Recombinant Human MAN1A1 His-tag Protein, CF

MAN1A1 is involved in the removal of 4 distinct alpha -1,2-linked mannose residues from Man9GlcNAc2 to produce Man5GlcNAc2, an essential step of N-glycan maturation.

1 μg/lane of Recombinant Human MAN1A1 His-tag (Catalog # 10665-GT) was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing a band at 63-65 kDa.

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

Recombinant Human MAN1A1 His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to remove alpha -mannose from the high mannose glycan Man-9.
  A distinct band is observed in the rhMAN1A1 digested sample on SDS-PAGE gel, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human MAN1A1 protein
  Pro63-Glu653, with a C-terminal 6-His tag
• Accession #: P33908.3
• N-terminal Sequence: Pro63
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, 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: 67.7 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: 63-65 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, NaCl and CaCl₂.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Digestion Buffer: 50 mM MES, 15 mM CaCl₂, 1 mg/mL BSA, pH 6.0
  • Labeling Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MnCl₂, pH 7.5
  • Recombinant Human MAN1A1 (rhMAN-1A1) (Catalog # 10665-GH)
  • Oligomannose-9 (Man-9) (Dextra Laboratories, Catalog # MC1131), 0.1 mg/mL stock in deionized water
  • Recombinant Human MGAT1 (rhMGAT1) (Catalog # 8334-GT)
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol, 50% deionized water
  • Recombinant Human FUT8 (rhFUT8) (Catalog # 5768-GT)
  • GDP-Cy5-Fucose (Catalog # ES301)
  • 15% SDS-PAGE gel
  • Reducing SDS-PAGE gel loading buffer
  • Fluorescent imager

  Digestion:

  1. Dilute rhMAN1A1 to 20 µg/mL in Digestion buffer.
  2. Dilute Man-9 to 20 µg/mL in Digestion Buffer.
  3. Combine 5 µL of 20 µg/mL Man-9, 5 µL of 20 µg/mL rhMAN1A1 and 10 µL of Digestion Buffer. Include a Control containing 5 µL of 20 µg/mL Man-9 and 15 µL of Digestion Buffer.
  4. Incubate at 37 °C for 2 hours.

  Labeling:

  1. Dilute rhMGAT1 to 100 µg/mL in Labeling Buffer.
  2. Dilute UDP-GlcNAc to 1 mM in Labeling Buffer.
  3. Dilute rhFUT8 to 100 µg/mL in Labeling Buffer.
  4. Dilute GDP-Cy5-Fucose to 0.05 mM in Labeling Buffer.
  5. Transfer 10 µL of each digestion to a new tube and add 5 µL of 100 µg/mL rhMGAT1, 5 µL of 1 mM UDP-GlcNAc, 5 µL of 100 µg/mL rhFUT8 and 5 µL of 0.05 mM GDP-Cy5-Fucose.
  6. Incubate at 37 °C for 60 minutes.
  7. Add 6 µL of Reducing SDS-PAGE gel loading buffer to each reaction.
  8. Load 12 µL of each reaction onto a 15% SDS-PAGE gel and perform electrophoresis.
  9. Analyze gel on a fluorescent imager.
  Per Reaction:
  • rhMAN1A1: 0.05 µg
  • Man-9: 0.05 µg
  • rhMGAT1: 0.5 µg
  • UDP-GlcNAc: 5 nmol
  • rhFUT8: 0.5 µg
  • GDP-Cy5-Fucose: 0.25 nmol

Notes

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

Alternate Names for Recombinant Human MAN1A1 His-tag Protein, CF

• HUMM3
• HUMM9
• MAN1A1
• MAN9

Background

N-glycan maturation in Golgi apparatus starts with high-mannose glycan Man-9 that is capped with four 1,2-alpha -linked mannose residues at its non-reducing ends. During the process, these mannose residues are removed to generate Man-5 oligomannose glycan, a precursor for complex and hybrid N-glycans (1). Failure of removing these mannose residues will result in the display of high-mannose glycans on cell surface and extracellular matrix. Increased levels of high-mannose glycans on cell surface are usually associated with disease progress such as tumorigenesis and viral infection (2). The removal of 1,2-alpha -linked mannose residues are catalyzed by 4 alpha -mannosidases, including MAN1A1, MAN1B1, MAN1A2 and MAN1C1, that have overlapping substrate specificity and slight differences in enzyme activity (3). MAN1A1 is also a tumor-suppressor (4) and low levels of expression of MAN1A1 correlate with poor prognosis in breast cancer patients (5, 6).

1. Oliveira-Ferrer, L. et al. (2014) Br. J. Cancer 110:753.
2. Moremen K.W. et al. (2012) Nat. Rev. Mol. Cell Biol. 13:448.
3. Moremen, K.W. and Nairn, A.V. (2014) Handbook of Glycosyltransferases and Related Genes p1297.
4. Liu, T. et al. (2014) PLoS One 9:e107941.
5. Milde-Langosch, K. et al. (2014) Breast Cancer Res. Treat. 145:295.
6. Karen Legler, et al. (2018) Br. J. Cancer 118:847.

Bioinformatics

• Uniprot:
  • P33908.3()