Recombinant Mouse RING finger and SPRY domain-containing protein 1 (Rspry1)

What is RSPRY1 and what domains does it contain?

RSPRY1 (Ring Finger And SPRY Domain Containing 1) is a protein coding gene that encodes a glycoprotein containing a RING-type zinc finger domain and an SPRY domain. The protein comprises 576 amino acids and is primarily located in the cytoplasm of skeletal muscle cells . The specific domains include:

• B.30/SPRY domain (amino acids 359-479)

• C3HC4-type RING finger domain (amino acids 526-565)

The RING finger domain typically functions in protein-protein interactions and potentially in ubiquitination processes, while the SPRY domain is involved in protein-protein interactions in diverse cellular contexts .

What are the known functions of RSPRY1 in normal physiology?

• It may be involved in the ubiquitination of target proteins, given its RING finger domain

• It likely plays a role in bone development, as demonstrated by strong protein localization in murine embryonic osteoblasts and periosteal cells during primary endochondral ossification

• It may regulate the FGF signaling pathway, similar to other SPRY domain-containing proteins

• It appears to have significant expression in cardiac tissue, particularly in the left ventricle and atrial appendage

Expression studies indicate RSPRY1 is present in multiple tissues, with particularly notable expression patterns during skeletal development, suggesting tissue-specific functions that may vary during developmental stages .

What are the most reliable methods for detecting RSPRY1 expression in mouse tissue samples?

Multiple complementary approaches are recommended for reliable RSPRY1 detection:

In situ hybridization:

• Use riboprobes targeting mouse Rspry1 exon 15 3' UTR (approximately 526 bp)

• This approach effectively visualizes spatial distribution of mRNA expression

RT-PCR:

• Can quantify expression levels across different tissues

• Primer design should target conserved regions for specificity

Immunohistochemistry (IHC):

• Affinity-purified antibodies such as rabbit polyclonal Novus antibody NBP1-92358 (directed against residues 87-233 of human RSPRY1) have shown good specificity

• For optimal results, compare antibody performance on both paraffin and frozen sections

• Validation through comparison with in situ hybridization results is recommended

ELISA:

• Commercial ELISA kits are available for mouse RSPRY1 quantification

• These typically detect RSPRY1 in serum, plasma and other biological fluids

• Detection range is approximately 0.156-10 ng/ml

What are the optimal conditions for working with recombinant mouse RSPRY1 protein?

Based on available product information for recombinant mouse RSPRY1:

Storage conditions:

• Lyophilized form can be stored at -20°C/-80°C for up to 12 months

• Liquid form can be stored at -20°C/-80°C for up to 6 months

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge vial before opening

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

Experimental considerations:

• Protein purity of >85% (as determined by SDS-PAGE) is suitable for most research applications

• For functional studies, consider that the recombinant protein may be partial length

• Tag type may vary depending on manufacturing process and should be considered in experimental design

How does RSPRY1 potentially interact with the MAPK-ERK signaling pathway?

RSPRY1 has been linked to the MAPK-ERK pathway, though the exact mechanisms remain to be fully elucidated. Current evidence suggests:

• RSPRY1 may function similarly to other SPRY domain-containing proteins (like Sprouty1, 2, and 4) that are known inhibitors of FGF signaling

• The SPRY domain typically functions as a protein interaction module and may:

  • Interact directly with components of the MAPK cascade

  • Modulate receptor tyrosine kinase signaling

  • Affect downstream signal transduction

• Knockout studies of related SPRY domain proteins provide potential insights:

  • Spry4 KO mice display dwarfism and abnormal digit development

  • Spry2/Spry4 double KO mice show severe craniofacial and limb development defects

Research approach recommendations:

• Phosphorylation studies to examine activation status of ERK1/2 in RSPRY1-deficient cells

• Co-immunoprecipitation assays to identify direct binding partners

• Pathway inhibitor studies to determine if RSPRY1 effects are dependent on MAPK activity

• Reporter assays to measure pathway activity in response to RSPRY1 manipulation

What methodologies are most effective for analyzing RSPRY1 mutations associated with skeletal dysplasias?

