Recombinant Pongo abelii Integrin beta-1 (ITGB1)

What is the structural composition of Pongo abelii ITGB1 and how does it compare to human ITGB1?

Pongo abelii ITGB1 is a transmembrane glycoprotein with a molecular weight of approximately 88.4 kDa, though its observed molecular weight in SDS-PAGE is typically between 100-130 kDa due to post-translational modifications . The protein consists of 798 amino acids structured into an extracellular domain, a transmembrane region, and a cytoplasmic tail. The extracellular domain contains multiple cysteine-rich repeats that form disulfide bonds critical for the protein's tertiary structure.

When comparing orangutan ITGB1 to human ITGB1, they share high sequence homology due to their evolutionary proximity. This conservation reflects the fundamental importance of ITGB1 in cellular functions across species. Researchers should note that while antibodies raised against human ITGB1 often cross-react with Pongo abelii ITGB1, epitope mapping may reveal subtle differences that could affect experimental outcomes in comparative studies.

Which alpha integrin subunits does Pongo abelii ITGB1 preferentially pair with, and how do these pairings affect ligand specificity?

Pongo abelii ITGB1 forms heterodimers with multiple alpha subunits (at least 12 different alpha chains in primates), each resulting in receptors with distinct ligand binding properties . These heterodimeric combinations determine the specificity for extracellular matrix proteins:

When designing experiments involving specific ITGB1 heterodimers, researchers should consider co-expressing the relevant alpha subunit or using cell lines that naturally express the alpha subunit of interest. The ligand binding specificity directly influences downstream signaling pathways and cellular responses.

What are the key post-translational modifications of Pongo abelii ITGB1 that affect its functionality?

Pongo abelii ITGB1, like its human counterpart, undergoes several post-translational modifications that significantly influence its function:

• N-glycosylation: Multiple N-glycosylation sites are present in the extracellular domain, contributing to the observed molecular weight discrepancy between the calculated (88 kDa) and observed (100-130 kDa) sizes in SDS-PAGE analysis . These glycosylations affect protein folding, stability, and ligand recognition.

• Phosphorylation: The cytoplasmic domain contains phosphorylation sites that regulate integrin activation state and interactions with cytoskeletal and signaling proteins.

• Proteolytic processing: Limited proteolysis can occur under certain conditions, generating fragments with distinct biological activities.

To study these modifications, researchers should consider:

• Enzymatic deglycosylation using PNGase F or Endo H to assess glycosylation patterns

• Phospho-specific antibodies to detect activation-dependent phosphorylation

• Protein phosphatase treatments to evaluate the role of phosphorylation in protein-protein interactions

• Protease inhibitor cocktails during protein extraction to prevent artifactual proteolytic processing

The conservation of these modification sites between human and orangutan ITGB1 suggests similar regulatory mechanisms, though species-specific differences may exist in glycosylation patterns.

What are the optimal expression systems for producing functional recombinant Pongo abelii ITGB1?

The production of functional recombinant Pongo abelii ITGB1 requires careful consideration of expression systems to ensure proper folding, post-translational modifications, and maintenance of native conformation. Based on available research data:

• Mammalian Expression Systems: HEK293 cells represent the preferred platform for expressing functional ITGB1, as they provide proper glycosylation and disulfide bond formation essential for the protein's structural integrity . These cells naturally express endogenous integrins, which can serve as a positive control in your expression studies.

• Insect Cell Systems: Baculovirus-infected insect cells (Sf9, High Five) offer an alternative that balances yield with post-translational modification capabilities. While glycosylation patterns differ from mammalian cells, they often produce correctly folded and functional ITGB1.

• Cell-Free Systems: In vitro cell-free expression systems have been used for producing ITGB1 domains, though these typically require refolding procedures and lack the complete post-translational modifications .

The methodological approach should include:

• Codon optimization for the selected expression system

• Addition of appropriate signal peptides for secretion or membrane targeting

• Inclusion of purification tags (His, GST, etc.) that minimally interfere with protein function

• Co-expression with relevant alpha subunits if studying heterodimer functionality

• Careful buffer selection during purification to maintain native conformation

Researchers should validate the functionality of recombinant ITGB1 through binding assays with known ligands and heterodimer formation assessment.

