GO:0106407 Glc2Man9GlcNAc2 oligosaccharide glucosidase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0106407 describes the molecular function of glucosidase II, which removes the second glucose from the Glc2Man9GlcNAc2 oligosaccharide on newly synthesized glycoproteins [1, 5].
• This activity is a key step in the endoplasmic reticulum (ER) N-glycan processing pathway, enabling glycoprotein folding and quality control [3, 5].
• The enzyme is a heterodimeric complex composed of a catalytic alpha subunit (GANAB) and a regulatory beta subunit (PRKCSH) [1, 5].
• Defects in glucosidase II are linked to diseases such as polycystic liver disease and congenital disorders of glycosylation.
• Research methods include synthetic glycan substrates, kinetic assays, and CRISPR-based gene editing to create knockout or point-mutation models [5, 6].
• EDITGENE provides custom cell models and screening services to study glucosidase II function and its role in disease.
Description
Glc2Man9GlcNAc2 oligosaccharide glucosidase activity (GO:0106407) is a molecular function that catalyzes the hydrolysis of the second glucose residue from the Glc2Man9GlcNAc2 oligosaccharide attached to nascent glycoproteins [1, 5]. This reaction is a critical step in the N-linked glycosylation processing pathway within the endoplasmic reticulum (ER), where it facilitates the interaction of glycoproteins with lectin chaperones and ensures proper folding. The enzyme responsible, glucosidase II, is conserved from yeast to humans and is essential for glycoprotein maturation and quality control [2, 8]. Researchers study this activity to understand fundamental glycobiology and its implications in diseases such as polycystic liver disease and congenital disorders of glycosylation. The availability of synthetic high-mannose glycans and kinetic models has enabled detailed substrate specificity analyses [5, 6]. Moreover, genetic tools like CRISPR-Cas9 allow the creation of knockout and point-mutation cell models to dissect the enzyme's physiological roles [1, 8].
Glc2Man9GlcNAc2 oligosaccharide glucosidase activity At A Glance
| GO ID | GO:0106407 |
|---|---|
| GO term | Glc2Man9GlcNAc2 oligosaccharide glucosidase activity |
| Ontology | molecular_function |
| Synonym | glucosidase II |
| Major function | Catalyzes the removal of the second glucose from Glc2Man9GlcNAc2 on glycoproteins |
| Reaction | Glc(2)Man(9)GlcNAc(2)-[protein] + H2O = GlcMan(9)GlcNAc(2)-[protein] + beta-D-glucopyranose |
| Localization | Endoplasmic reticulum |
| Subunits | Catalytic alpha subunit (e.g., GANAB) and regulatory beta subunit (e.g., PRKCSH) |
| Substrates | Glc2Man9GlcNAc2 oligosaccharide, synthetic high-mannose glycans |
What Is GO:0106407?
According to the Gene Ontology, GO:0106407 is defined as the catalysis of the reaction: Glc(2)Man(9)GlcNAc(2)-[protein] + H2O = GlcMan(9)GlcNAc(2)-[protein] + beta-D-glucopyranose. In simpler terms, it is the enzymatic removal of a glucose molecule from a specific N-linked oligosaccharide precursor on proteins, a process carried out by glucosidase II [1, 5].
Why Is Glc2Man9GlcNAc2 oligosaccharide glucosidase activity Important in Cell Biology?
Glc2Man9GlcNAc2 oligosaccharide glucosidase activity is essential for the N-glycan processing pathway, which ensures proper protein folding and quality control in the endoplasmic reticulum [3, 5]. This activity is required for the generation of monoglucosylated glycans that interact with lectin chaperones calnexin and calreticulin, facilitating glycoprotein folding. Dysregulation of this enzyme has been associated with diseases such as polycystic liver disease and congenital disorders of glycosylation. Understanding its mechanism and regulation is crucial for developing therapeutic strategies targeting glycosylation-related pathologies [1, 5].
• Essential for N-linked glycosylation processing and glycoprotein folding.
• Enables interaction with lectin chaperones calnexin and calreticulin.
• Mutations in glucosidase II subunits cause polycystic liver disease.
• Defects lead to congenital disorders of glycosylation.
