Recombinant Human MGAT4A His-tag, CF

Recombinant Human MGAT4A His-tag Protein (Catalog # 11496-GT) catalyzes a beta 1-4GlcNAc linkage to the alpha 1-3Man arm of N-glycans.

Recombinant Human MGAT4A His-tag Protein (Catalog # 11496-GT) activity was measured by its ability to transfer N-acetyl-alpha -D-glucosamine from UDP-N-acetyl-alpha -D-glucosamine to glycan Cy5-Fuc labeled N2f (GL304). Following treatment, a mobility shift was observed via SDS-PAGE gel.

2 μg/lane of Recombinant Human MGAT4A His-tag Protein (Catalog # 11496-GT) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 54-60 kDa, under reducing conditions.

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

Recombinant Human MGAT4A His-tag, CF Summary

• Additional Information: Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A
• Details of Functionality: Measured by its ability to transfer N-acetyl-alpha -D-glucosamine from UDP-N-Acetyl-alpha -D-glucosamine to glycan Cy5-Fuc labeled N2f. Able to convert >85% of the substrate glycan N2f, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human MGAT4A protein Leu93-Asn535, with a C-terminal 6-His tag
• Accession #: Q9UM21
• N-terminal Sequence: Leu93
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 52 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: 54-60 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 degreesC as supplied. 3 months, -20 to -70 degreesC 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 Tris, 10 mM MnCl₂, pH 7.0
  • Recombinant Human MGAT4A His-tag (rhMGAT4A) (Catalog # 11496-GT)
  • Cy5-Fuc labeled N2f (Cy5-N2f) (Catalog # GL304)
  • UDP-GlcNAc, 50 mM stock in 50% Ethanol
  • 17% SDS-PAGE Gel
  • Gel loading dye
  • Gel Imager with Cy5 fluorescent dye detection capability
  1. Dilute rhMGAT4A to 50 µg/mL with Assay Buffer.
  2. Create a Reaction Mix containing 0.02 µM Cy5-N2f and 1 mM UDP-GlcNAc in Assay Buffer.
  3. Combine 10 µL of rhMGAT4A and 10 µL of Reaction Mix. For a Control, combine 10 µL of Assay Buffer and 10 µL of Reaction Mix.
  4. Incubate reaction and control at 37 °C for 60 minutes.
  5. Add gel loading dye to each tube.
  6. Load half the volume of each reaction and control onto a 17% SDS-PAGE gel. Let samples migrate at least 80% down the gel before stopping.
  7. Acquire gel image and determine percent conversion of Cy5-N2f.
  Per Reaction:
  • rhMGAT4A: 0.5 µg
  • Cy5-N2f: 0.2 pmol
  • UDP-GlcNAc: 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 MGAT4A His-tag, CF

• alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A
• alpha-1,3-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase
• EC 2.4.1.145
• GlcNAc-T IVa
• GnT-4a
• GNT-IV
• GNT-IVA
• isoenzyme A
• isozyme A
• mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase
• MGAT4A
• N-Acetylglucosaminyltransferase IVa
• N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase IVa
• UDP-GlcNAc:a-1,3-D-mannoside b-1,4-acetylglucosaminyltransferase IV
• UDP-N-acetylglucosamine: alpha-1,3-D-mannosidebeta-1,4-N-acetylglucosaminyltransferase IVa
• UDP-N-acetylglucosamine:alpha1,3-d-mannosidebeta1,4-N-acetylglucosaminyltransferase

Background

Recombinant human Alpha-1,3-mannosyl-glycoprotein 4-beta -N-acetylglucosaminyltransferase A (MGAT4A), also known as GlcNAc-T IVa is a metal-dependent, golgi single-pass type II membrane protein that contains an N-terminal transmembrane region, a catalytic domain, and a C-terminal carbohydrate binding module that regulates its catalytic activity (1, 2). MGAT4A is one of several human MGAT enzymes involved in the initiation and synthesis of N-glycans, with each enzyme having unique specificity and preferences (3). MGAT4 has two human isozymes that catalyze formation of the beta 1-4 GlcNAc branch in the alpha 1-3 Mannose arm of N‑glycans (4-6). The two isozymes have different expression profiles in tissues and cancer cell lines with MGAT4A expression within gastrointestinal tissues such as the pancreas (2, 4, 5). Loss of MGAT4A in mouse models has been shown to induce type 2 diabetes (9,10). Expression and activity of MGAT4A is particularly important during differentiation and oncogenesis (7, 8) including within choriocarcinoma, invasive mole, and placental site trophoblastic tumors (11-13). The discovery of MGAT4A modulators may lead to new therapeutics for the treatment of these diseases (10). MGAT4A may also be useful as a tool for the glycoengineering of complex N-glycans. The activity of MGAT4A was demonstrated using a fluorescent gel shift assay.

1. Oka, N. et al. (2022) Glycobiology. 32:1153.
2. Nagae, M. et. al. (2022) Commun. Biol. 5:695.
3. Brockhausen, I. et. al. (1988) Biochemie 70:1521.
4. Yoshida, A. et. al. (1999) Glycobiology. 9:303.
5. Oguri, S. et. al. (2006) Glycoconj. J. 23:473.
6. Osada, N. et. al. (2022) J. Biol. Chem. 298:102400.
7. Nishikawa, A. et. al. (1990) Biochim. Biophys. Acta. 1035:313.
8. Nakao, H. et. al. (1990) Biochem. Biophys. Res. Commun. 172:1260.
9. Ohtsubo, K. et. al. (2005) Cell. 123:1307.
10. Ohtsubo, K. et. al. (2011) Nat. Med. 17:1067.
11. Mizuochi, T. et. al. (1983) J. Biol. Chem. 258:14126.
12. Endo, T. et. al. (1987) Cancer Res. 47:5242.
13. Niimi, K. et. al. (2012) Br. J. Cancer. 107:1969.

Bioinformatics

• Uniprot:
  • Q9UM21(Human)