Recombinant Human Isthmin-2 (ISM2)

What is the basic structure of human ISM2 protein?

Human Isthmin-2 (ISM2) is a secreted protein of approximately 63.9 kDa with several characteristic domains. The protein contains a hydrophobic signal peptide at the N-terminus, a centrally positioned thrombospondin type 1 repeat (TSR1) domain, and a C-terminal adhesin-associated domain in MUC4 and other proteins (AMOP) domain . The full-length mature protein spans amino acids 27-571, resulting in a molecular weight of approximately 68.6 kDa when expressed with tags . The protein's structure is evolutionarily conserved, suggesting important biological functions.

What are the known sequence motifs in ISM2 and their potential functions?

ISM2 contains several important sequence motifs with distinct functions:

• WSPW motif in the TSR1 domain - This motif is known to mediate inhibition of angiogenesis in endothelial cells and anti-proliferative activity .

• WSRL motif in the AMOP domain - This sequence has been implicated in autophagy induction, suggesting a potential role for ISM2 in cellular degradation and recycling processes .

Unlike ISM1 which contains a CSVTCG motif that facilitates CD36 interaction and a DGE motif that serves as an α2β1 ligand sequence, ISM2 has distinct motifs that may confer unique functional properties to the protein .

How does ISM2 compare to ISM1 in terms of structure and function?

While both ISM1 and ISM2 share similar domain architecture (signal peptide, TSR1 domain, and AMOP domain), they differ in several key aspects:

The most notable difference is in their TSR1 domains - ISM1 contains a WSLW motif while ISM2 has a WSPW motif, which may result in different functional properties despite both potentially having roles in angiogenesis regulation .

What expression systems are most effective for producing functional recombinant human ISM2?

E. coli expression systems have been successfully used to produce recombinant human ISM2 protein . The resulting protein exhibits greater than 85% purity as determined by SDS-PAGE analysis . When designing expression constructs, researchers should consider:

• Including the full-length mature protein (amino acids 27-571) to ensure complete functional domains

• Appropriate tags for purification and detection (e.g., N-terminal His tag and C-terminal Myc tag)

• Codon optimization for the chosen expression system to enhance protein yield

For functional studies, it's important to note that post-translational modifications may affect protein activity. While E. coli can produce high yields of protein, it lacks the machinery for certain post-translational modifications found in mammalian cells. For studies requiring these modifications, mammalian or insect cell expression systems may be more appropriate.

What purification strategies optimize yield and purity of recombinant ISM2?

For His-tagged recombinant human ISM2, a multi-step purification strategy is recommended:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin as the primary capture step

• Ion exchange chromatography as an intermediate purification step

• Size exclusion chromatography for final polishing and buffer exchange

This approach typically yields protein with greater than 85% purity as assessed by SDS-PAGE . For enhanced purity (>95%), additional chromatography steps may be necessary. The choice between liquid and lyophilized forms depends on the intended application - lyophilized forms offer greater stability for long-term storage but may require optimization of reconstitution conditions.

What buffer conditions are optimal for maintaining ISM2 stability?

Recombinant human ISM2 stability is influenced by buffer composition, pH, and additives. Based on available data, the following conditions are recommended :

For liquid formulations:

• Tris/PBS-based buffer

• 5-50% glycerol as a stabilizing agent

• pH 7.4-8.0

• Storage at -80°C for long-term or -20°C for medium-term use

For lyophilized formulations:

• Pre-lyophilization buffer: Tris/PBS-based buffer with 6% Trehalose

• pH 8.0

• Storage at -20°C with desiccant

• Reconstitution by brief centrifugation prior to opening, followed by careful addition of sterile water or buffer

What is the significance of the TSR1 domain in ISM2 function?

The TSR1 domain in ISM2 is of particular interest because this domain is found in multiple proteins with anti-angiogenic properties . The WSPW motif within ISM2's TSR1 domain is conserved in several other proteins including ADAMTS12, SBSPON, RSPO1, and SEMA5A . This heparin-binding motif has been reported to mediate inhibition of angiogenesis in endothelial cells and anti-proliferative activity of thrombospondins .

When designing experiments to investigate TSR1 domain function, researchers should consider:

• Domain-specific mutation studies (particularly targeting the WSPW motif)

• Recombinant expression of the isolated TSR1 domain to assess its independent activity

• Competitive binding assays with known TSR1-interacting proteins

How does the AMOP domain contribute to ISM2 activity?

