Recombinant Mouse ADAMTS-like protein 4 (Adamtsl4), partial

What is the structure and function of ADAMTS-like protein 4 in mice?

ADAMTS-like protein 4 (Adamtsl4) belongs to the ADAMTS-like protein family, which lacks the catalytic metalloproteinase domain present in ADAMTS proteins. Structurally, Adamtsl4 contains a spacer module and thrombospondin type 1 motifs. Functionally, Adamtsl4 plays a crucial role in fibrillin microfibril biogenesis and is necessary for stable anchorage of zonule fibers to the lens capsule, which is essential for proper lens positioning within the eye . Mutations in Adamtsl4 lead to ectopia lentis (lens displacement) and can affect the retinal pigment epithelium (RPE), suggesting its importance in ocular tissue development and maintenance .

How is Adamtsl4 gene expression regulated in different ocular tissues?

Adamtsl4 expression has been detected in multiple ocular tissues, with significant expression in the lens and RPE. Quantitative real-time PCR studies in mouse models with Adamtsl4 mutations have shown that nonsense mutations can lead to a 3.0-3.5-fold decrease in Adamtsl4 mRNA expression in lens and RPE-enriched RNA samples, suggesting that transcripts with premature stop codons undergo nonsense-mediated mRNA decay . The specific regulatory mechanisms controlling tissue-specific expression of Adamtsl4 during development remain an area of ongoing research, but its expression patterns correlate with its functional importance in zonular fiber development and RPE maintenance.

What phenotypes are associated with Adamtsl4 mutations in mouse models?

Mouse models with Adamtsl4 mutations, such as the tvrm267 model, demonstrate two primary phenotypes:

• Ectopia lentis (EL): Characterized by displacement of the crystalline lens due to weakened or broken zonular fibers that normally anchor the lens to the ciliary body.

• RPE defects: Notably, homozygous Adamtsl4 mutant mice exhibit focal RPE defects primarily in the inferior eye, termed the "splatter" phenotype. These RPE cells show reduced pigmentation, altered cellular morphology, and decreased expression of RPE-specific transcripts .

Additionally, some Adamtsl4 mutant mice demonstrate increased axial length compared to age-matched controls, correlating with the severity of the RPE phenotype .

How does Adamtsl4 contribute to zonular fiber development and stability at the molecular level?

Adamtsl4 is essential for the development and stability of zonular fibers, which anchor the lens to the ciliary body. Research using the Adamtsl4 tvrm267 mouse model suggests that Adamtsl4 specifically mediates the attachment of zonular fibers to the lens capsule rather than affecting fiber formation itself . At the molecular level, Adamtsl4 likely interacts with fibrillin microfibrils, which are major components of zonular fibers. The protein appears to function in organizing or stabilizing these microfibrils at their attachment points on the lens capsule. When Adamtsl4 is mutated or absent, the zonular fibers can still form but fail to maintain stable connections to the lens, resulting in progressive weakening and eventual breakage of these anchoring structures, leading to lens displacement (ectopia lentis) . Future research using recombinant Adamtsl4 could elucidate the specific protein-protein interactions mediating these attachments.

What are the cellular and molecular mechanisms behind the RPE defects observed in Adamtsl4-deficient mice?

The mechanisms underlying RPE defects in Adamtsl4-deficient mice involve cellular dedifferentiation and structural disorganization. Ultrastructural analysis of RPE cells in Adamtsl4 tvrm267 mice reveals:

• Disorganization along the apical border with areas lacking apical processes

• Loss of basal infoldings typically organized adjacent to Bruch's membrane

• Decreased pigmentation and loss of apical F-actin

• Reduction of apical ezrin expression

These changes collectively suggest cellular reorganization consistent with RPE dedifferentiation. The molecular pathway connecting Adamtsl4 deficiency to these changes may involve alterations in cell-extracellular matrix interactions, as Adamtsl4 is implicated in extracellular matrix organization. The regional specificity of RPE defects (primarily in the inferior eye) indicates potential interactions with other spatially regulated factors, which remain to be identified through further research using recombinant Adamtsl4 in rescue experiments.

