Recombinant Human Annexin A8-like protein 1 (ANXA8L1)

What is Recombinant Human Annexin A8-like Protein 1 (ANXA8L1)?

Recombinant Human Annexin A8-like Protein 1 (ANXA8L1) is a member of the annexin family of calcium-dependent phospholipid binding proteins. It functions as a membrane binding protein with diverse properties, including voltage-sensitive calcium channel activity, ion selectivity, and membrane fusion capabilities. ANXA8L1 is closely related to ANXA8L2 (Annexin A8-like protein 2) and shares significant structural and functional homology with other annexin family members. The recombinant form of ANXA8L1 typically contains a His-tag at the N-terminus and is expressed in expression systems such as E. coli for research applications . The protein has been implicated in various cellular processes and has garnered interest due to its overexpression in certain cancer types, suggesting potential roles in oncogenesis.

How does ANXA8L1 differ from other annexin family members?

While ANXA8L1 shares the core structural and functional properties of the annexin family, it has distinct features that differentiate it from other family members:

What are the optimal expression systems for producing recombinant ANXA8L1?

The optimal expression of recombinant ANXA8L1 can be achieved using both prokaryotic and eukaryotic expression systems, each offering distinct advantages depending on research needs:

Prokaryotic Expression (E. coli):
E. coli expression systems are commonly used for ANXA8L1 production due to their high yield, cost-effectiveness, and relatively simple protocols. The typical approach involves cloning the ANXA8L1 coding sequence into an expression vector containing a His-tag at the N-terminus, transformation into E. coli strains such as BL21(DE3), followed by IPTG-induced expression. This system allows for robust production of the protein with typical yields in the range of several milligrams per liter of culture .

Eukaryotic Expression (Pichia pastoris):
For applications requiring post-translational modifications, Pichia pastoris has proven effective for ANXA8L1 expression. This yeast-based system offers advantages for proteins that may require specific folding environments or modifications not available in bacterial systems. Research indicates that human ANXA8 expressed in P. pastoris maintains improved antigenic properties compared to the E. coli-expressed version .

The choice between these systems should be guided by specific research requirements:

• Use E. coli for high-yield applications where post-translational modifications are not critical

• Consider P. pastoris for applications requiring proper folding or when generating antigens for antibody production

• For structural studies, E. coli expression followed by careful refolding protocols may be sufficient

What purification strategies yield the highest purity ANXA8L1 preparations?

Achieving high-purity ANXA8L1 preparations typically involves a multi-step purification approach:

• Immobilized Metal Affinity Chromatography (IMAC): Using the His-tag engineered at the N-terminus of recombinant ANXA8L1, IMAC with Ni-NTA or similar resins serves as an effective initial purification step. Optimal elution is typically achieved with an imidazole gradient (20-250 mM) .

• Ion Exchange Chromatography: Following IMAC, ion exchange chromatography (typically anion exchange using Q-Sepharose) can separate ANXA8L1 from remaining contaminants based on charge differences. This step is particularly effective when performed at pH 7.4-8.0, where ANXA8L1 binds efficiently to the column.

• Size Exclusion Chromatography: A final polishing step using gel filtration (such as Superdex 200) separates any remaining impurities and aggregates based on molecular size, yielding preparations with >90% purity as commonly required for research applications .

The optimal buffer conditions for purified ANXA8L1 storage are:

• 20 mM Tris-HCl (pH 8.0)

• 0.1 M NaCl

• 10% glycerol

• 1 mM DTT

This buffer composition maintains protein stability during storage at -20°C or -80°C for long-term storage, or at 4°C for short-term use .

How can I verify the proper folding and functionality of purified ANXA8L1?

