PMM1 Human
Description
Glycosylation Support
PMM1 contributes to GDP-mannose synthesis but is non-essential for baseline glycosylation, as PMM2 compensates in most tissues . PMM1-deficient mice show normal glycosylation patterns, contrasting with PMM2-deficient mice, which exhibit severe congenital disorders of glycosylation (e.g., CDG-Ia) .
Ischemia-Induced Glc-1,6-P₂ Degradation
PMM1 is brain-specific and crucial for degrading Glc-1,6-P₂ during ischemia:
-
Mechanism: IMP (elevated during ischemia) binds PMM1, activating its phosphatase activity to hydrolyze Glc-1,6-P₂ to Glu-6-P, restoring glycolysis .
-
Evidence:
Tissue Distribution and Expression Patterns
PMM1 is ubiquitous in embryos but downregulated postnatally in most tissues except brain .
Comparative Kinetics
| Parameter | PMM1 | PMM2 |
|---|---|---|
| V<sub>max</sub> | Identical for Man-1-P and Glu-1-P | 20× higher for Man-1-P vs. Glu-1-P |
| Glc-1,6-P₂ Hydrolysis | 110× stimulation by IMP (K<sub>a</sub>=3 µM) | No significant activity |
Conditional Essentiality
-
Ischemia Model: PMM1 knockout mice show no baseline phenotype but fail to degrade Glc-1,6-P₂ during ischemia .
-
PGM2L1 Interaction: PMM1 antagonizes PGM2L1 (Glc-1,6-P₂ synthase) in cells, maintaining metabolic balance .
Clinical and Biotechnological Relevance
-
Disease Association: No PMM1-linked disorders are reported, unlike PMM2 (CDG-Ia) .
-
Therapeutic Potential: Recombinant PMM1 (produced in E. coli) is used in studies to model ischemic brain metabolism .
-
Diagnostic Utility: PMM1 activity assays could aid in understanding neurological ischemia but remain experimental .
Product Specs
For short-term storage (up to 2-4 weeks), the entire vial can be stored at 4°C. For extended storage, it is recommended to freeze the solution at -20°C. To ensure long-term stability, consider adding a carrier protein (0.1% HSA or BSA). Repeated freezing and thawing should be avoided.
Q&A
What is PMM1 and how was it initially characterized?
PMM1 is the human homologue of SEC53 or yeast phosphomannomutase. It was first cloned from a human liver cDNA library and encodes a protein of 262 amino acids with a predicted molecular mass of 29 kDa. The protein shares 54% identity with yeast phosphomannomutase . PMM1 belongs to the phosphohexomutase family of enzymes that catalyze the interconversion of hexose 6-phosphates and hexose 1-phosphates, a critical step in cellular metabolism .
The initial characterization involved expressing the human cDNA in Escherichia coli, which yielded an active phosphomannomutase that was subsequently purified to homogeneity. The gene was mapped to chromosome 22q13.1 using hybrid cell lines and fluorescence in situ hybridization techniques .
How does PMM1 expression vary across human tissues?
Northern blot analysis of human tissues has revealed a differential expression pattern for PMM1:
What is the molecular basis for PMM1's dual enzyme activity?
PMM1 exhibits a remarkable dual functionality, acting as both a mutase and a phosphatase depending on cellular conditions. The 1.93 Å resolution structure of PMM1 complexed with inosine monophosphate (IMP) has revealed the structural basis for this molecular switch .
When IMP binds to PMM1, it occupies the substrate recruitment site, inhibiting the mutase activity while simultaneously activating phosphatase activity. This phosphatase activation occurs with an IMP Kact value of 1.5 μM and results from the hydrolysis of the phospho-enzyme intermediate .
The structural analysis has identified key residues involved in this switch mechanism:
How do PMM1 and PMM2 differ despite their sequence similarity?
What experimental approaches are optimal for studying PMM1's phosphatase activity?
When investigating PMM1's phosphatase activity, researchers should consider the following methodological approach:
-
Enzyme purification: Express recombinant PMM1 in E. coli and purify to homogeneity using affinity chromatography .
-
Activity assays: Measure phosphatase activity using glucose 1,6-bisphosphate as substrate, with and without IMP as an activator. Monitor the conversion to glucose 6-phosphate using coupled enzyme assays .
-
Structural analysis: Employ T2 relaxation nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) to assess conformational changes upon IMP binding .