How do homozygous versus heterozygous Adamtsl4 mutations affect phenotype severity and progression?

Research using mouse models demonstrates that the inheritance pattern and zygosity of Adamtsl4 mutations significantly impact phenotype expression. In homozygous Adamtsl4 tvrm267 mice, both ectopia lentis and RPE defects are observed, with complete penetrance but variable expressivity of the RPE phenotype . The ectopia lentis phenotype progressively worsens with age, suggesting time-dependent weakening of zonular attachments.

Heterozygous carriers typically show milder or absent phenotypes, consistent with the autosomal recessive inheritance pattern observed in human ADAMTSL4-related eye disorders . This dose-dependent effect suggests that a threshold level of functional Adamtsl4 is necessary for normal ocular development and maintenance. Quantitative studies using recombinant Adamtsl4 could help establish this threshold and potentially inform therapeutic approaches.

What are the optimal methods for generating and purifying recombinant mouse Adamtsl4 for functional studies?

For generating functional recombinant mouse Adamtsl4:

• Expression System Selection:

  • Mammalian expression systems (HEK293 or CHO cells) are preferred over bacterial systems due to the requirement for proper post-translational modifications, particularly glycosylation patterns that may affect protein folding and function.

  • Use expression vectors containing strong promoters (CMV) and appropriate secretion signals to enhance yield.

• Construct Design:

  • Include a C-terminal tag (His6 or FLAG) for purification, positioned to minimize interference with functional domains.

  • Consider generating both full-length protein and domain-specific constructs to assess domain-specific functions.

  • For partial Adamtsl4 constructs, prioritize regions containing the thrombospondin type 1 repeats and the spacer domain, which are crucial for function .

• Purification Strategy:

  • Two-step purification using affinity chromatography followed by size exclusion chromatography.

  • Validate protein integrity using western blotting with domain-specific antibodies.

  • Assess proper folding using circular dichroism spectroscopy.

• Functional Validation:

  • Binding assays with potential interacting partners (e.g., fibrillin-1).

  • Cell adhesion assays using lens epithelial cells or RPE cells.

Proper storage in small aliquots at -80°C with cryoprotectants (10% glycerol) is recommended to maintain protein activity for longer periods.

What experimental approaches are most effective for studying Adamtsl4 interactions with extracellular matrix components?

Several complementary approaches are recommended for investigating Adamtsl4-ECM interactions:

• In vitro Binding Assays:

  • Solid-phase binding assays using purified recombinant Adamtsl4 and candidate ECM proteins (particularly fibrillin-1).

  • Surface plasmon resonance to determine binding kinetics and affinity constants.

  • Co-immunoprecipitation with tagged proteins to identify novel binding partners.

• Cell-Based Assays:

  • Cell adhesion assays using Adamtsl4-coated surfaces.

  • Immunofluorescence co-localization studies in relevant cell types (lens epithelial cells, RPE cells).

  • Proximity ligation assays to confirm protein-protein interactions in situ.

• Ex vivo Approaches:

  • Explant cultures of lens capsules or RPE sheets treated with recombinant Adamtsl4.

  • Immunohistochemistry on tissue sections from wild-type and Adamtsl4-mutant mice.

  • Atomic force microscopy to assess mechanical properties of ECM in the presence/absence of Adamtsl4.

• Advanced Imaging Techniques:

  • Super-resolution microscopy to visualize Adamtsl4 distribution within microfibrils.

  • Electron microscopy with immunogold labeling to precisely localize Adamtsl4 at zonular attachment sites.

These methodologies should be tailored based on the specific research question and combined for comprehensive characterization of Adamtsl4-ECM interactions.

How can researchers effectively quantify Adamtsl4 expression and distribution in ocular tissues?

For accurate quantification and localization of Adamtsl4 in ocular tissues:

• Transcript Quantification:

  • qRT-PCR with well-validated primers spanning exon-exon junctions to avoid genomic DNA amplification.