The AMOP domain of ISM2 contains a WSRL motif known to be involved in autophagy induction . This suggests a potential role for ISM2 in cellular degradation and recycling processes. Furthermore, based on studies of related proteins, the AMOP domain may be involved in:

• Protein-protein interactions

• Cell adhesion processes

• Extracellular matrix organization

The AMOP domain in ISM1 has been shown to contain an RKD motif that is selective in binding with the extracellular surface of αvβ5 integrins, which are involved in vascular permeability and cell migration . Whether ISM2's AMOP domain has similar integrin-binding properties remains to be fully characterized.

What are the potential roles of ISM2 in angiogenesis regulation?

While the specific role of ISM2 in angiogenesis remains elusive, several lines of evidence suggest it may have anti-angiogenic properties:

• ISM2 contains a TSR1 domain with a WSPW motif that has been associated with angiogenesis inhibition in other proteins .

• Many TSR1-domain-containing proteins, including ADAMTS family members, have demonstrated anti-angiogenic functions .

• The related protein ISM1 has established anti-angiogenic effects, inducing endothelial cell apoptosis via αvβ5 integrin when in soluble form .

To investigate ISM2's role in angiogenesis, researchers should consider employing:

• In vitro tube formation assays with endothelial cells

• Migration and proliferation assays with endothelial cells

• Ex vivo aortic ring assays

• In vivo assays such as chorioallantoic membrane (CAM) assay or matrigel plug assay

What receptors does ISM2 interact with and what signaling pathways does it activate?

While the specific receptors for ISM2 have not been fully characterized, insights may be gained from studies on ISM1. ISM1 interacts with two cell-surface receptors: the high-affinity glucose-regulated protein 78 kDa (GRP78) and the low-affinity αvβ5 integrin . ISM1-GRP78 interaction triggers a direct interaction between GRP78 and Src, leading to Src activation and subsequent phosphorylation of adherens junction proteins .

Based on structural similarities, potential ISM2 receptors may include:

• Integrins, particularly those recognizing the motifs present in the AMOP domain

• Cell surface proteoglycans that interact with the TSR1 domain

• Potentially GRP78, given the domain conservation between ISM1 and ISM2

To identify ISM2 receptors, researchers should consider:

• Co-immunoprecipitation studies followed by mass spectrometry

• Surface plasmon resonance to measure binding affinities

• Cell-based binding assays with candidate receptors

• Receptor blocking studies to assess functional relevance

How do post-translational modifications affect ISM2 function?

Post-translational modifications likely play critical roles in ISM2 function, similar to what has been observed for ISM1. For ISM1, C-mannosylation is required for N-glycosylation of the protein, and these modifications facilitate protein folding and stabilizing, particularly in the TSR1 domain .

Potential post-translational modifications of ISM2 may include:

• N-glycosylation, which could be essential for protein secretion and regulation of enzymatic activity

• C-mannosylation, particularly on tryptophan residues in the TSR1 domain

• Phosphorylation, which might regulate protein-protein interactions

To investigate the impact of these modifications:

• Use site-directed mutagenesis to disrupt potential modification sites

• Employ enzymatic deglycosylation to assess effects on protein activity

• Utilize tunicamycin treatment to inhibit N-glycosylation in expression systems

• Apply mass spectrometry to map and quantify specific modifications

How can researchers effectively block ISM2 function in experimental systems?

To study the physiological and pathological roles of ISM2, researchers often need to inhibit its function. Several approaches can be employed:

• Neutralizing antibodies specifically targeting functional domains (e.g., the TSR1 or AMOP domains)

• RNA interference (siRNA or shRNA) to knockdown ISM2 expression

• CRISPR-Cas9 mediated gene editing to create ISM2 knockout models

• Competitive antagonists based on key motifs (e.g., synthetic peptides mimicking the WSPW motif)

For receptor-focused approaches:

• Use antibodies against potential ISM2 receptors

• Apply peptide inhibitors targeting receptor-ISM2 binding interfaces

• Consider small molecule inhibitors of downstream signaling pathways

When designing these studies, it's important to include appropriate controls to distinguish between effects specific to ISM2 inhibition versus off-target effects.

What is known about ISM2's role in pathological conditions?

While specific information about ISM2 in pathological conditions is limited, insights can be drawn from studies on the ISM family. ISM1 has been implicated in acute lung injury (ALI), where it is significantly up-regulated in lipopolysaccharide (LPS)-treated mouse lung and contributes to pulmonary vascular hyperpermeability .

Given the structural similarities between ISM1 and ISM2, as well as the presence of domains associated with angiogenesis regulation, ISM2 may potentially play roles in:

• Vascular disorders and pathological angiogenesis

• Inflammatory conditions, particularly those affecting vascular integrity

• Cancer progression, potentially through modulation of tumor angiogenesis

• Metabolic disorders, as suggested by the broader roles of the ISM family in metabolism

Research approaches to explore ISM2's pathological roles should include:

• Expression analysis in disease tissues compared to healthy controls

• Correlation studies between ISM2 levels and disease progression

• Genetic association studies looking at ISM2 variants in patient populations

• Animal models with ISM2 manipulation (overexpression or knockout) in disease contexts

How can ISM2 be targeted for therapeutic purposes?

