Comparative Structures and Evolution of Vertebrate Carboxyl Ester Lipase (CEL) Genes and Proteins with a Major Role in Reverse Cholesterol Transport

Abstract

Bile-salt activated carboxylic ester lipase (CEL) is a major triglyceride, cholesterol ester and vitamin ester hydrolytic enzyme contained within pancreatic and lactating mammary gland secretions. Bioinformatic methods were used to predict the amino acid sequences, secondary and tertiary structures and gene locations for CEL genes, and encoded proteins using data from several vertebrate genome projects. A proline-rich and O-glycosylated 11-amino acid C-terminal repeat sequence (VNTR) previously reported for human and other higher primate CEL proteins was also observed for other eutherian mammalian CEL sequences examined. In contrast, opossum CEL contained a single C-terminal copy of this sequence whereas CEL proteins from platypus, chicken, lizard, frog and several fish species lacked the VNTR sequence. Vertebrate CEL genes contained 11 coding exons. Evidence is presented for tandem duplicated CEL genes for the zebrafish genome. Vertebrate CEL protein subunits shared 53-97% sequence identities; demonstrated sequence alignments and identities for key CEL amino acid residues; and conservation of predicted secondary and tertiary structures with those previously reported for human CEL. Phylogenetic analyses demonstrated the relationships and potential evolutionary origins of the vertebrate CEL family of genes which were related to a nematode carboxylesterase (CES) gene and five mammalian CES gene families.

Figure 1

Amino acid sequence alignments for human and other vertebrate CEL subunits. See Table 1 for sources of CEL sequences; * shows identical residues for CEL subunits;: similar alternate residues;. dissimilar alternate residues; N-Signal peptide residues are in red; N-glycosylation residues at 207NIT (human CEL) are in green; active site (AS) triad residues Ser, Asp, and His are in pink; O-glycosylation sites are in blue; disulfide bond Cys residues for human CEL (•); essential arginines which contribute to bile-salt binding are in red; helix (human CEL or predicted helix); sheet (human CEL) or predicted sheet; bold font shows known or predicted exon junctions; exon numbers refer to human CEL gene; CEL “loop” covering the active site (human CEL residues 136–143) are in green; Hu-human CEL; Co-cow CEL; Mo-mouse CEL; Op-opossum CEL; Ch-chicken CEL; Z1-zebrafish CEL1; Z2-zebrafish CEL2.

Figure 2

Amino acid alignments for C-terminal 11-residue repeat sequences for mammalian CEL subunits. Hydrophobic amino acid residues are shown in red; hydrophilic residues in green; acidic residues in blue; basic residues in pink; (squared T) refers to known O-glycosylation sites for human CEL; R refers to repeat number. P-proline; V-valine; T-threonine; G-glycine; D-aspartate; E-glutamate; S-serine; A-alanine; K-lysine; N-asparagine; note consistent PVPP start sequences.

Figure 3

Predicted tertiary structures for mouse CEL and zebrafish CEL1 subunits. The predicted mouse CEL and zebrafish CEL1 3-D structures were obtained using the SWISS MODEL web site http://swissmodel.expasy.org/ and based on the reported structure for bovine CEL (PDB: 1aqlB) [1]; the rainbow color code describes the 3-D structures from the N- (blue) to C-termini (red color); N refers to amino terminus; C refers to carboxyl terminus; specific alpha helices (αA … αN) and beta sheets (β1 … β13) were identified, as well as the active site region and the “loop” covering the active site.

Figure 4

Gene structures for the human and mouse CEL genes. Derived from AceView website http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/ [2]; the major isoform variant is shown with capped 5′- and 3′- ends for the predicted mRNA sequences; introns and exons are numbered; the length of the mRNAs (as kilobases or kb) and comparative expression levels with the average gene are shown; a CpG island (CpG51); several predicted transcription factor binding sites; and a MiRNA485-5p binding site were identified for the human CEL gene; the direction for transcription is shown; 3′UTR refers to 3′-untranslated region.

Figure 5

Comparative sequences for vertebrate 5′-flanking, 5′-untranslated, and coding regions for the CEL genes. Derived from the UCSC Genome Browser using the Comparative Genomics track to examine alignments and evolutionary conservation of CEL gene sequences; genomic sequences aligned for this study included primate (human and rhesus), nonprimate eutherian mammal (mouse, dog and elephant), a marsupial (opossum), a monotreme (platypus), bird (chicken), reptile (lizard), amphibian (frog), and fish (stickleback); conservation measures were based on conserved sequences across all of these species in the alignments which included the 5′flanking, 5′-untranslated (5′UTR), exons, introns, and 3′ untranslated (3′UTR) regions for the CEL gene; regions of sequence identity are shaded in different colors for different species; exons 1–11 are shown which are regions of conservation.

Figure 6

Comparative tissue expression for human and mouse CEL genes. Expression “heat maps” (GNF Expression Atlas 2 data) (http://biogps.gnf.org/) [3] were examined for comparative gene expression levels among selected human (GNF1H) and mouse (GNF1M) tissues for CEL showing high (red), intermediate (black), and low (green) expression levels. The results were derived from the human and mouse genome browsers (http://genome.ucsc.edu/) [4].

Figure 7

Phylogenetic tree of vertebrate CEL with human and mouse CES1, CES2, CES3, CES4, and CES5 amino acid sequences. The tree is labeled with the gene name and the name of the vertebrate. Note the major cluster for the vertebrate CEL sequences and the separation of these sequences from human and mouse CES1, CES2, CES3, CES4, and CES5 sequences. The tree is “rooted” with the CES sequence (T27C12) from a nematode (Caenorhabditis elegans). See Table 1 for details of sequences and gene locations. A genetic distance scale is shown (% amino acid substitutions). The number of times a clade (sequences common to a node or branch) occurred in the bootstrap replicates are shown. Only replicate values of 90 or more which are highly significant are shown with 100 bootstrap replicates performed in each case.