  • RNA-seq for comprehensive transcriptomic analysis and detection of alternative splicing.

  • In situ hybridization for spatial distribution within intact tissues.

• Protein Quantification:

  • Western blotting with validated antibodies for total protein quantification.

  • ELISA development using recombinant Adamtsl4 standards for precise quantification.

  • Mass spectrometry-based proteomics for absolute quantification and identification of post-translational modifications.

• Spatial Distribution Analysis:

  • Immunohistochemistry on paraffin or frozen sections with careful validation of antibody specificity.

  • Confocal microscopy with z-stack analysis for three-dimensional distribution.

  • Laser capture microdissection combined with qRT-PCR or proteomics for region-specific analysis.

• Developmental Studies:

  • Temporal expression analysis during key developmental windows.

  • Reporter gene constructs (e.g., Adamtsl4-GFP) for live imaging in model systems.

When analyzing data, researchers should normalize expression to appropriate reference genes or proteins that show stable expression in the tissues and conditions being studied. For spatial distribution studies, quantitative image analysis using software like ImageJ or CellProfiler is recommended for objective assessment .

What are the key differences between mouse and human ADAMTSL4 that researchers should consider when designing translational studies?

When designing translational studies, researchers should account for several important differences between mouse Adamtsl4 and human ADAMTSL4:

• Sequence Homology:

  • While mouse and human ADAMTSL4 share significant homology, there are structural differences that may influence protein-protein interactions and function.

  • Critical functional domains show higher conservation than spacer regions.

• Expression Patterns:

  • Subtle differences exist in tissue-specific expression patterns between species.

  • Whether ADAMTSL4 is expressed in the neuroretina remains unclear due to conflicting results in immunohistochemical studies .

• Mutation Effects:

  • The tvrm267 mouse model harbors a nonsense mutation (p.Gln609*) located near the C-terminal end of the spacer module, which differs from the distribution of known human mutations throughout the protein .

  • The European founder variant (c.767_786del) commonly found in human patients has different molecular consequences than the mouse tvrm267 mutation .

• Phenotypic Spectrum:

  • Humans with ADAMTSL4 mutations typically present with isolated ectopia lentis or ectopia lentis et pupillae .

  • The mouse model additionally shows RPE defects, which are not commonly reported in human patients .

• Genetic Background Effects:

  • The phenotypic expression of Adamtsl4 mutations in mice may be influenced by genetic background, similar to other ocular phenotypes.

How can Adamtsl4 mouse models be used to develop therapeutic approaches for ADAMTSL4-related eye disorders?

The Adamtsl4 tvrm267 mouse model provides valuable opportunities for developing therapeutic approaches for ADAMTSL4-related eye disorders:

• Gene Therapy Development:

  • AAV-mediated gene delivery to specific ocular tissues (lens epithelium, ciliary body, RPE).

  • CRISPR/Cas9-based gene editing to correct pathogenic mutations.

  • Evaluate therapeutic window during development for maximum efficacy.

• Protein Replacement Therapy:

  • Recombinant Adamtsl4 administration to ocular tissues through various delivery methods.

  • Engineered protein variants with enhanced stability or function.

  • Assess dose-response relationships and treatment frequency requirements.

• Pharmacological Interventions:

  • Small molecules that can stabilize zonular fibers or prevent their degradation.

  • Compounds that can suppress nonsense-mediated mRNA decay for nonsense mutations.

  • Drugs targeting downstream effects (e.g., RPE dedifferentiation).

• Surgical Innovations:

  • Development and refinement of lens stabilization techniques.

  • Testing novel materials that can mimic zonular fiber function.

• Therapeutic Evaluation Metrics:

  • Quantitative assessment of lens stability through imaging techniques.

  • RPE morphology and function evaluation using electroretinography and fundus imaging.

  • Cellular and molecular markers of treatment efficacy.

The mouse model allows longitudinal studies to determine optimal intervention timing, as treatment before zonular fiber degradation may prevent ectopia lentis, while later intervention might only halt progression .

