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 °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 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)