Recombinant Mouse Alpha-1,3/1,6-mannosyltransferase ALG2 (Alg2)

What is Alpha-1,3/1,6-mannosyltransferase ALG2 and what is its main function in glycosylation pathways?

Mouse ALG2 is a member of the glycosyltransferase 1 family that operates in the biosynthetic pathway of dolichol-linked oligosaccharides (LLOs), which serve as precursors for protein N-glycosylation. It possesses dual enzymatic activities, functioning as both an alpha-1,3-mannosyltransferase and an alpha-1,6-mannosyltransferase. Its primary role is catalyzing the addition of the second and third mannose residues to dolichol-linked oligosaccharide chains on the cytoplasmic face of the endoplasmic reticulum (ER) membrane .

Specifically, ALG2 mannosylates Man(1)GlcNAc(2)-dolichol diphosphate (M₁Gn₂-PDol) and Man(2)GlcNAc(2)-dolichol diphosphate to form the branched Man(3)GlcNAc(2)-dolichol diphosphate (M₃Gn₂-PDol) core oligosaccharide . This process is essential for the proper assembly of the complete oligosaccharide structure (Glc₃Man₉GlcNAc₂) that is eventually transferred to nascent proteins by oligosaccharyltransferase.

How does mouse ALG2 compare to human ALG2 in terms of sequence homology and functional differences?

Mouse ALG2 exhibits high sequence homology with human ALG2, showing approximately 86% antigen sequence identity . This high degree of conservation reflects the evolutionary importance of the enzyme's function in protein glycosylation across mammalian species.

Despite this similarity, there are species-specific differences in ALG2 structure and function. While the well-studied yeast ALG2 interacts with the ER membrane through four hydrophobic domains, human ALG2 (hALG2) associates with the ER via a single membrane-binding domain and demonstrates markedly higher stability in vitro . This structural divergence has implications for enzyme stability and activity in experimental settings, with human ALG2 showing higher specific mannosyltransferase activity than yeast ALG2 .

What experimental models are most suitable for studying ALG2 function?

Several experimental models can be employed to study ALG2 function:

• Recombinant protein systems: Purified recombinant ALG2 can be used for in vitro enzymatic assays to study its mannosyltransferase activities. Human ALG2 expressed in E. coli has shown increased stability compared to yeast ALG2, making it advantageous for kinetic studies .

• Cell culture models: Human cell lines (such as HEK293) and mouse cell lines can be used to study ALG2 function through gene knockdown, knockout, or overexpression approaches. Recent studies have used CRISPR-based approaches to deplete ALG2 in MCF10A mammary epithelial cells and MDA-MB-468 breast cancer cells to assess its role in cell proliferation .

• Yeast complementation assays: Yeast alg2 mutants can be complemented with mammalian ALG2 to assess functional conservation and study structure-function relationships. Suc2-fusion ALG2 proteins have been demonstrated to complement yeast alg2 mutants, confirming enzymatic activity of the fusion proteins .

What methods are available for detecting and quantifying ALG2 expression and activity?

Detection methods:

• Western blotting: Using specific antibodies against ALG2, such as those recognizing the 319-413 amino acid region in human ALG2 .

• Immunoprecipitation: For isolation and subsequent analysis of ALG2 from cellular extracts.

• RT-qPCR: For quantifying ALG2 mRNA expression levels.

Activity assays:

• Liquid chromatography-mass spectrometry (LC-MS): A quantitative kinetics assay has been developed for studying purified human ALG2, enabling distinction and quantitation of each of the two reactions catalyzed by ALG2 .

• In vitro mannosyltransferase assays: Using purified acceptor substrates (such as M₁Gn₂-pyrophosphate-phytanol), GDP-mannose donor, and neutral lipids to reconstitute ALG2 activity .

• Cellular glycomics analysis: Profiling N-linked glycans to detect alterations in glycosylation patterns resulting from ALG2 manipulation.

How can the dual mannosyltransferase activities of ALG2 be experimentally distinguished?

Distinguishing between the α1,3 and α1,6 mannosyltransferase activities of ALG2 requires sophisticated analytical approaches:

• LC-MS analysis: A liquid chromatography-mass spectrometry quantitative kinetics assay has been developed specifically for human ALG2 that can distinguish and quantitate each of the two mannosyltransferase reactions . This approach enables researchers to monitor the formation of both M₂Gn₂(α-1,3)-PDol and M₂Gn₂(α-1,6)-PDol intermediates, as well as the final M₃Gn₂-PDol product.

