Gene_locus Report for: human-PPT2

Mutations in palmitoyl protein thioesterase-1 (PPT1) have been found to cause the infantile form of neuronal ceroid lipofuscinosis, which is a lysosomal storage disorder characterized by impaired degradation of fatty acid-modified proteins with accumulation of amorphous granular deposits in cortical neurons, leading to mental retardation and death. Palmitoyl protein thioesterase-2 (PPT2) is a second lysosomal hydrolase that shares a 26% identity with PPT1. A previous study had suggested that palmitoyl-CoA was the preferred substrate of PPT2. Furthermore, PPT2 did not hydrolyze palmitate from the several S-palmitoylated protein substrates. Interestingly, PPT2 deficiency in a recent transgenic mouse model is associated with a form of neuronal ceroid lipofuscinosis, suggesting that PPT1 and -2 perform non-redundant roles in lysosomal thioester catabolism. In the current paper, we present the crystal structure of PPT2 at a resolution of 2.7 A. Comparisons of the structures of PPT1 and -2 show very similar architectural features; however, conformational differences in helix alpha4 lead to a solvent-exposed lipid-binding groove in PPT1. The limited space between two parallel loops (beta3-alphaA and beta8-alphaF) located immediately above the lipid-binding groove in PPT2 restricts the binding of fatty acids with bulky head groups, and this binding groove is significantly larger in PPT1. This structural difference accounts for the ability of PPT2 to hydrolyze an unbranched structure such as palmitoyl-CoA but not palmitoylcysteine or palmitoylated proteins. Furthermore, differences in fatty acid chain length specificity of PPT1 and -2, also reported here, are explained by the structure and may provide a biochemical basis for their non-redundant roles.

Palmitoylated proteins contain a 16-carbon saturated fatty acyl group that is post-translationally attached by a labile thioester bond. These modified proteins are mainly membrane-bound; the lability of the thioester bond allows the process to be reversible, a unique property of this modification. We report here that the gene for G14, located in the class III region of the human MHC, encodes a polypeptide with significant sequence similarity to mammalian palmitoyl protein thioesterase (PPT1), an enzyme that removes palmitate from palmitoylated proteins. The gene for G14, also known as PPT2, is transcribed as at least five different transcripts, which are expressed in different cell lines of the immune system. Immunoprecipitation of these mammalian cells, with an anti-G14 antiserum, showed a specific band of approx. 42 kDa in cell extracts and supernatants. Expression of the G14 cDNA in the baculovirus system revealed that it encoded a secreted glycosylated polypeptide with S-thioesterase activity. The enzymic activity of the recombinant G14 protein was further characterized in quantitative spectrophotometric assays, which revealed that it had the highest S-thioesterase activity for the acyl groups palmitic and myristic acid followed by other long-chain acyl substrates. The S-thioesterase activity of the G14 protein was found to be considerably higher in supernatants than in cell extracts, which was consistent with the protein's being secreted. The G14 polypeptide contains, in addition to an N-terminal lipase domain, a C-terminal domain common to the cytokine receptor superfamily, which might determine the substrate specificity and/or the protein target of the G14 protein.

Palmitoyl-protein thioesterase is a lysosomal hydrolase that removes long chain fatty acyl groups from modified cysteine residues in proteins. Mutations in this enzyme were recently shown to underlie the hereditary neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis, and lipid thioesters derived from acylated proteins were found to accumulate in lymphoblasts from individuals with the disorder. In the current study, we describe the cloning and expression of a second lysosomal thioesterase, palmitoyl-protein thioesterase 2 (PPT2), that shares an 18% identity with palmitoyl-protein thioesterase. Transient expression of a PPT2 cDNA led to the production of a glycosylated lysosomal protein with palmitoyl-CoA hydrolase activity comparable with palmitoyl-protein thioesterase. However, PPT2 did not remove palmitate groups from palmitoylated proteins that are substrates for palmitoyl-protein thioesterase. In cross-correction experiments, PPT2 did not abolish the accumulation of protein-derived lipid thioesters in palmitoyl-protein thioesterase-deficient cell lines. These results indicate that PPT2 is a lysosomal thioesterase that possesses a substrate specificity that is distinct from that of palmitoyl-protein thioesterase.