How should researchers design experiments to investigate ITGB1-mediated signaling pathways in Pongo abelii cells?

Investigating ITGB1-mediated signaling in Pongo abelii cells requires a multi-faceted experimental approach:

• Activation Strategies:

  • Immobilized ligand engagement (fibronectin, collagen, laminin)

  • Activating antibodies that induce conformational changes

  • Manganese (Mn²⁺) treatment to artificially activate integrins

• Signaling Pathway Assessment:
  ITGB1 activates several key pathways that should be monitored simultaneously:

  Pathway	Key Molecules to Assess	Recommended Detection Method
  FAK/Src	FAK (Y397), Src (Y416)	Phospho-specific immunoblotting
  PI3K/Akt	Akt (S473, T308), GSK-3β	Phospho-specific immunoblotting, kinase activity assays
  MAPK/ERK	ERK1/2 (T202/Y204)	Phospho-specific immunoblotting, kinase activity assays
  Rho GTPases	RhoA, Rac1, Cdc42	GTPase activity pull-down assays
  TGF-β	SMAD2/3 phosphorylation	Phospho-specific immunoblotting
  Wnt	β-catenin nuclear translocation	Subcellular fractionation, immunofluorescence

• Temporal Dynamics:
  Design time-course experiments (5 min, 15 min, 30 min, 1 hr, 4 hr, 24 hr) to capture both immediate signaling events and delayed transcriptional responses.

• Pathway Inhibition:
  Use specific inhibitors to distinguish pathway crosstalk:

  • FAK inhibitors (PF-573228)

  • PI3K inhibitors (LY294002, Wortmannin)

  • MEK inhibitors (U0126, PD98059)

  • Src family kinase inhibitors (PP2)

• RNA Interference:
  Design siRNA/shRNA targeting ITGB1 and key pathway components, including controls for knockdown efficiency validation via qPCR and western blotting.

When comparing Pongo abelii cells to human cells, researchers should be mindful of potential differences in pathway kinetics and feedback mechanisms, necessitating parallel experiments under identical conditions .

What isolation and purification methods yield the highest purity and activity for recombinant Pongo abelii ITGB1?

Obtaining high-purity, functionally active recombinant Pongo abelii ITGB1 requires strategic purification approaches:

• Affinity Chromatography Options:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged ITGB1

  • Glutathione-S-transferase (GST) affinity for GST-tagged constructs

  • Immunoaffinity chromatography using anti-ITGB1 antibodies (such as 12594-1-AP)

  • Ligand-based affinity using immobilized fibronectin or other integrin ligands

• Optimal Extraction Conditions:

  • Membrane proteins require careful solubilization

  • Use mild detergents (0.5-1% CHAPS, DDM, or Triton X-100)

  • Include protease inhibitors and phosphatase inhibitors

  • Maintain physiological pH (7.2-7.4) and ionic strength

  • Consider including divalent cations (1-2 mM Ca²⁺, Mg²⁺) to stabilize conformation

• Multi-step Purification Protocol:
  a. Cell lysis with appropriate detergent buffer
  b. Initial affinity purification step
  c. Ion exchange chromatography (typically anion exchange)
  d. Size exclusion chromatography for final polishing
  e. Quality control testing

• Functional Validation Methods:

  • Ligand binding assays using surface plasmon resonance

  • Heterodimer formation analysis with co-expressed alpha subunits

  • Cell adhesion rescue experiments in ITGB1-knockout cells

  • Integrin activation assays using conformation-specific antibodies

When working with membrane proteins like ITGB1, researchers must balance solubilization efficiency with maintaining native conformation. The purified protein should be stored with stabilizing agents (glycerol, specific detergents) and minimal freeze-thaw cycles to preserve activity.

How can researchers effectively study the role of Pongo abelii ITGB1 in mechanotransduction and cellular response to matrix stiffness?