• Linkage-specific enzymes: Utilizing enzymes that specifically cleave α1,3 or α1,6 mannose linkages to differentiate between the two activities.

• NMR spectroscopy: Nuclear magnetic resonance can be employed to determine the specific linkage configurations in glycan structures produced by ALG2.

What are the known disease associations with ALG2 dysfunction and how can they be modeled?

Defects in the ALG2 gene have been associated with congenital disorder of glycosylation type Ih (CDG-Ii), a rare inherited metabolic disorder characterized by abnormal glycosylation of proteins . This condition belongs to a broader category of congenital disorders of glycosylation (CDG) that can manifest with multisystemic effects, including neurological impairments, developmental delays, and dysmorphic features.

Disease modeling approaches:

• Patient-derived cells: Fibroblasts or induced pluripotent stem cells (iPSCs) from CDG-Ii patients can be used to study the cellular consequences of ALG2 mutations.

• CRISPR/Cas9 genome editing: Introduction of specific patient-associated mutations into cell lines or model organisms to recapitulate disease phenotypes.

• Conditional knockout models: Tissue-specific deletion of ALG2 in mice can help understand organ-specific effects of ALG2 deficiency.

• In vitro glycosylation assays: Comparing the enzymatic activities of wild-type and mutant ALG2 proteins to determine how specific mutations affect mannose transfer activities.

• Glycan profiling: Analysis of N-glycan structures in patient samples or disease models to identify characteristic alterations in glycosylation patterns associated with ALG2 dysfunction.

How do alterations in ALG2 expression affect cellular glycoprotein processing?

ALG2 deficiency impacts cellular glycoprotein processing at multiple levels:

• Impaired N-glycan precursor synthesis: Reduction in ALG2 function leads to accumulation of incomplete LLO intermediates (particularly M₁Gn₂-PDol or M₂Gn₂-PDol with only α1,6 mannose) .

• Effects on glycoprotein folding and stability: Recent studies have shown that depletion of the related enzyme ALG3 (which acts downstream of ALG2) results in reduced levels of phosphorylated EGFR and E-cadherin, indicative of improper protein folding, as well as the appearance of faster migrating bands on SDS-PAGE indicative of deglycosylated receptors . Similar effects would be expected with ALG2 depletion.

• Impact on cellular proliferation: Depletion of ALG3 significantly reduces cell proliferation in both wild-type mammary epithelial cells and PTEN-deficient, AKT-hyperactivated breast cancer cells . Given ALG2's position in the same pathway, similar proliferation defects might occur with ALG2 depletion.

• Substrate accumulation: ALG2 dysfunction may lead to regulation of the LLO biosynthetic pathway by controlling accumulation of M₂Gn₂(α-1,6) intermediate .

How does the membrane topology of ALG2 influence its enzymatic function?

The membrane topology of ALG2 is crucial for its enzymatic function and differs significantly between species:

Yeast ALG2 (ScAlg2) interacts with the ER membrane through four hydrophobic domains. In contrast, human ALG2 (hAlg2) associates with the ER via a single membrane-binding domain . This structural divergence has important implications:

• Enzyme stability: Human ALG2 demonstrates markedly increased stability compared to yeast ALG2, likely due to its reduced hydrophobicity. This property has been exploited to develop more sensitive in vitro mannosyltransferase assays .

• Substrate accessibility: The cytoplasmic orientation of ALG2's catalytic domain is essential for accessing both the dolichol-linked acceptor substrate embedded in the ER membrane and the soluble GDP-mannose donor. Topological studies using Suc2A fusion proteins have been employed to map the orientation of ALG2's N- and C-termini .

• Membrane microenvironment: The lipid composition of the ER membrane can influence ALG2 activity. Optimal in vitro mannosyltransferase assays of recombinant yeast ALG2 require addition of crude membranes or neutral bilayer lipids .

The simplified membrane association of human ALG2 may represent an evolutionary adaptation that enhances enzyme stability while maintaining proper positioning at the cytoplasmic face of the ER membrane, where dolichol-linked oligosaccharide synthesis begins.

