Recombinant Danio rerio Inositol monophosphatase 3 (impad1)
Description
Introduction to Recombinant Danio rerio Inositol Monophosphatase 3
Recombinant Danio rerio Inositol monophosphatase 3 (impad1) is a protein expressed and purified from engineered E. coli systems to match the native zebrafish protein sequence . The recombinant protein is typically fused to an N-terminal His tag to facilitate purification and experimental applications . The protein belongs to the inositol monophosphatase family, a group of enzymes that play critical roles in cellular signaling and phosphate metabolism.
Inositol monophosphatase 3 is encoded by the impad1 gene in zebrafish and has several synonyms in the scientific literature, including bpnt2, impa3, zgc:123256, IMP 3, and IMPase 3 . The zebrafish model provides valuable insights into the function of this protein, which has orthologs in humans and other vertebrates, making it relevant for comparative studies across species.
Physical Properties
The recombinant protein is typically provided as a lyophilized powder with high purity (greater than 90% as determined by SDS-PAGE) . The protein contains transmembrane regions, as indicated by its classification as a transmembrane protein in product specifications . This suggests that in its native environment, Inositol monophosphatase 3 may be associated with cellular membranes, which is consistent with its putative roles in signaling pathways.
Enzymatic Classification
Based on its human ortholog, Inositol monophosphatase 3 likely functions as a phosphatase, specifically catalyzing the hydrolysis of phosphate groups from various substrates . The human ortholog IMPAD1 is known to catalyze the hydrolysis of phosphoadenosine phosphate (PAP) to adenosine monophosphate (AMP) . While the specific enzymatic activities of the zebrafish protein haven't been fully characterized in the provided search results, it's reasonable to infer similar functionality based on sequence homology and conserved domains.
Substrate Specificity
The enzyme belongs to the inositol monophosphatase family, suggesting its ability to remove phosphate groups from inositol phosphates . This activity is crucial for the regulation of inositol-based second messengers in cellular signaling cascades. Although the zebrafish-specific substrate preferences aren't detailed in the search results, related inositol phosphatases in zebrafish show substrate specificity. For example, other zebrafish inositol phosphatases like Inpp5ka demonstrate high activity against specific phosphoinositides such as PtdIns(4,5)P2 .
Zebrafish Orthologs and Gene Duplication
The zebrafish genome, like other teleost fish, underwent a whole genome duplication event, resulting in multiple orthologs for many genes . While this specific phenomenon hasn't been explicitly documented for impad1 in the search results, it represents an important consideration when studying zebrafish proteins. For example, in the case of INPP5K (another inositol phosphatase), zebrafish possess two orthologs (inpp5ka and inpp5kb) with divergent functions .
Comparison with Human IMPAD1
The human ortholog of impad1, known as IMPAD1, has been more extensively characterized. The human protein:
-
Localizes to the Golgi apparatus
-
Catalyzes the hydrolysis of phosphoadenosine phosphate (PAP) to adenosine monophosphate (AMP)
-
Functions as a mitochondrial electron transport inhibitor that prevents reactive oxygen species (ROS) production
-
Plays a role in the formation of skeletal elements through endochondral ossification
Mutations in human IMPAD1 are associated with GRAPP-type chondrodysplasia with joint dislocations, indicating its importance in skeletal development . The zebrafish ortholog may serve as a valuable model for studying these functions, although specific differences might exist due to evolutionary divergence.
Expression and Localization
While the search results do not provide zebrafish-specific expression data for impad1, related inositol phosphatases in zebrafish show tissue-specific expression patterns. For instance, inpp5ka and inpp5kb show expression in the developing head, eyes, and tail of zebrafish embryos, with different abundance levels and some unique localization patterns .
Based on the human ortholog, Inositol monophosphatase 3 may be localized to cellular compartments including the cytosol, Golgi apparatus, Golgi lumen, nuclear body, and nucleus . The protein might also function as an integral component of membranes, consistent with its classification as a transmembrane protein .
