Recombinant Mouse ADAMTS-like protein 1 (Adamtsl1), partial

What is the structural composition of ADAMTSL1 and how does it compare to other ADAMTS family proteins?

ADAMTSL1 contains thirteen thrombospondin type 1 repeats (TSRs), four Immunoglobulin-like C2-type domains and a single PLAC (protease and lacunin) domain in its full-length form (1762 amino acids). It also exists as a short splice variant named punctin-1 (525 amino acids) containing only four TSRs. Unlike the prototypic ADAMTS proteins, ADAMTSL1 lacks the metalloproteinase and disintegrin domains but maintains the characteristic TSRs, which have a highly conserved three-layered fold stabilized by three disulfide bonds and stacked side chains of Trp and Arg residues near the N-terminus . These TSRs undergo two unusual post-translational modifications that can regulate secretion: C-mannosylation and O-fucosylation .

What are the current known biological functions of ADAMTSL1 in normal physiology?

ADAMTSL1 has been demonstrated to contribute to skeletal muscle homeostasis primarily through modulation of TGF-β bioavailability . High expression levels of ADAMTSL1 are detected in skeletal muscle tissue, suggesting tissue-specific functionality . Like other ADAMTSL family members (specifically ADAMTSL2, 3, and 6), ADAMTSL1 appears to be part of the extracellular matrix complex that regulates TGF-β bioavailability . This role in TGF-β regulation connects ADAMTSL1 to broader processes of tissue regeneration, extracellular matrix organization, and potentially organ development.

How can researchers effectively create and validate ADAMTSL1 knockout mouse models?

Researchers can develop ADAMTSL1 knockout models through constitutive excision of exon 1, as demonstrated in existing models . Validation of the knockout should include:

• Genotyping using specific primers to confirm deletion of the target exon

• RT-PCR and Western blot analyses to confirm absence of protein expression

• Evaluation of expected phenotypes, including age-related changes in muscle architecture

When using this model, researchers should be aware that ADAMTSL1+/- and ADAMTSL1-/- mice are born at expected Mendelian frequencies and appear healthy and fertile initially, with phenotypic manifestations becoming more apparent with age . Specifically, researchers should monitor for progressive muscle weakening, which becomes evident at approximately 12 months of age and can be observed as a typical hunchback posture in one-year-old knockout animals .

What methodological approaches are optimal for studying ADAMTSL1 function in muscle regeneration models?

To study ADAMTSL1's role in muscle regeneration, researchers should consider employing cardiotoxin (CTX) injection models in both wild-type and ADAMTSL1 knockout mice . The recommended protocol includes:

• Intramuscular injection of cardiotoxin in tibialis anterior or gastrocnemius muscles

• Tissue collection at multiple timepoints (5, 10, and 21 days post-injury)

• Histological analysis focusing on:

  • Fiber regeneration (H&E staining)

  • Lipid accumulation (Oil Red O staining)

  • Fibrosis development (Masson's trichrome staining)

Researchers should complement these in vivo approaches with in vitro analysis of satellite cells isolated from both wild-type and knockout animals to distinguish between intrinsic satellite cell defects and alterations in the regenerative microenvironment . ADAMTSL1 knockout models show defects in muscle regeneration characterized by lipid droplet accumulation, while isolated satellite cells maintain their ability to differentiate and fuse in vitro, suggesting that ADAMTSL1's primary role involves the regulation of the regenerative niche rather than direct effects on satellite cell function .

How can researchers accurately measure ADAMTSL1's impact on TGF-β signaling pathway activity?

Researchers investigating ADAMTSL1's effect on TGF-β signaling should employ multiple complementary approaches:

• Pathway activity assessment:

  • RNA-seq analysis of TGF-β target genes, specifically examining whether expression of canonical targets is altered in ADAMTSL1 knockout tissues

  • Quantification of phosphorylated SMAD2/3 levels via Western blot

  • Immunohistochemical analysis of nuclear SMAD localization

• TGF-β bioavailability measurement:

  • ELISA quantification of active versus latent TGF-β in tissue samples

  • Co-immunoprecipitation of ADAMTSL1 with known TGF-β sequestration complex members

• Validation in human samples:

  • Implementation of TGF-β pathway activity scoring in human samples with varying levels of ADAMTSL1 expression

Research has demonstrated that approximately 18% of major TGF-β pathway target genes are significantly altered in muscles from ADAMTSL1 knockout mice compared to wild-type mice . Additionally, TGF-β pathway activity strongly correlates with ADAMTSL1 expression in human muscle samples, particularly in mild dystrophy patients .

What experimental design best elucidates the molecular mechanisms by which ADAMTSL1 regulates TGF-β bioavailability?

To investigate the molecular mechanisms underlying ADAMTSL1's regulation of TGF-β:

• Protein interaction studies:

  • Perform proximity ligation assays to confirm in situ interactions between ADAMTSL1 and TGF-β complex components

  • Use domain deletion constructs to identify specific ADAMTSL1 regions responsible for TGF-β binding

• Extracellular matrix organization:

  • Implement second harmonic generation microscopy to assess collagen organization

  • Quantify ECM protein composition in wild-type versus knockout tissue using mass spectrometry

• Cell-specific contributions:

  • Use conditional knockout models to delete ADAMTSL1 from specific cell populations (myoblasts versus fibro-adipogenic progenitors)

  • Implement parabiosis or tissue grafting experiments to determine if circulating ADAMTSL1 can rescue local deficiencies

Evidence suggests that ADAMTSL1 functions similarly to other ADAMTSL family members (ADAMTSL2, 3, and 6) as part of the ECM complex regulating TGF-β bioavailability . This regulation appears to be particularly important in contexts of tissue regeneration and pathological fibrosis.