Several complementary approaches are recommended for comprehensive mutation analysis:

Genomic analysis:

• Autozygome/exome sequencing combination:

  • Particularly effective for consanguineous families

  • Has successfully identified homozygous frameshift mutations in RSPRY1

• Variant filtering strategies:

  • Focus on rare coding/splicing homozygous variants (MAF < 0.001)

  • Prioritize variants within critical autozygous intervals

  • Verify absence in population databases (dbSNP, 1000 Genomes, ExAC)

• Variant validation:

  • Use multiple bioinformatics algorithms (e.g., SIFT, PolyPhen-2, MutationTaster, CADD)

  • Confirm with Sanger sequencing

Functional validation:

• Nonsense-mediated decay (NMD) analysis:

  • RT-PCR on patient cells treated with/without cycloheximide

  • Western blotting to confirm protein loss

• 3D protein modeling:

  • For missense variants, analyze impact on domain structure

  • Though limited by incomplete structural information for RSPRY1

• Animal models:

  • Gene knockout or patient-specific mutations

  • Analyze skeletal development and FGF signaling pathway activity

How are RSPRY1 mutations linked to spondyloepimetaphyseal dysplasia (SEMD), and what methodologies best characterize this relationship?

RSPRY1 mutations have been definitively established as the cause of SEMD, Faden-Alkuraya type (OMIM #616723), a rare autosomal recessive disorder. This causative relationship has been established through:

Genetic evidence:

• Multiple independent families with biallelic RSPRY1 mutations show similar clinical phenotypes

• Different mutation types (frameshift, missense, canonical splice site) all produce similar skeletal manifestations

• "Matchmaking" systems connecting similar phenotypes across diverse populations (Saudi Arabia, Peru, Turkey, India) have reinforced the gene-disease association

Clinical findings consistent across patients include:

• Short stature

• Facial dysmorphism

• Progressive vertebral defects

• Small epiphysis

• Cupping and fraying of metaphyses

• Brachydactyly

• Short fourth metatarsals

• Intellectual disability

• Mild scoliosis

Recommended characterization approaches:

• Detailed radiographic analysis at different developmental stages to document progression

• Standardized phenotyping using established skeletal dysplasia classification systems

• Functional studies in patient-derived cells to identify dysregulated pathways

• Skeletal tissue-specific expression studies

A novel clinical finding recently associated with RSPRY1 mutations is joint dislocation, particularly affecting elbow joints, expanding the phenotypic spectrum of this disorder .

What experimental models are most suitable for studying RSPRY1-related skeletal development disorders?

Several experimental approaches can effectively model RSPRY1-related disorders:

Cellular models:

• Patient-derived primary cells:

  • Fibroblasts can be reprogrammed to study developmental processes

  • Osteoblasts can demonstrate direct impact on bone formation

• CRISPR/Cas9-engineered cell lines:

  • Introduction of patient-specific mutations into relevant cell types

  • Skeletal precursor cells (osteoblasts, chondrocytes) are particularly valuable

Animal models:

• Mouse models:

  • Complete knockout models to study loss-of-function

  • Knock-in models with patient-specific mutations

  • Advantage: Mouse and human RSPRY1 have high sequence homology

  • Challenge: Phenotype may differ from human presentation

• Zebrafish models:

  • Rapid skeletal development and transparent embryos facilitate visualization

  • Morpholino knockdown or CRISPR targeting can efficiently disrupt gene function

Developmental assays:

• Micromass cultures:

  • Primary mesenchymal cells can form cartilage nodules in vitro

  • Allows study of early chondrogenesis and RSPRY1 role

• Organ culture systems:

  • Ex vivo culture of embryonic bones

  • Permits analysis of RSPRY1 function in intact developing skeletal elements

Importantly, strong RSPRY1 protein localization has been demonstrated in murine embryonic osteoblasts and periosteal cells during primary endochondral ossification, making these tissues particularly relevant for research focus .

