GO:0042283 dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0042283 describes the enzyme activity that adds the second glucose residue to the lipid-linked oligosaccharide (LLO) precursor during N-linked glycosylation.
• The reaction transfers glucose from dolichyl phosphate glucose (Dol-P-Glc) to Glc1Man9GlcNAc2-PP-Dol, producing Glc2Man9GlcNAc2-PP-Dol.
• In humans, this activity is carried out by ALG8, an alpha-1,3-glucosyltransferase embedded in the endoplasmic reticulum membrane.
• Biallelic ALG8 mutations cause ALG8-CDG, a congenital disorder of glycosylation with a wide clinical spectrum ranging from mild to severe.
• ALG8 expression supports cancer stemness by promoting WNT/beta-catenin signaling through glycosylation in colon cancer.
• Studying GO:0042283 requires combining CRISPR knockout, point-mutation knock-in, and glycosylation profiling to link genotype to phenotype [1,2].
Description
GO:0042283, dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity, is a molecular function that catalyzes a specific step in the assembly of the lipid-linked oligosaccharide (LLO) precursor used for N-linked protein glycosylation. This activity adds the second glucose residue to the growing LLO, converting Glc1Man9GlcNAc2-PP-Dol to Glc2Man9GlcNAc2-PP-Dol by transferring glucose from dolichyl phosphate glucose (Dol-P-Glc). In humans, the enzyme responsible for this activity is ALG8, an endoplasmic reticulum membrane protein. Researchers study GO:0042283 because defects in LLO biosynthesis cause congenital disorders of glycosylation (CDGs), and ALG8-CDG is a recognized subtype with a broad phenotypic range. Beyond rare disease, ALG8-mediated glycosylation has been implicated in cancer biology, where it supports stemness and WNT/beta-catenin signaling in colon cancer cells. Understanding this activity at the molecular level helps explain how cells build the N-glycan precursor, how mutations alter protein folding and signaling, and how glycosylation pathways can be targeted in disease contexts [1,2].
dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity At A Glance
| GO ID | GO:0042283 |
|---|---|
| GO term | dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity |
| Ontology | molecular_function |
| Synonym | dolichyl-P-Glc:Glc1Man9GlcNAc2-PP-dolichyl glucosyltransferase activity |
| Major function | Adds the second glucose to the LLO precursor during N-linked glycosylation |
| Substrate | Dolichyl phosphate glucose (Dol-P-Glc) and Glc1Man9GlcNAc2-PP-Dol |
| Product | Glc2Man9GlcNAc2-PP-Dol |
| Human gene | ALG8 |
| Associated disease | ALG8-CDG |
What Is GO:0042283?
GO:0042283 is defined as the catalysis of the addition of the second glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. Specifically, it transfers glucose from dolichyl phosphate glucose (Dol-P-Glc) onto the lipid-linked oligosaccharide Glc1Man9GlcNAc2-PP-Dol, forming Glc2Man9GlcNAc2-PP-Dol.
Why Is dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity Important in Cell Biology?
GO:0042283 is important because it represents a critical step in N-linked glycosylation, a process that affects protein folding, stability, and cell signaling. Mutations in ALG8, the enzyme carrying this activity, cause ALG8-CDG, a congenital disorder of glycosylation with a wide clinical spectrum. Additionally, ALG8-mediated glycosylation has been linked to cancer stemness and WNT/beta-catenin signaling in colon cancer, highlighting its relevance beyond rare genetic disease.
• Required for proper assembly of the N-glycan precursor on the lipid carrier dolichol.
• Defects in ALG8 cause ALG8-CDG, a multisystem congenital disorder of glycosylation.
• ALG8 expression supports stemness in colon cancer through glycosylation of WNT/beta-catenin pathway components.
• The activity is essential for normal protein folding and quality control in the endoplasmic reticulum.
• It represents a potential therapeutic target in cancers with altered glycosylation.
• Studying this activity helps interpret variants of uncertain significance in ALG8.
• It provides a model for understanding other LLO glycosyltransferases.
• It links glycosylation defects to clinical phenotypes such as developmental delay and coagulopathy.
Molecular Mechanism of dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity
Substrate recognition and binding
In simple terms: The enzyme grabs the sugar donor and the growing lipid-linked sugar chain.
ALG8 recognizes dolichyl phosphate glucose (Dol-P-Glc) as the glucose donor and Glc1Man9GlcNAc2-PP-Dol as the acceptor substrate. The enzyme is embedded in the endoplasmic reticulum membrane, where it accesses the lipid-linked oligosaccharide.
Catalytic transfer of glucose
In simple terms: The enzyme moves a glucose molecule onto the sugar chain.
