Marine invertebrate lipases: Comparative and functional genomic analysis

Abstract

Lipases are key enzymes involved in lipid digestion, storage and mobilization of reserves during fasting or heightened metabolic demand. This is a highly conserved process, essential for survival. The genomes of five marine invertebrate species with distinctive digestive system were screened for the six major lipase families. The two most common families in marine invertebrates, the neutral an acid lipases, are also the main families in mammals and insects. The number of lipases varies two-fold across analyzed genomes. A high degree of orthology with mammalian lipases was observed. Interestingly, 19% of the marine invertebrate lipases have lost motifs required for catalysis. Analysis of the lid and loop regions of the neutral lipases suggests that many marine invertebrates have a functional triacylglycerol hydrolytic activity as well as some acid lipases. A revision of the expression profiles and functional activity on sequences in databases and scientific literature provided information regarding the function of these families of enzymes in marine invertebrates.

Introduction

With the advent of new sequencing tools, there is a trend for sequencing genomes in model and non-model organisms. This genomic revolution enable access to genome sequences in global databases and makes possible identification and comparison of genomic variation of relevant functions in animals. One of the relevant metabolic pathways is the study of the genes involved in lipid digestion. Lipids are the major source of metabolic energy and essential molecules for the formation of cell and tissue membranes. They are important in the physiology and reproductive process of marine animals and reflect the biochemical and ecological conditions of the marine environment (Sargent et al., 1995). Among lipids, neutral lipids are particularly important since they support larval development, especially embryogenesis and metamorphosis (Sewell, 2005).

Neutral lipids are hydrolyzed by lipases (E.C. 3.1.1.3) (Jaeger et al., 1994). These hydrolytic enzymes possess multifunctional properties such as broad substrate specificity and regio-specificity (Jaeger et al., 1994). Lipases are divided into six families, based on their sequences, which is characterized by an α/β hydrolase fold (CL0028; Zinke et al., 2002). These families are the neutral (Pfam: PF00151), acid (Pfam: PF04083), lipase 2 (Pfam: PF01674), lipase 3 (Pfam: PF01764), lipase with motif Gly-Asp-Ser-(Leu) also known as GDSL (Pfam: PF00657) and hormone sensitive lipases (HSL; Pfam: PF06350) (Derewenda, 1994, Holmquist, 2000). Another small family of lipases with an important role in lipid metabolism among vertebrates and invertebrates is the adipose triglyceride lipase (ATGL; Pfam: PF01734), which function mainly in adipose tissue (Holmes, 2012). All lipases contain a consensus sequence Gly-X-Ser-X-Gly (X: any amino acid) and their catalytic mechanism is a two-step mechanism based on catalytic residues Ser, Asp/Glu and His (Ollis et al., 1992). Among other special features, lipases possess an α-helix fragment or lid which covers the nucleophilic serine and regulates access to the active site (Ollis et al., 1992). In contrast to most lipases, the ATGL family is characterized by a patatin domain and their active site is composed only of Ser and Asp (Wilson et al., 2006).

In marine invertebrates, there is a poor understanding of lipid digestion. Most previous studies on digestive lipases used crude enzyme preparations (Hervant et al., 1999, Perera et al., 2008) and only a few digestive lipases have been isolated (Slim et al., 2007, Rivera-Perez et al., 2011a, Rivera-Perez et al., 2011b). These enzymes are tissue specific in the digestive gland (Johnston et al., 2004, Johnston and Joel, 2005, Moltschaniwskyj and Johnston, 2006, Rivera-Perez et al., 2011a), and they have been described as regio-specific and non-specific lipases. Only one intracellular lipase has been isolated and characterized in crustaceans (Rivera-Perez et al., 2011b). In contrast to mammals and insects, marine invertebrates are able to hydrolyze long-chain triglycerides (Rivera-Perez et al., 2011a, Forrellat-Barrios et al., 2004, Del Monte et al., 2002) which are associated with hydrophobicity of the lid. The lipases described so far function at pH 8.0 (Slim et al., 2007, Pasquevich et al., 2011, Rivera-Perez et al., 2011a), the same pH as human pancreatic lipase (Carriere et al., 2000). Another important characteristic of marine lipases is that they do not require colipase, a small protein cofactor, to display their activity (Rivera-Perez et al., 2011a), as has been described in mammals (Carriere et al., 2000).

Given the importance of the function of lipases in lipid metabolism, this paper focuses on functional genomic analysis of marine invertebrate lipases. Lipases belonging to the seven families of lipases described earlier were found in the genome of five marine invertebrates and one fresh water invertebrate: Pacific oyster Crassostrea gigas, owl limpet Lottia gigantea, purple sea urchin Strongylocentrotus purpuratus, starlet sea anemone Nematostella vectensis, the sponge Amphimedon queenslandica, and water flea Daphnia pulex. These species represent five phylums: Cnidaria (N. vectensis), Echinodermata (S. purpuratus), Crustacea (D. pulex), Mollusca (C. gigas and L. gigantea) and Porifera (A. queenslandica). These animals differ in their organs dedicated to food digestion, as well as their source of lipids and their storage.