• Target for antiviral and anticancer therapies due to role in viral envelope protein folding.
• Provides a model for studying ER quality control mechanisms.
• Kinetic properties inform drug design and enzyme inhibitor development.
• Conserved from yeast to plants and humans, facilitating comparative studies [1, 2, 8].
What Happens During Glc2Man9GlcNAc2 oligosaccharide glucosidase activity?
Substrate Recognition and Binding
In simple terms: The enzyme grabs the sugar chain on a new protein.
Glucosidase II specifically recognizes the Glc2Man9GlcNAc2 oligosaccharide attached to asparagine residues on nascent glycoproteins. The enzyme binds the terminal glucose residue and positions it for hydrolysis.
Catalytic Hydrolysis
In simple terms: The enzyme cuts off the second glucose.
The catalytic alpha subunit hydrolyzes the alpha-1,3-linked glucose, releasing beta-D-glucopyranose and leaving GlcMan9GlcNAc2 [1, 5]. This reaction is metal-independent and follows a kinetic model with substrate inhibition.
Product Release and Chaperone Interaction
In simple terms: The trimmed sugar chain now interacts with folding helpers.
The monoglucosylated glycan (GlcMan9GlcNAc2) is recognized by calnexin and calreticulin, which promote protein folding and prevent aggregation. This step is crucial for ER quality control.
Alternative Pathway: Endomannosidase
In simple terms: A backup enzyme can also remove glucose.
In glucosidase II-deficient cells, endomannosidase provides an alternative route for glucose removal, allowing some glycoproteins to mature.
Key Genes Involved in GO:0106407 Glc2Man9GlcNAc2 oligosaccharide glucosidase activity
The following genes and proteins are directly involved in Glc2Man9GlcNAc2 oligosaccharide glucosidase activity or its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| GANAB | Catalytic alpha subunit of glucosidase II | Mutations linked to polycystic liver disease; target for knockout studies |
| PRKCSH | Regulatory beta subunit of glucosidase II | Mutations cause polycystic liver disease; required for enzyme stability |
| CANX | Calnexin, lectin chaperone | Binds monoglucosylated glycans; part of ER quality control |
| CALR | Calreticulin, lectin chaperone | Binds monoglucosylated glycans; facilitates folding |
| UGGT | UDP-glucose:glycoprotein glucosyltransferase | Reglucosylates misfolded glycoproteins for chaperone re-entry |
| MAN1B1 | ER mannosidase I | Trims mannose residues; part of ER-associated degradation |
| EDEM1 | ER degradation-enhancing alpha-mannosidase-like protein | Targets misfolded glycoproteins for degradation |
| SEC61A1 | ER translocon subunit | Facilitates protein entry into ER; mutations cause tubulointerstitial kidney disease |
| RPN1 | Proteasome subunit | Indirect role in ERAD; not directly linked to glucosidase II |
| RPN2 | Proteasome subunit | Indirect role in ERAD; not directly linked to glucosidase II |
| DERL1 | Derlin-1, ERAD component | Retrotranslocates misfolded proteins; not directly linked |
| SEL1L | ERAD component | Recognizes misfolded glycoproteins; not directly linked |
| OS9 | ER lectin | Recognizes trimmed glycans for ERAD; not directly linked |
| XBP1 | Transcription factor | Regulates unfolded protein response; not directly linked |
| ATF6 | Transcription factor | Regulates UPR; not directly linked |
| ERN1 | ER stress sensor | Activates UPR; not directly linked |
| EIF2AK3 | PERK, ER stress kinase | Phosphorylates eIF2alpha; not directly linked |
How Is Glc2Man9GlcNAc2 oligosaccharide glucosidase activity Regulated?
Glucosidase II activity is regulated at multiple levels. The beta subunit (PRKCSH) is essential for stability and ER retention of the catalytic alpha subunit. Enzyme activity can be influenced by macromolecular crowding, which affects substrate accessibility and reaction rates. Additionally, the unfolded protein response (UPR) can modulate expression of glycosylation enzymes under ER stress. Kinetic studies reveal substrate inhibition and metal independence, suggesting regulation by substrate concentration.
