HSD17B14 Antibody

What is HSD17B14 and what are its known physiological functions?

Comparative enzyme activity studies have shown that HSD17B14 is approximately 50-2300 times more active toward L-fucose than β-estradiol. For instance, human HSD17B14 showed activity of 752.19 ± 9.67 nmol × min⁻¹ × mg⁻¹ protein toward 5 μM L-fucose compared to just 0.33 ± 0.58 nmol × min⁻¹ × mg⁻¹ protein toward 5 μM β-estradiol . This striking difference suggests that steroids are unlikely to be the physiological substrates for HSD17B14, despite its classification within the hydroxysteroid dehydrogenase family.

What types of HSD17B14 antibodies are currently available for research?

Several types of HSD17B14 antibodies are available for research applications:

• Rabbit polyclonal antibodies: These are common and suitable for various applications including Western blotting (WB) and immunohistochemistry on paraffin-embedded tissues (IHC-P) .

• Mouse monoclonal antibodies: These offer high specificity and are particularly useful for immunofluorescence staining and immunohistochemistry applications .

• Custom-made antibodies: Some researchers have generated their own antibodies for specific studies, such as the anti-HSD17B14 rabbit polyclonal antibody used in kidney research and shared between institutions .

The choice of antibody depends on the specific research application, species compatibility, and detection method. Most commercially available antibodies have been validated for human samples, with some also cross-reacting with mouse tissues .

In which tissues is HSD17B14 predominantly expressed?

HSD17B14 shows a distinctive tissue expression pattern that has provided clues to its physiological functions:

• Kidney: HSD17B14 is highly expressed in human kidney tissue, particularly in proximal tubular cells . This expression pattern has significance in research on diabetic kidney disease.

• Steroidogenic tissues: The protein is expressed in several classical steroidogenic tissues including breast, ovary, and testis, which initially supported the hypothesis of its role in sex steroid interconversion .

• Brain: Immunohistochemistry studies have demonstrated HSD17B14 expression in human brain tissue, specifically in the medulla, where it localizes to neuronal cytoplasm .

• Colon and stomach: HSD17B14 has been detected in gastrointestinal tissues, as demonstrated by immunohistochemical analysis .

Expression levels of HSD17B14 can change significantly in disease states. For example, its expression is positively correlated with eGFR (estimated glomerular filtration rate) and markedly decreased in kidneys from patients with diabetic kidney disease .

How has HSD17B14 been implicated in breast cancer prognosis and treatment response?

HSD17B14 has emerged as a potential predictive marker for tamoxifen response in estrogen receptor-positive breast cancer. In a study involving 847 post-menopausal women with lymph node-negative breast cancer, researchers found that tumoural expression levels of HSD17B14 could predict the outcome of adjuvant tamoxifen treatment .

Specifically, patients with estrogen-positive tumors demonstrating high HSD17B14 expression had significantly fewer local recurrences when treated with tamoxifen (HR 0.38; 95% C.I. 0.19–0.77, p = 0.007) compared to patients with lower tumoural HSD17B14 expression, for whom tamoxifen did not significantly reduce local recurrences (HR 1.19; 95% C.I. 0.54–2.59; p = 0.66) .

This predictive value was observed specifically for tamoxifen treatment outcomes, as no prognostic importance of HSD17B14 was seen for systemically untreated patients. The mechanism is hypothesized to involve HSD17B14's oestradiol-lowering properties, which may potentiate the anti-proliferative effects of tamoxifen .

What is the relationship between HSD17B14 genetic variants and diabetic kidney disease?

Gene-based testing of protein coding genetic variants in individuals with type 1 diabetes has revealed a significant association between HSD17B14 variants and protection against diabetic kidney disease (DKD). Carriers of rarer, disruptive alleles in HSD17B14 experienced net protection against loss of kidney function and development of end-stage kidney disease (ESKD) .

Through deep resequencing of HSD17B14 in 2239 individuals, researchers detected 20 coding variants within this gene. Several rare variants contributed to the observed protective effect, along with one common protective variant (rs35299026, R130W, with a frequency of approximately 5% in European cohorts) .

The protective association reached exome-wide significance in a meta-analysis involving 4196 subjects (P value=3.3 × 10⁻⁷). Paradoxically, while the genetic variants appear protective, HSD17B14 expression is typically high in healthy proximal tubules but downregulated in kidney injury and diabetic nephropathy .