Verifying the proper folding and functionality of purified ANXA8L1 requires multiple complementary approaches:

• Biophysical Characterization:

  • Circular Dichroism (CD) spectroscopy to assess secondary structure content

  • Thermal shift assays to determine protein stability and proper folding

  • Dynamic Light Scattering (DLS) to evaluate protein homogeneity and detect aggregation

• Functional Assays:

  • Calcium-dependent phospholipid binding assays using liposomes containing phosphatidylserine

  • Ion channel activity assessments using artificial lipid bilayers or patch-clamp techniques

  • Membrane fusion assays to evaluate ANXA8L1's capacity to promote membrane fusion events

• Structural Verification:

  • MALDI-TOF mass spectrometry to confirm the expected molecular weight of 39.4 kDa

  • Limited proteolysis followed by mass spectrometry to verify domain organization

  • X-ray crystallography or cryo-EM for detailed structural analysis (for advanced studies)

A comprehensive verification protocol should include at minimum:

• SDS-PAGE to assess purity (target >90%)

• Western blotting using anti-ANXA8L1 or anti-His-tag antibodies

• A functional assay confirming calcium-dependent membrane binding

What are the most sensitive methods for detecting ANXA8L1 in biological samples?

The detection of ANXA8L1 in biological samples can be accomplished through several complementary approaches, with varying degrees of sensitivity and specificity:

Immunological Methods:

• Sandwich ELISA: Recent developments have established highly sensitive sandwich ELISA protocols for ANXA8 detection, which can be adapted for ANXA8L1. Using specific monoclonal antibodies targeting different epitopes (similar to the E9 and B7 antibodies developed for ANXA8), this method can achieve detection limits as low as 0.065 ng/mL, making it suitable for detecting physiological concentrations in biological samples .

• Western Blotting: For detection in cell or tissue lysates, Western blotting using specific antibodies against ANXA8L1 provides reliable detection. The protocol typically involves:

  • SDS-PAGE separation of proteins

  • Transfer to PVDF membranes

  • Blocking with 5% non-fat dry milk

  • Incubation with primary anti-ANXA8L1 antibody (similar to the approach using ab111693 for ANXA8)

  • Detection with HRP-conjugated secondary antibody

Mass Spectrometry-Based Methods:
For unbiased detection and absolute quantification, LC-MS/MS approaches offer high specificity:

• Selected Reaction Monitoring (SRM) or Multiple Reaction Monitoring (MRM) targeting unique peptides derived from ANXA8L1

• Parallel Reaction Monitoring (PRM) for increased selectivity in complex samples

Practical Considerations for Method Selection:

• For routine screening of multiple samples: Sandwich ELISA

• For visual confirmation in tissues: Immunohistochemistry

• For absolute quantification: Mass spectrometry

• For relative expression across conditions: Western blotting or RT-qPCR (for mRNA)

How can I establish a reliable ELISA for ANXA8L1 quantification?

Establishing a reliable ELISA for ANXA8L1 quantification requires careful consideration of antibody selection, assay optimization, and validation:

1. Antibody Selection and Validation:

• Generate or obtain monoclonal antibodies targeting different epitopes of ANXA8L1

• Screen antibodies for specificity against related annexins (especially ANXA8, ANXA2, and ANXA5)

• Select antibody pairs that recognize distinct, non-overlapping epitopes

2. Assay Development Protocol:

• Coating: Capture antibody (e.g., similar to E9 for ANXA8) at 1-2 μg/mL in carbonate buffer (pH 9.6), 4°C overnight

• Blocking: 2-3% BSA in PBS, 1-2 hours at room temperature

• Sample Incubation: Diluted samples and standards, 2 hours at room temperature

• Detection: Biotinylated detection antibody (e.g., similar to B7 for ANXA8) followed by streptavidin-HRP

• Visualization: TMB substrate and stop solution (2N H₂SO₄)

• Quantification: Four-parameter logistic curve fitting

3. Assay Optimization Steps:

• Antibody concentration titration to determine optimal coating and detection concentrations

• Incubation time optimization for maximal sensitivity without background increase

• Buffer composition adjustments to minimize matrix effects

• Temperature control to ensure consistent performance

4. Validation Parameters:

• Specificity: Cross-reactivity testing against other annexins (particularly ANXA8, ANXA2, ANXA5)

• Sensitivity: Lower limit of detection (target <0.1 ng/mL based on ANXA8 ELISA performance)

• Precision: Intra-assay and inter-assay CV <15%

• Linearity: R² >0.98 across the detection range

• Recovery: 80-120% in spiked samples

A fully optimized ANXA8L1 ELISA should achieve performance metrics similar to those reported for ANXA8 detection (detection limit ~0.065 ng/mL) while maintaining specificity against other annexin family members .