Glc2Man9GlcNAc2 oligosaccharide glucosidase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| GANAB | Polycystic liver disease | Knockout or point-mutation in liver cell lines (e.g., HepG2) |
| PRKCSH | Polycystic liver disease | Knockout in HEK293T cells; assess polycystin-1 maturation |
| GANAB | Congenital disorders of glycosylation | Patient-derived fibroblasts or iPSCs with point mutations |
| PRKCSH | Cancer glycosylation | Overexpression in cancer cell lines; assess migration and invasion |
| GANAB | ER quality control | Knockout in HeLa cells; monitor glycoprotein folding |
Polycystic Liver Disease
Mutations in GANAB and PRKCSH, encoding glucosidase II subunits, cause autosomal dominant polycystic liver disease (ADPLD). Loss of glucosidase II function leads to defective maturation of polycystin-1, a protein critical for liver and kidney tubular structure.
Congenital Disorders of Glycosylation (CDG)
Defects in glucosidase II can result in CDG type II, characterized by broad clinical manifestations including developmental delay, seizures, and coagulopathy. The impaired N-glycan processing affects multiple organ systems.
Cancer
Altered glycosylation is a hallmark of cancer. Glucosidase II activity influences folding of oncogenic receptors and may affect tumor progression. Targeting this enzyme could disrupt cancer cell survival.
From Glc2Man9GlcNAc2 oligosaccharide glucosidase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of GANAB knockout on glycoprotein processing? | CRISPR knockout in HEK293T or HeLa cells |
| How do disease-associated point mutations affect glucosidase II activity? | Point mutation knock-in using CRISPR in cell lines |
| Can tagged GANAB be used to study subcellular localization? | Knock-in of fluorescent tag (e.g., GFP) at endogenous locus |
| What is the impact of PRKCSH overexpression on polycystin-1 maturation? | Overexpression in liver cell lines |
| How does glucosidase II deficiency affect ER stress? | Knockout in mouse embryonic fibroblasts |
| Can CRISPR library screening identify modifiers of glucosidase II function? | Genome-wide CRISPR knockout library in reporter cells |
How to Study the Glc2Man9GlcNAc2 oligosaccharide glucosidase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Synthetic glycan assay | Enzymatic hydrolysis of Glc2Man9GlcNAc2 | Substrate specificity and kinetics |
| Mass spectrometry | Glycan structures and processing intermediates | Profiling N-glycans in cells |
| Lectin blotting | Binding to specific glycans | Detecting monoglucosylated glycans |
| CRISPR knockout | Gene function loss | Studying GANAB/PRKCSH roles |
| CRISPR point mutation | Effect of specific variants | Modeling disease mutations |
| CRISPR knock-in | Tagged protein expression | Localization and interaction studies |
| Pulse-chase | Protein maturation and trafficking | Assessing glycoprotein folding |
| Immunoprecipitation | Protein-protein interactions | Chaperone binding |
Enzymatic Activity Assays
Glucosidase II activity can be measured using synthetic high-mannose glycans as substrates, with product analysis by HPLC or mass spectrometry. Kinetic parameters are determined by varying substrate concentrations.
Glycan Analysis
N-linked glycans can be profiled by mass spectrometry or lectin blotting to assess processing intermediates in wild-type and mutant cells.
CRISPR-Cas9 Genome Editing
Knockout, point mutation, and knock-in cell models are generated to study gene function and disease-associated variants.
Protein Folding and Trafficking Assays
Pulse-chase labeling and immunoprecipitation can monitor glycoprotein maturation and interaction with chaperones.
How CRISPR Can Be Used to Study GO:0106407 Glc2Man9GlcNAc2 oligosaccharide glucosidase activity
Knockout
CRISPR knockout of GANAB or PRKCSH eliminates glucosidase II activity, causing accumulation of Glc2Man9GlcNAc2 and triggering ER stress. These models are used to study glycoprotein maturation and disease mechanisms.
Point Mutation
Introducing disease-associated point mutations (e.g., in GANAB) via CRISPR allows assessment of specific effects on enzyme activity and substrate binding.
Knock-in
Knock-in of fluorescent or affinity tags at endogenous loci enables real-time imaging and proteomic analysis of glucosidase II subunits.
