Recombinant Human Zinc-alpha-2-glycoprotein (AZGP1)

What is the molecular structure of AZGP1 and how does this relate to its biological functions?

AZGP1 is a 40-kDa single-chain polypeptide secreted in various body fluids, including seminal plasma and breast cyst fluid. Its structural similarity to HLA class I molecules suggests evolutionary conservation of immune-related functions . The protein consists of 278 amino acids (positions 22-298 in the mature protein) with several significant structural features:

• Contains an MHC class I-like domain organization

• Features N-glycosylation sites, particularly at Asn-128 (Hex5HexNAc4)

• Structurally capable of binding polyunsaturated fatty acids, suggesting a functional role in lipid metabolism

Crystallographic data (PDB: 1T7W) reveals structural elements that contribute to its multiple biological functions, including immune regulation and adipocyte lipolysis . The protein's tertiary structure enables interactions with various molecular partners, explaining its diverse physiological roles.

Research methods for structural characterization typically include:

• X-ray crystallography for high-resolution structure determination

• Circular dichroism for secondary structure analysis

• Molecular docking studies to predict protein-protein interactions

• Mass spectrometry for post-translational modification mapping

For researchers pursuing structural studies, recombinant AZGP1 produced in wheat germ expression systems provides a reliable source for in vitro characterization .

Which experimental models are most appropriate for studying AZGP1 functions?

Several experimental models have proven effective for investigating different aspects of AZGP1 biology:

In vivo models:

• ob/ob mice: Particularly useful for studying metabolic functions and anti-diabetic properties

• Unilateral ureteric obstruction (UUO) mouse models: Ideal for investigating AZGP1's role in kidney fibrosis

• Orthotopic xenograft models: Effective for studying AZGP1's role in tumor progression using cells with manipulated AZGP1 expression

In vitro models:

• Human peripheral blood mononuclear cell (PBMC)-derived macrophages: Used to study M1/M2 polarization effects of AZGP1

• Primary adipose-derived stem/progenitor cells (ASPCs): Valuable for examining AZGP1's effects on adipogenic differentiation and fibrotic gene expression

• Cancer cell lines with variable AZGP1 expression: Useful for studying cell-autonomous and non-autonomous effects

Transgenic approaches:

• Conditional AZGP1 overexpression models (e.g., AZGP1-SLC34a1-CreERT2 mice): Allow tissue-specific and temporally controlled AZGP1 expression

• AZGP1 knockout models: Demonstrate loss-of-function effects across multiple systems

What are the validated methods for measuring AZGP1 expression in clinical and research samples?

Multiple complementary techniques have been established for reliable AZGP1 detection:

Protein-level detection:

• Immunohistochemistry (IHC): Effectively used for tissue localization and semi-quantitative analysis in breast cancer and prostate cancer specimens

• Western blotting: Suitable for quantitative analysis in cell and tissue lysates

• ELISA: Optimal for measuring AZGP1 concentrations in serum, plasma, and other body fluids

• Flow cytometry: Used for cellular-level quantification in immune cell studies

Gene expression analysis:

• Quantitative RT-PCR: Common approach for AZGP1 mRNA quantification using validated primers

• Microarray analysis: Used for comprehensive expression analysis in large cohorts

• RNA sequencing: Provides comprehensive transcriptomic context for AZGP1 expression

For exosomal AZGP1 detection, which has emerging diagnostic relevance in pancreatic cancer, specialized isolation protocols followed by qRT-PCR have been validated . The primer sequences that have shown high specificity are:

How does AZGP1 contribute to cancer biology, and what are the methodological approaches for studying these effects?

AZGP1 demonstrates context-dependent roles in cancer, with expression patterns varying by cancer type and subtype:

Breast cancer:

• Expression varies by molecular subtype, with unique implications in triple-negative breast cancer (TNBC)

• In TNBC, AZGP1 secretion inhibits adipogenesis and promotes transdifferentiation of adipose-derived stem/progenitor cells (ASPCs) into cancer-associated fibroblasts, facilitating tumor growth

• Gene set enrichment analysis (GSEA) reveals AZGP1 correlates with immune cell composition in breast cancer tissues

Prostate cancer:

• Serves as an independent prognostic biomarker, with absent/low AZGP1 expression predicting earlier PSA recurrence

• Validated in a prospective multicenter phase III study as a prognostic indicator

Pancreatic cancer:

• Exosomal AZGP1 shows promise as a diagnostic marker

Colorectal cancer:

• Upregulated in radioresistant tissue, potentially serving as a target for andrographolide to overcome radiation resistance

