Asparagine endopeptidase explained

Ec Number:3.4.22.34
Cas Number:149371-18-6
Legumain
Width:280px

Asparagine endopeptidase (AEP, mammalian legumain, δ-secretase;) is a proteolytic enzyme from C13 peptidase family which hydrolyses a peptide bond using the thiol group of a cysteine residue as a nucleophile (hence also called cysteine protease). It is also known as asparaginyl endopeptidase, citvac, proteinase B, hemoglobinase, PRSC1 gene product or LGMN (Homo sapiens), vicilin peptidohydrolase and bean endopeptidase. In humans it is encoded by the LGMN gene (previous symbol PRSC1).[1] [2] [3]

It hydrolyzes substrates at the C-terminus of asparagine residues. Discovered in 1996 in beans, its homologues have been identified in plants, protozoa, vertebrates, and helminths. The enzyme has been implicated in several human diseases such as cancer, atherosclerosis and inflammation .[4] It can be detected in spleen, liver, brain, testis tissue and heart[5] and the protein is mostly localised to lysosomes and endosomes. It is also interesting that AEP is activated in age-dependent manner.[6]

Distribution

This enzyme was originally identified in the vacuoles of legume seeds, and was subsequently identified the lysosomes of mammals and Schistosoma mansoni.[7] They are now known to be present in a range of plants and animals.[8] [9]

Activity

Reaction and specificity

This enzyme catalyses the following chemical reaction:

Hydrolysis of proteins and small molecule substrates at -Asn-Xaa- bondsBoth plant and animal legumains are most active in acidic environments.[10]

Prodomain processing

Legmains are produced as inactive precursor zymogens. their C-terminal domain binds over their active site (where a substrate would normally bind), inhibiting activity. Once in the acidic environment of the vacuole or lysosome, the prodomain is cleaved off to reveal the active enzyme.

Mechanism

Legumain is a cysteine protease from the C13 family of the CD clan of proteases (MEROPS).[11] It uses a catalytic triad of Cysteine-Histidine-Asparagine in its active site to perform covalent proteolysis of its substrate.

Activation

Asparagine endopeptidase is synthesized as an inactive zymogen.[12] AEP and other cysteine peptidase are activated when pH changes from neutral to acidic. It undergoes autoproteolytic maturation for catalytic activation. It appears to be autocatalytically cleaved after asparagine or aspartate residue. Activation begins at pH 4.5. The chemical structure at this point shows that breaks which occurs at pH 4.5 can be healed under the basic crystallization conditions. C-terminal fragments (≈13 kDa) generated during autoproteolysis can gradually re-ligated to form the proenzyme when the pH is increased towards pH 7.5, which means that proteolytic activation of AEP can be reversible.

Biological roles

Antimicrobial activity

In plant Oldenlandia affinis it generates antimicrobial cyclic peptides which are important for defence against pathogens in plants.[13] [14] The herb has been used in native African medicine to accelerate childbirth.

Animals

Innate immune system

There are many regulators which affect immune system and help to keep it balanced. If the immune system is too active there is a danger of developing an autoimmune disease, while passive immune system will lead to infections or cancer. Antigen presenting is a key role in activation of immune system. It has been discovered that AEP plays role in this critical moment. AEP is involved in presenting of foreign and self proteins using MHCII protein complex.[15] The role of AEP in immunity is not clear, but it seems that it is connected with checkpoint inhibitors such as PD-1, which downregulates AEP which is key to shifting the balance between cancer fighting cells and regulatory T cells. In the absence of AEP, inhibitory checkpoints may not have a beneficial response. Measuring of this enzyme in patients could predict which one of them may provide better response to treatment.[16]

Signalling

In innate immunity TLRs play an important role. These TLRs (mainly TLR7 and TLR9) can be proteolytically activated by AEP.[17] The reduction of proinflammatory cytokines by stimulating TLR9 was found in myeloid cells and plasmacytoid dendritic cells which lacked AEP.[18] Enzyme is also important in processing of influenza virus and immune response using TLR7. AEP plays a critical role in TLR processing. and AEP can initiate removal of invariant chain in MHC-II complex, which can critically influence peptide generation and activity of MHCII.[19]

Disease

Neurodegenerative diseases

AEP is activated during brain ischemia or brain acidosis and epilepsia seizure. It digests SET protein, which is an inhibitor of DNase, leading to DNA damage and causing damage of the brain. Increased activity of AEP in brain is also observed in patients with Alzheimer's disease and Parkinson's disease (PD). AEP cleaves tau protein [20] and amyloid precursor protein. In patients with PD, alpha synuclein is cut by AEP into toxic chunks.,[21] which are hypothesized to possibly play a role in the initiation or pathogenesis of PD [22]

Alzheimer's disease

Active AEP was found at increased levels and translocated to the cytoplasm of neuronal cells of AD patients.[23] In AD the plaques are composed of amyloid beta, intracellular neurofibrillary tangles and tau protein. The dysfunction of APP proteolysis and the abnormal phosphorylation of tau lead to the formation of neuritic plaques and neurofibrillary tangles (NFTs), respectively, causing neuronal degeneration and dementia[24] It also play a crucial role in behavior disorders connected with AD such as anxiety and depression. It also plays a role in stroke. Since stroke elicits acidity in the brain AEP become active due to low pH level. Then it cleaves SET which causes death of brain cells.[25] Targeting of AEP might help to prevent onset of AD symptoms. Development of AEP-selective inhibitors (such as Cbz-L-Ala-L-Ala-AzaAsnchloromethylketone and aza-peptidyl AEP inhibitors) is crucial for helping with diseases.

Notes and References

  1. Book: Asparaginyl endopeptidase . Handbook of Proteolytic Enzymes . Hara-Nishimura I . 1998 . 746–749 . Barrett AJ, Rawlings ND, Woessner JF . Academic Press . London .
  2. Book: Schistosome legumain . Handbook of Proteolytic Enzymes . Dalton JP, Brindley PJ . 1998 . 749–754 . Barrett AJ, Rawlings ND, Woessner JF . Academic Press . London .
  3. Chen JM, Rawlings ND, Stevens RA, Barrett AJ . Identification of the active site of legumain links it to caspases, clostripain and gingipains in a new clan of cysteine endopeptidases . FEBS Letters . 441 . 3 . 361–5 . December 1998 . 9891971 . 10.1016/S0014-5793(98)01574-9 . 46043484 . free .
  4. Zhang Z, Xie M, Ye K. October 2016. Asparagine endopeptidase is an innovative therapeutic target for neurodegenerative diseases. Expert Opinion on Therapeutic Targets. 20. 10. 1237–45. 10.1080/14728222.2016.1182990. 5315368. 27115710.
  5. Chen JM, Dando PM, Stevens RA, Fortunato M, Barrett AJ . Cloning and expression of mouse legumain, a lysosomal endopeptidase . The Biochemical Journal . 335 (Pt 1) . 1 . 111–7 . October 1998 . 9742219 . 10.1042/bj3350111 . 1219758 .
  6. Zhao L, Hua T, Crowley C, Ru H, Ni X, Shaw N, Jiao L, Ding W, Qu L, Hung LW, Huang W, Liu L, Ye K, Ouyang S, Cheng G, Liu ZJ . Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage . Cell Research . 24 . 3 . 344–58 . March 2014 . 24407422 . 3945893 . 10.1038/cr.2014.4 .
  7. Chen JM, Dando PM, Rawlings ND, Brown MA, Young NE, Stevens RA, Hewitt E, Watts C, Barrett AJ . Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase . The Journal of Biological Chemistry . 272 . 12 . 8090–8 . March 1997 . 9065484 . 10.1074/jbc.272.12.8090 . free .
  8. Müntz K, Shutov AD . Legumains and their functions in plants . en . Trends in Plant Science . 7 . 8 . 340–4 . August 2002 . 12167328 . 10.1016/S1360-1385(02)02298-7 .
  9. Shutov AD, Blattner FR, Kakhovskaya IA, Müntz K . New aspects of the molecular evolution of legumains, Asn-specific cysteine proteinases . Journal of Plant Physiology . 169 . 3 . 319–21 . February 2012 . 22196948 . 10.1016/j.jplph.2011.11.005 .
  10. Chen JM, Fortunato M, Barrett AJ . Activation of human prolegumain by cleavage at a C-terminal asparagine residue . The Biochemical Journal . 352 Pt 2 . 2 . 327–34 . December 2000 . 11085925 . 1221463 . 10.1042/bj3520327 .
  11. Web site: C13 family. merops.sanger.ac.uk. MEROPS - the Peptidase Database. 2016-06-09.
  12. Li DN, Matthews SP, Antoniou AN, Mazzeo D, Watts C . Multistep autoactivation of asparaginyl endopeptidase in vitro and in vivo . The Journal of Biological Chemistry . 278 . 40 . 38980–90 . October 2003 . 12860980 . 10.1074/jbc.m305930200 . free .