Based on the known properties of ISM2 and related proteins, several therapeutic strategies could be developed:

• Anti-angiogenic therapy: If ISM2's anti-angiogenic properties are confirmed, recombinant ISM2 or peptides derived from its functional domains could potentially be used to inhibit pathological angiogenesis in conditions such as cancer or diabetic retinopathy.

• Targeted delivery: Fusion proteins combining ISM2 domains with cytotoxic agents could potentially deliver therapeutic payloads to cells expressing ISM2 receptors.

• Modulation of vascular permeability: If ISM2 affects vascular permeability similar to ISM1, antagonists could potentially be developed to prevent vascular leakage in conditions like acute lung injury.

• Immunomodulation: Given the potential roles of the ISM family in immunity , therapeutic approaches targeting ISM2 might be relevant for inflammatory or autoimmune conditions.

For any therapeutic development, researchers should consider:

• Specificity to minimize off-target effects

• Delivery methods appropriate for the target tissue

• Pharmacokinetic and pharmacodynamic properties

• Potential immune responses to recombinant proteins

What are the optimal methods for detecting and quantifying ISM2 in biological samples?

Accurate detection and quantification of ISM2 in biological samples is crucial for understanding its expression patterns and potential roles in physiological and pathological processes. Recommended approaches include:

For protein detection and quantification:

• ELISA with antibodies specific to ISM2 (avoid cross-reactivity with ISM1)

• Western blotting with validation using recombinant ISM2 as a positive control

• Mass spectrometry-based proteomics for unbiased detection and absolute quantification

• Immunohistochemistry or immunofluorescence for tissue localization studies

For mRNA expression analysis:

• Quantitative real-time PCR with carefully designed primers specific to ISM2

• RNA sequencing for global expression profiling and splice variant detection

• In situ hybridization for spatial expression analysis in tissues

When developing these assays, researchers should validate specificity using:

• Recombinant ISM2 protein standards

• Positive and negative control tissues based on known expression patterns

• ISM2 knockout or knockdown samples as negative controls

What are the best approaches for studying ISM2 function in in vitro systems?

To investigate ISM2 function in controlled laboratory settings, several in vitro approaches are recommended:

Cell-based assays:

• Endothelial cell tube formation assays to assess angiogenic effects

• Cell migration and invasion assays to evaluate effects on cell motility

• Apoptosis assays to determine if ISM2 induces programmed cell death similar to ISM1

• Vascular permeability assays using endothelial cell monolayers

• Reporter assays to identify downstream signaling pathways

Protein interaction studies:

• Co-immunoprecipitation to identify binding partners

• Surface plasmon resonance to measure binding kinetics

• Proximity ligation assays to visualize protein interactions in situ

• Yeast two-hybrid or mammalian two-hybrid screens for unbiased interaction discovery

When designing these experiments, researchers should consider:

• Using appropriate concentrations of recombinant ISM2 (typically 10-500 ng/mL based on studies with related proteins)

• Including positive controls (e.g., known angiogenesis inhibitors)

• Testing both soluble and immobilized forms, as these may have different effects as observed with ISM1

• Examining time-dependent responses to distinguish between immediate signaling events and secondary effects

What animal models are most suitable for in vivo studies of ISM2 function?

In vivo studies are essential for understanding the physiological roles of ISM2. Based on approaches used for related proteins, recommended animal models include:

Genetic models:

• Conditional knockout mice to study tissue-specific effects and avoid developmental lethality

• Inducible expression systems to examine the effects of ISM2 overexpression

• Knockin models with tagged ISM2 for in vivo tracking and interaction studies

Disease models:

• Angiogenesis models (e.g., oxygen-induced retinopathy, tumor xenografts)

• Acute lung injury models to examine effects on vascular permeability, building on findings with ISM1

• Developmental biology models to investigate potential roles in embryonic development

• Metabolic disease models, given the reported involvement of the ISM family in metabolism

For administration of recombinant ISM2:

• Local delivery (e.g., intradermal, intraocular) to assess effects on specific tissues

• Systemic delivery to evaluate organ-specific responses and biodistribution

• Controlled release systems for sustained exposure in chronic models

When administering recombinant ISM2, researchers should monitor for potential systemic effects similar to those observed with ISM1, such as enhanced vascular permeability in the lungs .