What is the relationship between ALG2 and the AKT signaling pathway?

Emerging research suggests a potential connection between ALG2 and the AKT signaling pathway:

Recent in silico data mining has revealed that α-1,3/1,6-mannosyltransferase ALG2 harbors an AKT substrate consensus sequence . This finding suggests that ALG2 might be directly phosphorylated by AKT, potentially linking glycosylation processes to growth factor signaling and cellular metabolism.

The functional significance of this potential phosphorylation remains to be fully elucidated, but studies on the related enzyme ALG3 provide some insights:

• Impact on cell proliferation: Depletion of ALG3 significantly reduces cell proliferation in both wild-type cells and PTEN-deficient, AKT-hyperactivated breast cancer cells .

• Receptor glycosylation: ALG3 depletion affects the glycosylation and phosphorylation status of growth factor receptors like EGFR and HER3 .

These findings suggest a potential bidirectional relationship:

• AKT may regulate ALG2 activity through phosphorylation

• ALG2-mediated glycosylation may influence the function of components in the AKT signaling pathway

Further research is needed to confirm whether ALG2 is indeed phosphorylated by AKT and to determine how this phosphorylation affects its mannosyltransferase activities and cellular function.

How can advanced structural biology techniques be applied to study ALG2's catalytic mechanism?

Advanced structural biology techniques can provide crucial insights into ALG2's dual catalytic functions:

• X-ray crystallography: Obtaining crystal structures of ALG2 in complex with substrates, products, or inhibitors could reveal the molecular basis for its ability to catalyze both α1,3 and α1,6 mannose additions. Challenges include the membrane association of the enzyme and potential conformational flexibility.

• Cryo-electron microscopy (cryo-EM): This technique could be particularly valuable for visualizing ALG2 in its native membrane environment, potentially in complex with other components of the glycosylation machinery.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach can map conformational changes and substrate binding sites by measuring the exchange rates of backbone amide hydrogens with deuterium from the solvent.

• Site-directed mutagenesis combined with activity assays: Systematic mutation of conserved residues can help identify amino acids essential for each of the two mannosyltransferase activities.

• Molecular dynamics simulations: Computational approaches can model the dynamics of substrate binding and catalysis, potentially revealing how ALG2 can accommodate different substrate orientations to form distinct glycosidic linkages.

The development of more stable recombinant versions of human ALG2, which shows greater stability than yeast ALG2 , provides new opportunities for detailed structural analysis of this important enzyme.

What strategies can be employed to develop specific inhibitors or modulators of ALG2 activity?

Developing specific inhibitors or modulators of ALG2 activity could have significant research and potential therapeutic applications:

Target-based approaches:

• Substrate analogs: Design of GDP-mannose analogs or modified oligosaccharide acceptor substrates that compete with natural substrates.

• Transition state mimics: Development of compounds that mimic the transition state of the mannosyl transfer reaction.

• Allosteric modulators: Identification of compounds that bind to regulatory sites and selectively modulate either the α1,3 or α1,6 mannosyltransferase activity.

Screening approaches:

• High-throughput screening: Using the developed LC-MS quantitative kinetics assay for human ALG2 to screen compound libraries for inhibitors or modulators.

• Fragment-based drug discovery: Identification of small molecule fragments that bind to ALG2, followed by fragment linking or growing to develop more potent and selective compounds.

• Virtual screening: Computational docking of compounds to ALG2 structural models to identify potential inhibitors.

Considerations for selectivity:

• Distinguishing between α1,3 and α1,6 activities: Development of compounds that selectively inhibit one activity while sparing the other could be valuable research tools.

• Species selectivity: Exploiting structural differences between human/mouse and yeast ALG2 to develop selective inhibitors.

• Glycosyltransferase family selectivity: Ensuring specificity for ALG2 over other glycosyltransferases involved in N-glycan biosynthesis.

What are the recommended protocols for recombinant expression and purification of mouse ALG2?

Expression systems:

• E. coli expression: Human ALG2 expressed in E. coli has demonstrated increased stability and higher mannosyltransferase specific activity compared to yeast ALG2 . A similar approach may be suitable for mouse ALG2, with appropriate optimization.

• Mammalian expression: For applications requiring mammalian post-translational modifications, expression in HEK293 or CHO cells may be preferable.