What approaches can differentiate between ANXA8L1 and closely related proteins like ANXA8?

Differentiating between ANXA8L1 and closely related proteins such as ANXA8 presents a significant challenge due to their structural similarities. Several approaches can be employed to achieve reliable discrimination:

Immunological Discrimination:

• Epitope Mapping and Antibody Development:

  • Identify unique peptide sequences in ANXA8L1 not present in ANXA8

  • Generate monoclonal antibodies against these unique epitopes

  • Validate antibody specificity through Western blotting against recombinant ANXA8L1 and ANXA8

• Two-Site Immunoassays with Differential Recognition:

  • Develop sandwich ELISA formats where at least one antibody recognizes an ANXA8L1-specific epitope

  • Implement competitive ELISA formats to quantify the relative abundance of each protein

Mass Spectrometry-Based Approaches:

• Peptide-Centric Discrimination:

  • Identify proteotypic peptides unique to ANXA8L1 not shared with ANXA8

  • Develop SRM/MRM methods targeting these unique peptides

  • Include internal standard peptides for absolute quantification

• Top-Down Proteomics:

  • Analyze intact proteins to detect subtle mass differences between ANXA8L1 and ANXA8

  • Perform electron transfer dissociation (ETD) to identify isoform-specific fragmentation patterns

Experimental Validation Strategy:
To ensure reliable discrimination, implement a multi-tiered approach:

When implementing these approaches, always include positive controls (recombinant ANXA8L1 and ANXA8) and negative controls (other annexin family members) to validate the specificity of your discrimination method .

How does ANXA8L1 contribute to cancer progression mechanisms?

ANXA8L1 has emerged as a significant factor in cancer progression, particularly in renal cell carcinoma (RCC), through several interconnected mechanisms:

1. Cell Cycle Regulation and Proliferation:
Bioinformatic analyses and functional studies have revealed that ANXA8L1 significantly impacts cell cycle regulation and DNA replication. Knockdown of ANXA8 (closely related to ANXA8L1) affects the expression of key cell cycle regulators, including CDC6, CDK2, CHEK1, and CCNB1 . These proteins are critical for controlling cell cycle checkpoints and progression, suggesting that elevated ANXA8L1 expression may promote uncontrolled cell proliferation in cancer cells.

3. Comparative Analysis with Other Annexins:
Research has revealed differential expression patterns among annexin family members in RCC:

This pattern suggests that different annexins may play opposing roles in cancer progression, with ANXA8L1 and ANXA8 potentially functioning as oncogenic drivers .

4. Potential Molecular Mechanisms:
While the exact molecular pathways through which ANXA8L1 promotes cancer progression remain under investigation, several possibilities exist:

• Membrane remodeling and enhanced cellular motility

• Regulation of calcium-dependent signaling pathways that promote survival

• Interaction with extracellular matrix components to facilitate invasion

• Modulation of cellular responses to chemotherapeutic agents

Further research is needed to fully elucidate these mechanisms, but the current evidence strongly supports ANXA8L1 as a potential therapeutic target and prognostic marker in RCC and potentially other cancer types.

What experimental approaches can effectively knock down or inhibit ANXA8L1 in research models?