How does ADAMTSL1 expression relate to rhabdomyosarcoma progression and prognosis?

ADAMTSL1 expression is significantly lower in rhabdomyosarcoma (RMS) samples compared to normal skeletal muscle tissues across multiple independent cohorts . Research should focus on:

• Expression analysis:

  • Quantify ADAMTSL1 expression levels in different RMS subtypes (embryonal versus alveolar)

  • Correlate expression with clinical outcomes and treatment response

• Functional studies:

  • Implement ADAMTSL1 overexpression in RMS cell lines to assess effects on:

    • Proliferation and apoptosis

    • Myogenic differentiation capacity

    • TGF-β pathway activity

    • Invasive and metastatic properties

• Prognostic value assessment:

  • Develop standardized scoring systems incorporating ADAMTSL1 expression

  • Validate in prospective clinical cohorts

ADAMTSL1 expression behaves as a strong prognostic factor particularly in the aggressive fusion-positive RMS and correlates with a neural-like phenotype of tumor cells resulting from gain of SMAD2/3/4 targets . This suggests that ADAMTSL1 may have potential as a biomarker and therapeutic target in certain RMS subtypes.

What is the relationship between ADAMTSL1 variants and developmental disorders?

ADAMTSL1 variants have been identified in families affected by developmental disorders including:

• Ocular abnormalities:

  • Developmental glaucoma

  • Myopia

  • Retinal defects

• Associated conditions:

  • Craniofacial and dental anomalies

  • Auditory deficits

  • Brain abnormalities

  • Renal defects

  • Limb anomalies

Research has identified a heterozygous c.124T>C, p.(Trp42Arg) variant in a three-generation family with these conditions . This mutation affects a tryptophan residue in the conserved TSR domain, potentially disrupting the structural stability of the protein.

The functional consequences of this variant should be investigated through:

• Expression studies to assess protein stability and secretion

• Structural analysis to determine effects on TSR folding

• Functional assays measuring interactions with TGF-β pathway components

• Animal models replicating the specific variant

How can researchers reconcile the seemingly contradictory roles of ADAMTSL1 in development, regeneration, and disease contexts?

The multifaceted functions of ADAMTSL1 across different biological contexts present a significant challenge for researchers. To reconcile these apparent contradictions:

• Tissue-specific function analysis:

  • Implement tissue-specific conditional knockout models

  • Compare transcriptional responses to ADAMTSL1 deficiency across tissues

  • Identify tissue-specific interaction partners using proximity-dependent biotinylation

• Developmental stage considerations:

  • Create inducible knockout models to delete ADAMTSL1 at different developmental timepoints

  • Compare acute versus chronic loss of function

• Context-dependent activity:

  • Examine ADAMTSL1 function under homeostatic versus injury/stress conditions

  • Evaluate potential compensatory mechanisms in different contexts

• Dose-dependent effects:

  • Create hypomorphic alleles to study partial loss of function

  • Implement overexpression models to identify gain-of-function phenotypes

ADAMTSL1 appears to function as part of the TGF-β regulatory network, which has well-documented context-dependent effects. In muscle regeneration, ADAMTSL1 positively regulates TGF-β bioavailability , while its reduced expression in RMS correlates with disease progression, suggesting potential tumor suppressor functions in specific contexts .

What are the most effective approaches for analyzing post-translational modifications of recombinant ADAMTSL1?

Analyzing post-translational modifications of recombinant ADAMTSL1 requires sophisticated techniques:

• Glycosylation analysis:

  • Mass spectrometry to identify C-mannosylation and O-fucosylation sites

  • Enzymatic deglycosylation assays to assess functional consequences

  • Site-directed mutagenesis of predicted modification sites

• Expression systems selection:

  • Compare mammalian, insect, and cell-free expression systems

  • Assess differences in modification patterns between systems

  • Validate against native protein isolated from tissues

• Functional consequences:

  • Stability assays comparing modified versus unmodified protein

  • Binding assays to determine effects on interactions with ECM components

  • Cellular assays measuring secretion efficiency and extracellular retention

TSRs in ADAMTSL1 undergo C-mannosylation and O-fucosylation, which regulate protein secretion and function . These modifications are particularly important to consider when producing recombinant protein for experimental use, as improper modification can significantly impact functionality.

What are the most promising future research directions for ADAMTSL1?

Based on current knowledge, the most promising research directions include:

• Therapeutic applications:

  • Development of ADAMTSL1-based therapies for muscle degenerative disorders

  • Exploration of ADAMTSL1 as a biomarker in RMS diagnosis and prognosis

  • Investigation of ADAMTSL1 supplementation for conditions associated with fibrosis

• Molecular mechanisms:

  • Identification of specific ADAMTSL1 binding partners in the ECM

  • Elucidation of the precise mechanisms by which ADAMTSL1 regulates TGF-β availability

  • Characterization of the functional differences between ADAMTSL1 splice variants

• Translational research:

  • Establishment of correlations between ADAMTSL1 variants and human pathologies

  • Development of screening methods for ADAMTSL1-related developmental disorders

  • Investigation of ADAMTSL1's role in age-related muscle weakness