Recombinant Human MANBA/Beta-Mannosidase His-tag Protein, CF

2 μg/lane of Recombinant Human MANBA Protein (Catalog # 10520-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 110 kDa.

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

Recombinant Human MANBA/Beta-Mannosidase His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze 4-methylumbelliferyl Beta-D-mannopyranoside.
  The specific activity is >100 pmol/min/μg, as measured under the described conditions.
• Source: Human embryonic kidney cell, HEK293-derived human MANBA/Beta-Mannosidase protein
  Ala19-Tyr879 & Glu20 - Ser878, with C-terminal 6-His tag
• Accession #: O00462.3
• N-terminal Sequence: Ala19 & Glu20
• Protein/Peptide Type: Recombinant Proteins
• 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: 99.9 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: 110 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: 25 mM MES, pH 5.0
  • Recombinant Human MANBA (rhMANBA) (Catalog # 10520-GH)
  • Substrate: 4-Methylumbelliferyl-beta -D-mannopyranoside (Sigma, Catalog # M0905), 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 rhMANBA to 8 µg/mL in Assay Buffer.
  2. Dilute Substrate to 1 mM in Assay Buffer.
  3. Load 50 μL of 8 µg/mL rhMANBA into the plate, and start the reaction by adding 50 μL of 1 mM Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of 1 mM Substrate.
  4. Read at excitation and emission wavelengths of 365 nm and 445 nm, respectively, (top read) in kinetic mode for 5 minutes.
  5. 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 4-Methylumbelliferone (Sigma, Catalog # M1381).

  Per Well:
  • rhMANBA: 0.4 µg
  • Substrate: 0.5 mM

Notes

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

Alternate Names for Recombinant Human MANBA/Beta-Mannosidase His-tag Protein, CF

• beta-mannosidase
• EC 3.2.1.25
• Lysosomal beta A mannosidase
• MANB1
• MANB1mannanase
• MANBA
• Mannanase
• Mannase
• mannosidase, beta A, lysosomal

Background

MANBA is a lysosomal beta -mannosidase in the degradation pathway of N-glycans and catalyzes the removal of the innermost beta (1-4)-linked mannose residue (1). Mutations of MANBA can lead to cellular storage and secretion in the urine of the disaccharide, Man( beta 1–4)GlcNAc (2), and the symptoms of lysosomal storage disease (LSD) beta -mannosidosis, associated with mental retardation, behavioral problems including hyperactivity and aggressiveness, developmental delay, frequent infections, and hearing loss (3). Angiokeratoma corporis diffusum is another symptom of beta ‐mannosidosis that is characterized by involuntary eye movements (4) and can be found in other LSDs such as Fabry disease, beta ‐galactosidosis, fucosidosis, aspartylglucosaminuria, and galactosialidosis (5). LSDs are progressive disorders and are among the most common genetic disorders affecting children (6). Patients with LSDs are usually treated with enzyme replacement therapy (ERT) in which recombinant enzymes are administered to them for direct uptake into the lysosome (7). MAMBA belongs to the glycosyl hydrolase (GH) 2 family, in which enzymes are classified on the basis of sequence similarity (8). The only other human GH2 enzyme is beta ‐glucuronidase, deficiency of which leads to Sly syndrome (9). GH2 family members are characterized by a catalytic TIM barrel (10), in which the catalytic acid-base and nucleophile residues lie on beta ‐strands 4 and 7, respectively. Human beta ‐mannosidase is sensitive to pH with optima between 4.5 and 5.0 (11).

1. Alkhayat, A. H. et al. (1998) Hum Molec Genet 7:75.
2. Hokke, C.H. et al. (1990). J Inherit Metab Dis 13:273.
3. Wenger, D.A. et al. (1986). N Engl J Med 315:1201.
4. Gytz, H. et al. (2019) FEBS J. 286:1319.
5. Rodriguez‐Serna, M. et al. (1996) Arch Dermatol 132:1219.
6. Hawkins‐Salsbury, J.A. et al. (2011) Hum Mol Genet 20:R54.
7. Wyatt, K. et al. (2012) Health Technol Assess 16:1. 
8. Cantarel, B.L. et al. (2009). Nucleic Acids Res 37:D233.
9. Sly, W.S. et al. (1973). J. Pediatr. 82:249.
10. Henrissat, B. et al. (1995). Proc Natl Acad Sci USA 92:7090.
11. Percheron, F. et al. (1992). Biochimie 74:5.

Bioinformatics

• Uniprot:
  • O00462.3()