• Insect cell expression: Baculovirus-infected insect cells can offer a compromise between bacterial and mammalian systems, providing some eukaryotic processing with higher protein yields.

Purification strategies:

• Affinity tags: Addition of His, FLAG, or GST tags to facilitate purification. The position of the tag should be carefully considered to avoid interference with enzymatic activity.

• Membrane protein considerations: As ALG2 associates with membranes, purification protocols may need to include detergents or lipid nanodiscs to maintain protein stability and activity.

• Quality control: Verification of purified ALG2 activity using the LC-MS quantitative kinetics assay to ensure that both mannosyltransferase activities are preserved.

How can ALG2 knockout/knockdown models be generated and validated?

Genetic modification approaches:

• CRISPR/Cas9 genome editing: Generation of ALG2 knockout cell lines using guide RNAs targeting critical exons. This approach has been successfully employed for the related enzyme ALG3 in MCF10A mammary epithelial cells and MDA-MB-468 breast cancer cells .

• RNA interference: siRNA or shRNA targeting ALG2 mRNA for transient or stable knockdown.

• Conditional knockout models: Cre-loxP or similar systems for tissue-specific or inducible deletion of ALG2 in mice.

Validation methods:

• Genotyping: PCR-based verification of genomic modifications.

• Expression analysis: Confirmation of reduced ALG2 mRNA and protein levels by RT-qPCR and Western blotting.

• Functional validation:

  • Altered glycosylation patterns of cellular glycoproteins

  • Accumulation of LLO intermediates (M₁Gn₂-PDol)

  • Reduced cell proliferation, as observed with ALG3 depletion

  • Changes in glycoprotein folding and stability, similar to effects seen with ALG3 depletion on EGFR and E-cadherin

• Rescue experiments: Reintroduction of wild-type or mutant ALG2 to confirm specificity of observed phenotypes.

How is ALG2 function regulated in response to cellular stress and metabolic changes?

The regulation of ALG2 in response to cellular stress and metabolic changes remains an area ripe for investigation:

• ER stress response: As a component of the N-glycosylation machinery in the ER, ALG2 function may be modulated during ER stress and the unfolded protein response (UPR).

• Metabolic regulation: The discovery that ALG2 harbors an AKT substrate consensus sequence suggests potential regulation by growth factor signaling and cellular metabolism.

• Substrate availability: ALG2 activity depends on the availability of GDP-mannose, which links glycosylation to carbohydrate metabolism. Under conditions of altered GDP-mannose levels, the preference for α1,3 versus α1,6 mannose addition can be shifted .

• Lipid environment: Changes in ER membrane composition during stress conditions could affect ALG2 association with the membrane and its enzymatic activity.

Research approaches to investigate these regulatory mechanisms could include:

• Phosphoproteomics to identify stress-induced modifications of ALG2

• Metabolic labeling to track changes in ALG2-dependent glycosylation under different cellular conditions

• Lipidomics to correlate changes in ER membrane composition with ALG2 activity

What is the potential role of ALG2 in cancer biology and therapeutic implications?

Emerging evidence suggests potential roles for ALG2 in cancer biology:

• Cell proliferation: Studies on the related enzyme ALG3 have shown that its depletion significantly reduces cell proliferation in both wild-type mammary epithelial cells and PTEN-deficient, AKT-hyperactivated breast cancer cells . Similar effects might be expected with ALG2 manipulation.

• AKT signaling connection: The presence of an AKT substrate consensus sequence in ALG2 suggests potential integration with oncogenic signaling pathways.

• Receptor glycosylation: Proper glycosylation is essential for the folding, stability, and function of growth factor receptors like EGFR and HER3. ALG3 depletion affects the glycosylation and phosphorylation status of these receptors , and ALG2 manipulation would likely have upstream effects on the same process.

Therapeutic implications:

• Combination approaches: Inhibition of ALG2 could potentially sensitize cancer cells to other therapies, particularly those targeting AKT signaling or growth factor receptors.

• Biomarker potential: Alterations in ALG2 expression or N-glycan structures could serve as biomarkers for certain cancer types or for predicting response to therapy.

• Target vulnerability: Cancer cells with hyperactivated AKT signaling might exhibit differential sensitivity to ALG2 inhibition compared to normal cells.