MGAT2 Human, Sf9

What is MGAT2 and what is its role in glycosylation pathways?

MGAT2 (Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase) is an enzyme that catalyzes a crucial step in the conversion of oligomannose to complex N-glycans . It belongs to the family of glycosyltransferases, which are responsible for the synthesis and modification of glycan structures attached to proteins and lipids. The enzyme plays a vital role in the N-glycosylation pathway, which is essential for proper protein folding, stability, and function within the cell. In terms of biological significance, the N-glycans produced through MGAT2 activity are critical for various cellular processes including cell-cell recognition, protein trafficking, and immune system function .

The MGAT2 gene in humans is structurally unique in that it contains no introns in its coding sequence, which has important implications for genetic studies and mutation analysis . This characteristic makes it particularly susceptible to certain types of mutations, as alternative splicing cannot compensate for genetic defects in the coding region.

What are the structural characteristics of MGAT2 Human, Sf9?

MGAT2 Human expressed in Sf9 cells is a single, glycosylated polypeptide chain containing 427 amino acids (residues 30-447), with a molecular mass of approximately 49.3kDa . When analyzed by SDS-PAGE, the protein typically appears at approximately 40-57kDa due to its glycosylation status . The enzyme has three distinct domains characteristic of glycosyltransferases: a short N-terminal cytoplasmic domain, a hydrophobic non-cleavable signal-anchor domain, and a C-terminal catalytic domain .

The commercially available recombinant form of MGAT2 Human from Sf9 cells is typically expressed with a 6-amino acid His tag at the C-terminus to facilitate purification . This design allows for efficient purification using proprietary chromatographic techniques while maintaining the enzyme's functional integrity. The amino acid sequence contains specific regions critical for substrate binding and catalytic activity, including conserved motifs characteristic of glycosyltransferase family members .

How is MGAT2 Human, Sf9 produced and purified?

MGAT2 Human, Sf9 is produced using a baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells . This expression system is preferred for producing mammalian glycosyltransferases as it allows for proper folding and post-translational modifications that are closer to the native human protein than bacterial expression systems would provide. The production process involves creating a recombinant baculovirus containing the human MGAT2 gene, infecting Sf9 cells with this virus, and allowing the cells to express the recombinant protein .

For purification, the expressed MGAT2 protein, which contains a C-terminal 6x His tag, is isolated using chromatographic techniques . While the exact proprietary methods are not disclosed in the available literature, typical approaches include affinity chromatography utilizing the His tag (nickel or cobalt-based matrices), followed by additional purification steps such as ion exchange or size exclusion chromatography to achieve high purity. The final product is a sterile filtered colorless solution with purity greater than 90% as determined by SDS-PAGE analysis .

What are the optimal storage conditions for MGAT2 Human, Sf9?

For optimal preservation of MGAT2 Human, Sf9 enzyme activity, specific storage conditions must be followed based on intended use timeframes . For short-term storage (when the entire vial will be used within 2-4 weeks), the protein can be stored at 4°C in its formulation buffer without significant loss of activity. For longer-term storage, it is recommended to store the protein frozen at -20°C .

The standard formulation of commercial MGAT2 protein solution (0.25mg/ml) contains 20mM Tris-HCl (pH 7.5), 10% glycerol, 0.15M NaCl, and 1mM DTT . This buffer composition is designed to maintain protein stability during storage. For extended long-term storage, it is specifically recommended to add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) to prevent protein loss due to adsorption to storage vessel surfaces and to further stabilize the enzyme . Multiple freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of enzymatic activity .

What methods can be used to assess MGAT2 enzyme activity?

Several methodological approaches exist for measuring MGAT2 enzyme activity, each with specific advantages for different research questions. A novel cell-based assay using high-resolution LC/MS has been developed to overcome limitations of traditional methods . This technique monitors the incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) to trace cellular DAG synthesis activity driven by MGAT2. This approach offers significantly reduced background interference and increased sensitivity compared to traditional TLC-based methods .