This suggests a complex relationship between HSD17B14 function and kidney health, with researchers proposing that HSD17B14 may represent a druggable therapeutic target for DKD treatment .

How do HSD17B14 expression patterns change in disease states, particularly in kidney injury models?

HSD17B14 expression shows notable changes in various disease states, particularly in kidney pathologies:

These expression pattern changes provide important clues about the functional roles of HSD17B14 in disease processes and potential mechanisms of protection or susceptibility.

What are the recommended protocols for using HSD17B14 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of HSD17B14, researchers should follow these methodological guidelines:

• Sample preparation:

  • Use formalin-fixed, paraffin-embedded tissue sections

  • Deparaffinize sections with xylene and ethanol

  • Perform antigen retrieval using pressure cooking or heat-induced epitope retrieval with appropriate buffer (e.g., Antigen Retrieval Reagent-Basic)

• Antibody application:

  • Block sections with 3% BSA-PBS

  • Apply primary anti-HSD17B14 antibody at optimized concentration (typically 1/30 to 15 μg/mL)

  • Incubate either overnight at 4°C or for 1 hour at room temperature

  • After washing with PBS, apply appropriate secondary antibody

• Detection systems:

  • For chromogenic detection: Use HRP-DAB staining systems (e.g., Vector ABC Elite Kit followed by Vector DAB kit or Anti-Mouse HRP-DAB Cell & Tissue Staining Kit)

  • Counterstain with hematoxylin for nuclear visualization

  • For immunofluorescence: Use fluorophore-conjugated secondary antibodies

• Validation controls:

  • Include positive control tissues (kidney, brain medulla, or breast tissues)

  • Include negative controls by omitting primary antibody

  • Consider double-staining with other markers for co-localization studies (e.g., with KIM-1 for injured kidney tubules)

Researchers should optimize antibody dilutions for their specific applications and tissues, as recommended by antibody manufacturers .

What methods are used to validate the specificity of HSD17B14 antibodies?

Validation of HSD17B14 antibodies is crucial for ensuring reliable research results. Several complementary approaches are typically employed:

• Recombinant protein validation:

  • Testing against recombinant HSD17B14 protein

  • Using HSD17B14-transfected cells as positive controls and vector-only transfected cells as negative controls

• Peptide-neutralizing assays:

  • Pre-incubating the antibody with the immunizing peptide to confirm specificity

  • Signal should be eliminated or significantly reduced when the antibody is neutralized

• Western blot analysis:

  • Verification of correct molecular weight band detection (predicted size: 28 kDa)

  • Comparison of expression in control versus overexpressing systems (e.g., HSD17B14 overexpression lysate from HEK-293T cells)

• Knockout/knockdown controls:

  • Using tissues from knockout models (e.g., mouse model lacking Hsd17b14) as negative controls

  • Using RNA interference to reduce expression as validation

• Cross-reactivity assessment:

  • Testing against related proteins, particularly other hydroxysteroid dehydrogenases

  • Evaluating species cross-reactivity for antibodies intended for use across multiple species

For example, in one study, the researchers generated and validated an antibody targeting the HSD17B14 antigen using HSD17B14-transfected cells and a peptide-neutralizing assay before applying it to analyze tissue microarrays with tumors from 912 post-menopausal women .

How can HSD17B14 enzyme activity be accurately measured in research samples?

Measuring HSD17B14 enzyme activity requires precise methodologies tailored to its catalytic properties:

• Radiometric assays:

  • Radio-high pressure liquid chromatography (radio-HPLC) methods

  • Using radiolabeled substrates (e.g., [³H]-estradiol) and measuring conversion to products

  • This approach was used to show that cells transiently expressing HSD17B14 oxidize both oestradiol and testosterone to oestrone and androstenedione, respectively

• Spectrophotometric NAD+/NADH assays:

  • Monitoring NAD+ reduction or NADH oxidation at 340 nm

  • Particularly useful for the newly discovered L-fucose dehydrogenase activity

  • Comparative studies showed HSD17B14 enzymes were about 50-2300 times more active toward 5 μM L-fucose than toward 5 μM β-estradiol

• Recombinant protein expression systems:

  • Expression in E. coli for purified enzyme activity studies

  • For example, human HSD17B14 (hHSD17B14), rabbit HSD17B14 (rbHSD17B14), and rat HSD17B14 (rHSD17B14) were expressed in E. coli to compare their activities toward L-fucose and β-estradiol

• Kinetic parameter determination:

  • Measuring reaction rates at different substrate concentrations

  • Calculating Km and kcat values

  • The kcat value for estradiol was reported to be very low (0.08 min⁻¹), supporting the hypothesis that steroids may not be physiological substrates

When designing activity assays, researchers should consider the recent findings suggesting L-fucose may be a more relevant substrate than steroids for HSD17B14 .

