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Contents

Introduction

The lysosome is a membrane-bound organelle which is present in animal cells. Those are acidic vesicles and contain more than fifty digestive enzymes such as proteases, nucleases, glycosidases, sulfatases, lipases, phosphatases phospholipases and esterases. The lumen’s pH of 4,5 is optimal for the hydrolytic enzymes. Indeed, acidic pH is important for the degradation of intracellular and extracellular compounds.[1][2] [3]

Particularly, there are enzymes able to cleave phospholipids on a specific site: phospholipases. These phospholipases are classified into four types, phospholipase A1, A2, C and D depending on the specific cleavage site of the substrate. The Phospholipase A2 cleaves the acyl-ester bonds on sn-2 position of glycerophospholipids and they produce free fatty acids.[4] [5]


Substrate specificity depending on the phospholipase family
Substrate specificity depending on the phospholipase family


Structure

The crystal structure was performed by 1,83 Angstrom spacing. The researchers used a protein secreted from HEK293S GnTI- cells. Those cells come from embryonic kidney of human and do not express N-acetylglucosaminyltransferase I. This line of cells is used to overexpress a wide variety of mammalian membrane proteins.

The lysosomal phospholipase A2 is composed of 380 amino acids. The protein contains both alpha helix and beta strand therefore, belongs to the alpha/beta hydrolase superfamily. [6]

The protein is divided into . First, the from Leucine 18 to Proline 81, contains six beta strand and two alpha helices. The from Glycine 198 to Threonine 288 then, from Glycine 301 to Glycine 324 presents five alpha helix and four beta strand. Finally, the from Alanine 1 to Glutamine 17, from Glycine 82 to Glycine 197, from Methionine 289 to Threonine 300, then, from Aspartate 325 to the end of the protein.

The lysosomal phospholipase A2 has a in the alpha/beta hydrolase domain at conserved topological positions: Serine 165, Aspartate 327 and Histidine 359. Some of particularity of the protein are observed in the protein structure: there is a between Cysteine 32 and Cysteine 56; a from Aspartate 211 to Proline 220 and this characteristic loop is a conserved element in different species. This loop undergoes a conformational change upon contact with its substrate to allow the substrate to access the active site of the protein. [7]

Finally, the quater structure of the protein is the assembly of four chains : A, B, C and D.

Function

Lysosomal phospholipase A2 degrades glycerophospholipids by hydrolysis, but also plays a role in cellular phospholipid homeostasis.

Tissue distribution: The highest level of lysosomal phospholipase A2 activity was found in alveolar macrophages. It was 40 times higher in alveolar macrophages than other tissues, such as monocytes and peritoneal macrophages. [8]

Mechanism

The lysosomal phospholipase A2 activity is strongly dependent on pH and is the highest at pH 4.5, a pH commonly found in lysosomes and in local inflammation events. Bis Monoacylglycerol Phosphate (BMP) inducing a negative charge on lysosomal membrane, the electrostatic repulsion is weakened between lysosomal phospholipase A2 and the membrane at low pH. The protein has a global basic electrostatic surface that is complementary of the acidic inner membrane of the lysosomal membrane. The structure shows a hydrophobic surface including Tyrosine 30, Leucine 31, Leucine 50 and Valine 52 on the membrane-binding domain which would be sufficient to bring the protein to the lipid bilayers.

After docking on membrane, a phospholipid, substrate of the enzyme, enters into the active site. The catalytic triad is located such a way to cleave the acyl group of the phospholipids. The serine is able to act as a nucleophile cleave the acyl-ester bonds. The histidine of the catalytic triad is placed to protonate the lysophospholipid after the cleavage.[9]

Ligands

Lysosomal phospholipase A2 has 5 types of  :

- : N-acetylglucosamine sugars are observed at Asn66, Asn240, Asn256 and Asn365 (found on Asn-X-Ser/Thr motifs) and are N-glycosylation sites. NAG are useful for a post-translational modification : glycosylation.

- : 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (or HEPES) is observed at Ser33, a tyrosine kinase phosphorylation site. EPE is a zwitterion, its dissociation in water decreases as the heat decreases, allowing to maintain enzyme structure and function at low temperature.