Mutations in palmitoyl protein thioesterase-1 (PPT1) have been found to cause the infantile form of neuronal ceroid lipofuscinosis, which is a lysosomal storage disorder characterized by impaired degradation of fatty acid-modified proteins with accumulation of amorphous granular deposits in cortical neurons, leading to mental retardation and death. Palmitoyl protein thioesterase-2 (PPT2) is a second lysosomal hydrolase that shares a 26% identity with PPT1. A previous study had suggested that palmitoyl-CoA was the preferred substrate of PPT2. Furthermore, PPT2 did not hydrolyze palmitate from the several S-palmitoylated protein substrates. Interestingly, PPT2 deficiency in a recent transgenic mouse model is associated with a form of neuronal ceroid lipofuscinosis, suggesting that PPT1 and -2 perform non-redundant roles in lysosomal thioester catabolism. In the current paper, we present the crystal structure of PPT2 at a resolution of 2.7 A. Comparisons of the structures of PPT1 and -2 show very similar architectural features; however, conformational differences in helix alpha4 lead to a solvent-exposed lipid-binding groove in PPT1. The limited space between two parallel loops (beta3-alphaA and beta8-alphaF) located immediately above the lipid-binding groove in PPT2 restricts the binding of fatty acids with bulky head groups, and this binding groove is significantly larger in PPT1. This structural difference accounts for the ability of PPT2 to hydrolyze an unbranched structure such as palmitoyl-CoA but not palmitoylcysteine or palmitoylated proteins. Furthermore, differences in fatty acid chain length specificity of PPT1 and -2, also reported here, are explained by the structure and may provide a biochemical basis for their non-redundant roles.

Palmitoylated proteins contain a 16-carbon saturated fatty acyl group that is post-translationally attached by a labile thioester bond. These modified proteins are mainly membrane-bound; the lability of the thioester bond allows the process to be reversible, a unique property of this modification. We report here that the gene for G14, located in the class III region of the human MHC, encodes a polypeptide with significant sequence similarity to mammalian palmitoyl protein thioesterase (PPT1), an enzyme that removes palmitate from palmitoylated proteins. The gene for G14, also known as PPT2, is transcribed as at least five different transcripts, which are expressed in different cell lines of the immune system. Immunoprecipitation of these mammalian cells, with an anti-G14 antiserum, showed a specific band of approx. 42 kDa in cell extracts and supernatants. Expression of the G14 cDNA in the baculovirus system revealed that it encoded a secreted glycosylated polypeptide with S-thioesterase activity. The enzymic activity of the recombinant G14 protein was further characterized in quantitative spectrophotometric assays, which revealed that it had the highest S-thioesterase activity for the acyl groups palmitic and myristic acid followed by other long-chain acyl substrates. The S-thioesterase activity of the G14 protein was found to be considerably higher in supernatants than in cell extracts, which was consistent with the protein's being secreted. The G14 polypeptide contains, in addition to an N-terminal lipase domain, a C-terminal domain common to the cytokine receptor superfamily, which might determine the substrate specificity and/or the protein target of the G14 protein.

Palmitoyl-protein thioesterase-2 (PPT2) is a homolog of PPT1, the enzyme that is deficient in the lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis (NCL). As a first step toward determining whether mutations in the gene encoding PPT2 (PPT2) are associated with any of the molecularly uncharacterized forms of NCL, we report here the structure and chromosomal localization of human PPT2. PPT2 spans about 10 kb and is composed of nine exons. One major (2.0 kb) and two minor (7.0 and 2.8 kb) mRNAs are transcribed from the gene, and the larger transcripts appear to be messenger RNAs in which PPT2 exons are spliced into a downstream gene encoding a homolog of human latent transforming growth factor-beta binding protein (human LTBP). PPT2 is located in the human major histocompatibility class III locus on chromosome 6p21.3, a position that rules out PPT2 as the causative gene in any of the NCLs at defined chromosomal loci. No mutations were detected by SSCP analysis in a preliminary analysis of 12 subjects referred with a suspected diagnosis of infantile NCL who had normal PPT activity. However, five single nucleotide polymorphisms were found in unrelated normal individuals. These polymorphisms (and a microsatellite discovered within PPT2) will aid in the further delineation of the possible role of PPT2 in lysosomal storage disorders of unknown etiology.

Palmitoyl-protein thioesterase is a lysosomal hydrolase that removes long chain fatty acyl groups from modified cysteine residues in proteins. Mutations in this enzyme were recently shown to underlie the hereditary neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis, and lipid thioesters derived from acylated proteins were found to accumulate in lymphoblasts from individuals with the disorder. In the current study, we describe the cloning and expression of a second lysosomal thioesterase, palmitoyl-protein thioesterase 2 (PPT2), that shares an 18% identity with palmitoyl-protein thioesterase. Transient expression of a PPT2 cDNA led to the production of a glycosylated lysosomal protein with palmitoyl-CoA hydrolase activity comparable with palmitoyl-protein thioesterase. However, PPT2 did not remove palmitate groups from palmitoylated proteins that are substrates for palmitoyl-protein thioesterase. In cross-correction experiments, PPT2 did not abolish the accumulation of protein-derived lipid thioesters in palmitoyl-protein thioesterase-deficient cell lines. These results indicate that PPT2 is a lysosomal thioesterase that possesses a substrate specificity that is distinct from that of palmitoyl-protein thioesterase.