What is the current understanding of Adamtsl4's role in cancer research and potential therapeutic applications?

Recent research has revealed emerging roles for ADAMTSL family proteins, including Adamtsl4, in cancer biology:

While this represents an emerging area of research, the connections between Adamtsl4's known functions in ECM organization and its potential roles in cancer biology warrant further investigation using recombinant proteins and genetic models.

What methods are most appropriate for classifying and analyzing ADAMTSL4 genetic variants in research settings?

For effective classification and analysis of ADAMTSL4 variants:

• Variant Identification and Annotation:

  • Next-generation sequencing targeting the ADAMTSL4 gene or as part of whole-exome/genome sequencing.

  • Proper annotation against canonical transcripts (NM_019032 for DNA and NP_061905 for protein) .

  • Use of standardized nomenclature following HGVS guidelines.

• Variant Classification Methodology:

  • Loss of function (LoF) variants should be clearly defined as those that introduce premature stop codons (nonsense), shift the transcriptional reading frame (frameshift), or alter essential splice-site nucleotides .

  • Non-LoF variants, particularly missense variants, require careful functional assessment.

  • Apply ACMG/AMP guidelines for variant interpretation in research contexts.

• Functional Assessment Approaches:

  • mRNA stability assays to assess nonsense-mediated decay.

  • Recombinant protein expression to evaluate variant effects on protein stability and function.

  • Cell-based assays measuring ECM interactions and microfibril formation.

• Population Frequency Analysis:

  • Comparison with population databases (gnomAD, 1000 Genomes) to assess variant rarity.

  • Consideration of population-specific founder variants, such as the European c.767_786del frameshift variant .

• Genotype-Phenotype Correlation:

  • Systematic documentation of clinical phenotypes associated with specific variants.

  • Statistical approaches to identify associations between variant types and phenotypic features.

Researchers should maintain awareness that certain ADAMTSL4 mutations may have variable expressivity, and thorough phenotyping is essential for accurate genotype-phenotype correlation studies .

How can researchers establish reliable genotype-phenotype correlations in ADAMTSL4-related disorders?

Establishing reliable genotype-phenotype correlations requires systematic approaches:

• Standardized Phenotyping Protocol:

  • Comprehensive ocular examination including assessment of lens position, anterior chamber depth, and iris configuration.

  • Fundus imaging to detect potential RPE abnormalities similar to those observed in mouse models .

  • Optical coherence tomography to assess retinal structure.

  • Biometric measurements including axial length, which has been associated with ADAMTSL4 mutations in both humans and mice .

• Molecular Classification:

  • Categorize variants based on molecular consequences (complete loss of function vs. partial function).

  • Consider the specific protein domains affected by mutations.

  • Assess potential founder effects and population-specific variants .

• Statistical Approaches:

  • Multivariate analysis to control for confounding factors like age and environmental influences.

  • Machine learning algorithms to identify subtle phenotypic patterns associated with specific genotypes.

  • Meta-analysis of published cases to increase statistical power.

• Longitudinal Studies:

  • Track phenotypic progression over time to identify age-dependent effects.

  • Document interventions and their outcomes to assess genotype-specific responses to treatment.

• Mouse Model Validation:

  • Generate mouse models with human-specific mutations using CRISPR/Cas9 technology.

  • Compare phenotypes between human patients and corresponding mouse models.

  • Use recombinant wildtype and mutant proteins to assess functional differences in vitro.

This multifaceted approach can help establish whether specific ADAMTSL4 variants predict particular clinical features, progression rates, or complications, ultimately informing personalized management strategies .

What are the critical controls needed when designing experiments with recombinant Adamtsl4?

When designing experiments with recombinant Adamtsl4, include these essential controls:

• Protein Quality Controls:

  • Denatured protein control to confirm structure-dependent functions.

  • Tagged protein control without Adamtsl4 sequence to exclude tag-specific effects.

  • Concentration-matched non-related protein control (e.g., BSA) to control for non-specific protein effects.

