Organization of the lipoprotein lipase gene of red sea bream Pagrus major

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Abstract

Lipoprotein lipase (LPL) is a key enzyme of lipid deposition and metabolism. To investigate the mechanism of lipid deposition in fish, as a first step, we have characterized the LPL gene of a marine teleost red sea bream Pagrus major by cDNA and genomic structure analysis. The red sea bream LPL gene encodes 511 amino acids and spans approximately 6.3 kb of the genome. The coding region is organized into ten exons and nine introns. In comparison with the LPL of other animals, the deduced amino acid sequence shows a high degree of similarity with a conservation of functional domains, e.g. catalytic triad, N-glycosylation sites, lipid and heparin binding regions. The 1.1 kb of 5′ flanking region contains two CCAAT, sequences homologous to Oct-I site and response elements for hormones including glucocorticoid, insulin and thyroid hormone. The results of the present study will facilitate further study of the function and regulation of the LPL in non-mammalian vertebrates.

Introduction

Lipoprotein lipase (LPL) plays an important role for plasma lipoprotein metabolism. LPL is an N-linked glycoprotein binding to heparan sulfate and located on the capillary endothelium. LPL hydrolyzes triacylglycerols present in plasma lipoproteins and supplies free fatty acids for storage in adipose tissue, or for oxidation in other tissues (Nilsson-Ehle et al., 1980). The LPL protein contains multiple functional domains required for secretion, glycosylation, catalysis, lipid and heparin binding. Structural analyses of the cDNA have revealed that these functional domains and sites are highly conserved among different species (Wion et al., 1987, Enerbäck et al., 1987, Senda et al., 1987, Cooper et al., 1989, Zechner et al., 1991, Edwards et al., 1993, Raisonnier et al., 1995). The mammalian and avian LPL genes are organized into ten exons and nine introns (Deeb and Peng, 1989, Kirchgessner et al., 1989, Enerbäck and Bjursell, 1989, Zechner et al., 1991, Cooper et al., 1992), and the multiple functional domains in LPL are postulated to be confined to specific exons (Auwerx et al., 1992, Hide et al., 1992): for example, the signal peptide for secretion is assigned to exon 1, the functional N-glycosylation site to exon 2, catalytic serine and a lipid binding region to exon 4, a putative heparin binding region to exon 6 and 3′untranslated sequence to exon 10.

LPL is synthesized in various tissues. LPL gene expression is regulated in response to physiological, nutritional and developmental state of animals in tissue-specific manner (Enerbäck et al., 1988, Cooper et al., 1989, Semenkovich et al., 1989, Tavangar et al., 1992). The structural and functional analyses of the promoter region have identified the existence of cis-regulatory elements in this region. Deletion and reporter assays (Previato et al., 1991, Lu and Bensadoun, 1993, Tanuma et al., 1995) have identified positive and negative regulatory elements for tissue-specific (Zhang and Bensadoun, 1999) and differentiation-linked expression (Enerbäck et al., 1992). Moreover, the structural analyses have identified potential cis-regulatory elements in 5′ flanking regions (Hua et al., 1991, Cooper et al., 1992, Bey et al., 1998, Volpe et al., 1994). These findings have provided insight into the mechanisms for transcriptional regulation.

In fish, partial cDNAs of zebrafish LPL gene and partial genomic structures of zebrafish and rainbow trout LPL gene have been reported (Arnault et al., 1996). And the complete cDNA sequence data for rainbow trout LPL gene are available under Genbank/EMBL accession number AJ224693 (submitted to the data base by Lindberg, A. and Olivecrona, G.). However, information for the entire genomic structure or regulatory region is not available. Our research group is interested in body lipid deposition and metabolism in fish with reference to the regulation of adiposity in cultured species. Elucidation of the structure, function, and the regulation of LPL gene expression in primitive vertebrates may enable us to better understand and eventually manipulate lipid deposition in cultured fish. To study the role of LPL in lipid deposition in fish, as a first step, we characterized the LPL gene of red sea bream, Pagrus major, a marine teleost. This species appears a good experimental animal to investigate the adiposity in fish, because this species can build up remarkable body fat depots (Oku and Ogata, 2000).

In the present study, we isolated and sequenced the LPL cDNA of red sea bream adipose tissue, followed by genomic structure analysis. The entire nucleotide sequences with all introns and 1.1 kb of 5′flanking region were determined. The information for the entire genomic structure and transcriptional regulation of red sea bream were elucidated.

Section snippets

Fish

Red sea bream (Pagrus major) were purchased from a local dealer (Nissin Marine Tech, Aichi, Japan) and cultured in the wet laboratory of National Research Institute of Aquaculture (Nansei, Mie, Japan) until they were killed. Body weights of fish used in the polymerase chain reaction, the cDNA analysis and the genomic structure analysis were 25, 200 and 500 g, respectively.

Extraction of RNA

RNA for the polymerase chain reaction (PCR) was prepared with Quickprep Micro mRNA Purification Kit (Pharmacia, Uppsala,

cDNA and genomic structure analysis

By screening of cDNA library of red sea bream visceral adipose tissue, a cDNA clone for the LPL (RSBcLPL08) was isolated and the nucleotide sequence was determined. The 2946 bp cDNA clone, which contained 113 bp of 5′ and 1.3 kb of 3′untranslated sequences, encoded 511 amino acid residues (Fig. 2).

Using the cDNA clone as a probe, a genomic clone containing the LPL gene (RSBgLPL06) was isolated and the nucleotide sequence of coding region with all introns and 1.1 kb of 5′flanking region were

Acknowledgments

The authors thank Dr Tohru Suzuki, Dr Jeffery Silverstein and Mrs Izumi Okai for their helpful assistance. This work was supported by Biodesign Program of Ministry of Agriculture, Forestry, and Fisheries of Japan (BDP-01-IV-1-11).

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