TPO Human, CHO

What are the optimal conditions for expressing recombinant human TPO in CHO cells?

Recombinant human thyroid peroxidase (TPO) can be efficiently expressed in Chinese hamster ovary (CHO) cells using specific approaches to maximize yield and functionality. The most successful method involves:

• Amplifying full-length TPO cDNA (including the native signal peptide sequence) by PCR from a human fetal liver cDNA library

• Cloning the amplified product into a mammalian expression vector under control of the SV40 early promoter and enhancer

• Establishing stable transfection in CHO cells, followed by clone selection based on TPO expression levels

When properly expressed, rhTPO secreted from CHO cells typically appears as a broad band on SDS-PAGE (characteristic of heavily glycosylated proteins) with an average molecular mass of 85 kDa. The protein demonstrates biological activity both in vitro and in vivo, making this expression system ideal for research applications .

How can researchers confirm the enzymatic activity of human TPO expressed in CHO cells?

Multiple complementary approaches should be employed to comprehensively assess TPO enzymatic activity:

Beyond these direct activity assays, functional assessment of expressed TPO can be demonstrated through:

• JAK/STAT signal transduction pathway activation

• In vivo testing via platelet count elevation in treated mice

• Immunological detection via Western blot or flow cytometry

Always include appropriate controls (positive and negative) to ensure signal specificity, as some assays (particularly Luminol) can detect peroxidase activity even in cell lines lacking TPO expression .

What is the significance of the propeptide region in human TPO protein structure and function?

The propeptide region of human TPO (amino acids 21-108) has been extensively studied through deletion experiments, yielding surprising results regarding its functional relevance:

Engineered recombinant human TPO lacking the propeptide (TPOΔpro) demonstrates:

• Plasma membrane localization comparable to wild-type TPO

• Preserved enzymatic activity

• Retained recognition by autoantibodies from AITD patients

• Maintained N-glycosylation patterns

• Proper intracellular trafficking

• Reduced susceptibility to intramolecular proteolysis

Molecular modeling and dynamics simulations confirm these experimental observations, pointing to a redundant role for the propeptide sequence in TPO function and structure. This finding has significant implications for structural studies, as TPOΔpro provides a more homogeneous preparation that remains enzymatically active and retains the major conformational determinants recognized by autoantibodies .

What methodologies can detect TPO expression in transfected CHO cells?

Several complementary techniques can verify successful human TPO expression in CHO cells:

Molecular Methods:

• RT-PCR or qPCR for TPO mRNA quantification

• Northern blotting for mRNA expression analysis

Protein Detection:

• Western blot analysis: Under non-reducing conditions, human TPO typically appears as a broad immunoreactive band of ~200 kDa and a doublet of ~110 kDa

• Flow cytometry using TPO-specific antibodies can confirm surface expression

• Cell surface protein biotinylation followed by protein extraction and immunoblotting

Functional Assessment:

• Peroxidase activity assays (AUR, Luminol)

• Immunoreactivity with sera from patients with autoimmune thyroid diseases

FACS analysis of TPO-expressing CHO cells incubated with Hashimoto's thyroiditis patient sera shows approximately 100-fold greater fluorescence compared to controls, providing a sensitive method to detect functionally expressed protein .

How does glycosylation affect human TPO function when expressed in CHO cells?

Glycosylation plays critical roles in TPO protein processing and function:

• Structural Role: N-linked glycosylation contributes to proper folding and stability of the TPO protein, influencing the formation and maintenance of its dimeric structure

• Enzymatic Function: Glycosylation creates the appropriate microenvironment for the active site, potentially affecting substrate binding and catalytic efficiency

• Cellular Trafficking: Glycosylation signals guide TPO through the secretory pathway, facilitating correct localization to the plasma membrane

• Immunological Aspects: Interestingly, experiments with tunicamycin (which inhibits N-glycosylation) have shown no decrease in TPO immunoreactivity, suggesting that glycosylation is not essential for recognition by autoantibodies

When expressed in CHO cells, human TPO typically appears as a heterogeneous protein due to variable glycosylation, visualized as a broad band on SDS-PAGE. This pattern is characteristic of properly processed TPO and confirms successful post-translational modification in the CHO expression system .

How do TPO autoantibodies from Hashimoto's thyroiditis patients interact with recombinant human TPO?