What are the most promising approaches for studying RSPRY1's interaction with the FGF signaling pathway?

The potential interaction between RSPRY1 and FGF signaling presents a compelling research direction, with several methodological approaches showing promise:

Molecular interaction studies:

• Proximity ligation assays to detect RSPRY1-FGF receptor interactions in situ

• Surface plasmon resonance to quantify direct binding between purified RSPRY1 and FGF pathway components

• FRET/BRET analysis to detect proximity in living cells

Signaling pathway analysis:

• Phosphoproteomic profiling of RSPRY1-deficient cells with/without FGF stimulation

• Transcriptomic analysis to identify altered gene expression patterns in FGF-responsive genes

• Reporter assays using FGF-responsive elements to quantify pathway activity

Comparative studies with Sprouty proteins:

• Domain swap experiments between RSPRY1 and Sprouty proteins to determine functional conservation

• Rescue experiments in Sprouty-deficient models using RSPRY1

• Co-expression studies to identify potential redundancy or competition

This research direction is particularly compelling given that:

• Sprouty proteins (which also contain SPRY domains) are established inhibitors of FGF signaling

• Spry4 KO mice show dwarfism and digit abnormalities reminiscent of RSPRY1-associated phenotypes

• Spry2/Spry4 double KO mice exhibit severe craniofacial and limb development defects through enhanced FGF signaling

How can advanced protein structural analysis contribute to understanding RSPRY1 function?

Despite RSPRY1's important role in skeletal development, its three-dimensional structure remains unsolved, presenting both challenges and opportunities for structural biology approaches:

Current structural knowledge:

• RSPRY1 contains B.30/SPRY domain (aa 359-479) and C3HC4-type RING finger domain (aa 526-565)

• No solved three-dimensional structure or suitable modeling template is currently available (UniProt entry: Q96DX4)

• Critical residues like Cys551 in the RING domain appear functionally important based on pathogenic variants

Recommended structural analysis approaches:

• Cryo-electron microscopy (cryo-EM):

  • Particularly suitable for flexible proteins like RSPRY1

  • Can resolve domain organization without crystallization

  • May reveal conformational changes upon binding partners

• Solution NMR for domain characterization:

  • Individual domains (RING, SPRY) could be expressed and analyzed separately

  • Allows dynamic studies of domain interactions with partners

• Cross-linking mass spectrometry:

  • Can identify spatial relationships between domains

  • Helpful for proteins resistant to crystallization

• Molecular dynamics simulations:

  • Predict impact of disease-causing mutations on protein stability

  • Model conformational changes in different cellular environments

• AlphaFold2 or RoseTTAFold predictions:

  • Deep learning approaches show promise for predicting structures of proteins lacking templates

  • Can be validated with limited experimental data

Understanding RSPRY1's structure would significantly advance our ability to:

• Predict functional consequences of clinical mutations

• Design targeted therapeutics

• Identify potential binding partners

• Clarify its role in signaling pathways

What are the most significant knowledge gaps in RSPRY1 research that present opportunities for breakthrough discoveries?

Despite recent advances in understanding RSPRY1, several critical knowledge gaps remain:

• Molecular function:

  • The precise biochemical activity of RSPRY1 remains undefined

  • Whether it functions primarily as an E3 ubiquitin ligase, scaffold protein, or through other mechanisms is unknown

• Developmental regulation:

  • Temporal and spatial regulation of RSPRY1 expression during skeletal development is incompletely characterized

  • Upstream regulators of RSPRY1 expression remain to be identified

• Pathway integration:

  • How RSPRY1 interfaces with established skeletal development pathways (FGF, BMP, Wnt) needs clarification

  • Potential role in non-skeletal tissues where it's expressed (heart, muscle) is unexplored

• Structure-function relationships:

  • How the RING and SPRY domains cooperate functionally is undefined

  • Protein interaction network and binding partners remain largely unknown

• Genotype-phenotype correlations:

  • Why certain mutations cause specific clinical features

  • Whether different mutation types result in varied disease severity