Several experimental approaches can be employed to effectively knock down or inhibit ANXA8L1 in research models, each with specific advantages and applications:

1. RNA Interference (RNAi) Approaches:

shRNA-Mediated Knockdown:
Lentiviral vectors expressing short hairpin RNAs (shRNAs) targeting ANXA8L1 provide stable, long-term knockdown. The protocol typically involves:

• Design of target-specific shRNA sequences (typically 21-29 nucleotides)

• Cloning into lentiviral vectors

• Viral packaging in 293T cells

• Transduction of target cells (e.g., 769-P renal carcinoma cells for RCC studies)

• Selection with puromycin (typically 8 μg/ml) to establish stable knockdown cell lines

siRNA-Mediated Knockdown:
For transient knockdown experiments, synthetic small interfering RNAs (siRNAs) offer a straightforward approach:

• Transfection of target cells with siRNA duplexes using lipid-based reagents

• Typically achieves 70-90% knockdown within 24-72 hours

• Useful for acute experiments without the need for selection

2. CRISPR/Cas9 Gene Editing:

For complete knockout studies, CRISPR/Cas9 provides a powerful approach:

• Design of guide RNAs targeting exonic regions of ANXA8L1

• Delivery via plasmid transfection or lentiviral transduction

• Selection and clonal isolation of edited cells

• Validation by sequencing and protein expression analysis

3. Pharmacological Inhibition Strategies:

While specific ANXA8L1 inhibitors are not yet widely available, related approaches include:

• Calcium chelators to disrupt calcium-dependent functions

• Peptide-based inhibitors designed to interfere with membrane binding

• Small molecule screening to identify potential inhibitors

4. Validation and Analysis Methods:

Regardless of the knockdown approach, comprehensive validation is essential:

• Western blot analysis to confirm protein reduction (using antibodies such as anti-ANXA8 antibody ab111693)

• qRT-PCR to verify mRNA reduction

• Functional assays to assess the impact on cellular phenotypes:

  • Cell cycle analysis using flow cytometry

  • Proliferation assays (MTT, BrdU incorporation)

  • Migration and invasion assays

  • Calcium signaling measurements

5. Experimental Design Considerations:

When designing knockdown experiments, include appropriate controls (scrambled shRNA/siRNA or non-targeting guide RNAs) and validate knockdown efficiency before proceeding with functional analyses .

What role does ANXA8L1 play in leukocyte recruitment and inflammatory processes?

ANXA8L1, as a member of the annexin family, appears to share functional properties with annexin A8 (anxA8) in regulating leukocyte recruitment and inflammatory processes. The current understanding suggests several key mechanisms:

1. Regulation of Endothelial Cell Surface Adhesion Molecules:
Studies on annexin A8 have demonstrated its critical role in controlling leukocyte adhesion to activated endothelium. Loss of annexin A8 in human umbilical vein endothelial cells (HUVEC) significantly decreases the cell surface presentation of crucial adhesion molecules, particularly P-selectin and CD63 . Given the structural and functional similarities between ANXA8L1 and anxA8, it is reasonable to hypothesize that ANXA8L1 may similarly regulate endothelial adhesion molecule trafficking and presentation.

2. Impact on Leukocyte Rolling and Adhesion:
The reduction in P-selectin and CD63 surface expression caused by anxA8 deficiency directly translates to functional consequences, with reduced leukocyte rolling and adhesion observed both in vitro and in inflammatory-activated endothelial venules of anxA8-deficient mice . This suggests that annexins in this family, including ANXA8L1, play significant roles in the initial steps of the leukocyte adhesion cascade during inflammation.

3. Molecular Mechanism of Action:
The underlying mechanism appears to involve the regulation of intracellular trafficking pathways:

• Weibel-Palade Body (WPB) Composition: AnxA8 deficiency leads to reduced CD63 content in WPBs, the specialized secretory organelles of endothelial cells that store P-selectin .

• Trafficking from Multivesicular Endosomes: AnxA8 facilitates the proper transport of CD63 from late multivesicular endosomes to WPBs. In its absence, CD63 is retained in intraluminal vesicles, preventing its incorporation into WPBs .

• P-selectin Stability Regulation: Following WPB exocytosis, reduced CD63 surface levels result in enhanced P-selectin re-internalization, reducing the duration of the adhesive surface phenotype of endothelial cells .