  • Both full-length and domain-specific constructs to map functional regions.

• Genetic Controls:

  • Wild-type cells/tissues alongside Adamtsl4-null models.

  • Heterozygous models to assess dose-dependent effects.

  • Rescue experiments using recombinant protein in Adamtsl4-deficient models.

  • Species-appropriate controls when comparing mouse and human proteins.

• Experimental Condition Controls:

  • Time-course experiments to capture dynamic processes.

  • Tissue/cell type-specific controls to account for context-dependent functions.

  • Environmental condition variations (pH, temperature, ion concentrations) that might affect protein function.

  • Parallel experiments with known ADAMTS family members to distinguish family-wide vs. Adamtsl4-specific effects.

• Analytical Controls:

  • Quantification using multiple methodologies (e.g., both protein abundance and activity measurements).

  • Technical replicates to assess experimental variability.

  • Biological replicates to account for individual variation.

  • Blinded analysis to prevent observer bias.

These controls help ensure experimental rigor and facilitate interpretation of results, particularly important given the complex roles of Adamtsl4 in different ocular tissues .

How should researchers account for potential functional differences between recombinant and native Adamtsl4 in their experimental design?

Researchers must consider several key differences between recombinant and native Adamtsl4:

• Post-translational Modifications:

  • Native Adamtsl4 undergoes specific glycosylation patterns that may not be fully replicated in recombinant systems.

  • Characterize glycosylation profiles of recombinant proteins using mass spectrometry and compare to native protein when possible.

  • Consider using tissue-specific cell lines for recombinant production to better match the native cellular environment.

• Structural Considerations:

  • Recombinant tags may affect protein folding or function, necessitating comparison of N-terminal, C-terminal, and untagged versions.

  • Partial recombinant constructs lack context of the full protein structure, potentially altering functional properties.

  • Analytical ultracentrifugation or size exclusion chromatography with multi-angle light scattering can assess proper oligomerization states.

• Experimental Design Adjustments:

  • Include tissue extracts containing native Adamtsl4 as positive controls when possible.

  • Perform parallel experiments with different expression systems to identify system-specific artifacts.

  • Consider using knockin approaches that maintain native expression regulation rather than recombinant overexpression.

  • Validate key findings from recombinant studies in physiological contexts using genetic models.

• Quantitative Considerations:

  • Titrate recombinant protein concentrations to identify physiologically relevant ranges.

  • Compare activity per mole of protein rather than mass concentration when assessing functional differences.

  • Account for potential differences in protein stability and half-life in experimental timelines.

By systematically addressing these considerations, researchers can maximize the translational relevance of findings obtained using recombinant Adamtsl4 proteins .

How might Adamtsl4 research inform our understanding of other extracellular matrix-related disorders?

Adamtsl4 research offers valuable insights into broader ECM biology:

• Mechanistic Parallels:

  • The role of Adamtsl4 in microfibril organization provides a model for understanding other ECM assembly disorders.

  • Research on how Adamtsl4 mutations affect zonular fiber stability may inform mechanisms in other fibrillinopathies like Marfan syndrome.

  • The tissue-specific effects of Adamtsl4 deficiency (lens zonules vs. RPE) illustrate how context influences ECM protein function .

• Methodological Applications:

  • Techniques developed to study Adamtsl4-fibrillin interactions can be applied to other ECM protein complexes.

  • Imaging approaches used to visualize microfibril disruption in Adamtsl4 models can be extended to other ECM disorders.

  • Quantitative assays measuring ECM biomechanical properties in Adamtsl4-deficient tissues provide templates for similar studies.

• Therapeutic Implications:

  • Successful approaches for restoring Adamtsl4 function might inspire similar strategies for other ECM proteins.

  • Understanding tissue-specific requirements for Adamtsl4 may inform targeted delivery approaches for other ECM therapeutics.

  • Insights into cellular responses to ECM defects (e.g., RPE dedifferentiation) may be relevant to multiple disorders .