For a traditional in vitro enzyme activity assay, researchers typically measure the transfer of N-acetylglucosamine from UDP-GlcNAc (the donor substrate) to the acceptor substrate (a mannose-containing oligosaccharide). The reaction products can be detected using radioisotope-labeled substrates or fluorescently tagged substrates with appropriate separation techniques. When designing MGAT2 activity assays, it's critical to consider the enzyme's preference for specific stereoisomers of substrates. Studies have shown that recombinant MGAT2 enzyme selectively acylates 2-monoacylglycerol with higher efficiency than other stereoisomers .

How does MGAT2 compare structurally and functionally to other MGAT family members?

MGAT2 belongs to a family of monoacylglycerol acyltransferases that includes MGAT1 and MGAT3, each with distinct tissue expression patterns and functional roles. MGAT3, identified as a novel MGAT, is highly homologous to both MGAT1 and MGAT2, but with key differences in tissue distribution and substrate specificity . While MGAT2 is widely expressed in various tissues, MGAT3 expression in humans is restricted to the gastrointestinal tract, with highest levels found in the ileum .

At the subcellular level, both MGAT2 and MGAT3 are localized to the endoplasmic reticulum, which corresponds to their roles in lipid and glycan metabolism . From a functional perspective, MGAT2 plays a broader role in N-glycan processing, while MGAT3 appears to serve as the intestinal MGAT involved in dietary fat absorption. Recombinant MGAT3 enzyme has been shown to selectively acylate 2-monoacylglycerol with higher efficiency than other stereoisomers, similar to MGAT2 but with tissue-specific implications .

What are the implications of MGAT2 mutations in disease states?

Mutations in the MGAT2 gene have significant clinical implications, particularly in the development of carbohydrate-deficient glycoprotein syndrome type II (CDG2A, also known as CDGS2) . This rare inherited metabolic disorder is characterized by abnormal glycosylation of proteins due to defective N-glycan processing. The absence of introns in the MGAT2 gene coding sequence makes it particularly vulnerable to mutations, as alternative splicing cannot compensate for genetic defects .

Patients with MGAT2 mutations typically present with developmental delay, intellectual disability, dysmorphic features, and various systemic abnormalities. At the molecular level, these mutations lead to the production of incomplete or truncated N-glycans on glycoproteins, affecting their function and stability. Diagnostic approaches for identifying MGAT2-related disorders include analysis of serum transferrin glycosylation patterns, which show characteristic abnormalities in N-glycan structures. Genetic testing targeting the MGAT2 gene can confirm the diagnosis, and functional assays using patient-derived cells can assess the impact of specific mutations on enzyme activity .

How can MGAT2 be targeted for obesity and metabolic disorder therapies?

MGAT2 has emerged as a promising therapeutic target for addressing obesity and related metabolic disorders, including type 2 diabetes . The enzyme plays a critical role in intestinal fat absorption and energy metabolism, making it a logical intervention point for modulating fat processing. Inhibition of MGAT2 could potentially reduce dietary fat absorption and improve metabolic parameters in individuals with obesity and metabolic syndrome.

The cell-based assay described in the literature provides a valuable methodology for screening, developing, and evaluating MGAT2 inhibitors . This high-resolution LC/MS-based approach offers advantages over traditional methods by allowing assessment of MGAT2-mediated enzyme activity in a cellular context that more closely mimics the physiological environment. Using this assay, researchers have characterized several MGAT2 inhibitors from different chemotypes . When designing studies to evaluate MGAT2 inhibitors, researchers should consider using murine secretin tumor cell-1 line of enteroendocrine origin to construct human MGAT2-expressing recombinant cell lines, as this approach has been validated for inhibitor screening .

What are the considerations for experimental design when working with MGAT2 Human, Sf9?

When designing experiments with MGAT2 Human, Sf9, several methodological considerations are essential for obtaining reliable and reproducible results. First, the formulation buffer components must be considered for their potential interference with downstream applications. The standard formulation (20mM Tris-HCl (pH 7.5), 10% glycerol, 0.15M NaCl, and 1mM DTT) may need adjustment depending on the experimental system .

For enzyme activity assays, researchers should determine the optimal enzyme concentration, substrate concentration, pH, temperature, and incubation time to ensure linear reaction kinetics. The choice of detection method will depend on the specific research question, with options including radioisotope-based assays, fluorescence-based approaches, or mass spectrometry. For inhibitor screening, the cell-based assay using high-resolution LC/MS offers advantages in terms of physiological relevance and sensitivity .