Methodological approaches:

• Loss-of-function studies: siRNA or CRISPR-Cas9 targeting of AZGP1 in cancer cell lines demonstrates functional outcomes (e.g., MDA-MB-468 cells with AZGP1 depletion show reduced tumor growth)

• Orthotopic models: Injection of AZGP1-manipulated cancer cells into relevant anatomical sites (e.g., mammary fat pad for breast cancer studies)

• Microenvironment analysis: Assessment of tumor-adjacent stroma using markers like Picro Sirius Red staining and α-SMA expression to evaluate AZGP1-mediated effects on fibrosis

• Receptor binding studies: Investigation of potential signaling mechanisms through receptor identification

Research has shown that AZGP1 depletion in MDA-MB-468 cells significantly reduces tumor growth in orthotopic xenografts, with corresponding decreases in fibrosis markers in surrounding adipose tissue (Picro Sirius Red and α-SMA staining) . This effect appears to be mediated through paracrine rather than cell-autonomous mechanisms, as AZGP1 depletion does not affect proliferation of these cells in vitro .

What is the current evidence regarding AZGP1's role in metabolic disorders and what research methodologies are employed?

AZGP1 demonstrates significant metabolic effects that position it as a potential therapeutic target for metabolic disorders:

Anti-diabetic properties:

• Recombinant human ZAG administration (50 μg daily, intravenous) in ob/ob mice induces:

  • Progressive body weight loss (3.5g in 5 days)

  • 0.4°C rise in body temperature (suggesting increased energy expenditure)

  • 17% decrease in glucose levels

  • 25% decrease in triglycerides

  • 62% decrease in non-esterified fatty acids

  • 36% reduction in plasma insulin levels

  • 4-fold increase in pancreatic insulin

  • 53% decrease in area under the glucose tolerance curve

Mechanisms of action:

• Increases glucose and lipid utilization by muscle and brown adipose tissue

• Induces 2-fold increase in brown adipose tissue weight

• Enhances expression of uncoupling proteins-1 and -3

• Increases skeletal muscle mass through enhanced protein synthesis and decreased degradation

• Promotes adipocyte lipolysis

Cardiovascular implications:

• Higher AZGP1 levels associate with lower risk of mortality and cardiovascular events in older adults

• Has been incorporated into prediction models for mortality and cardiovascular outcomes

Research methodologies:

• Metabolic phenotyping: Glucose tolerance tests, insulin measurements, and lipid profiling

• Tissue-specific analyses: Assessment of brown adipose tissue activation and skeletal muscle metabolism

• Protein turnover studies: Measuring rates of protein synthesis and degradation

• Transgenic approaches: Conditional overexpression or knockout models

• Prospective cohort studies: Evaluation of AZGP1 as a predictive biomarker for cardiovascular outcomes

How does AZGP1 influence kidney fibrosis progression, and what experimental designs best capture this relationship?

AZGP1 demonstrates protective effects against kidney fibrosis through several mechanisms:

Experimental evidence:

• In unilateral ureteric obstruction (UUO) mouse models, two therapeutic approaches have been validated:

  • Systemic administration of recombinant AZGP1

  • Conditional overexpression of AZGP1 in proximal tubular cells using AZGP1-SLC34a1-CreERT2 mice

Mechanistic insights:

• AZGP1 appears to influence tubular cell lipid metabolism, establishing a novel link between metabolic regulation and fibrotic processes

• AZGP1-deficient mice show enhanced susceptibility to fibrosis, confirming its protective role

Experimental designs for studying AZGP1 in kidney fibrosis:

• UUO model: The standard approach involving surgical ligation of one ureter to induce progressive fibrosis

• Conditional expression systems: Using Cre/loxP technology with tamoxifen induction (e.g., SLC34a1-CreERT2 system for proximal tubule specificity)

• Histological assessments: Quantification of fibrotic markers through histochemical staining and immunohistochemistry

• Molecular analyses: Evaluation of fibrotic gene expression profiles and pathway activation

• Metabolomic profiling: Assessment of lipid metabolism alterations in response to AZGP1 manipulation

For successful conditional overexpression, the following protocol has been validated:

• Tamoxifen administration 14 days before UUO surgery

• Intraperitoneal injection of tamoxifen (20 mg/mL) every other day, 4 times total

• Confirmation of recombination using reporter systems (e.g., crossing with Ai14 reporter mice)

What is the current understanding of AZGP1's immunomodulatory functions and how can these be studied?