-
Mutagenesis studies: Perform site-directed mutagenesis of key residues (particularly Arg180 and Arg183) to confirm their role in IMP binding and phosphatase activation .
-
Controls: Include parallel experiments with PMM2 as a negative control for IMP-induced phosphatase activity .
A rigorous experimental design should include multiple trials to confirm data consistency, clearly defined controls, and appropriate statistical analyses, following the principles outlined in the experimental design process .
How can researchers effectively use siRNA techniques to study PMM1 function?
RNA interference using siRNA is a powerful approach for investigating PMM1 function in cellular contexts. When designing siRNA experiments for PMM1:
-
Selection of siRNAs: Utilize pre-designed siRNA sets that target multiple regions of the PMM1 transcript to ensure effective knockdown. Commercial options include PMM1 Human Pre-designed siRNA sets containing three designed siRNAs for the PMM1 gene .
-
Controls: Include appropriate negative controls (non-targeting siRNA) and positive controls (siRNA targeting housekeeping genes) to validate experimental quality .
-
Transfection optimization: Optimize transfection conditions based on cell type, using fluorescently labeled siRNA (e.g., FAM-labeled) to monitor transfection efficiency .
-
Knockdown validation: Confirm PMM1 knockdown at both mRNA level (qRT-PCR) and protein level (Western blot).
-
Functional assays: Assess the impact of PMM1 knockdown on:
-
Cellular hexose phosphate levels
-
Response to ischemic conditions
-
Glycolytic flux
-
Protein glycosylation patterns
-
-
Rescue experiments: Perform rescue experiments with wild-type and mutant PMM1 constructs to confirm specificity and explore structure-function relationships.
This methodological approach follows the principles of experimental design by clearly defining variables, controls, and measurement strategies 6.
What is the evidence for PMM1's role during brain ischemia?
PMM1's phosphatase activity appears to play a crucial neuroprotective role during brain ischemia:
-
Biochemical mechanism: During ischemic conditions, PMM1 converts glucose 1,6-bisphosphate to glucose 6-phosphate in the presence of IMP, effectively rescuing glycolysis when oxygen supply is limited .
-
IMP as activator: Ischemia leads to increased IMP levels, which activates PMM1's phosphatase function with high affinity (Kact = 1.5 μM) .
-
Conformational changes: IMP binding to PMM1 favors an enzyme conformation that is catalytically competent for water attack at the phosphoaspartyl intermediate, as confirmed by T2 relaxation NMR and SAXS analyses .
-
Isoform specificity: This function is specific to PMM1 and not found in PMM2, suggesting evolutionary specialization of PMM1 for this protective role .
The data supporting this role highlights PMM1 as a potential therapeutic target for ischemic brain injuries and stroke, though further research using appropriate animal models is needed to fully validate this hypothesis.
How does PMM1's function differ from PMM2 in relation to congenital disorders of glycosylation?
While mutations in PMM2 are strongly associated with carbohydrate-deficient glycoprotein syndrome type 1 (CDG1 or Jaeken disease), PMM1 has not been implicated in this disorder despite its similar enzymatic activity in vitro:
What are the best approaches for distinguishing between PMM1 and PMM2 activities in complex biological samples?
Distinguishing between PMM1 and PMM2 activities in cell lysates or tissue samples presents a methodological challenge due to their similar mutase activities. Recommended approaches include:
-
IMP-based differential assays: Measure phosphomannomutase activity with and without IMP. The IMP-induced phosphatase activity will be predominantly attributed to PMM1 .
-
Immunodepletion: Use isoform-specific antibodies to sequentially deplete either PMM1 or PMM2 from samples before activity measurements.
-
Thermal stability profiling: Exploit potential differences in thermal stability between the two isoforms to selectively inactivate one before enzymatic assays.
-
Mass spectrometry: Employ targeted proteomics to quantify each isoform in samples using isoform-specific peptides.
-
Gene editing controls: Generate CRISPR/Cas9 knockout cell lines for either PMM1 or PMM2 to serve as specificity controls for activity assays .
These methodological approaches should be validated using recombinant proteins and optimized for specific sample types to ensure reliable discrimination between the two isoforms.
How can researchers investigate the regulation of PMM1 gene expression?
Understanding the transcriptional and post-transcriptional regulation of PMM1 requires a systematic approach:
-
Promoter analysis: Characterize the PMM1 promoter region to identify transcription factor binding sites and regulatory elements.
