Molecular cloning and characterization of Leukocyte cell-derived chemotaxin Genes in rock bream (Oplegnathus fasciatus) and diskabalone (Haliotis discus discus)

Abstract

Leukocyte cell-derived chemotaxin (LECT)는인간의 T cell line에서분리가되었으며호중구의주화성기능을갖고있다고처음보고되었다. 인간의 LECT2의경우133개의아미노산서열로이루어져있으며16 kDa의단백질과 3개의이황화결합구조를갖고있다.인간의 LECT2는성인과태아의간에서모두명확하게발현되었으며세포성장과분화및손상과치료과정과같은면역반응및다양한기능을갖고있는단백질이다. chondromodulin-II는 LECT2와동일하며, 이는소의단백질로써연골세포와골아세포를자극시켜활성화를일으킨다. LECT의중요한기능으로는케모카인의유도와이에따른반응에주로관여를한다고알려져있으며이밖에도다양한면역관련경로에관여를하여세포의보호와종양성장을억제한다. 이연구에서는돌돔의 GS-FLX database와전복의 normalized cDNA library로부터 LECT2와 LECT1 유전자를분리하고 RbLECT2와 AbLECT1의전체유전자서열을확인하였으며 NCBI BLAST를통하여알려져있는다른 LECT유전자서열들과비교하여분석하였다. 그리고이미밝혀져있는다른관계된유전자들과의유사관계를밝히기위하여 ClustalW pairwise, multiple analysis와 phylogenetic analysis를통하여 RbLECT2와 AbLECT1 유전자의특성을분석하였다. 또한각조직별로유전자의발현을알아보기위하여돌돔에서는근육, 아가미, 간, 비장, 신장, 피부, 혈액, 심장, 뇌, 소화관에서 LECT2유전자의발현정도를측정하였으며전복의경우에는간췌장,혈구,아가미, 근육, 소화관, 외투막에서 LECT1유전자의발현정도를측정하였다. In vivo에서돌돔의 LECT2 유전자발현분석을위해박테리아와 LPS를돌돔에주입하고그조직특이적 mRNA 발현을확인하기위하여주입후 3, 6, 12, 24, 48시간마다간조직으로부터 mRNA를분리하여 RT-PCR을통해발현수준을측정하였다.그리고전복의 LECT1 유전자발현분석을위해박테리아복합체를전복의근육내로주입하고, 그조직특이적 mRNA 조절을확인하기위해서주입후 3, 6, 12, 24, 48시간마다혈액세포로부터 mRNA를분리하여real time RT-PCR을통해발현수준을측정하였다. 비교를위하여 PBS 처리가된그룹을 control로사용하였다. 돌돔의 LECT2의유전자서열은456 bp (151 amino acids)의 open reading frame을포함하는 768 bp로확인되었다. 돌돔 LECT2 유전자를분석한결과 Peptidase_M23 domain과 N-myristoylation sites를포함하고있었다. 돌돔의 LECT2 단백질은대서양연어 LECT2, 무지개송어 LECT2, 북부민물꼬치고기 LECT2와 70% 이상아미노산서열이유사성을나타내었다. 계통학적분석에의해돌돔의 LECT2 단백질은다른어류의 LECT2 단백질과계통상거리가가까운것으로확인되었다. 돌돔의각조직내에서 LECT2의발현을real time RT-PCR을통하여분석한결과, 간에서가장높은발현량을보였고그다음으로아가미와피부순으로발현량이많았으며근육과신장에서는 LECT2 발현량이모든조직중에서가장낮은발현량을나타내어조직특이적으로 LECT2가발현되었다.LECT2의박테리아와 LPS를통한면역반응과 mRNA의발현양상을관찰하기위하여각각의병원체를돌돔내로주입한후에각시간별로 LECT2의발현수준을측정한결과, 박테리아를주입한경우에는 48시간까지점진적으로발현수준이증가하는것을확인하였다. 돌돔의 LECT2 mRNA는병원체에대해서발현수준이control에비하여그수준이상승하였고다른어류에서도비슷한양상을보였다. Peptidase_M23 domain과병원체주입에의한 LECT2 mRNA 발현수준상승과관련하여다른어류나포유동물의 LECT2 단백질들과높은유사성을나타내었고이는공통적인조상에서나누어졌음을시사한다. 전복의 AbLECT1의전체유전자는 576 bp open reading frame(192 amino acids)을포함하여전체 705 bp가확인되었고예상되는분자량과isoelectric point는각각 21kDa과 8.9를나타내었다. AbLECT1의 N-말단에는신호서열을포함하고있었으며 16-17 아미노산잔기사이에서절단되는것으로확인되었다. AbLECT1는 BRICHOS domain과 Cys active site motifs를포함하였고이 motif는포유동물에서도나타난바있다. AbLECT2에서 BRICHOS domain 서열이확인되었고이는대부분의 LECT1 단백질들의공통적인 motif이다. 전복의 LECT1은Lactobacillus jensenii의발현유전자와비슷하였으나같은 motif를갖지않았으며지브라피쉬의 LECT1이 BLASTp에서가장유사하였다. 무척추동물에서는아직 LECT1이아직보고가되어있지않기때문에어류와포유류의 LECT1을ClustalW multiple analysis를통하여비교분석하였으며 AbLECT1은인간의 LECT1과 15.5%의유사성을띄었으며 AbLECT1의BRICHOS domain의분석을위하여소, 인간, 닭, 지브라피쉬와비교를하였다. RT-PCR 발현분석을통해박테리아를전복내로주입한후각조직별로 LECT1의발현량을분석하였으며외투막에서가장높은발현량을나타내었다. LECT1의면역적발현수준을보기위하여박테리아을주입후 48시간째혈구세포의 LECT1의발현수준이증가함을확인하였다. 결론적으로본연구에서는돌돔과전복으로부터 chemotactic 기능을갖고있는 LECT 유전자를분리하여그염기서열의특성을분석하고조직발현분석을수행하였으며이연구들은척추동물과무척추동물의 LECT를통한면역조절시스템에대한면역학적연구에새로운포석이될것이다.

Leukocyte cell-derived chemotaxin-2 (LECT2) was first isolated from the culture fluid of the human (Homo sapiens) T cell line SKW-3 and was shown to have neutrophil chemotactic activity. Human LECT2, a 16-kDa basic protein, consisting of 133 amino acid residues and three intramolecular disulphide bonds, is specifically expressed in the adult and fetal livers.Although it was originally demonstrated to have a chemotactic function in vitro, recent data sustain a further multifunctional role of LECT2 that extends from cell growth, differentiation, damage/repair process and carcinogenesis to autoimmune diseases. The in vivo function of LECT2 protein still remains obscure. In order to study the phylogeny of LECT2, a full-length cDNA clone of LECT2 gene, 768 bp in size, was isolated in Rock bream (Oplegnathus fasciatus). Its deduced amino acid sequence of 151 residues, presents 73, 56, 45 and 42 % overall identity to Atlantic salmon, Zebra fish, Mouse and Human LECT2 proteins, respectively. In contrast to mammalian LECT2 protein, trout LECT2 protein reveals potential N-myristoylation site and Peptidase_M23 superfamilymotif (152AGAATCC-GACCCC448). Phylogenetic analysis shows that Rock bream LECT2 is clustered with the known homologous proteins. RbLECT2 mRNA was significantly (p < 0.05) up-regulatedafter infection with a bacteria and LPS in rock bream liver. A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively.Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone.