Recombinant Human Legumain/Asparaginyl Endopeptidase, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Legumain/Asparaginyl Endopeptidase, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, N-carbobenzyloxy-Ala-Ala-Asn-7-amido-4-methylcoumarin (Z-AAN-AMC). The specific activity is >250 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Legumain/Asparaginyl Endopeptidase protein
  Ile18-Tyr433, with an N-terminal 7-His tag
• Accession #: AAH03061.1
• N-terminal Sequence: His
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: LGMN
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 49 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 60 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Activation Buffer: 50 mM Sodium Acetate, 100 mM NaCl, pH 4.0
  • Assay Buffer: 50 mM MES, 250 mM NaCl, pH 5.0
  • Recombinant Human Legumain/Asparaginyl Endopeptidase (rhLegumain) (Catalog # 2199-CY)
  • Substrate: Z-Ala-Ala-Asn-AMC (Bachem, Catalog # I-1865), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhLegumain to 100 µg/mL in Activation Buffer.
  2. Incubate for 2 hours at 37 °C.
  3. Dilute rhLegumain to 1 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM in Assay Buffer.
  5. Load into a black well plate 50 µL of 1 ng/µL rhLegumain and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
  6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  7. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

  Per Well:
  • rhLegumain: 0.050 µg
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Legumain/Asparaginyl Endopeptidase, CF

• AEP
• Asparaginyl Endopeptidase
• cysteine protease 1
• Legumain
• LGMN
• LGMN1
• Protease, cysteine 1
• protease, cysteine, 1 (legumain)
• PRSC1EC 3.4.22.34

Background

Legumain is a lysosomal cysteine protease whose activity is found in several tissues tested (1, 2). Legumain plays a pivotal role in the endosomal/lysosomal degradation system because the Legumain deficiency causes the accumulation of pro cathepsins B, H and L, another group of lysosomal cysteine proteases (3). Over-expression of Legumain in tumors is significant for invasion/metastasis (4). Also known as Asparaginyl Endopeptidase, it specifically cleaves peptide bonds with Asn at the P1 position. Nevertheless, it also cleaves peptide bonds with Asp at the P1 position. Auto-activation of pro Legumain involves both types of the cleavage, which result in the removal of the pro peptides in both C- and N-termini (5). In addition, Legumain activates pro MMP-2 and processes bacterial antigens for MHC class II presentation and pro thymosin alpha to thymosin alpha ₁ and thymosin alpha ₁₁, two acidic peptides with immunoregulatory properties (6‑8). Human Legumain is synthesized as a 433 amino acid precursor with a signal peptide (residues 1‑17). The pro enzyme (residues 18‑433) was expressed with an N-terminal His tag. This activity of Legumain can be inhibited by recombinant human Cystatins C and E/M and recombinant mouse Cystatin C (Catalog # 1196-PI, 1286-PI and 1238-PI, respectively).

1. Chen, J.-M. et al. (1997) J. Biol. Chem. 272:8090.
2. Tanaka, T. et al. (1996) Cytogenet. Cell Genet. 74:120.
3. Shirahama-Noda, K. et al. (2003) J. Biol. Chem. 278:33194.
4. Liu, C. et al. (2003) Cancer Res. 63: 2957.
5. Li D.N. et al. (2003) J. Biol. Chem. 278:38980.
6. Chen, J.M. et al. (2001) Biol. Chem. 382:777.
7. Schwarz, G. et al. (2002) Biol. Chem. 383:1813.
8. Sarndeses, C.S. et al. (2003) J. Biol. Chem. 278:13286.

Publications for Legumain/Asparaginyl Endopeptidase (2199-CY)(8)

Publications using 2199-CY

• M Buša, Z Matoušková, P Bartošová-, P Pachl, P ?ezá?ová, RM Eichenberg, P Deplazes, M Horn, S Štefani?, M Mareš An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily The Journal of Biological Chemistry, 2023-02-01;0(0):102970. 2023-02-01 [PMID: 36736427] (Bioassay, N/A)
• H Sun, W Ma, S Duan, J Huang, R Jia, H Cheng, B Chen, X He, K Wang An endogenous stimulus detonated nanocluster-bomb for contrast-enhanced cancer imaging and combination therapy Chemical Science, 2021-08-17;12(36):12118-12129. 2021-08-17 [PMID: 34667577] (Enzyme Assay)
• K Schlegel, K Awwad, RG Heym, D Holzinger, A Doell, S Barghorn, TR Jahn, C Klein, Y Mordashova, M Schulz, L Gasparini N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain Acta Neuropathol Commun, 2019-11-13;7(1):177. 2019-11-13 [PMID: 31722749] (Enzyme Assay, N/A)
• B Prabucka, M Mielecki, M Chojnacka, W Bielawski, M Czarnocki-, S Orzechowsk Structural and functional characterization of the triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 and its dimerization-dependent inhibitory activity Phytochemistry, 2017-06-24;142(0):1-10. 2017-06-24 [PMID: 28654769] (Bioassay)
• TJ Perlenfein, JD Mehlhoff, RM Murphy Insights into the mechanism of cystatin C oligomer and amyloid formation and its interaction with beta-amyloid J. Biol. Chem., 2017-05-09;0(0):. 2017-05-09 [PMID: 28487367] (Bioassay, Human)
• Mortensen S, Kidmose R, Petersen S, Szilagyi A, Prohaszka Z, Andersen G Structural Basis for the Function of Complement Component C4 within the Classical and Lectin Pathways of Complement. J Immunol, 2015-04-24;194(11):5488-96. 2015-04-24 [PMID: 25911760] (Enzyme Assay, Human)
• Briggs JJ, Haugen MH, Johansen HT, Riker AI, Abrahamson M, Fodstad O, Maelandsmo GM, Solberg R Cystatin E/M suppresses legumain activity and invasion of human melanoma. BMC Cancer, 2010-01-15;10(1):17. 2010-01-15 [PMID: 20074384] (Western Blot)
• Clerin V, Shih HH, Deng N, Hebert G, Resmini C, Shields KM, Feldman JL, Winkler A, Albert L, Maganti V, Wong A, Paulsen JE, Keith JC, Vlasuk GP, Pittman DD Expression of the cysteine protease legumain in vascular lesions and functional implications in atherogenesis. Atherosclerosis, 2008-02-21;0(0):. 2008-02-21 [PMID: 18377911] (Bioassay, Human)

Bioinformatics

• Gene Symbol: LGMN
• Uniprot:
  • AAH03061.1()