Abstract

Lipase I (enzyme name LIPI or LPDL) (gene name LIPI [human] or Lipi [mouse]) is a phospholipase which generates 2-acyl lysophosphatidic acid (LPA), a lipid mediator required for maintaining homeostasis of diverse biological functions and in activating cell surface recaptors. Bioinformatic methods were used to predict the amino acid sequences, secondary and tertiary structures and gene locations for LIPI genes and encoded proteins using data from several mammalian genome projects. LIPI is located on human chromosome 21 and is distinct from other phospholipase A1-like genes (LIPH and PS-PLA1). Mammalian LIPI genes contained 10 (human) or 11 (mouse) coding exons transcribed predominantly on the negative DNA strand. Mammalian LIPI protein subunits shared 61% - 99% sequence identities and exhibited sequence alignments and identities for key LIPI amino acid residues as well as extensive conservation of predicted secondary and tertiary structures with those previously reported for pancreatic lipase (PL), with “N-signal peptide”, “lipase” and “plat” structural domains. Comparative studies of mammalian LIPI sequences with LIPH, PS-PLA1 and pancreatic lipase (PL) confirmed predictions for LIPI N-terminal signal peptides (residues 1 - 15); predominantly conserved mammalian LIPI N-glycosylation sites (63NNSL and 396NISS for human LIPI); active site “triad” residues (Ser159; Asp183; His253); disulfide bond residues (238 - 251; 275 - 286; 289 - 297; 436 - 455); and a 12 residue “active site lid”, which is shorter than for other lipases examined. Phylogenetic analyses supported a hypothesis that LIPI arose from a vertebrate LIPH gene duplication event within a mammalian common ancestral genome. In addition, LIPI, LIPH and PL-PLA1 genes were distinct from the vascular lipase (LIPG, LIPC and LPL) and pancreatic lipase (PL) gene families.

Keywords

Mammalian LIPI Genes and Proteins; Amino Acid Sequence; Lipase I; Evolution; Phylogeny

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1. INTRODUCTION

Lipase I (LIPI; E.C.2.7.11.34), also known as phosphatidic acid-selective phospholipase A1β, PA-PLA1β, cancer-testis antigen 17 (CT17) or LPD lipase (PLDL), is a membrane-associated phospholipase catalyzing the production of fatty acids and lysophosphatidic acid (LPA) from phosphatidic acid [1,2]. LPA is a lipid mediator involved in diverse biological functions in the body [3]. Most of these functions are mediated by G protein-coupled receptors (GPCRs) specific to LPA, for which six GPCRs have been described [4]. A second membrane associated phosphatidic acid-selective phospholipase, lipase H (LIPH; E.C.2.7.11.30) (also known as phosphatidic acid-selective phospholipase A1α or PA-PLA1α), has also been identified as playing a major role in hair follicle cells, with autosomal recessive hypotrichosis (ARH) resulting from LIPH mutations, leading to the woolly hair hypotrichosis phenotype in human populations and sparse hair on the scalp [5-7]. A third membrane associated phosphatidic acid-selective phospholipase, phosphatidylserine specific phospholipase A1 (PS-PLA1), has also been described which hydrolyzes fatty acids at the sn-1 position of phosphatidylserine and 1-acyl-2-lysophosphatidylserine [8-10].

Human LIPI and mouse Lipi genes are expressed in the testis with LIPI found at the connecting piece of primary spermatocytes [1,2], although a specific role for LIPI in LPA signalling has not as yet been reported [10]. LIPI has been identified as a Ewing tumor-associated cancer/testis antigen (called CTA17), which is also expressed in testis and thyroid tissues [11,12]. The human LIPI gene spans more than 98 kilobases of DNA, comprises 10 coding exons on the reverse strand and is localized on chromosome 21 near a gene encoding the RNA binding motif protein 11 (RBM11) [13,14]. Mouse Lipi spans more than 45 kilobases of DNA, encodes 11 coding exons on the reverse strand of chromosome 16, and is also located near the gene encoding RNA binding motif protein 11 (RBM11) [2,14]. Mutations of the human LIPI (or LPDL) and mouse Lipi (or Lpdl) genes have been associated with dyslipidemia in humans and hypertriglyceridemia in mice [2]. In addition, a monosomic deletion model for the mouse chromosome 16 Lipi-Usp25 region exhibited a significant increase in fat mass, thickened subcutaneous fat and liver steatosis, when fed a high-fat diet [15]. Hesse and coworkers [16] have recently identified two LIPH/LIPI-like genes in the chicken genome with wide tissue expression profiles.

