Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2, CF

MGAT2 (Catalog # 10711-GT) recognizes an unmodified GlcNAc residue installed by MGAT1 (8334-GT) in the alpha 3 arm of the trimannose core of N-glycans and transfers a second GlcNAc residue to the alpha 6 arm of the structure to generate product glycan M2. MGAT2 can tolerate core-6 fucose and its Cy5 derivatives.

1 μg/lane of Recombinant Human MGAT2 His-tag (Catalog # 10711-GT) was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing bands at 46-54 kDa.

Lane 1 contained substrate glycan M1N1f' (GL302). In the presence of rhMGAT2, the glycan was modified and a mobility shift was observed.

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

Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2, CF Summary

• Additional Information: HA-tag
• Details of Functionality: Measured by its ability to modify the glycan Cy5-labeled M1N1f and thereby creating a band shift.
  Able to convert >50% of the substrate glycan M1N1f to the product glycan, as measured under the described conditions.
• Source: Human embryonic kidney cell, HEK293-derived human N-Acetylglucosaminyltransferase 2/MGAT2 protein
  Asn34-Gln447, with N-terminal HA tag
• Accession #: Q10469.1
• N-terminal Sequence:

  Tyr of HA tag

• 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: 48.7 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: 46-54 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: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 10 mM CaCl₂, 10 mM MnCl₂, pH 7.5
  • Recombinant Human MGAT2 (rhMGAT2) (Catalog # 10711-GT)
  • Uridine 5-diphospho-N-acetylglucosamine (UDP-GlcNAc) (Sigma, Catalog # U4375), 50 mM stock in 50% Ethanol
  • Uridine 5-diphosphogalactose disodium salt (UDP-Gal) (Sigma, Catalog # U4500), 10 mM stock in deionized water
  • Cy5-labeled M1N1f (Catalog # GL301)
  • Recombinant Human B4GALT1 (rhB4GALT1) (Catalog # 3609-GT)
  • 15% SDS-PAGE gel and SDS-PAGE reagents
  • Fluorescent imager
  1. Dilute rhMGAT2 to 10 µg/mL in Assay Buffer.
  2. Create Reaction Mixture A containing 0.130 µM Cy5-labeled M1N1f and 1.5 mM UDP-GlcNAc in Assay Buffer.
  3. Combine 10 µL of 10 µg/mL rhMGAT2 and 10 µL of Reaction Mixture A. Prepare a control by combining 10 µL of Assay Buffer and 10 µL of Reaction Mixture A.
  4. Incubate the reaction and control at 37 °C for 30 minutes.
  5. After incubation, heat at 90 °C for 2 minutes. Cool on ice and quickly spin down condensation.
  6. Create Reaction Mixture B containing 50 µg/mL rhB4GALT1 and 1.5 mM UDP-Gal in Assay Buffer.
  7. Add 10 µL of Reaction Mixture B to the reaction and control.
  8. Incubate the reaction and control at 37 °C for 20 minutes.
  9. Add 6 µL of the reducing sample buffer to reaction and control.
  10. Load 15 µL per lane of each reaction and control on a 15% SDS-PAGE gel and perform SDS-PAGE. Let samples run down approximately 80% of the length of the gel.
  11. Visualize the gel with a fluorescent imager and calculate percent conversion.

  Per Reaction:

  • rhMGAT2: 0.1 µg
  • Cy5-labeled M1N1f: 1.3 pmol
  • UDP-GlcNAc: 0.5 mM
  • rhB4GALT1: 0.5 µg
  • UDP-Gal: 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 N-Acetylglucosaminyltransferase2/MGAT2, CF

• alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase
• Beta-1,2-N-acetylglucosaminyltransferase II
• CDG2A
• EC 2.4.1.143
• GlcNAc-T II
• GLCNACTII
• GNT2
• GNT-IICDGS2
• Mannoside acetylglucosaminyltransferase 2
• mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase
• MGAT2
• N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase II
• UDP-N-acetylglucosamine:alpha-6-D-mannosidebeta-1,2-N-acetylglucosaminyltransferase II

Background

Mannosylglycoprotein N-acetyl-glucosaminyltransferase 2 (MGAT2) is a key enzyme for the synthesis of complex type N-glycan. It specifically recognizes an unmodified GlcNAc residue installed by MGAT1 in the alpha 3 arm of the trimannosy core of N-glycans and transfers a second GlcNAc residue to the alpha 6 arm of the structure, thereby initiating the conversion of oligomannose type N-glycan to the complex type N-glycan (1-3). MGAT2 is a Golgi resident type II membrane protein with a typical domain structure of glycosyltransferases, containing a short N-terminal cytoplasmic domain, a hydrophobic non-cleavable signal-anchor domain, and a C-terminal catalytic domain (4). MGAT2 Mutations lead to type II carbohydrate-deficient glycoprotein syndrome (5) and abnormal brain development (6). Additionally, MGAT2 maybe involved in glycoantigen presentation and T cell activation (7). The activity of recombinant MGAT2 is demonstrated in an electrophoretic gel mobility shift assay using a fluorophore-labeled core N-glycan as the substrate.

1. Moremen, K.W. and Haltiwanger, R.S. (2019) Nat. Chem. Biol. 15:853.
2. Kadirvelraj, R. et al. (2018) Proc. Natl. Acad. Sci. USA 115:4637.
3. Schachter, H. (1986). Biochem. Cell Biol. 64:163.
4. Paulson, J.C. and Colley, K.J. (1989). J. Biol. Chem. 264:17615.
5. Wang, Y. et al. (2001) Glycobiology 11:1051.
6. Tan, J. et al. (1996) Am. J. Hum. Genet. 59:810.
7. Ryan, S.O. et al. 2014 Glycobiology 24:262.

Bioinformatics

• Uniprot:
  • Q10469.1()