TPO autoantibodies from Hashimoto's thyroiditis patients show distinctive interaction patterns with recombinant human TPO expressed in CHO cells:

Recognition Patterns:

• Western blot analysis reveals that under non-reducing conditions, Hashimoto's sera recognize a broad immunoreactive band of ~200 kDa and a doublet of ~110 kDa

• The intensity of this recognition generally correlates with anti-microsomal antibody (anti-MSA) titer

• All 36 Hashimoto's sera tested in one study reacted with these bands, while normal sera were non-reactive

Conformational Dependence:

• Western blots under reducing conditions show significantly diminished signals

• Some sera with lower anti-MSA titers become negative under reducing conditions

• The 200-kDa broad band disappears, and the 110-kDa doublet converts to a single band

• This demonstrates that many TPO autoepitopes are conformational and dependent on proper protein folding

Glycosylation Independence:

• Studies using tunicamycin to inhibit N-glycosylation show no decrease in TPO immunoreactivity

• This indicates that recognition by autoantibodies is largely independent of glycosylation patterns

These findings have important implications for understanding the nature of the autoimmune response in Hashimoto's thyroiditis and for developing more specific diagnostic tests.

What are the key differences between assays for measuring TPO activity?

Different assays for measuring TPO activity have distinct characteristics that affect their application in research:

Despite their different specificity profiles, both assays identify similar peroxidation inhibitors when screening compounds. This suggests that while the Luminol assay lacks specificity for TPO, it can still be useful for preliminary screening if followed by confirmation with the more specific AUR assay.

For the most reliable assessment of TPO activity and inhibition, researchers should consider using the AUR assay as the primary method, while the Luminol assay can serve as a complementary approach with appropriate controls to account for its lower specificity .

How does the dimeric structure of TPO influence its enzymatic activity?

The dimeric structure of TPO has profound implications for its enzymatic function:

Under non-reducing conditions, TPO appears as a broad band of approximately 200 kDa on Western blots, consistent with its dimeric structure. When this quaternary structure is disrupted (as observed under reducing conditions), both enzymatic activity and autoantibody recognition are significantly diminished .

Understanding this unique dimeric architecture provides insights into TPO's specialized function in thyroid hormone biosynthesis and its role as an autoantigen in thyroid autoimmune diseases.

What challenges exist in purifying membrane-bound TPO while maintaining its native conformation?

Purifying membrane-bound TPO presents several significant technical challenges:

Solubilization Complexities:

• As a membrane-bound enzyme, TPO requires detergents for solubilization

• Harsh detergents may disrupt the native conformation and dimeric structure

• Finding the optimal detergent combination and concentration that efficiently solubilizes TPO without denaturing it requires extensive optimization

Structural Preservation:

• Maintaining the dimeric structure throughout purification is crucial

• Evidence shows that reducing conditions significantly diminish both enzymatic activity and autoantibody binding

• Purification methods must preserve the conformational determinants critical for immunological studies

Heterogeneity Considerations:

• TPO is heavily glycosylated, appearing as a broad band on SDS-PAGE

• This glycosylation heterogeneity complicates purification and characterization

• Different glycoforms may have different properties but are difficult to separate effectively

Proteolytic Susceptibility:

• TPO is prone to proteolysis during extraction and purification

• TPOΔpro (lacking the propeptide) provides an advantage as it is less susceptible to intramolecular proteolysis

• This modification yields more homogeneous preparations suitable for structural studies

Conventional chromatography approaches can be successful when optimized specifically for TPO. One effective strategy involves a three-step chromatography process after secretion from CHO cells, resulting in biologically active purified protein .

What approaches are recommended for studying TPO inhibition by environmental chemicals?

Optimizing approaches for studying TPO inhibition by environmental chemicals requires several methodological considerations:

Cell Model Selection:

• Develop cell lines that stably overexpress human TPO protein, such as HEK-TPOA7

• Select models based on high and stable TPO gene and protein expression

• Validate TPO activity in the chosen system before inhibition studies

Assay Optimization:

• The AUR assay demonstrates specificity to TPO activity and is preferred for specific measurements

• Follow OECD guidance on good in vitro method practices

• Standardize conditions for reproducible results

• Include appropriate positive and negative controls

Chemical Diversity:

• Test compounds with different structures from various use categories

• Include widespread environmental contaminants to assess potential impact on thyroid hormone synthesis

• Establish dose-response relationships for identified inhibitors

Research has demonstrated that over half of tested chemicals with diverse structures can cause TPO inhibition, including prevalent environmental contaminants. This suggests a potentially significant impact of environmental chemicals on thyroid hormone synthesis, with implications for thyroid function and associated disorders .