There have been few biochemical and structural studies of mammalian LIPI, however two reports have described amino acid sequences for human and mouse LIPI, derived from cDNA sequences, encoding 460 and 476 amino acids, respectively, containing N-terminal and lipase domains in each case [1,2]. Human LIPI (also called LPDL) exhibited 71% identity with mouse LIPI, and ~44% identity with LIPH and ~35% identity with vascular lipase sequences [lipoprotein lipase (LPL), endothelial lipase (EL) and hepatic lipase (HL)]. In common with the human and mouse LIPH amino acid sequences, the LIPI sequences were characterized by shortened “active site lid” motifs and exhibited a higher affinity for heparin binding than for LIPH.

This paper reports the predicted gene structures and amino acid sequences for several mammalian LIPI genes and proteins, the predicted secondary and tertiary structures for mammalian LIPI protein subunits, and the structural, phylogenetic and evolutionary relationships for these genes and enzymes with the vertebrate LIPH and PL-PLA1 gene families and with vertebrate vascular and pancreatic lipase gene families.

2. METHODS

2.1. Mammalian LIPI Gene and Protein Identification

BLAST (Basic Local Alignment Search Tool) studies were undertaken using web tools from the National Center for Biotechnology Information (NCBI) (http://blast. ncbi.nlm.nih.gov/Blast.cgi) [17]. Protein BLAST analyses used human and mouse LIPI amino acid sequences previously described [1,2] (Table 1). Non-redundant protein sequence databases for several mammalian and other vertebrate genomes were accessed from sources previously described [18]. Predicted LIPI-like protein sequences were obtained in each case and subjected to analyses of predicted protein and gene structures.

BLAT (BLAST-Like Alignment Tool) analyses were subsequently undertaken for each of the predicted vertebrate LIPI amino acid sequences using the University of California Santa Cruz (UCSC) Genome Browser [http:// genome.ucsc.edu/cgi-bin/hgBlat] [14] with the default settings to obtain the predicted locations for each of the vertebrate LIPI genes, including predicted exon boundary locations and gene sizes. This browser was also used to show alignments of LIPI genes from several vertebrate genomes (called Multiz alignments). Structures for human and mouse LIPI isoforms were obtained using the AceView website to examine predicted gene and protein structures [19]. Mammalian LIPI sequences were aligned using the ClustalW2 multiple sequence alignment program [20].

2.2. Predicted Structures and Properties of Mammalian LIPI Protein Subunits

Predicted secondary and tertiary structures for mammalian LIPI-like subunits were obtained using the PSIPRED v2.5 web site tools [21] and the SWISS MODEL web tools [swissmodel.expasy.org], respectively [22]. The reported tertiary structure for horse pancreatic lipase (PDB: 1hpl) [23] served as the reference for the predicted human, mouse and rat LIPI tertiary structures, with a modeling range of residues 10 to 447. Theoretical isoelectric points and molecular weights for mammalian LIPI subunits were obtained using Expasy web tools (http://au.expasy.org/tools/pi_tool.html). SignalP 3.0 web tools were used to predict the presence and location of signal peptide cleavage sites (http://www.cbs. dtu.dk/services/SignalP/) for each of the predicted mammalian LIPI sequences [24]. The NetNGlyc 1.0 Server was used to predict potential N-glycosylation sites for mammalian LIPI subunits (http://www.cbs.dtu.dk/services/ NetNGlyc/).

