysis. Results showed that both fgLH1 and fgLH3 mRNAs were almost constitutively expressed in tissues, but highly expressed in muscle and ovary, respectively. However, fgLH2 mRNA was detected only in RT-PCR, indicating that expression level of fgLH2 is very low in tissues. It may be that low expression level of fgLH2 contributes to the fewer contents of stable collagen cross-links in tiger puffer tissues. To further investigate expression profiles of fgLHs, we examined gene expressions in embryos during development. In embryos, expression profiles differ among three fgLHs, indicating that there are functional differences among the three fgLHs. This is the first report that examined gene expression patterns of three LHs in emrbyos and adult tissues in teleost.

Expression and distribution of fugu TIMP-2s (fgTIMP-2a and fgTIMP-2b) mRNAs in tissues and embryos.

In teleosts, two distinct types of TIMP-2s occur, TIMP-2a and TIMP-2b, but little is known about their locations and quantitative expressions. Here, we examined pufferfish (Takifugu rubripes) TIMP-2a (fgTIMP-2a) and TIMP-2b (fgTIMP-2b) quantities and locations in fugu adult tissues and embryos. To compare the quantitative expression of fgTIMP-2s, we performed a quantitative real-time PCR (qPCR). FgTIMP-2a mRNA was constitutively expressed and significant differences in expression were not observed among adult tissues. Whereas, fgTIMP-2b mRNA was significantly differently expressed in ordinary muscle and gill compared to the expression level in whole blood (P<0.05). Although significant difference was not observed between brain and other tissues, both fgTIMP-2s mRNAs were abundant in the brain. In addition, we examined embryos during development using qPCR. Both fgTIMP-2s mRNAs gradually increased during embryonic development from 48 hpf. However, fgTIMP-2b mRNA was obviously abundant compared to fgTIMP-2a mRNA in embryos. We also examined the specific mRNA distribution in embryos. The fgTIMP-2s mRNAs showed the same distribution during development. Both fgTIMP-2s are expressed in adult fugu tissues and embryos but their expression levels clearly differ, suggesting that there is a predominance of fgTIMP-2b over fgTIMP-2a in vivo.

Molecular cloning and expression of gelatinases (MMP-2 and MMP-9) in the pufferfish Takifugu rubripes.

To determine the metabolism location of the extra-cellular matrix proteins in fugu (Takifugu rubripes), we cloned the cDNAs of the fugu gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, and examined their expressions in various adult tissues using a quantitative real-time PCR. The expression profiles of fugu gelatinases were different among tissues. FgMMP-9 mRNA was abundant in tissues that contain blood cells abundantly where fgMMP-2 mRNA was little expressed. We also examined the expression of these genes in fugu embryos during development using a whole mount in situ hybridization. Fugu MMP-2 mRNA was expressed in the pharyngeal area and mesenchyme in embryos at 80 hours post fertilization (hpf). While fugu MMP-9 mRNA was expressed in the vent at 140 hpf and the caudal end of the fin fold at 172 hpf. Although fugu MMP-2 mRNA was expressed in the pectoral fin bud at 120 hpf, fugu MMP-9 mRNA did not appear in this tissue until 10 days post fertilization (dpf). These data show expression profiles differ between the fugu gelatinases and suggest expressions of these genes are controlled at the matrix protein degradation site in fugu embryos during development.

Expression of fugu TIMP-3 and -4 genes in adult tissues and embryos.

The tissue inhibitors of metalloproteinases (TIMPs) are involved in various processes of extra-cellular matrix (ECM) metabolism by inhibiting matrix metalloproteinases (MMPs). However, the fundamental information for these genes is little known in fish. Previously, we report cDNA cloning and gene expressions of two fugu (Takifugu rubripes) TIMP-2s. Here, we cloned cDNA of fugu TIMP-3 and performed an expression analysis of TIMP-3 and -4 mRNA in fugu adult tissues using a quantitative real-time PCR. The expression level of TIMP-3 mRNA was constitutive in all tissues, while TIMP-4 was significantly higher in the brain (P=0.05). Further, we performed a whole mount in situ hybridization in fugu embryos at different stages. In early stages, TIMP-3 mRNA was abundant in the somites and the caudal end of the notochord. At hatching larvae, the TIMP-3 mRNA was abundant in the pectoral fin, dorsal and ventral fin fold along the entire antero-posterior axis. TIMP-3 may be involved in axis elongation and somitogenesis. TIMP-4 mRNA was expressed in the tail bud, at the midbrain-hindbrain boundary and in the diencephalon from 72 to 104 hpf. This indicates TIMP-4 is highly expressed in the brain matrix in vivo.

cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei.

A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.