How can researchers address issues of protein solubility and stability when working with recombinant ISM2?

Recombinant ISM2, like many complex proteins, can present challenges related to solubility and stability. Effective strategies include:

For improving solubility during expression and purification:

• Optimize expression conditions (temperature, induction time, media composition)

• Use solubility-enhancing fusion tags (e.g., SUMO, MBP, or thioredoxin)

• Consider co-expression with molecular chaperones

• Test different cell lysis conditions and buffers with various additives (e.g., mild detergents, reducing agents)

For enhancing stability of purified protein:

• Use Tris/PBS-based buffers with 5-50% glycerol for liquid formulations

• Consider lyophilization with 6% Trehalose as a stabilizing agent

• Maintain pH in the range of 7.4-8.0

• Add protease inhibitors to prevent degradation

• Aliquot and store at -80°C to avoid freeze-thaw cycles

When handling reconstituted lyophilized protein, brief centrifugation prior to opening is recommended to collect all material .

How can researchers differentiate between the effects of ISM1 and ISM2 in experimental systems?

Distinguishing between ISM1 and ISM2 effects is crucial for accurate functional characterization. Recommended approaches include:

• Use highly specific antibodies validated for lack of cross-reactivity

• Design siRNA or shRNA with confirmed specificity for either ISM1 or ISM2

• Create single-knockout models for each protein separately

• Employ domain-specific recombinant proteins to identify unique functional regions

• Conduct rescue experiments with one protein in the background of the other's depletion

When analyzing experimental data:

• Look for differential tissue expression patterns

• Compare temporal dynamics of responses

• Examine protein-specific downstream signaling events

• Consider potential compensatory mechanisms when one protein is depleted

What controls should be included in ISM2 functional studies to ensure valid interpretations?

Proper controls are essential for robust experimental design and valid interpretation of results. Key controls for ISM2 functional studies include:

For protein activity assays:

• Heat-inactivated ISM2 to control for non-specific protein effects

• Structurally similar but functionally distinct proteins (e.g., mutated versions of ISM2)

• Domain-specific fragments to identify active regions

• Concentration gradients to establish dose-dependent relationships

For gene expression studies:

• Non-targeting siRNA or shRNA controls

• Wild-type controls for genetic models

• Empty vector controls for overexpression studies

• Housekeeping genes for normalization

For receptor interaction studies:

• Competitive binding with known ligands

• Cell lines lacking putative receptors

• Blocking antibodies against candidate receptors

• Downstream signaling inhibitors to confirm pathway specificity

Additional considerations:

• Include both positive and negative controls for each experimental system

• Validate key findings using multiple independent approaches

• Consider both acute and chronic effects, as these may differ

What are the most promising avenues for future ISM2 research?

Several key areas warrant further investigation to advance our understanding of ISM2 biology:

• Receptor identification and characterization: Determining the specific cell surface receptors for ISM2 and their tissue distribution would significantly enhance our understanding of ISM2's biological functions.

• Comparative analysis with ISM1: Detailed structure-function comparisons between ISM1 and ISM2, particularly focused on their unique motifs (WSLW in ISM1 versus WSPW in ISM2), could reveal selective pharmacological targeting opportunities.

• Role in disease processes: Investigation of ISM2 expression and function in various pathological conditions, particularly those involving vascular abnormalities, inflammation, or metabolic dysfunction.

• Developmental roles: Given the expression of ISM family members during embryonic development, studies exploring ISM2's contributions to organogenesis and tissue patterning could reveal novel developmental functions.

• Post-translational modification analysis: Comprehensive characterization of ISM2's post-translational modifications and their impact on protein function, stability, and interactions.

What emerging technologies might advance ISM2 research?

Several cutting-edge technologies offer promising approaches for deepening our understanding of ISM2:

• CRISPR-Cas9 genome editing: Generation of precise knockin and knockout models to study ISM2 function in various contexts, including disease models and development.

• Single-cell transcriptomics: Analysis of cell-specific ISM2 expression patterns in heterogeneous tissues and during developmental processes.

• Cryo-electron microscopy: Determination of high-resolution structures of ISM2 alone and in complex with its receptors to inform structure-based drug design efforts.

• Spatial transcriptomics and proteomics: Mapping the tissue-specific expression patterns of ISM2 at high resolution to identify potential sites of action.

• Organoid systems: Utilization of 3D tissue models to investigate ISM2's role in tissue organization and function in near-physiological conditions.

• Antibody engineering: Development of highly specific monoclonal antibodies for detection, neutralization, and potentially therapeutic applications targeting ISM2.

• Computational approaches: Application of machine learning and molecular dynamics simulations to predict ISM2 interactions and functional properties based on sequence and structural features.