How can TPO expressed in CHO cells contribute to understanding autoimmune thyroid diseases?

Recombinant human TPO expressed in CHO cells provides valuable tools for investigating autoimmune thyroid diseases:

Autoantibody Analysis:

• TPO-expressing CHO cells serve as reliable substrates for flow cytometry assays with sera from Hashimoto's thyroiditis patients

• Studies show approximately 100-fold greater fluorescence when these cells are incubated with Hashimoto's sera compared to controls

• Western blot analysis using extracted antigen can detect immunoreactive bands that correlate with anti-microsomal antibody titers

Epitope Mapping:

• Modified versions of TPO (such as TPOΔpro) allow investigation of specific domains' contributions to autoantibody recognition

• Flow cytometry, immunocytochemistry, and ELISA can assess antibody binding to conformational epitopes

• These approaches help identify immunodominant regions that could be targeted for therapeutic intervention

Longitudinal Insights:

• Studies in both mice and humans indicate that thyroglobulin antibodies (TgAbs) precede TPO antibodies in the development of autoimmune thyroid disease

• This temporal relationship, observed in relatives of probands with juvenile Hashimoto's thyroiditis, provides insights into disease progression

• TPO-expressing cells offer systems to study this sequential development of autoimmunity

These applications make recombinant TPO an essential resource for immunological research in autoimmune thyroid diseases, with potential implications for diagnostic and therapeutic advancements.

What modifications to human TPO expression can enhance protein yield and stability?

Several strategic modifications can enhance the yield and stability of human TPO expressed in CHO cells:

Sequence Optimization:

• Removing the propeptide sequence (amino acids 21-108) creates TPOΔpro, which is less susceptible to intramolecular proteolysis

• This modification yields more homogeneous protein preparations while maintaining enzymatic activity and autoantibody recognition

• Codon optimization for CHO expression can improve translation efficiency

Expression System Enhancements:

• Utilizing mammalian expression vectors with strong promoters (such as the SV40 early promoter and enhancer)

• Incorporating optimal signal sequences to enhance secretion or membrane localization

• Screening for high-producing clones to maximize yield

Purification Improvements:

• Implementing conventional chromatography steps specifically optimized for TPO

• Using gentle solubilization methods appropriate for this membrane-bound protein

• Developing purification strategies that maintain the native dimeric structure

Stability Considerations:

• Adding stabilizing agents during purification and storage

• Modifying regions prone to aggregation or degradation

• Controlling proteolytic processing through protease inhibitors

The successful expression of TPOΔpro as a membrane-anchored, enzymatically active form that maintains recognition by patients' autoantibodies represents a significant advancement for obtaining substantial quantities of homogeneous TPO preparations suitable for crystallization, structural studies, and immunological investigations .

What is the relationship between TPO dysfunction and diabetes mellitus?

The relationship between thyroid peroxidase dysfunction and diabetes mellitus represents an important area of clinical endocrinology research:

Epidemiological Association:

• Thyroid dysfunction is more common in patients with diabetes than in the general population

• The NHANES III study reported a higher prevalence of thyroid disorders in subjects with diabetes compared to those without diabetes, especially in patients with positive anti-TPO antibodies

Type-Specific Relationships:

• Autoimmune thyroid dysfunction occurs in 17% to 30% of adults with type 1 diabetes, reflecting shared autoimmune mechanisms

• In type 2 diabetes, subclinical and overt hypothyroidism are the most common forms of thyroid dysfunction, with prevalence ranging from 6% to 20%

Risk Factors:

• Female sex, older age, obesity, TPO antibody positivity, and hospitalization increase the risk of developing hypothyroidism in type 2 diabetes

• Patients with T2D >65 years of age show a significantly increased risk of hypothyroidism (OR, 4.2)

• The risk differs between males and females (OR, 4.82 vs 2.60), and between obese and non-obese patients (OR, 2.56 vs 3.11)

Clinical Implications:

• Preexisting diabetes mellitus is exacerbated by hyperthyroidism

• Insulin treatment should be adjusted in patients with diabetes after the occurrence of thyroid dysfunction

• Hyperglycemia should be reevaluated in hyperthyroid subjects after controlling thyroid dysfunction

A meta-analysis of 36 articles confirmed a higher pooled prevalence of subclinical hypothyroidism in patients with T2D compared to healthy controls (1.93-fold increased risk; 95% CI, 1.66 to 2.24), demonstrating the significant association between these disorders .