Studying ITGB1-mediated mechanotransduction requires specialized experimental approaches that capture the mechanical aspects of cell-matrix interactions:

• Substrate Preparation Techniques:

  • Polyacrylamide hydrogels of defined stiffness (1-100 kPa range)

  • PDMS substrates with tunable elastic modulus

  • Micropatterned adhesive islands to control cell spreading

  • Functionalization with specific ECM proteins (fibronectin, collagen, laminin)

• Force Measurement and Application Methods:

  • Atomic force microscopy (AFM) for measuring cellular traction forces

  • Magnetic twisting cytometry for applying localized forces to ITGB1 receptors

  • Traction force microscopy using fluorescent beads embedded in substrates

  • Molecular tension sensors incorporating FRET-based reporters

• Mechanosensitive Signaling Analysis:
  Monitor key mechanosensitive molecules that directly interact with ITGB1:

  Mechanosensitive Element	Activation Indicator	Role in ITGB1 Signaling
  Talin	Unfolding of rod domains	Links ITGB1 to actin cytoskeleton
  Vinculin	Conformational activation	Reinforces integrin-cytoskeleton connections
  YAP/TAZ	Nuclear translocation	Transcriptional regulation of mechanosensitive genes
  RhoA	GTP-bound state	Cytoskeletal tension regulation
  Cav1	Phosphorylation state	Mechanosensing through caveolae

• Live Cell Imaging Applications:

  • TIRF microscopy to visualize focal adhesion dynamics

  • FRET-based reporters for studying protein-protein interactions

  • Optogenetic approaches to locally activate integrins

• Transcriptional Profiling:
  Compare gene expression profiles on substrates of varying stiffness to identify ITGB1-dependent mechanosensitive genes.

To specifically study Pongo abelii ITGB1, researchers can use CRISPR/Cas9 to knock out endogenous ITGB1 in cell lines and rescue with the orangutan variant, then compare mechanical responses to cells expressing human ITGB1 .

What approaches should researchers use to investigate the interactome of Pongo abelii ITGB1 and how it differs from human ITGB1?

Comprehensively mapping the ITGB1 interactome requires multi-modal approaches that capture both stable and transient interactions:

• Proximity-Based Labeling Methods:

  • BioID: Fusion of ITGB1 with biotin ligase (BirA*) to biotinylate proximal proteins

  • APEX2: Fusion with engineered ascorbate peroxidase for proximity-dependent biotinylation

  • TurboID: Enhanced biotin ligase for faster labeling kinetics
    These methods capture the spatial proteome around ITGB1 in living cells under various conditions.

• Co-Immunoprecipitation Strategies:

  • Standard IP using anti-ITGB1 antibodies validated for Pongo abelii (e.g., 12594-1-AP)

  • Tandem affinity purification using tagged ITGB1 constructs

  • Cross-linking assisted IP to capture transient interactions

  • Analyze precipitates using mass spectrometry for unbiased discovery

• Domain-Specific Interaction Mapping:

  • Generate constructs expressing specific domains of ITGB1

  • Yeast two-hybrid screening with domain-specific baits

  • Peptide arrays to map linear interaction motifs

  • In vitro pull-down assays with recombinant domains

• Comparative Analysis Workflow:

  • Perform parallel interactome studies in cells expressing human vs. Pongo abelii ITGB1

  • Use SILAC or TMT labeling for quantitative proteomics comparison

  • Analyze data with specialized software (e.g., String-DB, Cytoscape) to visualize network differences

  • Validate key differential interactions using targeted biochemical assays

• Functional Validation:

  • siRNA knockdown of identified interactors

  • Competitive peptide inhibition of specific interactions

  • Structure-based mutagenesis of interaction interfaces

  • Correlation of interactome changes with functional outcomes

The cytoplasmic tail of ITGB1 contains multiple protein-binding motifs, including the NPXY motifs that interact with phosphotyrosine-binding (PTB) domain proteins such as talin and kindlin, which are critical for integrin activation and signaling .

How can researchers develop organoid models to study ITGB1 function in Pongo abelii tissue development and disease?