Use as a Research Tool
Recombinant Danio rerio Inositol monophosphatase 3 serves as an important research tool for:
-
Studying phosphoinositide signaling pathways
-
Investigating the role of inositol phosphatases in development
-
Understanding evolutionary conservation of enzyme function across species
-
Exploring potential disease mechanisms related to phosphate metabolism
Zebrafish as a Model Organism
Zebrafish represent an excellent model organism for studying human diseases due to their genetic similarity to humans, transparent embryos allowing easy visualization of development, and rapid life cycle . For conditions linked to IMPAD1 mutations in humans, zebrafish models could provide valuable insights into disease mechanisms and potential therapeutic approaches.
Reconstitution Protocol
For experimental use, the following reconstitution protocol is recommended:
-
Briefly centrifuge the vial before opening to bring contents to the bottom
-
Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL
-
Add 5-50% glycerol (final concentration) for long-term storage
The default final concentration of glycerol is typically 50% for optimal stability.
Product Specs
Please note that we will prioritize shipping the format currently in stock. However, if you have specific format requirements, kindly indicate them in your order notes, and we will accommodate your request.
All of our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please communicate with us in advance as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What is Inositol monophosphatase 3 (impad1) in Danio rerio?
Inositol monophosphatase 3 (impad1) is a critical enzyme in zebrafish (Danio rerio) involved in phosphate metabolism pathways. It belongs to the inositol monophosphatase family with EC number 3.1.3.25. The protein is encoded by the impad1 gene (also known as impa3) and is annotated under ORF name zgc:123256. This enzyme catalyzes the dephosphorylation of inositol phosphate, playing a crucial role in cellular signaling pathways and phosphate homeostasis . The full-length protein consists of 341 amino acids and has been identified with UniProt accession number Q2YDR3 .
Unlike the related enzyme IMPDH (Inosine monophosphatase dehydrogenase), which is involved in purine metabolism and has been extensively studied in zebrafish photoreceptors, impad1 functions primarily in the inositol signaling pathway, which is essential for numerous cellular processes including neurodevelopment and metabolic regulation .
How does impad1 compare to other phosphatases in zebrafish?
Inositol monophosphatase 3 (impad1) belongs to a family of phosphatases in zebrafish that includes multiple variants. Unlike other phosphatases such as IMPDH1a (Inosine monophosphate dehydrogenase), which forms prominent protein filaments in photoreceptor cells and undergoes diurnal expression changes, impad1 has distinct functional characteristics .
While IMPDH1a in zebrafish is primarily expressed in rod and cone photoreceptors and is involved in guanine production through purine metabolism, impad1 has a broader expression pattern and functions in inositol phosphate metabolism. This distinction highlights the specialized roles of different phosphatases in zebrafish development and physiology .
The functional differences between impad1 and other phosphatases like IMPDH are reflected in their distinct roles in metabolic pathways. While IMPDH enzymes regulate guanine nucleotide pools critical for photoreceptor function, impad1 influences inositol-based signaling pathways, which have broader implications for development and cellular communication .
What approaches are recommended for generating and validating impad1 knockout models in zebrafish?
When generating impad1 knockout models in zebrafish, CRISPR-Cas9 technology has emerged as the method of choice due to its efficiency and precision. Based on successful approaches with related genes like SAMHD1, the following methodology is recommended:
-
Target site selection: Design guide RNAs targeting early exons of impad1, preferably within the first one-third of the coding sequence to ensure complete loss of function. Multiple guide RNAs should be designed and validated for specificity using in silico tools.
-
Microinjection protocol: Inject 1-2 cell stage zebrafish embryos with a mixture containing Cas9 mRNA (or protein) and guide RNAs. A typical injection contains 150-300 pg of Cas9 mRNA and 50-100 pg of each guide RNA.
-
Founder identification: Screen F0 embryos using T7 endonuclease assays or direct sequencing to identify successful editing events. Raise potential founders to adulthood.
