Phylogenetic and expression analysis of the angiopoietin-like gene family and their role in lipid metabolism in pigs

Abstract

Objective: The objective of this study was to investigate the phylogenetic and expression analysis of the angiopoietin-like (ANGPTL) gene family and their role in lipid metabolism in pigs.

Methods: In this study, the amino acid sequence analysis, phylogenetic analysis, and chromosome adjacent gene analysis were performed to identify the ANGPTL gene family in pigs. According to the body weight data from 60 Jinhua pigs, different tissues of 6 pigs with average body weight were used to determine the expression profile of ANGPTL1-8. The ileum, subcutaneous fat, and liver of 8 pigs with distinct fatness were selected to analyze the gene expression of ANGPTL3, ANGPTL4, and ANGPTL8.

Results: The sequence length of ANGPTLs in pigs was between 1,186 and 1,991 bp, and the pig ANGPTL family members shared common features with human homologous genes, including the high similarity of the amino acid sequence and chromosome flanking genes. Amino acid sequence analysis showed that ANGPTL1-7 had a highly conserved domain except for ANGPTL8. Phylogenetic analysis showed that each ANGPTL homologous gene shared a common origin. Quantitative reverse-transcription polymerase chain reaction analysis showed that ANGPTL family members had different expression patterns in different tissues. ANGPTL3 and ANGPTL8 were mainly expressed in the liver, while ANGPTL4 was expressed in many other tissues, such as the intestine and subcutaneous fat. The expression levels of ANGPTL3 in the liver and ANGPTL4 in the liver, intestine and subcutaneous fat of Jinhua pigs with low propensity for adipogenesis were significantly higher than those of high propensity for adipogenesis.

Conclusion: These results increase our knowledge about the biological role of the ANGPTL family in this important economic species, it will also help to better understand the role of ANGPTL3, ANGPTL4, and ANGPTL8 in lipid metabolism of pigs, and provide innovative ideas for developing strategies to improve meat quality of pigs.

Keywords: Angiopoietin-like Protein; Evolution; Expression; Fat Metabolize; Jinhua Pig.

Figure 1

Multiple sequence alignment of representative pig angiopoietin-like (ANGPTL) protein sequences. The conserved residues are shaded. Specifically, the black part is highly similar, the gray part is less similar, and the non-color covered part has no similarity.

Figure 2

Phylogenetic analysis of pig angiopoietin-like (ANGPTL) genes and their representative sequences from human and animals. The topology was constructed using the amino acid sequences by the Neighbor-Joining method with 1,000 bootstrap repeats. Only the branches with a bootstrap value greater than 50 are displayed at branching points.

Figure 3

Comparison of homologous genomic regions of pig ANGPTL3 with humans, mice, and several other animals. The genetic environment of the pig ANGPTL3 gene was characterized, and the homologous genes of humans, mice, and other animals were identified. The horizontal line represents the chromosome fragments, the arrow box represents the gene, and the arrowhead points in the direction of predicted gene transcription. Only genes conserved across species are displayed. The same color is of the gene homologs, and they are presented according to their order in the chromosome. The gene names and symbols are: PATJ, PATJ crumbs cell polarity complex component); L1TD1, LINE1 type transposase domain containing 1; KANK4, KN motif and ankyrin repeat domains 4; USP1, ubiquitin-specific peptidase 1; DOCK6, dedicator of cytokinesis 6; ANGPTL3, angiopoietin-like 3; ATG4C, autophagy related 4C cysteine peptidase; FOXD3, forkhead box D3; ALG6, ALG6 alpha-1,3-glucosyltransferase; ITGB3BP, integrin subunit beta 3 binding protein; EFCAB7, EF-hand calcium binding domain 7.

Figure 4

Comparison of homologous genomic regions of pig ANGPTL4 with humans, mice, and several other animals. The genetic environment of the pig ANGPTL4 gene was characterized, and the homologous genes of humans, mice, and other animals were identified. The horizontal line represents the chromosome segment, the arrow box represents the gene, and the arrowhead points in the direction of predicted gene transcription. Only genes preserved across species will appear. The same color is of the gene homologs, and they are presented according to their order in the chromosome. The gene names and symbols are: CERS4, ceramide synthase 4; CD320, CD320 molecule; DUFA7, NADH:ubiquinone oxidoreductase subunit A7; RPS28, Ribosomal protein S28; KANK3, KN motif, and ankyrin repeat domains 3; ANGPTL4, angiopoietin-like 4; Rab11B, RAB11B, member RAS oncogene family; MARCH2, membrane-associated ring finger (C3HC4) 2; HNRNPM, heterogeneous nuclear ribonucleoprotein M; PRAM1, PML-RARA regulated adaptor molecule 1; ZNF414, zinc finger protein 414.

Figure 5

Comparison of homologous genomic regions of pig ANGPTL8 with humans, mice, and several other animals. The genetic environment of the pig ANGPTL8 gene was characterized, and the homologous genes of humans, mice, and other animals were identified. The horizontal line represents the chromosome segment, the arrow box represents the gene, and the arrowhead points in the direction of predicted gene transcription. Only genes preserved across species will appear. The same color is of the gene homologs, and they are presented according to their order in the chromosome. The gene names and symbols are: SMARCA4, SWI/SNF related, matrix associated, actin-dependent regulator of chromatin, subfamily a, member 4; LDLR, low density lipoprotein receptor; SPC24, SPC24 component of NDC80 kinetochore complex; KANK2, KN motif and ankyrin repeat domains 2; DOCK6, dedicator of cytokinesis 6; ANGPTL8, angiopoietin-like 8; SPAN16, sperm peptide antigen; RAB3D, RAB3D, member RAS oncogene family; TMEM250, Transmembrane protein 250; CCDC159, Coiled-coil domain containing 159; PLPPR2, phospholipid phosphatase related 2.

Figure 6

Relative expression of ANGPTL1–8 in different tissues, including tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, trachea, lung, heart, longissimus muscle, subcutaneous fat, liver, kidney, brain, pancreas, and spleen of Jinhua pigs. The indicated tissue segments were collected from 6 Jinhua pigs at 270 days of age following by RNA isolation, reverse transcription and RT-qPCR. ANGPTL8, angiopoietin-like 8. Data was expressed as mean±standard error of the mean (n = 6).

Figure 7

Body weight (A) and backfat thickness (B) of Jinhua pigs in the H and L groups. Jinhua pigs with similar body weight and significantly different backfat thickness were weighed individually and the backfat thickness of each pig was measured at 270 days of age. H, the H group consisted of pigs with relatively high backfat thickness; L, the L group was consisted of pigs with relatively low backfat thickness. ANGPTL, angiopoietin-like. Data was expressed as mean±standard error of the mean (n = 8) and analyzed by one-way analysis of variance analysis followed by an unpaired two-tailed student’s t-test.

Figure 8

The expression of ANGPTL3, ANGPTL4, and ANGPTL8 in the liver (A, B, and C), subcutaneous fat (D), and ileum (E) of Jinhua pigs in the H and L groups. The liver, subcutaneous fat, and ileum segments were collected from 16 Jinhua pigs at 270 days of age following by RNA isolation, reverse transcription and RT-qPCR. H, the H group consisted of pigs with relatively high backfat thickness; L, the L group was consisted of pigs with relatively low backfat thickness. Data was expressed as mean±standard error of the mean (n = 8) and analyzed by one-way analysis of variance analysis followed by an unpaired two-tailed student’s t-test.