-
Reporter assays: Construct reporter gene systems with the PMM1 promoter to assess transcriptional activity under various conditions, particularly hypoxia and metabolic stress.
-
ChIP sequencing: Identify transcription factors that bind to the PMM1 promoter in different tissues and under different physiological conditions.
-
RNA stability assays: Investigate post-transcriptional regulation by measuring PMM1 mRNA stability under various conditions.
-
miRNA studies: Identify potential miRNA binding sites in PMM1 mRNA and validate their functional impact using luciferase reporter assays.
-
Epigenetic profiling: Analyze DNA methylation and histone modifications in the PMM1 gene locus across tissues with differential expression.
This multi-faceted approach can provide insights into why PMM1 shows strong expression in liver, heart, brain, and pancreas but lower expression in skeletal muscle , potentially revealing tissue-specific regulatory mechanisms.
What emerging technologies might advance PMM1 research?
Several cutting-edge technologies hold promise for deepening our understanding of PMM1 biology:
-
Cryo-EM for dynamic structural analysis: Capture PMM1 in different conformational states during the catalytic cycle, particularly the transition between mutase and phosphatase activities.
-
Single-cell metabolomics: Track PMM1's impact on cellular metabolism at the single-cell level, especially during ischemic conditions.
-
Organ-on-chip models: Develop microfluidic systems that mimic tissue-specific environments to study PMM1 function in physiologically relevant contexts.
-
AI-driven protein engineering: Apply machine learning algorithms to design PMM1 variants with enhanced phosphatase activity for potential therapeutic applications in ischemic conditions.
-
In vivo metabolic imaging: Develop tools to visualize PMM1 activity in living tissues, particularly during ischemic events in the brain.
These technological approaches could overcome current limitations in understanding the spatiotemporal dynamics of PMM1 function in complex biological systems.
How might comparative analysis of PMM1 across species inform human PMM1 research?
Evolutionary analysis of PMM1 across species can provide valuable insights into its functional specialization:
-
Phylogenetic analysis: Trace the divergence of PMM1 and PMM2 across evolutionary history to identify when their functional specialization occurred.
-
Structural conservation: Compare the conservation of key residues (e.g., Arg180, Arg183) across species to identify functionally important domains.
-
Expression pattern comparison: Analyze tissue-specific expression patterns across species to identify conserved regulatory mechanisms.
-
Animal models: Develop appropriate animal models with genetic modifications in PMM1 to study its function in vivo, considering species-specific differences.
-
Functional assays across species: Compare the IMP-induced phosphatase activity of PMM1 homologs from different species to understand the evolutionary conservation of this function. This comparative approach could reveal whether PMM1's role in ischemic protection is a recent evolutionary adaptation in humans or a conserved function across species, informing both basic research and potential therapeutic applications.
Product Science Overview
PMM1 catalyzes the reversible conversion of mannose-6-phosphate (Man-6-P) to mannose-1-phosphate (Man-1-P). This reaction is vital for the synthesis of GDP-mannose, which is necessary for several critical cellular processes . GDP-mannose is essential for:
- N-glycosylation of proteins: This process is crucial for proper protein folding and stability.
- Synthesis of microbial cell wall polysaccharides: These polysaccharides are important for the structural integrity of microbial cells.
- Biosynthesis of L-ascorbic acid (vitamin C) in plants: This vitamin is a key antioxidant that protects cells from oxidative damage .
In humans, there are two isozymes of phosphomannomutase: PMM1 and PMM2. These isozymes are encoded by separate genes and have distinct but overlapping functions. PMM1 is primarily involved in mannose metabolism, while PMM2 has additional roles in glycosylation processes .
The evolutionary significance of PMM1 is highlighted by its conservation across different species. In many organisms, a single gene encodes both PMM and phosphoglucomutase (PGM) activities. This bifunctional enzyme is an example of evolutionary convergence, where similar functions have evolved independently in different lineages .
PMM1 is of particular interest in medical research due to its role in metabolic pathways. Mutations in the PMM2 gene are known to cause congenital disorders of glycosylation (CDG), a group of inherited metabolic disorders that affect glycosylation. While PMM1 mutations are less commonly associated with disease, understanding its function can provide insights into related metabolic disorders .
Recombinant human PMM1 is produced using genetic engineering techniques. This involves inserting the human PMM1 gene into a suitable expression system, such as bacteria or yeast, to produce the enzyme in large quantities. Recombinant PMM1 is used in research to study its structure, function, and potential therapeutic applications .