Organoid models represent powerful systems for studying ITGB1 functions in tissue-specific contexts:

• Organoid Establishment Protocol:

  • Source appropriate Pongo abelii stem cells or primary tissue

  • Optimize ECM composition (Matrigel, collagen, synthetic matrices)

  • Determine growth factor cocktails for specific tissue differentiation

  • Establish reliable quantitative metrics for organoid growth and differentiation

• ITGB1 Manipulation Strategies:

  • CRISPR/Cas9 editing of ITGB1 in stem cells prior to organoid formation

  • Inducible shRNA systems for temporal control of ITGB1 knockdown

  • Small molecule integrin inhibitors at different developmental stages

  • Function-blocking antibodies against specific ITGB1-containing heterodimers

• Analysis Methods for ITGB1-Dependent Phenotypes:

  Analytical Approach	Measurement Parameters	Relevance to ITGB1 Function
  Live imaging	Organoid growth kinetics, morphogenesis	Developmental roles of ITGB1
  Immunofluorescence	Cell polarity, ECM deposition patterns	Structural organization
  Single-cell RNA-seq	Cell-type specific transcriptional profiles	Differentiation effects
  Electron microscopy	Cell-ECM interface ultrastructure	Adhesion complex formation
  Mechanical testing	Organoid stiffness, viscoelasticity	Tissue biomechanical properties

• Disease Modeling Applications:

  • Introduce disease-associated ITGB1 mutations

  • Create cancer models by combining ITGB1 alterations with oncogene activation

  • Study fibrosis by manipulating ITGB1-TGF-β interactions

  • Model inflammatory responses through cytokine challenge

• Comparative Evolutionary Insights:

  • Parallel organoid systems with human and Pongo abelii ITGB1

  • Cross-species transplantation experiments

  • Analysis of species-specific ECM interactions

Researchers should be mindful that organoid ECM composition dramatically influences ITGB1-dependent behaviors. The self-organization of organoids relies heavily on proper integrin-ECM interactions, making these models particularly valuable for studying ITGB1 functions in tissue architecture development .

How should researchers address inconsistent results when studying ITGB1 activation states in Pongo abelii samples?

Inconsistent results in ITGB1 activation studies often stem from technical factors that can be systematically addressed:

• Activation State Assessment Methods:

  • Conformation-specific antibodies must be validated for Pongo abelii ITGB1

  • Flow cytometry provides quantitative single-cell data on activation states

  • Immunofluorescence microscopy allows spatial analysis of activation

  • FRET-based sensors can detect conformational changes in real-time

• Common Sources of Variability:

  Variability Source	Troubleshooting Approach	Preventive Measures
  Divalent cation concentrations	Titrate Mn²⁺, Ca²⁺, Mg²⁺	Standardize buffer compositions
  Cell confluency variations	Seed at consistent densities	Quantify and report cell density
  Serum factors	Use serum-free conditions	Pre-deplete integrin-binding proteins
  Mechanical stimuli	Control substrate stiffness	Standardize cell handling
  Temperature fluctuations	Maintain consistent temperature	Include temperature controls

• Experimental Controls:

  • Positive controls: Mn²⁺ treatment (1mM) for maximal activation

  • Negative controls: EDTA treatment (5mM) for integrin inactivation

  • Isotype controls for antibody specificity

  • Human ITGB1 for cross-species comparison

• Standardized Analysis Framework:

  • Establish clear quantitative metrics for activation states

  • Use ratiometric measurements (active/total ITGB1)

  • Implement automated image analysis algorithms

  • Apply appropriate statistical tests for small sample sizes

• Validation Approaches:

  • Orthogonal methods to confirm activation state

  • Ligand binding assays to verify functional consequences

  • Signaling readouts (FAK/Src phosphorylation) as downstream indicators

When comparing results across laboratories, researchers should develop a standardized protocol that specifies critical parameters affecting ITGB1 activation states. This promotes reproducibility and facilitates meaningful comparative studies between human and Pongo abelii ITGB1 .

What are the most common pitfalls in ITGB1 co-immunoprecipitation experiments and how can they be overcome?

