MGAT2 Human

What is the primary function of MGAT2 in human glycosylation pathways?

MGAT2 plays a critical role in the synthesis of complex-type N-glycan structures, representing the committed step in this process. It specifically extends a GlcNAcβ1,2- linkage on the Man-α1,6Manβ- arm of the trimannosyl N-glycan core. The enzyme employs UDP-GlcNAc as a sugar donor and follows a Mn²⁺-dependent inverting catalytic mechanism to generate the second GlcNAcβ1,2- branch from the trimannosyl glycan core . This enzymatic activity is essential for the proper processing of oligomannose to complex N-glycans, which are crucial for various cellular functions, particularly immune system development and functionality .

How is the MGAT2 gene structured and where is it expressed in human tissues?

The MGAT2 gene is located on chromosome 14 at chromosomal band q21. The genomic reference is NG_008920.1, and the transcript reference is NM_002408.3 . While the search results don't provide comprehensive information about tissue-specific expression patterns, studies indicate that MGAT2 is expressed in hepatic tissues, as demonstrated by its role in hepatic lipid metabolism . Additionally, the importance of MGAT2 in immune function suggests significant expression in immune system tissues .

What are the recommended techniques for measuring MGAT2 enzymatic activity in different tissue samples?

For measuring MGAT2 enzymatic activity, researchers typically prepare crude mitochondrial membrane fractions and conduct in vitro assays using specific substrates and inhibitors. As demonstrated in HepG2 cell studies, MGAT activity can be assessed by treating isolated membrane fractions with MGAT2-specific inhibitors. The standard approach involves measuring the reduction in MGAT activity following inhibitor treatment. In the referenced study, the MGAT2 inhibitor reduced MGAT2 activity by approximately 80% while having negligible effects on related enzymes like MGAT3 and DGAT1 .
When designing such experiments, it's crucial to include appropriate controls for related enzymes with potentially overlapping activities. For instance, DGAT1 has been shown to account for approximately 90% of in vitro MGAT activity in HepG2 cells, which could confound results if not controlled for properly .

How can researchers distinguish between MGAT2 activity and other similar glycosyltransferases in experimental settings?

Distinguishing MGAT2 activity from other glycosyltransferases requires a combination of specific inhibitors and carefully designed substrates. The use of selective small molecule inhibitors is a primary approach. In research settings, MGAT2 inhibitors with high specificity (approximately 80% reduction in MGAT2 activity with negligible effects on MGAT3 and DGAT1) can be employed alongside inhibitors for related enzymes .
Substrate specificity is another critical factor. MGAT2 has restricted substrate specificity, requiring both the Man-α1,6Manβ- acceptor arm and an unmodified GlcNAc-β1,2Man-α1,3Manβ- recognition arm for its action . Designing assays that leverage this specificity can help distinguish MGAT2 activity from related enzymes.

What are the clinical manifestations of MGAT2-CDG, and how does MGAT2 deficiency affect the immune system?

MGAT2-CDG (Congenital Disorder of Glycosylation type IIa) is characterized by profound global developmental disability, hypotonia, early onset epilepsy, and various multisystem manifestations . The immunological phenotype associated with MGAT2 deficiency includes persistent hypogammaglobulinemia and T-cell proliferation abnormalities .
Clinical reports have documented one patient with MGAT2-CDG who had normal IgM and IgA levels but decreased IgG levels until age 6, when the deficiency resolved spontaneously. This patient did not have a history of frequent infections. Other reported cases have shown scalp psoriasis, suggesting potential immune dysregulation .
Experimental evidence from mouse models with myeloid-specific knockout of Mgat2 has demonstrated deficient IgG antibody production in response to both infection and vaccination. Additionally, these models showed an induced, autoimmune-mediated depletion of naïve T-cells and decreased T-cell activity .

How does MGAT2 contribute to hepatic lipid metabolism and what implications does this have for metabolic disorders?

MGAT2 plays a significant role in hepatic lipid metabolism, specifically contributing to triacylglycerol (TG) synthesis and secretion. Research has demonstrated that MGAT2 is more influential than MGAT3 in this process . In experimental settings using HepG2 cells, inhibition of MGAT2 has been shown to reduce MGAT activity by approximately 24%, while inhibition of MGAT3 resulted in about a 35% decrease .
When investigating lipid metabolism pathways, researchers should note that MGAT2 functions in coordination with other enzymes. For instance, when HepG2 cells were incubated with 2-monoacylglycerol (2-MG) and [³H]oleate in the presence of both MGAT2 and MGAT3 inhibitors, the formation of radiolabeled diacylglycerol (DG) and TG was reduced by approximately 40% . This suggests that MGAT2 is part of a complex enzymatic network regulating hepatic lipid metabolism, with potential implications for metabolic disorders characterized by dysregulated lipid handling.

What is the structural basis for MGAT2 substrate recognition and catalysis?