4. Implications for Inflammatory Conditions:
The regulatory role of anxA8 and potentially ANXA8L1 in leukocyte recruitment has significant implications for various inflammatory conditions. The compromised leukocyte adhesiveness observed in anxA8-deficient models suggests that these proteins may be important modulators of inflammatory responses, potentially serving as targets for anti-inflammatory therapeutic approaches .

5. Research Model Considerations:
When investigating the role of ANXA8L1 in inflammatory processes, several model systems have proven valuable:

Further research is needed to fully elucidate the specific roles of ANXA8L1 distinct from ANXA8 in inflammatory processes, but the existing evidence strongly suggests its involvement in the regulation of leukocyte recruitment through modulation of endothelial adhesion molecule trafficking and presentation .

How can ANXA8L1 be exploited as a biomarker or therapeutic target in oncology?

ANXA8L1 shows significant potential as both a biomarker and therapeutic target in oncology, particularly in renal cell carcinoma (RCC). A comprehensive translational research strategy should consider multiple approaches:

Biomarker Development Strategy:

• Diagnostic Applications:

  • Development of highly sensitive ELISA-based blood tests for early detection

  • Integration of ANXA8L1 in multi-marker panels to improve specificity and sensitivity

  • Implementation of immunohistochemical protocols for tissue diagnostics

• Prognostic Applications:

  • Correlation of ANXA8L1 expression levels with patient outcomes in large clinical cohorts

  • Multivariate analysis to establish independent prognostic value beyond existing markers

  • Development of risk stratification algorithms incorporating ANXA8L1 expression data

• Predictive Biomarker Potential:

  • Assessment of ANXA8L1 expression correlation with response to specific therapeutic approaches

  • Longitudinal monitoring to evaluate treatment efficacy and disease progression

  • Integration into companion diagnostic strategies for targeted therapies

Therapeutic Target Development:

• Direct Targeting Approaches:

  • Development of monoclonal antibodies against extracellular or membrane-associated epitopes

  • Design of small molecule inhibitors targeting functional domains

  • Exploration of aptamer-based therapeutics with high specificity for ANXA8L1

• RNA-Based Therapeutics:

  • Design of siRNA or antisense oligonucleotides for ANXA8L1 knockdown

  • Development of miRNA-based approaches to regulate ANXA8L1 expression

  • Exploration of CRISPR-based therapies for specific targeting in cancer cells

• Combination Strategy Development:

  • Identification of synthetic lethal interactions with ANXA8L1 dependency

  • Exploration of synergistic effects with existing therapies

  • Investigation of ANXA8L1 inhibition impact on tumor microenvironment

Research Roadmap for ANXA8L1-Targeted Therapy Development:

What are the challenges in developing specific antibodies against ANXA8L1 and how can they be overcome?

Developing specific antibodies against ANXA8L1 presents several significant challenges due to its structural similarity with other annexin family members, particularly ANXA8. A comprehensive strategy to overcome these challenges includes:

1. Challenges in Epitope Selection:

• High Sequence Homology: ANXA8L1 shares substantial sequence identity with ANXA8 and other annexin family members, limiting the number of unique epitopes.

• Conserved Functional Domains: The core annexin repeats are highly conserved across the family, complicating the identification of ANXA8L1-specific regions.

• Conformational Epitopes: Many potentially unique epitopes may be conformational rather than linear, making immunization with peptides less effective.

2. Advanced Strategies for Antibody Development:

Computational Epitope Mapping:

• Utilize advanced bioinformatics algorithms to identify regions with maximal sequence divergence from other annexins

• Perform structural modeling to identify surface-exposed regions unique to ANXA8L1

• Design peptides or protein fragments optimized for immunogenicity while maintaining specificity

Immunization Approaches:

• Differential Immunization Strategy: Immunize with full-length ANXA8L1 followed by depletion of cross-reactive antibodies using related annexins

• Genetic Immunization: DNA immunization with ANXA8L1 expression constructs can present the protein in its native conformation

• Sequential Immunization: Prime with ANXA8L1-unique peptides followed by boosting with full-length protein

