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. 2012 May 11;287(20):16158-67.
doi: 10.1074/jbc.M112.347260. Epub 2012 Mar 9.

Reversible lysine acetylation regulates activity of human glycine N-acyltransferase-like 2 (hGLYATL2): implications for production of glycine-conjugated signaling molecules

Dominik P Waluk  1 , Filip Sucharski, Laszlo Sipos, Jerzy Silberring, Mary C Hunt
Affiliations

Reversible lysine acetylation regulates activity of human glycine N-acyltransferase-like 2 (hGLYATL2): implications for production of glycine-conjugated signaling molecules

Dominik P Waluk et al. J Biol Chem. .

Abstract

Lysine acetylation is a major post-translational modification of proteins and regulates many physiological processes such as metabolism, cell migration, aging, and inflammation. Proteomic studies have identified numerous lysine-acetylated proteins in human and mouse models (Kim, S. C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho, Y., Xiao, H., Xiao, L., Grishin, N. V., White, M., Yang, X. J., and Zhao, Y. (2006) Mol. Cell 23, 607-618). One family of proteins identified in this study was the murine glycine N-acyltransferase (GLYAT) enzymes, which are acetylated on lysine 19. Lysine 19 is a conserved residue in human glycine N-acyltransferase-like 2 (hGLYATL2) and in several other species, showing that this residue may be important for enzyme function. Mutation of lysine 19 in recombinant hGLYATL2 to glutamine (K19Q) and arginine (K19R) resulted in a 50-80% lower production of N-oleoyl glycine and N-arachidonoylglycine, indicating that lysine 19 is important for enzyme function. LC/MS/MS confirmed that Lys-19 is not acetylated in wild-type hGLYATL2, indicating that Lys-19 requires to be deacetylated for full activity. The hGLYATL2 enzyme conjugates medium- and long-chain saturated and unsaturated acyl-CoA esters to glycine, resulting in the production of N-oleoyl glycine and also N-arachidonoyl glycine. N-Oleoyl glycine and N-arachidonoyl glycine are structurally and functionally related to endocannabinoids and have been identified as signaling molecules that regulate functions like the perception of pain and body temperature and also have anti-inflammatory properties. In conclusion, acetylation of lysine(s) in hGLYATL2 regulates the enzyme activity, thus linking post-translational modification of proteins with the production of biological signaling molecules, the N-acyl glycines.

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Figures

FIGURE 1.
FIGURE 1.
Lysine 19 is a conserved residue in GLYATs in several species. Alignment of the first ∼24 amino acids of GLYATs in the indicated species was carried out using the ClustalW method. The conserved lysine 19 residue is shown in bold (note this residue is an arginine in hGLYATL1). There is a serine (Ser-20) adjacent to lysine 19 in most species. L1, like-1; L2, like-2.
FIGURE 2.
FIGURE 2.
Lysine 19 in human GLYATL2 expressed in HEK293 cells is not acetylated. Fragmentation CID mass spectrum of the triply ionized KSLEK*SIPESIKVYGAIF (751.9 m/z) peptide (K* indicates lysine at position 19 in the protein sequence) of the hGLYATL2 wild-type protein expressed in HEK293 cells.
FIGURE 3.
FIGURE 3.
Lysine 19 in human GLYATL2 is acetylated/deacetylated in response to treatment with the deacetylase inhibitor NAM. A, recombinant hGLYATL2 was produced in E. coli and affinity-purified as outlined under “Experimental Procedures.” Mass spectrum of the KSLEKSIPESIKVY peptide (K indicates lysine at position 19 in the protein sequence) from the wild-type hGLYATL2 protein, with different levels of ionization. The peptide targeted for MS/MS analysis is marked with a star. B, fragmentation ETD tandem mass spectrum of the KSLEK*SIPESIKVY peptide from the hGLYATL2 wild-type protein; lysine 19 is not acetylated. C, recombinant hGLYATL2 was produced in the presence of 5 mm deacetylase inhibitor NAM, and ETD MS/MS analysis was carried out on the KSLEKSIPESIKVY peptide from the NAM-treated hGLYATL2. The peptide chosen for further MS/MS analysis is marked with a star. D, CID tandem mass spectrum of the peptide shows an acetylated peptide with the sequence KSLEKAcSIPESIKVY, where KAc indicates an acetylated lysine at position 19 (Lys-19).
FIGURE 4.
FIGURE 4.
Quantitative analysis of acetylation levels in the KSLEKSIPESIKVY peptide of hGLYATL2 by LC/MS/MS. The peptides KSLEKSIPESIKVY where lysine 19 is not acetylated elute earlier than the same peptides with modified lysine 19. The ratio of acetylated/non-acetylated lysines in the KSLEKSIPESIKVY peptide in wild-type hGLYATL2 and NAM-treated hGLYATL2 were calculated by measuring the area under the peaks.
FIGURE 5.
FIGURE 5.
Lysine 19 is an important residue in regulation of hGLYATL2 enzyme activity. A, ∼1 μg of hGLYATL2 (wild type), K19Q, and K19R mutant proteins was incubated with 50 μm C18:1-CoA, 50 μm C20:4-CoA, 50 mm glycine with the addition of BSA in a molar ratio of 1:5.5 BSA:acyl-CoA as outlined under “Experimental Procedures.” A, N-oleoyl glycine conjugates formed. B, N-arachidonoyl glycine conjugates formed and were quantified using ESI-MS. The experiment was repeated four times (three times for the K19Q mutant), and the mean ± S.D. is shown. C, recombinant K19Q and K19R proteins (∼1 μg) were incubated for 2 min at various concentrations of oleoyl-CoA (5–100 μm) (C) or N-arachidonoyl-CoA (5–100 μm) (D) with the addition of BSA in a molar ratio of 1:5.5 BSA:acyl-CoA in the presence of glycine (50 mm). N-Arachidonoyl glycine was added as an internal standard (5 μm) to reactions where N-oleoyl glycine is the product formed, and N-oleoyl glycine was added as an internal standard (5 μm) to reactions where N-arachidonoyl glycine is the product formed. Samples were purified on Evolute columns, analyzed by ESI-MS, and quantified according to the internal standard. Km (μm) and Vmax (nmol/min/mg) were calculated using Sigma Plot Enzyme Kinetics program. The experiments were repeated twice, and one representative experiment is shown.
FIGURE 6.
FIGURE 6.
Acetylation/deacetylation of hGLYATL2 regulates enzyme activity. Wild-type hGLYATL2, K19Q, and K19R mutant proteins were produced in BL21(DE3) pLysS cells in the absence/presence of 5 mm NAM, a deacetylase inhibitor. ∼1 μg of hGLYATL2 (wild type), K19Q, and K19R mutant proteins was incubated with 50 μm oleoyl-CoA, arachidonoyl-CoA, 50 mm glycine with the addition of BSA in a molar ratio of 1:5.5 BSA:acyl-CoA as outlined under “Experimental Procedures,” and the N-oleoyl glycine (A) or N-arachidonoyl glycine (B) conjugates formed were quantified using ESI-MS. The experiment was repeated four times (three times for NAM-treated K19Q and K19R mutants), and the mean ± S.D. is shown.
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