2.3. Phylogenetic Studies and Sequence Divergence

Alignments of mammalian LIPI, human, mouse and zebrafish LIPH (lipoprotein H) and human, mouse, rat, chicken and zebrafish PS-PLA1, HL (hepatic lipase), LPL (lipoprotein lipase), EL (endothelial lipase) protein sequences, as well as human, mouse and frog pancreatic lipases (PL) sequences (Table 1s) were assembled using BioEdit v.5.0.1, as previously described [25]. Alignment ambiguous regions were excluded prior to phy

logenetic analysis yielding alignments of 379 residues for comparisons of sequences (Table 1). Evolutionary distances were calculated using the Kimura option as previously described [18]. Phylogenetic trees were constructed from evolutionary distances using the neighborjoining method [25]. Tree topology was examined by the boot-strap method (100 bootstraps were applied) of resampling and only values that were highly significant (≥95) are shown [26].

3. RESULTS AND DISCUSSION

3.1. Alignments of Human and Other Mammalian LIPI Subunits

The deduced amino acid sequences for orangutan (Pongo abelii), gibbon (Nomascus leucogenys), marmotset (Callithrix jacchus) and rat (Rattus norvegicus) LIPI subunits are shown in Figure 1 together with the previously reported sequences for human (Homo sapiens) and mouse (Mus musculus) LIPI subunits (Table 1) [1,2].

Alignments of the human and other mammalian LIPI subunits examined showed between 61% - 99% sequence identities, suggesting that these are products of the same family of genes and proteins (Table 2). The amino acid sequence for human and other primate LIPI subunits contained 460 residues while other mammalian LIPI subunits contained 476 (mouse and rat) to 452 residues

(dog LIPI) (Figure 1; Table 1). The mouse and rat LIPI sequences differed in length from the primate LIPI sequences as a result of 16 additional residues at the amino terminus end. Several key amino acid residues for vertebrate LIPI were recognized (sequence numbers refer to human LIPI) (Figures 1 and 1s). These included the catalytic triad for the active site (Ser159; Asp183; His253) forming a charge relay network for substrate hydrolysis, similar to human lipoprotein lipase, other lipases and carboxylesterases [27,28]; a hydrophobic N-terminus signal peptide (residues 1 - 15), similar to that reported for rat hepatic lipase [29]; and disulfide bond forming residues (Cys238/Cys251; Cys275/Cys286; Cys289/ Cys297; and Cys436/Cys455) [1,2]. Identical residues were observed for each of the mammalian LIPI subunits for the active site triad and disulfide bond forming residues, however the N-terminus 15-residue signal peptide underwent some changes in sequence but retained predicted signal peptide properties (Figures 1 and 1s; Table 1). Two of the N-glycosylation sites predicted for human LIPI at Asn63-Asn64-Ser65 and at Asn396-397Ile- 398Ile (designated as sites 3 and 9, respectively) were retained for most of the 11 mammalian LIPI sequences examined, although predicted N-glycolsylation sites were observed at other positions for some sequences, including Asn23-Thr24-Thr52 (site 1) for mouse LIPI; Asn34-Arg35-Thr36 (site 2) for rat LIPI; Asn61-Phe62-

Ser63 and Asn71-Phe72-Ser73 (site 4) for dog and panda LIPI, respectively (Table 3). Given the reported role of the N-glycosylated carbohydrate group in contributing to the stability and maintaining catalytic efficiency of a related enzyme (human carboxylesterase or CES1) [30], this property may be shared by mammalian LIPI as well. Human LIPI Ser25, which has been previously reported to undergo phosphorylation in response to DNA damage [31], has also been retained (but with Thr41 for mouse LIPI) for each of the mammalian LIPI sequences examined (Figure 1).

