Elsevier

Gene

Volume 557, Issue 2, 25 February 2015, Pages 146-153
Gene

Cloning of porcine GPIHBP1 gene and its tissue expression pattern and genetic effect on adipose traits

https://doi.org/10.1016/j.gene.2014.12.017Get rights and content

Highlights

  • Porcine GPIHBP1 gene sequence characteristics.

  • Tissue expression pattern.

  • Single nucleotide polymorphisms and genetic effect.

Abstract

Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism and is transported by glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) from the interstitial spaces to the capillary lumen. Here, we cloned a cDNA and the genomic locus of the porcine GPIHBP1 gene, and investigated its tissue expression pattern and its genetic effects on adipose traits. Porcine GPIHBP1 exhibits a four-exon/three-intron structure, including a 543 bp open reading frame that encodes 180 amino acids. The porcine GPIHBP1 protein shows 49%–65% homology and shares the major conserved structural domains of GPIHBP1 proteins in other mammals. Porcine GPIHBP1 mRNA levels were high in the adipose tissue, muscle and lung, and higher mRNA levels were observed in sows compared to boars in adipose tissues of the inner and outer layers of subcutaneous fat, abdominal fat, and suet fat. The mRNA expression pattern of porcine GPIHBP1 and LPL genes was similar in most tissues except for the lung. Thirty six single nucleotide polymorphisms (SNPs) were found in the porcine GPIHBP1 gene. Association analyses showed that the g.-255G>C and g.-626T>G SNPs are associated with intramuscular fat content, and that the g.-1557T>C and g.-1948G>A SNPs are associated with back fat thickness. In conclusion, porcine GPIHBP1 mRNA is abundantly expressed in the adipose tissue, muscle and lung, and gender affects GPIHBP1 mRNA expression levels; furthermore, four GPIHBP1 SNPs are genetic factors affecting adipose traits.

Introduction

Lipoprotein lipase (LPL) is a key enzyme for plasma lipid metabolism that is responsible for catalyzing triglyceride-rich lipoprotein hydrolysis. LPL is produced by parenchymal cells, such as myocytes and adipocytes, and then secreted into the surrounding interstitial spaces; however, LPL hydrolyzes triglycerides in plasma lipoproteins at the surface of the capillary lumen (Havel and Kane, 2001, Olivecrona and Olivecrona, 2009, Wang and Eckel, 2009). For decades, the mechanism for the movement of LPL from the interstitial spaces across endothelial cells to its site of action in the capillary lumen was unknown, but this issue was recently solved. It is now clear that a capillary endothelial cell protein, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), is an important binding site for LPL on endothelial cells and is responsible for transporting LPL from the interstitial spaces across endothelial cells to the capillary lumen (Beigneux et al., 2007, Davies et al., 2010).

GPIHBP1 belongs to the lymphocyte antigen 6 (Ly6) family of proteins and is expressed highly in the heart, skeletal muscle, and adipose tissues (Beigneux et al., 2007). GPIHBP1 gene sequences have been reported by mammalian genome sequencing projects in human, mouse and rat. These genes contain four exons encoding 166–236 amino acids (Gerhard et al., 2004, Gibbs et al., 2004, Thierry-Mieg and Thierry-Mieg, 2006). The mature GPIHBP1 protein contains at least four noteworthy functional domains (Beigneux et al., 2007, Gin et al., 2007): an N-terminal signal peptide; an impressive acidic amino acid domain at the amino terminus which is important for the binding of LPL and chylomicrons; a cysteine-rich Ly6 motif which plays a role in binding LPL (Franssen et al., 2010, Olivecrona et al., 2010); and a C-terminal region that includes a hydrophobic domain, which is replaced by a glycosylphosphatidylinositol (GPI) anchor within the endoplasmic reticulum that binds GPIHBP1 to the endothelial cell surface (Fisher, 2010, Nosjean et al., 1997, Ory, 2007). To date, there have been no reports on the sequence and structure of the porcine GPIHBP1 gene or protein.

The finding of chylomicronemia in Gpihbp1−/− mice (Beigneux et al., 2007) indicates that human GPIHBP1 mutations might play an important role in blood triglyceride metabolism. In recent years, some clinically significant GPIHBP1 mutations causing chylomicronemia have been reported in human (Beigneux et al., 2009a, Charrière et al., 2011, Coca-Prieto et al., 2011, Franssen et al., 2010, Olivecrona et al., 2010, Wang and Hegele, 2007). The mutations may attenuate and even abolish the ability of GPIHBP1 to bind LPL, thereby affecting plasma lipid metabolism. However, there have been no reports of GPIHBP1 genetic variation in other mammalian species. It is, therefore, of interest to determine whether there are also important polymorphisms in porcine GPIHBP1 as well as in human GPIHBP1.

Here, we cloned and characterized porcine GPIHBP1 cDNA and genomic sequences. We also investigated its expression pattern in tissues by comparing GPIHBP1 with LPL expression and by performing a comparative study between boars and sows using quantitative reverse transcription and polymerase chain reaction (qRT-PCR). In addition, we detected single nucleotide polymorphisms (SNPs) in the porcine GPIHBP1 gene and investigated their genetic effects on two important economic adipose traits, intramuscular fat (IMF) content and back fat thickness (BFT), which can be used for marker-assisted selection (MAS) in pig breeding systems.

Section snippets

Animals and samples

This study was conducted in compliance with the requirements of the Animal Ethics Committee of Sichuan Agricultural University. To clone the cDNA and the genomic locus of porcine GPIHBP1, adipose tissues from Yanan pigs (210 days old), a local Chinese breed, were sampled for total RNA and DNA isolation. To study the tissue expression pattern of GPIHBP1, six Yanan boars and six Yanan sows at 210 days old were selected and slaughtered. Seventeen fresh tissue samples, including the six adipose

Nucleotide sequence comparison of cDNA and genomic DNA

We obtained a 690 bp cDNA of the porcine GPIHBP1 gene (accession number KP121338 in GenBank), including an entire 543 bp open reading frame (ORF) coding for a protein of 180 amino acids (Supplementary Fig. S1). A 1984 bp genomic GPIHBP1 fragment was obtained and, by comparing cDNA and genomic sequences, we determined that it included four exons and three introns (accession number KP121339 in GenBank). The sequence and structure of porcine GPIHBP1 are shown in Supplementary Fig. S2.

Analysis of amino acid sequences and protein domains

The porcine

Discussion

Most mammalian GPIHBP1 genes show an identical or similar gene structure, having four exons and three introns; however, they encode different length amino acid sequences with some deletions or insertions. Although pig GPIHBP1 shares a moderate homology with GPIHBP1 in other mammals, it has conserved domains common to the known GPIHBP1 proteins in other mammals. An N-terminal signal peptide was found in pig GPIHBP1, which participates in trafficking of the GPIHBP1 protein from the endoplasmic

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT13083) and the International Science & Technology Cooperation Program of China (2014DFA31260). We thank the graduate students and teachers at the Laboratory of Zoology, and Swine Genetics and Breeding of Sichuan Agricultural University.

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