Co-immunoprecipitation (co-IP) of ITGB1 presents specific challenges due to its membrane localization and complex formation tendencies:

• Antibody Selection Issues:

  Pitfall	Solution	Validation Method
  Poor antibody specificity	Test multiple antibodies (e.g., 12594-1-AP)	Western blot with ITGB1-knockout controls
  Antibody interferes with protein interactions	Map epitopes relative to interaction domains	Competition assays with peptides
  Low antibody affinity for native conformation	Use native-state IP conditions	Compare IP efficiency under various conditions
  Antibody cross-reactivity with other integrins	Pre-absorb with recombinant proteins	Mass spectrometry validation

• Membrane Protein Solubilization Challenges:

  • Different detergents affect interaction preservation differently

  • Systematic testing of detergents (CHAPS, digitonin, NP-40, DDM)

  • Detergent concentration optimization (typically 0.3-1%)

  • Consider membrane fractionation before solubilization

• Complex Stability Considerations:

  • Some ITGB1 interactions are transient or weak

  • Use chemical crosslinking (DSP, formaldehyde) to capture transient interactions

  • Optimize salt concentration to maintain specific interactions

  • Include phosphatase inhibitors to preserve phosphorylation-dependent interactions

• Background Reduction Strategies:

  • Pre-clear lysates with control IgG and protein A/G beads

  • Use highly specific magnetic beads with low non-specific binding

  • Include competing peptides to reduce non-specific binding

  • Implement stringent washing protocols with validation

• Control Experiments:

  • IgG-only controls to identify non-specific binders

  • ITGB1-knockout cells as negative controls

  • Known ITGB1 interactors as positive controls

  • Reciprocal IPs to confirm interactions

When studying Pongo abelii ITGB1, researchers should verify antibody cross-reactivity with the orangutan protein, possibly using recombinant protein controls. The highly conserved nature of ITGB1 suggests that many antibodies developed against human ITGB1 will recognize the orangutan ortholog, but this should be experimentally confirmed .

How can researchers resolve data discrepancies when comparing Pongo abelii ITGB1 with human ITGB1 in functional assays?

When comparing functional properties between species orthologs, systematic approaches are needed to identify true biological differences versus technical artifacts:

• Expression Level Normalization:

  • Quantify protein expression by western blot with cross-reactive antibodies

  • Implement FACS-based sorting for equivalent expression

  • Use inducible systems to achieve matched expression levels

  • Account for differences in antibody affinity between species orthologs

• Experimental Design for Cross-Species Comparisons:

  Potential Discrepancy	Controlled Experimental Approach	Analytical Consideration
  Cell context differences	Express both orthologs in same null background	Account for endogenous integrins
  Heterodimer formation efficiency	Co-express with identical alpha subunits	Quantify alpha-beta pairing
  Post-translational modification differences	Analyze glycosylation, phosphorylation profiles	Consider species-specific modification enzymes
  Ligand binding preferences	Use concentration gradients of multiple ligands	Calculate binding constants for each
  Signaling kinetics differences	Perform detailed time-course experiments	Model kinetic parameters

• Statistical Analysis Framework:

  • Implement mixed-effects models to account for experimental batch effects

  • Use bootstrapping approaches for robust comparison

  • Calculate effect sizes rather than relying solely on p-values

  • Perform power analysis to ensure adequate sample sizes

• Orthogonal Validation Methods:

  • Structure-function studies with chimeric proteins

  • Domain swap experiments to identify functionally divergent regions

  • CRISPR/Cas9 knock-in of species-specific domains

  • In silico structural modeling to predict functional differences

• Biological Context Consideration:

  • Cell type-specific effects due to different cellular machinery

  • Species-specific ECM composition and architecture

  • Evolutionary adaptations in integrin-dependent processes

  • Differential regulation of expression and activation

When publishing comparative data, researchers should clearly report all normalization methods, control experiments, and statistical approaches used to ensure reproducibility. The evolutionary distance between humans and orangutans, while relatively small, may reveal subtle functional adaptations in ITGB1 that reflect species-specific requirements for cell-ECM interactions .

How can Pongo abelii ITGB1 research inform therapeutic approaches for human integrin-related disorders?