AZGP1's structural similarity to HLA class I molecules suggests an evolutionary role in immune regulation, which has been explored through various methodological approaches:

Established immune functions:

• Influences macrophage polarization, affecting the balance between pro-inflammatory M1 and anti-inflammatory M2 phenotypes

• Potentially modulates HLA class I/II expression on immune cells

• May influence PD-L1 expression on immune cells, affecting immune checkpoint regulation

Research methodologies:

• Flow cytometry analysis: Evaluating M1/M2 polarization markers (CD86, CD80/CD163, MRC1) and HLA class I/II expression on macrophages

• CIBERSORTx analysis: Digital cytometry tool used to estimate immune cell composition in tissues based on gene expression data

• Gene set enrichment analysis (GSEA): Identification of biological processes associated with AZGP1 expression

• In vitro polarization models: Using primary PBMC-derived macrophages to study direct effects of AZGP1 on immune cell phenotypes

The gating strategy for assessing PD-L1 or CD86 positivity has been described in studies investigating AZGP1's role in breast cancer immunity . This approach allows for measurement of percentage of positive cells within specific immune cell fractions.

Flow cytometry has also been used to evaluate the action of AZGP1 in M1, M2, and non-polarization models based on the expression of surface antigens, providing a means to assess immunomodulatory functions .

How does AZGP1 expression correlate with tumor immune microenvironment and what techniques can characterize these relationships?

AZGP1 expression in tumors demonstrates complex relationships with immune infiltration patterns:

Observed correlations:

• AZGP1 expression correlates with specific immune cell compositions in breast cancer tissues

• May influence the recruitment and functional phenotype of tumor-infiltrating immune cells

• In triple-negative breast cancer (TNBC), AZGP1 secretion indirectly shapes the immune microenvironment by promoting fibrosis and cancer-associated fibroblast development

Research techniques:

• Computational deconvolution methods:

  • CIBERSORTx for immune cell composition estimation from bulk tissue expression data

  • Gene Set Enrichment Analysis (GSEA) to screen biological processes associated with AZGP1

• Flow cytometry approaches:

  • Direct analysis of 11 types of immune cells in breast cancer tissues

  • Assessment of functional markers like PD-L1 and CD86 on immune cell subsets

• Spatial analysis techniques:

  • Immunohistochemistry for co-localization studies of AZGP1 with immune markers

  • Multiplex immunofluorescence for higher-dimensionality immune profiling

  • Spatial transcriptomics for integrated analysis of AZGP1 and immune signatures

• Functional assays:

  • Co-culture systems to assess AZGP1's effects on immune cell migration and function

  • Cytokine profiling to characterize secretory phenotypes associated with AZGP1 expression

These techniques can be integrated to provide a comprehensive view of how AZGP1 shapes the tumor immune microenvironment, potentially identifying new therapeutic targets or prognostic indicators.

What are the optimal production and purification strategies for generating research-grade recombinant human AZGP1?

The production of high-quality recombinant human AZGP1 requires careful consideration of expression systems and purification strategies:

Expression systems:

• Wheat germ cell-free system: Produces full-length human AZGP1 (amino acids 22-298) suitable for multiple applications including SDS-PAGE, ELISA, and Western blotting

• Mammalian expression systems: Provide proper post-translational modifications, particularly glycosylation, which may be critical for certain functional studies

• E. coli-based systems: Higher yield but lacks glycosylation; may be suitable for structural studies requiring large quantities

Purification approaches:

• Affinity chromatography using tagged constructs (His-tag, GST-tag)

• Ion exchange chromatography

• Size exclusion chromatography for final polishing

Quality control parameters:

• SDS-PAGE analysis for purity assessment (typically visualized with Coomassie Blue staining)

• Mass spectrometry for molecular weight confirmation and PTM mapping

• Functional assays to confirm biological activity

For verification of purified recombinant AZGP1, researchers should consider:

• Western blot confirmation using validated anti-AZGP1 antibodies

• Activity assays (e.g., lipolysis induction in adipocytes)

• Assessment of endotoxin levels for in vivo applications

• Glycosylation analysis if studying immune functions

What are the potential therapeutic applications of recombinant AZGP1 based on current research?