• Developmental Biology Connections:

  • The role of Adamtsl4 in ocular development illuminates general principles about how ECM proteins guide tissue morphogenesis.

  • Temporal aspects of Adamtsl4 function may parallel critical developmental windows in other ECM-dependent processes.

This cross-pollination between Adamtsl4 research and broader ECM biology accelerates progress in understanding and treating a wide range of connective tissue disorders.

What emerging technologies and approaches show promise for advancing Adamtsl4 research?

Several cutting-edge technologies are poised to transform Adamtsl4 research:

• Advanced Imaging Technologies:

  • Super-resolution microscopy (STORM, PALM) to visualize Adamtsl4 distribution within microfibrils at nanometer resolution.

  • Live-cell imaging with tagged Adamtsl4 to track dynamic interactions during fibril assembly.

  • Correlative light and electron microscopy (CLEM) to link protein localization with ultrastructural features.

  • Expansion microscopy to physically enlarge specimens for improved visualization of Adamtsl4-associated structures.

• Single-cell Technologies:

  • Single-cell RNA-seq to identify cell populations expressing Adamtsl4 and map expression heterogeneity.

  • Single-cell proteomics to correlate Adamtsl4 protein levels with cellular phenotypes.

  • Spatial transcriptomics to map Adamtsl4 expression patterns within intact tissues.

• Genome Editing Advances:

  • Base editing and prime editing for precise correction of Adamtsl4 mutations.

  • CRISPR activation/interference systems to modulate endogenous Adamtsl4 expression.

  • Conditional knockout models with tissue-specific and temporal control.

• Biomaterial and Organoid Applications:

  • 3D bioprinting incorporating Adamtsl4 to generate biomimetic ECM environments.

  • Organoid models of lens and RPE development to study Adamtsl4 function in simplified but physiologically relevant systems.

  • Microfluidic organ-on-chip platforms to study Adamtsl4 function under mechanical stress.

• Computational Approaches:

  • Molecular dynamics simulations of Adamtsl4-fibrillin interactions.

  • Machine learning algorithms to predict functional consequences of Adamtsl4 variants.

  • Systems biology models integrating Adamtsl4 into broader ECM interaction networks.

These technologies will enable researchers to address previously intractable questions about Adamtsl4 function and potentially accelerate therapeutic development for ADAMTSL4-related disorders .

What are the most promising therapeutic strategies targeting Adamtsl4 for ocular disorders?

Several therapeutic strategies show particular promise for ADAMTSL4-related disorders:

• Gene Therapy Approaches:

  • AAV-mediated gene replacement therapy targeting lens epithelial cells and ciliary body epithelium.

  • Advantages: One-time treatment with potential long-term expression; can be delivered before significant zonular damage occurs.

  • Current challenges: Optimal serotype selection for targeting relevant tissues; preventing immune responses; determining ideal intervention timing.

• mRNA Therapeutics:

  • Delivering synthetic Adamtsl4 mRNA to affected tissues for transient protein expression.

  • Advantages: Avoids genomic integration concerns; dosage can be adjusted as needed; potentially repeatable.

  • Current challenges: Delivery to target tissues; maintaining stability; achieving sustained therapeutic levels.

• Protein Replacement Therapy:

  • Direct administration of recombinant Adamtsl4 protein to the eye.

  • Advantages: Avoids genetic manipulation; dose can be precisely controlled; potentially applicable to all mutation types.

  • Current challenges: Protein production at scale; delivery method optimization; determining treatment frequency.

• Small Molecule Approaches:

  • Compounds that can suppress nonsense-mediated decay for patients with nonsense mutations.

  • Advantages: Oral administration possibility; potential for broad application across different mutations.

  • Current challenges: Specificity; potential off-target effects; efficacy in ocular tissues.

• Combinatorial Approaches:

  • Early gene therapy followed by protein supplementation as needed.

  • Advantages: Addresses both immediate needs and long-term maintenance; personalized based on mutation type and disease stage.

  • Current challenges: Determining optimal combination and sequence; regulatory complexity.