MGAT2 employs a sophisticated structural mechanism for substrate recognition and catalysis. The enzyme exhibits a GT-A Rossmann-like fold and uses conserved divalent cation-dependent substrate interactions with the UDP-GlcNAc donor . The structural basis for MGAT2's highly specific substrate recognition involves:

• A catalytic subsite that binds the Man-α1,6- monosaccharide acceptor

• A distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate "recognition arm"
  This modular architecture provides the enzyme with its characteristic substrate selectivity and catalytic efficiency. The substrate binding mechanism employs both conserved and convergent catalytic subsite modules. Notably, the recognition arm interactions in MGAT2 are similar to the enzyme-substrate interactions observed in Golgi α-mannosidase II (MAN2A1), despite these being different classes of enzymes (glycosyltransferase vs. glycoside hydrolase) .
  Crystal structures have revealed that D347 is positioned to act as the catalytic base for deprotonating the nucleophilic hydroxyl, consistent with the predicted inverting catalytic mechanism. Additional stabilizing interactions include H-bonding between the GlcNAc residue and the side chains of W346 and E259, with further H-bond interactions predicted between N318 and Y294 and the GlcNAc acetyl group of the donor .

How do mutations in MGAT2 affect enzyme structure and function at the molecular level?

While the search results don't provide specific details about how different mutations affect MGAT2 structure and function at the molecular level, we can infer some principles based on the structural information provided.
Given the modular architecture of MGAT2 with its distinct catalytic subsite and exosite pocket, mutations could potentially affect:

• The binding affinity for the UDP-GlcNAc donor or the Man-α1,6- acceptor at the catalytic subsite

• The recognition of the GlcNAc-β1,2Man-α1,3Manβ- "recognition arm" at the exosite pocket

• The coordination of the Mn²⁺ ion essential for the catalytic mechanism

• The positioning of key catalytic residues like D347, which acts as the catalytic base
  Kinetic analysis of alanine mutants for substrate-interacting residues has confirmed the importance of specific amino acids in the catalytic mechanism, though the search results don't provide the detailed findings of these studies .

What are the optimal recombinant expression systems for producing functional human MGAT2 for structural and functional studies?

For the successful expression of functional human MGAT2, a recombinant expression platform utilizing mammalian cells has proven effective. Specifically, researchers have generated a secreted form of the human MGAT2 catalytic domain (residues 29-447) by replacing the NH₂-terminal membrane anchor with a fusion peptide cassette designed to target secretion of the recombinant fusion protein in mammalian cells .
The expression protocol involves:

• Transiently transfecting HEK293S (GnTI-) cells with the MGAT2 construct

• Purifying the expressed protein using Ni²⁺-NTA chromatography

• Concurrent cleavage of fusion tags

• Trimming of glycan structures to a single GlcNAc residue

• Further purification steps
  This approach yields an enzyme preparation with kinetic constants for GlcNAc transfer that are similar to those of the intact fusion protein, indicating preservation of catalytic activity . This expression system is particularly suitable for structural studies, as it has successfully produced protein amenable to crystallization and structure determination.

How can researchers design inhibitors specific to MGAT2 without cross-reactivity with related glycosyltransferases?

Designing highly specific MGAT2 inhibitors requires detailed understanding of the enzyme's unique structural features. Based on the structural data, effective inhibitor design should target:

• The unique exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- "recognition arm"

• The specific configuration of the catalytic subsite that binds the Man-α1,6- monosaccharide acceptor

• Unique residues involved in substrate binding that differ from related enzymes like MGAT3 and DGAT1
  Experimental validation of inhibitor specificity is crucial. In the referenced studies, inhibitor specificity was assessed by measuring activity against MGAT2, MGAT3, and DGAT1. A highly specific MGAT2 inhibitor reduced MGAT2 activity by approximately 80% while having negligible effects on MGAT3 and DGAT1 activities .
  Researchers should also consider the possible compensatory mechanisms when inhibiting MGAT2. For instance, in HepG2 cells, inhibition of both MGAT2 and MGAT3 resulted in only a 35% decrease in MGAT activity, suggesting that other enzymes (particularly DGAT1) can compensate for MGAT function in certain contexts .

What specific immune cell populations and functions are most affected by MGAT2 deficiency?

MGAT2 deficiency has significant effects on both humoral and cellular immunity. In terms of humoral immunity, persistent hypogammaglobulinemia has been observed in patients with MGAT2-CDG . One patient was noted to have normal IgM and IgA levels but decreased IgG levels until age 6, indicating a specific effect on certain antibody classes .
For cellular immunity, T-cell function appears to be particularly affected. Clinical reports have documented T-cell proliferation abnormalities in MGAT2-deficient patients. Specifically, one patient showed extremely low proliferative responses to the mitogens Concanavalin A (ConA) and pokeweed mitogen (PWM), which are typical T-cell stimulators .
Interestingly, the measured proliferation to Candida, normally a poor stimulatory antigen, was consistently 5-6 fold higher in this patient's cells compared to controls, suggesting a dysregulated rather than uniformly suppressed immune response .
Mouse models with myeloid-specific knockout of Mgat2 have shown an induced, autoimmune-mediated depletion of naïve T-cells and decreased T-cell activity, further supporting the critical role of MGAT2 in maintaining normal T-cell function .