Recombinant A. Muciniphila M60 domain-containing peptidase

Recombinant A. Muciniphila M60 domain-containing peptidase His-tag Protein (Cat # 11479-M6) cleaves glycoproteins N-terminally to glycosylated Ser/Thr residues that are near but not next to the cleavage residue. Specificity requires two adjacent truncated O-glycans and M60 peptidase cleaves between the two residues. * represents the residue for which M60 peptidase has recognition. Sialyated glycan substrates in diagram are less preferred.

Recombinant A. Muciniphila M60 domain-containing peptidase His-tag Protein (Cat # 11479-M6) is measured by its ability to digest Recombinant Human CA125/MUC16 Protein, CF (5609-MU) and detected via SDS-PAGE gel.

2 μg/lane of Recombinant A. Muciniphila M60 domain-containing peptidase His-tag Protein (Catalog # 11479-M6) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 52-57 kDa, under reducing conditions.

• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant A. Muciniphila M60 domain-containing peptidase Summary

• Details of Functionality: Measured by its ability to digest MUC16.
  >90% of MUC16 is digested by rAm M60 peptidase, as measured under the described conditions.
• Source: E. coli-derived akkermansia muciniphila M60 domain-containing protein
  Ala21-Glu506 with an N-terminal Met and C-terminal 6-His tag
• Accession #: WP_012419679.1
• N-terminal Sequence: Met & Ala21
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 56 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: 52-57 kDa, under reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 50 mM MES, pH 6.5
  • Recombinant A. Muciniphila M60 domain-containing peptidase His-tag (rAm M60 peptidase) (Catalog # 11479-M6)
  • Substrate: Recombinant Human CA125/MUC16 Protein (rhMUC16) (Catalog # 5609-MU)
  • 4-20% SDS-PAGE Gel Gel loading dye
  • Gel Imager/Densitometer
  1. Dilute rAm M60 peptidase to 25 µg/mL with Assay Buffer.
  2. Dilute rhMUC16 to 100 µg/mL with Assay Buffer.
  3. Combine 10 µL of rAm M60 peptidase and 10 µL of rhMUC16 in a tube.
  4. Create a negative control by adding 10 µL of rhMUC16 and 10 µL of Assay Buffer.
  5. Incubate reactions and controls at 37 °C for 24 hours.
  6. Add gel loading dye to all tubes.
  7. Load the total reaction volume on a 4-20% SDS-PAGE gel and run down 80% of the gel, minimally.
  8. Stain the gel and acquire image.
  9. Use densitometry to calculate the percent digestion of rhMUC16 by rAm M60 peptidase.
  Per Reaction:
  • rAm M60 peptidase: 0.25 μg
  • rhMUC16: 1 μg

Notes

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

Alternate Names for Recombinant A. Muciniphila M60 domain-containing peptidase

• Akkermansia muciniphila ATCC BAA-835 Peptidase M60 domain-containing protein
• Akkermansia muciniphila ATCC BAA-835 Peptidase M60
• Amuc_0627
• conserved hypothetical protein [Akkermansia mucini
• conserved hypothetical protein [Akkermansia muciniphila ATCC BAA-835]
• M60 family metallopeptidase [Akkermansia muciniphi
• M60 family metallopeptidase [Akkermansia muciniphila]
• muciniphila M60 domain-containing

Background

Recombinant A. Muciniphila M60 domain-containing peptidase (Am M60 peptidase), also referred to as AM0627, is a zinc-dependent mucin-targeting protease from Akkermansia muciniphila, a gut symbiont found within the human and mouse gut mucus layer. Am M60 peptidase is classified as an enzyme within the large M60-like family of metalloproteases containing gluzincin motifs from organisms known to inhabit mucosal host environments (1,2). Am M60 peptidase is a secreted, monomeric protein with a signal sequence, an immunoglobulin-like fold domain, and catalytic domain that contains the zinc-binding site within the gluzincin motif (2,3). Am M60 peptidase is capable of cleaving glycopeptides between adjacent O-glycosylated threonine or serine, with a preference towards desialylated substrates (4). Glycopeptides with adjacent non-physiological truncated O-glycan structure that promote tumorigenesis and metastasis, known as tumor-associated carbohydrate antigens, serve as optimal substrates for Am M60 peptidase (3,5). A. muciniphilia relies on host-derived mucin as an energy source and leads to production of short chain fatty acids (SCFA) via mucin-degradation byproducts. SCFA regulate inflammation and provide hosts with health benefits for diseases such as inflammatory bowel disease, obesity, autism, and cancer (2, 6-9). Mucin-degrading enzymes such as Am M60 peptidase play a prominent role in host mucin degradation and consequently Am M60 peptidase may be of interest as a therapeutic target, a diagnostic tool to detect and monitor disease progression, and/or useful as a tool to further study mucins and O-glycoproteins (2-4, 10).  

1. Rawlings, N.D. et al. (2014) Nucl. Acids Res. 42: D503.
2. Shon, D.J. et. al. (2022) J. Biol. Chem. 298: 101917.
3. Taleb, V.  et. al. (2022) Nat. Commun. 13: 4324.
4. Shon, D.J. et. al. (2020) Proc. Natl. Acad USA 117: 21299.
5. Kudelka, M.R. et. al. (2015) Adv. Cancer Res. 126:53.
6. Yamada, T.  et al. (2019) EbioMedicine 48: 513.
7. Hansson, G.C. et. al. (2020) Annu. Rev. Biochem. 89: 769.
8. Pruss, K.M. et. al. (2021) ISME J. 15: 577.
9. Molaaghaee-Rouzbahani, S. et. al. (2023) Sci. Rep. 13:3237.
10. Labourel, A. et. al. (2023) Essays Biochem. 67:331.

Bioinformatics

• Uniprot:
  • WP_012419679.1()