- : (4s)-2-methyl-2,4-pentanediol is found at Asp 13, Asp211 which are glycosylation sites, and Arg214

-PO4 : Phosphate, observed at His347, a tyrosine kinase phosphorylation site and Glu346, a glycosylation site.

- : Chloride are found at His347 (tyrosine kinase site), Gln348 and Gln294 (glycosylation site).

[10][11]

Disease's treatment

Implication of lysosomal phospholipase A2 in detoxification :

Lipid oxidation products and in particular oxidized phospholipids (OxPL) are increasingly recognized as inducers of chronic inflammation characteristic of atherosclerosis. Atherosclerosis is a chronic inflammatory disease characterized by accumulation of monocytes and T-cells due to lipid abnormalities. Increased levels of phospholipids’ oxidation products have been detected in different organs and pathological states, including atherosclerotic vessels. They can integrate the lipid membranes of cells and lipoproteins, act as ligands and may cause local membrane disruption. Indeed, they stimulate production of chemokines and adhesion of monocytes to endothelial cells. Truncated oxidized-glycerophospholipids (ox-PLs) are bioactive lipids resulting from oxidative stress. They are generated by the oxidation of polyunsaturated fatty acid residues, which are usually present in the phospholipids at the sn-2 position (cleave position of lysosomal phospholipase A2). Since the ox-PLs are transferred to lysosomes, the lysosomal phospholipase A2 plays an important role in the degradation of them. In fact, lysosomal phospholipase A2 preferentially hydrolyses truncated ox-PCs compared to non-oxidized phospholipids with two long acyl chains (like DOPC) under acidic conditions.[12][13][14]


References

  1. Abe A, Hiraoka M, Shayman JA. A role for lysosomal phospholipase A2 in drug induced phospholipidosis. Drug Metab Lett. 2007 Jan;1(1):49-53. PMID:19356018
  2. Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644
  3. Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
  4. Shayman JA, Kelly R, Kollmeyer J, He Y, Abe A. Group XV phospholipase A(2), a lysosomal phospholipase A(2). Prog Lipid Res. 2011 Jan;50(1):1-13. doi: 10.1016/j.plipres.2010.10.006. Epub, 2010 Nov 11. PMID:21074554 doi:http://dx.doi.org/10.1016/j.plipres.2010.10.006
  5. ATCC: The Global Bioresource Center. Available at: https://www.lgcstandards-atcc.org/. (Accessed: 22nd January 2017)
  6. http://www.uniprot.org/uniprot/Q8NCC3
  7. Glukhova A, Hinkovska-Galcheva V, Kelly R, Abe A, Shayman JA, Tesmer JJ. Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase. Nat Commun. 2015 Mar 2;6:6250. doi: 10.1038/ncomms7250. PMID:25727495 doi:http://dx.doi.org/10.1038/ncomms7250
  8. Hiraoka M, Abe A, Lu Y, Yang K, Han X, Gross RW, Shayman JA. Lysosomal phospholipase A2 and phospholipidosis. Mol Cell Biol. 2006 Aug;26(16):6139-48. PMID:16880524 doi:http://dx.doi.org/10.1128/MCB.00627-06
  9. Glukhova A, Hinkovska-Galcheva V, Kelly R, Abe A, Shayman JA, Tesmer JJ. Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase. Nat Commun. 2015 Mar 2;6:6250. doi: 10.1038/ncomms7250. PMID:25727495 doi:http://dx.doi.org/10.1038/ncomms7250
  10. http://www.rcsb.org/pdb/explore.do?structureId=4x90
  11. http://www.ebi.ac.uk/pdbe-site/pdbemotif/?tab=boundmolecule&pdb=4x90&ligandCode3letter=cl
  12. Bochkov VN. Inflammatory profile of oxidized phospholipids. Thromb Haemost. 2007 Mar;97(3):348-54. PMID:17334500
  13. Abe A, Hiraoka M, Ohguro H, Tesmer JJ, Shayman JA. Preferential hydrolysis of truncated oxidized glycerophospholipids by lysosomal phospholipase A2. J Lipid Res. 2016 Dec 19. pii: jlr.M070730. PMID:27993948 doi:http://dx.doi.org/10.1194/jlr.M070730
  14. http://dx.doi.org/10.5772/50461

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