Comparative studies between human and Pongo abelii ITGB1 provide valuable insights for therapeutic development:

• Evolutionary Conservation Analysis:

  • Identify highly conserved regions as potential critical functional domains

  • Map species-specific variations that may correlate with disease resistance

  • Use comparative genomics to identify regulatory elements conserved across primates

• Structure-Function Insights for Drug Development:

  Structural Feature	Evolutionary Conservation	Therapeutic Relevance
  Ligand binding sites	Highly conserved	Direct targeting for competitive inhibition
  Activation regulatory regions	Moderately conserved	Allosteric modulation of activation state
  Cytoplasmic signaling motifs	Highly conserved	Intracellular signaling intervention
  Glycosylation sites	Variable conservation	Species-specific regulation
  Cysteine-rich domains	Highly conserved	Structural integrity targets

• Disease-Relevant Functional Differences:

  • Cancer research: Compare invasive and migratory behavior mediated by each ortholog

  • Fibrosis models: Evaluate TGF-β response differences between species

  • Inflammatory disorders: Assess immune cell adhesion and transmigration

  • Developmental disorders: Compare embryonic roles in model systems

• Therapeutic Screening Platforms:

  • Parallel screening against human and Pongo abelii ITGB1

  • Identify compounds with species-specific effects

  • Utilize evolutionary insights to predict off-target effects

  • Develop assays that reflect species-specific regulatory mechanisms

• Translational Research Applications:

  • Xenograft models using Pongo abelii cells in humanized models

  • Comparative tissue engineering with species-specific integrins

  • Gene therapy approaches informed by functional conservation

  • Biomarker development based on cross-species validation

The high degree of conservation between human and orangutan ITGB1 makes comparative studies particularly valuable for identifying critical functional elements that could be targeted therapeutically. Additionally, understanding species-specific differences may reveal natural regulatory mechanisms that could be exploited for therapeutic intervention in human disease contexts .

What methodological approaches best elucidate ITGB1's role in cancer progression using Pongo abelii models?

Investigating ITGB1's role in cancer using Pongo abelii models requires sophisticated experimental approaches:

• Cell Line Model Development:

  • Generate Pongo abelii cell lines with CRISPR-engineered ITGB1 mutations

  • Create isogenic lines with wild-type and mutant ITGB1

  • Develop fluorescent reporter systems for ITGB1 activity

  • Establish 3D culture systems that mimic tumor microenvironments

• Cancer-Relevant Functional Assays:

  Cancer Hallmark	Experimental Approach	ITGB1-Specific Parameters
  Invasion/Migration	Transwell assays, 3D invasion	ECM-specific invasion patterns
  Proliferation	Growth curves, cell cycle analysis	Adhesion-dependent proliferation
  Anoikis resistance	Suspension culture viability	Signaling in detached conditions
  Drug resistance	Therapy response profiling	Adhesion-mediated drug resistance
  Stemness	Tumorsphere formation	Cancer stem cell marker correlation

• Signaling Pathway Analysis:

  • Focus on cancer-relevant ITGB1 signaling cascades:

    • FAK/Src in invasion and survival

    • PI3K/Akt in proliferation and metabolism

    • MAPK/ERK in growth and differentiation

    • Rho GTPases in cytoskeletal reorganization

    • Wnt/β-catenin in stemness

• In Vivo Xenograft Approaches:

  • Orthotopic implantation of ITGB1-manipulated cells

  • Patient-derived xenografts with ITGB1 interventions

  • Metastasis tracking via bioluminescence imaging

  • Drug treatment studies targeting ITGB1-dependent pathways

• Translational Relevance Assessment:

  • Correlation with human cancer genomic databases

  • Tissue microarray analysis of ITGB1 in cancer progression

  • Identification of species-specific ITGB1-dependent vulnerabilities

  • Preclinical testing of ITGB1-targeting approaches

The abnormal expression of ITGB1 is closely associated with the development, progression, and poor prognosis of various tumors, including lung, breast, prostate, stomach, colorectal, pancreatic, and esophageal cancers . Comparative studies using Pongo abelii models can reveal conserved mechanisms of ITGB1-driven cancer progression, potentially identifying new therapeutic targets with cross-species validation.