3.2. Alignments of Human LIPI and Other Lipase Subunits

Alignments of human LIPI, lipase H (LIPH) [32], phosphatidylserine specific phospholipase A1 (PS-PLA1) [8-10], endothelial lipase (EL) [18,33], hepatic lipase (HL) [34], lipoprotein lipase (LPL) [35] and pancreatic lipase (PL) [36] sequences are shown in Figure 2. The following key amino acid residues were observed for each of these lipases consistent with those observed for the mammalian LIPI sequences (see Table 1). These included the N-terminal signal peptide sequences, which were distinct for each lipase but retained the property as signal peptides; the active site triad residues aligning with LIPI Ser159, Asp183 and His253; the disulfide cysteine residues reported for mammalian LIPH and LIPI sequences (human LIPI Cys238/Cys251; Cys275/Cys286; Cys289/Cys297; and Cys436/Cys455), although human PS-PLA1 did not contain the last disulfide pair, and additional disulfide bonds were observed for EL, LPL and HL (corresponding to human EL Cys64/Cys77) and for human PL (corresponding to Cys20/Cys26 and Cys107/ Cys118); distinct N-glycosylation sites were predominantly observed for each of the lipase sequences examined; the active site “lid” sequences showed that LIPI, LIPH and PS-PLA1 exhibited fewer residues in each case (12 amino acids), in comparison with the other lipases (EL [19 residues]; LPL and HL [22 residues]; and PL [23 residues]; and a high basic amino acid content region was observed for human LIPI residues (Arg299 →Arg321] aligning with a heparin binding site (human EL 324Lys→333Lys) reported for human EL which binds the enzyme to heparan sulfate proteoglycans on the luminal side of endothelial cells [37,38]. The latter region for human LIPI may explain the enhanced heparin binding reported for LIPI, in compareson with LIPH, and the higher isoelectric point (pI) values observed for mammalian LIPI proteins (Table 1) [1].

3.3. Predicted Secondary and Tertiary Structures of Mammalian LIPI Subunits

Predicted secondary structures for mammalian LIPI sequences were compared in Figure 1, and similar α- helix and β-sheet structures were observed for all of the mammalian LIPI subunits examined. Consistent structures were particularly apparent near key residues or functional domains including the β-sheet and α-helix structures near the active site Ser159 (β4/α3) and His253 (α4) residues; and the conserved disulfide bonds at Cys275/Cys286 (near α4) and Cys436/Cys455 (near β14). Proximate to the active site histidine (His253) and lo-

cated between two residues (Cys238/Cys251) forming disulfide intramolecular bonds are 12 amino acids forming the proposed “active site lid” structure for vertebrate LIPI [1,2]. In addition, eight β-sheets were observed at the LIPI C-terminus end, which is consistent with PLAT domain structures previously reported for horse pancreatic lipase (PL) [23]. It is apparent from these studies that the LIPI subunits examined have highly similar seconddary structures.

The tertiary structure for horse pancreatic lipase (PTL) is from [28]; predicted mouse, rat and human LIPI 3-D structures were obtained using the SWISS MODEL web site http://swissmodel.expasy.org and based on the reported structure for horse PTL (PDB: 1hpl); 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; the “lipase” and “plat” domains, the active site region and the “lid” covering the active site are shown.

Figure 3 describes predicted tertiary structures for human, mouse and rat LIPI sequences, in comparison with horse pancreatic lipase (PL) [23]. Identification of specific structures within the predicted LIPI sequences were based on the reported structure for horse PL which identifies a sequence of twisted β-sheets interspersed with several α-helical structures which are typical of the alphabeta hydrolase super-family. The active site LIPI triad was centrally located which is similar to that observed for other lipases [18,23,39] and carboxylesterase (human CES1) [40]. The major difference between LIPI and other lipases examined (see Figures 2 and 3) is the much smaller size of the “lid” region at positions 239 - 251, which may act as a surface loop that partially covers the opening to the catalytic triad and allows access to the

Conflicts of Interest

The authors declare no conflicts of interest.

References