-
Stable line establishment: Outcross F0 mosaic adults with wild-type fish and screen F1 offspring to identify germline transmission of mutations. Sequence the target region to identify specific indel mutations.
-
Validation strategy:
When characterizing the impad1 mutant model, comprehensive assessment should include:
-
Neurological phenotype analysis (brain development, locomotion)
-
Expression analysis of related genes using qPCR
-
Assessment of cellular death in relevant tissues
How can researchers optimize experimental design for consistent toxicity screening using impad1 as a marker?
Optimizing experimental design for consistent toxicity screening using impad1 as a marker requires careful consideration of multiple protocol parameters that influence reproducibility. Based on extensive comparative studies of zebrafish protocols, researchers should address the following key factors:
-
Chorion status: Determine whether embryos should be dechorionated prior to compound exposure. This parameter significantly affects compound uptake and has been identified as a major source of variability across laboratories. For impad1-related studies, dechorionation is recommended for more consistent exposure effects .
-
Exposure scenario: Choose between static (single exposure) versus static renewal (repeated exposure) protocols. Data shows that protocols with similar parameters (such as static exposure) can achieve active call concordance as high as 86% with negligible potency differences for developmental toxicity screening .
-
Standardized developmental timing: Initiate exposures at consistent developmental stages, preferably at early developmental points (6 hpf) for neurodevelopmental markers like impad1 .
-
Exposure duration and assessment timepoints: Standardize the duration of exposure (recommended 5 days for developmental toxicity) and consistent endpoints for measurement .
-
Container format and volume: Use standardized plate formats (96-well recommended) and consistent exposure volumes to minimize variability .
The table below summarizes critical protocol parameters that should be standardized for consistent impad1-based toxicity screening:
| Parameter | Recommendation | Impact on Variability |
|---|---|---|
| Fish strain | 5D Tropical zebrafish | Moderate |
| Exposure scenario | Static | High |
| Exposure initiation | 6 hpf | High |
| Chorion status | Dechorionated | Very High |
| Duration | 5 days for developmental toxicity | High |
| Container format | 96-well plate | Moderate |
| Assessment method | Quantitative PCR for impad1 expression | Moderate |
When implementing these protocol standardizations, researchers can expect to reduce inter-laboratory variability in potency estimations from an average 5.8-fold difference to less than 2-fold for neurodevelopmental markers like impad1 .
What role might impad1 play in cholesterol metabolism and neurodevelopment?
Recent research indicates potential links between phosphatase enzymes and cholesterol metabolism in zebrafish models of neurological disorders. While direct evidence for impad1's role in cholesterol metabolism is still emerging, several lines of investigation suggest important connections:
-
Phosphoinositide signaling and cholesterol regulation: Inositol monophosphatases like impad1 generate precursors for phosphoinositide signaling pathways, which interact with cholesterol metabolism regulatory networks. Dysregulation of related pathways has been observed in zebrafish models of neurological disorders .
-
Neurodevelopmental implications: In zebrafish models of Aicardi-Goutières syndrome (AGS), significant dysregulation of cholesterol biosynthesis was identified through qPCR analysis. Similar mechanisms may involve impad1, as phosphoinositide pathways interact with cholesterol regulation .
-
Potential therapeutic targets: Understanding impad1's role in cholesterol metabolism could reveal new therapeutic approaches for neurodevelopmental disorders. Research with zebrafish SAMHD1 mutants demonstrated that cholesterol dysregulation may play a role in disease pathophysiology, suggesting similar investigations with impad1 could be valuable .
-
Experimental approach for investigation:
This research direction holds promise for understanding fundamental connections between inositol phosphate metabolism, cholesterol regulation, and neurodevelopment in zebrafish models.
What methods are most effective for studying functional interactions between impad1 and other components of inositol signaling pathways?
Studying functional interactions between impad1 and other components of inositol signaling pathways requires a multi-faceted approach combining biochemical, genetic, and imaging techniques:
These methodologies will provide comprehensive insights into how impad1 functionally interacts with other components of inositol signaling networks in zebrafish.