How does the recent discovery of HSD17B14 as an L-fucose dehydrogenase change our understanding of its biological role?

The recent identification of HSD17B14 as an L-fucose dehydrogenase represents a paradigm shift in understanding this enzyme's primary biological function. This discovery challenges the long-held view of HSD17B14 as primarily involved in steroid metabolism .

Key implications of this finding include:

• Substrate specificity reassessment: While HSD17B14 can oxidize steroids like estradiol, it shows dramatically higher activity toward L-fucose. Activity measurements revealed human HSD17B14 was 2279 times more active toward L-fucose than β-estradiol, rabbit HSD17B14 was 954 times more active, and rat HSD17B14 was 52 times more active .

• Metabolic pathway involvement: Instead of primarily functioning in steroid metabolism, HSD17B14 may play a crucial role in fucose metabolism, potentially linking it to glycoprotein and glycolipid processing.

• Evolutionary considerations: The conservation of HSD17B14 across species with diverse steroid hormone systems but similar carbohydrate metabolism supports its primary role in carbohydrate rather than steroid metabolism.

• Nomenclature reconsideration: This functional discovery raises questions about whether HSD17B14 should continue to be classified primarily as a hydroxysteroid dehydrogenase or be reclassified to reflect its L-fucose dehydrogenase activity.

This finding explains the previously observed poor kinetic performance with steroid substrates and opens new avenues for understanding HSD17B14's role in both physiological and pathological contexts .

What are the implications of HSD17B14 as a potential therapeutic target in diabetic kidney disease?

The identification of HSD17B14 as a potential therapeutic target for diabetic kidney disease (DKD) has significant implications for future treatment strategies :

• Genetic evidence: Carriers of disruptive HSD17B14 alleles show protection against kidney function loss in diabetes, suggesting that pharmacological inhibition might replicate this protective effect .

• Expression patterns: HSD17B14 is highly expressed in proximal tubular cells but downregulated in kidney injury, suggesting a complex relationship between its expression and kidney health .

• Mechanistic considerations: The discovery of HSD17B14 as an L-fucose dehydrogenase raises questions about how this enzymatic activity connects to kidney protection. Potential mechanisms might involve:

  • Alterations in glycoprotein metabolism

  • Changes in fucosylation patterns of key kidney proteins

  • Effects on inflammatory or fibrotic pathways relevant to DKD progression

• Druggability assessment: As an enzyme with a well-defined catalytic site, HSD17B14 represents a potentially druggable target. Small molecule inhibitors or modulators could be developed to mimic the protective effects observed with genetic variants .

• Personalized medicine approach: Genetic testing for HSD17B14 variants could potentially identify diabetes patients at lower risk for DKD progression, allowing for tailored monitoring and treatment strategies.

How might HSD17B14 antibodies be utilized in predicting tamoxifen response in breast cancer patients?

HSD17B14 antibodies show promise as tools for developing predictive assays for tamoxifen response in breast cancer patients:

• Immunohistochemical scoring systems: Researchers could develop standardized scoring systems based on HSD17B14 staining intensity and distribution in tumor tissues. In previous research, high tumoural expression of HSD17B14 was significantly associated with improved response to tamoxifen in terms of local recurrence-free survival (HR 0.38; 95% C.I. 0.19–0.77, p = 0.007) .

• Combined biomarker panels: HSD17B14 expression could be incorporated into multi-marker panels alongside established predictors such as estrogen receptor status, progesterone receptor status, and HER2 expression to enhance predictive accuracy.

• Automated image analysis: Digital pathology and automated quantification of HSD17B14 staining could standardize assessment and reduce inter-observer variability.

• Circulating tumor cell (CTC) analysis: HSD17B14 antibodies might be utilized to assess expression in CTCs, potentially offering a less invasive method for monitoring treatment response.

• Functional assays: Beyond simple detection, antibodies could be used in functional assays to assess HSD17B14 activity in tumor samples, which might correlate more directly with tamoxifen response.

The development of such predictive tools could significantly advance personalized medicine approaches in breast cancer treatment, allowing clinicians to identify patients most likely to benefit from tamoxifen therapy while sparing others from unnecessary treatment and potential side effects .