The alpha-1,3-glucosyltransferase activity transfers glucose from Dol-P-Glc to the terminal mannose of Glc1Man9GlcNAc2-PP-Dol, forming an alpha-1,3 linkage and yielding Glc2Man9GlcNAc2-PP-Dol.
Role in N-linked glycosylation pathway
In simple terms: This step helps build the sugar tree that will be attached to proteins.
This activity is part of the sequential assembly of the lipid-linked oligosaccharide precursor in the endoplasmic reticulum. The addition of the second glucose is a prerequisite for subsequent addition of the third glucose and for efficient transfer of the oligosaccharide to nascent proteins.
Regulation and disease relevance
In simple terms: When this enzyme is faulty, glycosylation goes wrong and can cause disease.
Mutations in ALG8 reduce or abolish this activity, leading to ALG8-CDG with a wide clinical spectrum. In cancer, ALG8 expression can be upregulated and supports stemness through glycosylation of WNT/beta-catenin signaling components.
Key Genes Involved in GO:0042283 dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity
The following genes and proteins are directly or indirectly involved in the dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity and related N-linked glycosylation pathways.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ALG8 | Catalyzes the addition of the second glucose to the LLO precursor | Mutations cause ALG8-CDG; expression linked to cancer stemness [1,2] |
| ALG6 | Adds the first glucose to the LLO precursor | Related glucosyltransferase in the same pathway |
| ALG10 | Adds the third glucose to the LLO precursor | Downstream enzyme in LLO assembly |
| ALG1 | Adds the first mannose in LLO synthesis | Upstream step in N-glycan precursor assembly |
| ALG2 | Adds second mannose | Upstream step in LLO synthesis |
| ALG3 | Adds third mannose | Upstream step in LLO synthesis |
| ALG9 | Adds sixth and seventh mannose | Upstream step in LLO synthesis |
| ALG12 | Adds eighth mannose | Upstream step in LLO synthesis |
| DOLK | Synthesizes dolichol phosphate | Provides carrier for LLO |
| DPM1 | Synthesizes dolichol phosphate mannose | Provides mannose donor for LLO |
| MPDU1 | Synthesizes dolichol phosphate glucose | Provides glucose donor for ALG8 |
| DDOST | Component of oligosaccharyltransferase complex | Transfers LLO to proteins |
| STT3A | Catalytic subunit of oligosaccharyltransferase | Transfers glycan to nascent proteins |
| RPN1 | Subunit of oligosaccharyltransferase | Stabilizes complex |
| RPN2 | Subunit of oligosaccharyltransferase | Stabilizes complex |
| MAGT1 | Subunit of oligosaccharyltransferase | Facilitates glycan transfer |
| WNT3A | WNT ligand | Glycosylation affects WNT signaling |
| CTNNB1 | Beta-catenin | Effector of WNT signaling affected by glycosylation |
How Is dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity Regulated?
The expression and activity of ALG8, which carries GO:0042283, can be regulated at the transcriptional level in cancer, where it supports stemness through glycosylation of WNT/beta-catenin signaling components. In congenital disorders, mutations in ALG8 reduce enzymatic activity, leading to a wide clinical spectrum. However, specific upstream regulators such as mTOR or the integrated stress response have not been directly linked to this activity in the provided literature.
dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ALG8 | ALG8-CDG | Patient-derived fibroblasts; CRISPR knock-in of patient mutations |
| ALG8 | Colon cancer stemness | Colon cancer cell lines with ALG8 knockout or overexpression |
| ALG8 | WNT/beta-catenin signaling | Reporter assays in cancer cells |
| ALG6 | ALG6-CDG | Knockout cell models |
| ALG10 | ALG10-CDG | Knockout cell models |
ALG8-CDG (congenital disorder of glycosylation)
Biallelic mutations in ALG8 cause ALG8-CDG, a congenital disorder of glycosylation with a wide clinical spectrum. Molecular findings suggest an explanation for a milder phenotype in the first-described patient.
Colon cancer and cancer stemness
ALG8 fuels stemness through glycosylation of the WNT/beta-catenin signaling pathway in colon cancer. This links GO:0042283 activity to cancer progression and potential therapeutic targeting.
From dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of ALG8 affect LLO assembly? | ALG8 knockout cell lines |
| Do patient mutations reduce enzymatic activity? | Point-mutation knock-in of ALG8 variants |
| Can wild-type ALG8 rescue glycosylation defects? | Knock-in of wild-type ALG8 |
| How does ALG8 overexpression affect cancer stemness? | Overexpression of ALG8 in colon cancer cells |
| What proteins interact with ALG8? | Tagged knock-in of ALG8 for immunoprecipitation |
| Does ALG8 glycosylation affect WNT signaling? | Knockout and overexpression with WNT reporter assays |
How to Study the dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Mass spectrometry | Glycan structures | Analyze LLO and N-glycans in ALG8 mutants |
| Lectin blotting | Specific glycan epitopes | Detect glycosylation changes |
| CRISPR knockout | Gene function loss | Study ALG8 loss in cell models |
| CRISPR knock-in | Specific mutations | Model ALG8-CDG patient variants |
| RNA-seq | Transcriptome changes | Identify pathways affected by ALG8 |
| Proteomics | Protein abundance and interactions | Find ALG8 interaction partners |
| WNT reporter assay | WNT/beta-catenin activity | Link ALG8 to signaling |
| Sphere formation assay | Stemness potential | Assess cancer stemness |
Glycosylation profiling
Mass spectrometry and lectin blotting can assess LLO and N-glycan structures to determine the impact of ALG8 mutations or knockout on glycosylation [1,2].
CRISPR-based genetic screens
Genome-wide CRISPR knockout screens can identify genes that modify the effects of ALG8 loss, revealing pathway interactions.
Transcriptomics and proteomics
RNA-seq and proteomics can measure changes in gene expression and protein abundance upon ALG8 manipulation, linking GO:0042283 to downstream signaling.
Functional assays for stemness
Sphere formation and WNT reporter assays can test how ALG8-mediated glycosylation affects cancer stemness.
How CRISPR Can Be Used to Study GO:0042283 dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity
Knockout
CRISPR knockout of ALG8 can eliminate GO:0042283 activity, causing LLO accumulation and glycosylation defects. This model is useful to study the consequences of ALG8 loss in cancer and rare disease [1,2].
Point Mutation
Point mutations identified in ALG8-CDG patients can be introduced via CRISPR to model specific clinical phenotypes and assess residual enzymatic activity.
Knock-in
Knock-in of wild-type or tagged ALG8 allows rescue experiments and interaction studies to confirm the role of GO:0042283 in glycosylation.
Overexpression
Overexpression of ALG8 can enhance glycosylation and promote stemness in cancer cells, providing a gain-of-function model to study WNT/beta-catenin signaling.
How EDITGENE Supports dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity Research
Researchers studying dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in glycosylation, disease, or cancer stemness. EDITGENE provides CRISPR-based cell model services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity research.
Frequently Asked Questions About dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase activity
What is GO:0042283?
GO:0042283 is the molecular function that adds the second glucose to the lipid-linked oligosaccharide precursor during N-linked glycosylation.
What does dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase do?
It transfers glucose from dolichyl phosphate glucose to Glc1Man9GlcNAc2-PP-Dol, forming Glc2Man9GlcNAc2-PP-Dol.
Which gene encodes this activity in humans?
ALG8 encodes the enzyme responsible for this activity.
What diseases are associated with ALG8 mutations?
Mutations in ALG8 cause ALG8-CDG, a congenital disorder of glycosylation with a wide clinical spectrum.
How is ALG8 linked to cancer?
ALG8 fuels stemness through glycosylation of the WNT/beta-catenin signaling pathway in colon cancer.
What is the substrate of ALG8?
The substrates are dolichyl phosphate glucose and Glc1Man9GlcNAc2-PP-Dol.
What is the product of the ALG8 reaction?
The product is Glc2Man9GlcNAc2-PP-Dol.
How can I study GO:0042283 in the lab?
You can use CRISPR knockout, point mutation knock-in, and glycosylation profiling to study this activity [1,2].
What model systems are used for ALG8-CDG?
Patient fibroblasts and CRISPR knock-in cell lines expressing patient mutations are commonly used.
Does ALG8 affect WNT signaling?
Yes, ALG8-mediated glycosylation supports WNT/beta-catenin signaling in colon cancer.
Conclusion
GO:0042283 represents a key enzymatic step in N-linked glycosylation, carried out by ALG8 in humans. Its importance spans rare congenital disorders such as ALG8-CDG and cancer biology, where it promotes stemness through WNT/beta-catenin signaling [1,2]. Understanding this activity through CRISPR models and glycosylation profiling can reveal new therapeutic opportunities and improve diagnosis of glycosylation disorders.
References
- 1. Wu X et al.. 2022. ALG8 Fuels Stemness Through Glycosylation of the WNT/Beta-Catenin Signaling Pathway in Colon Cancer.. DNA Cell Biol 41(12):1075-1083 PMID: 36454274
- 2. Vuillaumier-Barrot S et al.. 2019. Wide clinical spectrum in ALG8-CDG: clues from molecular findings suggest an explanation for a milder phenotype in the first-described patient.. Pediatr Res 85(3):384-389 PMID: 30420707