Selection and Screening Methodologies:

• Counter-Selection Approach: Similar to the strategy used for ANXA8, implement screening against ANXA8L1 with counter-screens against ANXA8, ANXA2, and ANXA5

• Epitope Binning: Identify antibodies binding to non-overlapping epitopes for sandwich assay development

• High-Throughput SPR Screening: Employ surface plasmon resonance to characterize binding kinetics and epitope competition

3. Validation and Characterization Framework:

4. Alternative Approaches When Traditional Methods Fail:

• Recombinant Antibody Engineering: Use phage display technology to select highly specific single-chain variable fragments (scFvs)

• Aptamer Development: RNA or DNA aptamers can offer high specificity for distinguishing between closely related proteins

• Nanobody Technology: Camelid single-domain antibodies may access unique epitopes due to their smaller size and distinct paratope structure

By implementing this comprehensive strategy, researchers can overcome the challenges in developing specific antibodies against ANXA8L1, enabling the creation of sensitive and specific detection tools similar to those developed for ANXA8 that achieved detection limits in the sub-nanogram range (approximately 0.065 ng/mL) .

How does post-translational modification affect ANXA8L1 function in different cellular contexts?

Post-translational modifications (PTMs) likely play crucial roles in regulating ANXA8L1 function across different cellular contexts, although this area remains underexplored compared to other annexin family members. A comprehensive investigation of ANXA8L1 PTMs would address several key aspects:

1. Types of PTMs Potentially Affecting ANXA8L1:

Phosphorylation:

• Annexin family proteins are known targets of various kinases, including PKC, tyrosine kinases, and mitogen-activated protein kinases

• Phosphorylation could modulate calcium sensitivity, membrane binding properties, and protein-protein interactions

• Key residues likely include serine, threonine, and tyrosine residues within the annexin repeats and the N-terminal region

S-Glutathionylation and Oxidation:

• Cysteine residues in annexins can undergo oxidative modifications in response to cellular stress

• These modifications may alter membrane binding properties and potentially act as redox sensors

• Such modifications could be particularly relevant in cancer contexts characterized by elevated oxidative stress

SUMOylation and Ubiquitination:

• These modifications could regulate ANXA8L1 stability, subcellular localization, and turnover

• They may be particularly important in stress responses and cell cycle progression

2. Methodological Approaches to Study ANXA8L1 PTMs:

3. Cellular Context-Dependent Regulation:

Cancer Cells vs. Normal Cells:
Different PTM patterns in cancer cells compared to normal cells could contribute to ANXA8L1's association with worse outcomes in renal cell carcinoma . Key investigations should focus on:

• Comparative PTM profiling between normal kidney and RCC tissues

• Correlation of specific PTMs with disease progression and treatment response

• Identification of cancer-specific PTM enzymes as potential therapeutic targets

Inflammatory Contexts:
Given annexin A8's role in leukocyte recruitment , ANXA8L1 PTMs may regulate inflammatory responses through:

• Dynamic modifications in response to inflammatory cytokines

• Regulation of calcium-dependent trafficking of adhesion molecules

• Modulation of interactions with components of the endosomal sorting machinery

Cell Cycle-Dependent Regulation:
ANXA8L1's involvement in cell cycle progression suggests PTMs may regulate its function throughout the cell cycle:

• Cell cycle phase-specific modification patterns

• Regulation by cyclin-dependent kinases

• Coordination with DNA replication machinery

4. Research Strategy and Future Directions:

A comprehensive research strategy should incorporate:

• Systematic mapping of all PTMs on ANXA8L1 across different cellular states

• Generation of modification-specific antibodies for each key PTM

• Development of cellular models expressing PTM-deficient mutants

• Integration of PTM data with interactome analyses to define functional consequences

• In vivo studies examining how PTM patterns correlate with disease states

This multi-dimensional approach would significantly advance understanding of how post-translational modifications regulate ANXA8L1's diverse functions across cellular contexts, potentially revealing new therapeutic opportunities and improved biomarker applications.