  13. Gillon AD, Saska I, Jennings CV, Guarino RF, Craik DJ, Anderson MA. February 2008. Biosynthesis of circular proteins in plants. The Plant Journal. 53. 3. 505–15. 10.1111/j.1365-313x.2007.03357.x. 18086282. free.
  14. Craik DJ. February 2012. Host-defense activities of cyclotides. Toxins. 4. 2. 139–56. 10.3390/toxins4020139. 22474571. 3317112. free .
  15. Matthews SP, Werber I, Deussing J, Peters C, Reinheckel T, Watts C. March 2010. Distinct protease requirements for antigen presentation in vitro and in vivo. Journal of Immunology. 184. 5. 2423–31. 10.4049/jimmunol.0901486. 20164435. free.
  16. News: Enzyme AEP's importance to immunity discovered. 2018-08-28.
  17. Maschalidi S, Hässler S, Blanc F, Sepulveda FE, Tohme M, Chignard M, van Endert P, Si-Tahar M, Descamps D, Manoury B . Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation . PLOS Pathogens . 8 . 8 . e1002841 . 2012-08-16 . 22916010 . 3420946 . 10.1371/journal.ppat.1002841 . free .
  18. Sepulveda FE, Maschalidi S, Colisson R, Heslop L, Ghirelli C, Sakka E, Lennon-Duménil AM, Amigorena S, Cabanie L, Manoury B . Critical role for asparagine endopeptidase in endocytic Toll-like receptor signaling in dendritic cells . Immunity . 31 . 5 . 737–48 . November 2009 . 19879164 . 10.1016/j.immuni.2009.09.013 . free .
  19. Manoury B, Mazzeo D, Li DN, Billson J, Loak K, Benaroch P, Watts C . Asparagine endopeptidase can initiate the removal of the MHC class II invariant chain chaperone . Immunity . 18 . 4 . 489–98 . April 2003 . 12705852 . 10.1016/s1074-7613(03)00085-2 . free .
  20. Chen . C . Zhou . Y . Wang . H . Alam . A . Kang . SS . Ahn . EH . Liu . X . Jia . J . Ye . K . Gut inflammation triggers C/EBPβ/δ-secretase-dependent gut-to-brain propagation of Aβ and Tau fibrils in Alzheimer's disease. . The EMBO Journal . 1 September 2021 . 40 . 17 . e106320 . 10.15252/embj.2020106320 . 34260075. 8408610 .
  21. Zhang . Z . Kang . SS . Liu . X . Ahn . EH . Zhang . Z . He . L . Iuvone . PM . Duong . DM . Seyfried . NT . Benskey . MJ . Manfredsson . FP . Jin . L . Sun . YE . Wang . JZ . Ye . K . Asparagine endopeptidase cleaves α-synuclein and mediates pathologic activities in Parkinson's disease. . Nature Structural & Molecular Biology . August 2017 . 24 . 8 . 632–642 . 10.1038/nsmb.3433 . 28671665. 5871868 .
  22. Ahn . EH . Kang . SS . Liu . X . Chen . G . Zhang . Z . Chandrasekharan . B . Alam . AM . Neish . AS . Cao . X . Ye . K . Initiation of Parkinson's disease from gut to brain by δ-secretase. . Cell Research . January 2020 . 30 . 1 . 70–87 . 10.1038/s41422-019-0241-9 . 31649329. 6951265 .
  23. Basurto-Islas G, Grundke-Iqbal I, Tung YC, Liu F, Iqbal K. June 2013. Activation of asparaginyl endopeptidase leads to Tau hyperphosphorylation in Alzheimer disease. The Journal of Biological Chemistry. 288. 24. 17495–507. 10.1074/jbc.M112.446070. 3682549. 23640887. free .
  24. Zhang Z, Song M, Liu X, Kang SS, Kwon IS, Duong DM, Seyfried NT, Hu WT, Liu Z, Wang JZ, Cheng L, Sun YE, Yu SP, Levey AI, Ye K. November 2014. Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer's disease. Nature Medicine. 20. 11. 1254–62. 10.1038/nm.3700. 4224595. 25326800.
  25. Gao J, Li K, Du L, Yin H, Tan X, Yang Z. July 2018. Deletion of asparagine endopeptidase reduces anxiety- and depressive-like behaviors and improves abilities of spatial cognition in mice. Brain Research Bulletin. 142. 147–155. 10.1016/j.brainresbull.2018.07.010. 30030107. 51705806 .

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