Recombinant AZGP1 shows therapeutic potential across multiple disease contexts:

Metabolic disorders:

• Anti-diabetic properties in mouse models suggest applications in type 2 diabetes

• Daily administration (50 μg, IV) demonstrated significant metabolic improvements in ob/ob mice:

  • Improved glucose tolerance (53% reduction in glucose AUC)

  • Reduced insulin resistance (36% reduction in plasma insulin)

  • Enhanced glucose utilization

  • Increased muscle mass through improved protein metabolism

Fibrotic kidney disease:

• Systemic administration of recombinant AZGP1 shows anti-fibrotic effects in the UUO model

• Reduces progression of kidney fibrosis, potentially through effects on tubular cell lipid metabolism

Cancer:

• Context-dependent effects require careful consideration

• Potential therapeutic applications in cancer types where AZGP1 has tumor-suppressive functions

• In TNBC, targeting AZGP1 rather than supplementing it may be beneficial due to its promotion of a pro-tumorigenic microenvironment

Therapeutic delivery considerations:

• Intravenous administration has been validated in mouse models

• Potential for development of modified versions with improved half-life

• Tissue-specific targeting strategies may enhance efficacy while reducing off-target effects

Challenges for therapeutic development:

• Optimization of dosing regimens based on pharmacokinetic/pharmacodynamic studies

• Assessment of potential immunogenicity of recombinant protein

• Development of cost-effective production methods for clinical-grade material

What are the methodological approaches for investigating post-translational modifications of AZGP1 and their functional significance?

Post-translational modifications (PTMs) of AZGP1, particularly glycosylation, may significantly impact its biological functions:

Known PTMs:

• N-glycosylation, particularly at Asn-128 (Hex5HexNAc4)

• Potential variable glycosylation patterns between tissue sources and disease states

Research approaches:

• Mass spectrometry-based methods:

  • LC-MS/MS for glycan profiling

  • Glycopeptide analysis for site-specific glycosylation mapping

  • Comparative glycomics between AZGP1 from different sources

• Glycan engineering:

  • Site-directed mutagenesis of glycosylation sites

  • Expression in systems with different glycosylation capabilities

  • Enzymatic remodeling of glycans on purified protein

• Functional assessment:

  • Binding studies comparing differently glycosylated forms

  • In vitro activity assays with glycosylation variants

  • In vivo studies comparing native vs. deglycosylated AZGP1

Research evidence:

• AZGP1 from cancer patient samples may show altered glycosylation patterns compared to healthy individuals

• In salivary AZGP1, 22 glycan structures have been identified, with five unique to lung cancer samples

• Glycosylation may affect the immunomodulatory functions of AZGP1 due to its structural similarity to HLA class I

This area remains underexplored but represents a promising direction for understanding tissue-specific and disease-specific functions of AZGP1.

How can molecular docking studies enhance our understanding of AZGP1 interactions and potential therapeutic targeting?

Molecular docking studies provide valuable insights into AZGP1's interaction partners and potential for therapeutic targeting:

Validated approaches:

• Protein-protein docking:

  • X-ray crystal structures of AZGP1 (PDB: 1T7W) have been used for docking studies

  • GRAMM Docking Web Server has been validated for protein-protein interaction prediction

  • Visualization tools like PyMOL are commonly used to generate protein-protein interaction figures

• Small molecule docking:

  • Molecular docking between AZGP1 and potential therapeutic compounds (e.g., andrographolide)

  • Identifies binding sites and interaction energies

• Methodology considerations:

  • Removal of water molecules and addition of polar hydrogen atoms manually in AutoDockTools-1.5.7 to ensure accurate docking results

  • Validation of docking predictions through experimental binding assays

Research applications:

• Identification of AZGP1 as a target for andrographolide in overcoming radiation resistance in colorectal cancer

• Investigation of protein-protein interactions between AZGP1 and other molecules like EGF

• Structure-based design of molecules that could modulate AZGP1 functions

By combining computational approaches with experimental validation, researchers can develop a more comprehensive understanding of AZGP1's interaction network and identify novel therapeutic strategies.

What are the current knowledge gaps and promising future research directions for AZGP1?

Despite significant advances, several key knowledge gaps remain in AZGP1 research:

Receptor identification:

• The cell surface receptor(s) for AZGP1 remain poorly characterized

• Identification would enable better understanding of signaling mechanisms

Tissue-specific functions:

• The diverse and sometimes contradictory effects of AZGP1 across different tissues need further clarification

• Context-dependent functions in disease processes require more detailed investigation

PTM characterization:

• Comprehensive mapping of AZGP1 glycosylation patterns across tissues and disease states

• Functional significance of these modifications

Future research directions:

• Single-cell approaches to delineate cell type-specific responses to AZGP1

• Development of small molecule modulators of AZGP1 function

• Systems biology approaches to integrate AZGP1 into broader metabolic and immune networks

• Investigation of AZGP1 as a biomarker across multiple disease contexts

• Structural biology studies to identify functional domains for targeted therapeutic development