cDNA Cloning, Tissue Distribution, and Identification of the Catalytic Triad of Monoglyceride Lipase

Abstract

Monoglyceride lipase catalyzes the last step in the hydrolysis of stored triglycerides in the adipocyte and presumably also complements the action of lipoprotein lipase in degrading triglycerides from chylomicrons and very low density lipoproteins. Monoglyceride lipase was cloned from a mouse adipocyte cDNA library. The predicted amino acid sequence consisted of 302 amino acids, corresponding to a molecular weight of 33,218. The sequence showed no extensive homology to other known mammalian proteins, but a number of microbial proteins, including two bacterial lysophospholipases and a family of haloperoxidases, were found to be distantly related to this enzyme. By means of multiple sequence alignment and secondary structure prediction, the structural elements in monoglyceride lipase, as well as the putative catalytic triad, were identified. The residues of the proposed triad, Ser-122, in a GXSXG motif, Asp-239, and His-269, were confirmed by site-directed mutagenesis experiments. Northern blot analysis revealed that monoglyceride lipase is ubiquitously expressed among tissues, with a transcript size of about 4 kilobases.The sequential hydrolysis of stored triglycerides in adipose tissue is the result of a combined action of two lipases, hormone-sensitive lipase and monoglyceride lipase (MGL 1 ; EC 3.1.1.23). Hormone-sensitive lipase catalyzes the first and ratelimiting step, the hydrolysis of triglycerides, and also the subsequent hydrolysis of di-and monoglycerides (1). Hormonesensitive lipase has a marked, although not absolute, preference for the primary ester bond of glyceride substrates. It has been shown that MGL is required to obtain a complete degradation of monoglycerides to fatty acids and glycerol, i.e. in the absence of MGL there is an accumulation of monoglycerides (mainly 2-monoglycerides) (2). The main physiological role for MGL is probably to assure complete hydrolysis of monoglycerides formed during the lipolysis of stored triglycerides of the adipocyte. Another role for the enzyme could be to catalyze the hydrolysis of 2-monoglycerides formed as a result of lipoprotein lipase-catalyzed hydrolysis of triglycerides from chylomicrons and very low density lipoproteins. Lipoprotein lipase has monoglyceride-hydrolyzing activity, with an absolute preference for the primary ester bond (3). This lipase could therefore catalyze the hydrolysis of 1(3)-monoglycerides, which are formed through isomerization from 2-monoglycerides. However, since the rate of isomerization at pH 7.4 is low, it is more likely that a substantial fraction of the 2-monoglycerides, formed through the action of lipoprotein lipase, is transported into the adipocyte and hydrolyzed by MGL (4). It should be pointed out that besides these two enzymes, there is no evidence for any other monoglyceride-hydrolyzing activity of adipose tissue.MGL has been extensively purified from rat adipose tissue in our laboratory (5). The limited amounts of purified enzyme obtained have been used to study some of its enzymological and biochemical propertie...

The endocannabinoid system: a general view and latest additions

“…This(ese) enzyme(s), known as monoacylglycerol lipase(s) (MAGLs), is(are) found in both membrane and cytosolic fractions and also recognize(s) other unsaturated monoacylglycerols, such as, for example, mono-oleoyl-glycerol, which indeed acts as a competitive inhibitor for 2-AG inactivation by intact cells (Ben-Shabat et al, 1998;Di Marzo et al, 1998a). A MAGL with enzymatic properties and subcellular distribution very similar to this(ese) roughly characterized enzyme(s) has been cloned (Karlsson et al, 1997;Dinh et al, 2002). As previously found for FAAH (Egertova et al, 1998), rat MAGL is expressed in brain regions with high cannabinoid CB 1 receptor density, such as the hippocampus, but, unlike FAAH, it occurs in presynaptic neurons and is likely to be coexpressed with CB 1 receptors (Dinh et al, 2002).…”

The evolution and comparative neurobiology of endocannabinoid signalling

“…(c) The phylogenetic distribution of monoacylglycerol lipase MAGL was originally discovered on account of its role in fat metabolism [79] and subsequently, it was proposed that MAGL may regulate 2-AG levels in the brain [20]. Submission of human MAGL as a query sequence in BLAST searches of the GenBank protein database reveals orthologues in a wide range of animal species, including deuterostomian invertebrates, protostomian invertebrate and basal invertebrates such as cnidarians (Nematostella vectensis) and placozoans (Trichoplax adhaerens).…”

Regulation of adipose tissue lipolysis revisited

“…MGL hydrolyses the 1(3) and 2-ester bonds of monoacylglycerols at equal rates but has no affinity for DAG, TAG or cholesteryl esters. Site-directed mutagenesis has confirmed the importance of Ser 122 , Asp 239 and His 269 in the lipase and esterase activities of MGL (52) . MGL is not thought to be rate limiting in lipolysis because of its abundance (53) .…”