Overexpression
Overexpression of GANAB or PRKCSH can rescue knockout phenotypes or model gain-of-function effects in cancer and liver disease.
How EDITGENE Supports Glc2Man9GlcNAc2 oligosaccharide glucosidase activity Research
Researchers studying Glc2Man9GlcNAc2 oligosaccharide glucosidase activity-related genes often need to determine whether a candidate gene is causally involved in glycoprotein processing, ER quality control, or disease. EDITGENE provides custom CRISPR-edited cell models and screening services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for Glc2Man9GlcNAc2 oligosaccharide glucosidase activity research.
Frequently Asked Questions About Glc2Man9GlcNAc2 oligosaccharide glucosidase activity
What is Glc2Man9GlcNAc2 oligosaccharide glucosidase activity?
It is the enzymatic removal of the second glucose from the Glc2Man9GlcNAc2 oligosaccharide on glycoproteins, catalyzed by glucosidase II [1, 5].
What genes are involved in Glc2Man9GlcNAc2 oligosaccharide glucosidase activity?
The main genes are GANAB (catalytic alpha subunit) and PRKCSH (regulatory beta subunit).
What is the role of glucosidase II in protein folding?
It generates monoglucosylated glycans that interact with calnexin and calreticulin to promote proper protein folding.
Which diseases are associated with glucosidase II deficiency?
Polycystic liver disease and congenital disorders of glycosylation.
How can I measure glucosidase II activity?
Using synthetic high-mannose glycans as substrates and analyzing products by HPLC or mass spectrometry.
What is the substrate specificity of glucosidase II?
It specifically hydrolyzes the alpha-1,3-linked glucose on Glc2Man9GlcNAc2, with some activity on maltose [4, 5].
Can CRISPR be used to study glucosidase II?
Yes, CRISPR knockout, point mutation, and knock-in models are valuable for dissecting its function.
What are the kinetic properties of glucosidase II?
It follows a kinetic model with substrate inhibition and is metal-independent.
Is glucosidase II conserved across species?
Yes, it is found in yeast, plants, and mammals [1, 2, 8].
What is the alternative pathway for glucose removal?
Endomannosidase can remove glucose in glucosidase II-deficient cells.
Conclusion
Glc2Man9GlcNAc2 oligosaccharide glucosidase activity (GO:0106407) is a fundamental molecular function in glycoprotein processing, mediated by the heterodimeric enzyme glucosidase II. Its role in ER quality control and association with diseases like polycystic liver disease and CDG make it a significant research target. Advanced CRISPR models and biochemical assays continue to elucidate its mechanism and regulation, offering potential therapeutic avenues.
References
- 1. Taylor MA et al.. 2000. A potato alpha-glucosidase gene encodes a glycoprotein-processing alpha-glucosidase II-like activity. Demonstration of enzyme activity and effects of down-regulation in transgenic plants.. Plant J 24(3):305-16 PMID: 11069704
- 2. Kilker RD Jr et al.. 1981. Partial purification from Saccharomyces cerevisiae of a soluble glucosidase which removes the terminal glucose from the oligosaccharide Glc3Man9GlcNAc2.. J Biol Chem 256(10):5299-603 PMID: 7014569
- 3. Moore SE et al.. 1992. Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells.. J Biol Chem 267(12):8443-51 PMID: 1533222
- 4. Alonso JM et al.. 1993. Characterization of the maltase activity of glucosidase II from rat liver. Kinetic model.. Biol Chem Hoppe Seyler 374(10):977-82 PMID: 8297500
- 5. Totani K et al.. 2006. Substrate specificity analysis of endoplasmic reticulum glucosidase II using synthetic high mannose-type glycans.. J Biol Chem 281(42):31502-8 PMID: 16940048
- 6. Alonso JM et al.. 1991. Glucosidase II from rat liver microsomes. Kinetic model for binding and hydrolysis.. Biochem J 278 ( Pt 3)(Pt 3):721-7 PMID: 1898361
- 7. Totani K et al.. 2008. Effects of macromolecular crowding on glycoprotein processing enzymes.. J Am Chem Soc 130(6):2101-7 PMID: 18205357
- 8. Runge KW et al.. 1986. A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues.. J Biol Chem 261(33):15582-90 PMID: 3536907
