Recombinant Human Zinc transporter 4 (SLC30A4)
Description
Introduction to Recombinant Human Zinc Transporter 4 (SLC30A4)
Recombinant Human Zinc transporter 4 (SLC30A4) is a protein belonging to the SLC30 family of zinc transporters, also known as ZnT proteins. It is specifically engineered and produced through recombinant DNA technology to mimic the natural human SLC30A4 protein for research and medical applications. SLC30A4 is a critical component of the cellular machinery responsible for maintaining zinc homeostasis in the human body . The protein is artificially expressed in various expression systems, commonly E. coli or mammalian cell lines such as HEK-293, and is often tagged with affinity markers like histidine (His) to facilitate purification and identification .
As a member of the ZnT family, SLC30A4 plays a crucial role in reducing intracellular zinc availability by either exporting zinc out to the extracellular space or sequestering cytoplasmic zinc into intracellular compartments when cellular zinc levels are elevated . This function is essential for maintaining appropriate zinc levels, as zinc is a vital micronutrient involved in numerous biological processes including enzyme activity, protein structure, and cellular signaling.
Expression Systems and Tags
Recombinant Human SLC30A4 can be produced using different expression systems, each offering specific advantages for various research applications:
The protein is commonly fused with affinity tags to facilitate purification and detection:
| Tag Type | Position | Benefits |
|---|---|---|
| His tag | N-terminal | Single-step affinity purification, antibody detection |
| GST tag | N-terminal | Enhanced solubility, purification option |
These expression systems and tag configurations offer researchers flexibility in producing recombinant SLC30A4 protein tailored to specific experimental needs, ranging from structural studies to functional analyses.
Role in Zinc Homeostasis
SLC30A4 is a critical component of the zinc homeostasis machinery in human cells. As part of the ZnT family, it functions in opposition to the ZIP (SLC39) family of transporters to maintain proper zinc balance within cells . While ZIP transporters increase cytoplasmic zinc concentrations when cellular zinc is depleted, ZnT proteins like SLC30A4 contribute to cytoplasmic zinc balance by:
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Exporting zinc out to the extracellular space
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Sequestering cytoplasmic zinc into intracellular compartments when cellular zinc levels are elevated
This bidirectional control mechanism ensures that cells maintain optimal zinc concentrations, preventing both zinc deficiency and toxicity, which could otherwise lead to cellular dysfunction or death.
Cellular Localization and Transport Mechanisms
SLC30A4 is a membrane-bound protein with multiple transmembrane domains that facilitate zinc transport across cellular barriers . Research findings indicate that SLC30A4 is particularly important in mammary gland tissue, where it plays a role in zinc secretion into milk . The protein's dysfunction has been associated with reduced zinc secretion into milk, demonstrating its physiological relevance in specialized tissues .
The transport mechanism of SLC30A4 involves facilitated diffusion, enabling zinc ions to move across membranes according to concentration gradients . This process is energy-efficient and allows for rapid response to changing zinc concentrations within the cellular environment.
Laboratory Applications
Recombinant Human SLC30A4 protein has several important laboratory applications:
The availability of high-purity recombinant proteins (typically >90% as determined by SDS-PAGE) ensures reliable and reproducible results in these applications .
Key Research Findings
Research utilizing recombinant SLC30A4 has contributed significantly to our understanding of zinc transport mechanisms and related disorders:
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Analysis of zinc transporter gene expression in mammary gland disorders has revealed that SLC30A4 dysfunction leads to reduced zinc secretion into milk, highlighting its importance in maternal-infant zinc nutrition .
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Studies have shown unique tissue-specific expression patterns of SLC30A4, with differential responsiveness to dietary zinc deficiency and excess, suggesting tissue-specific regulatory mechanisms .
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Genetic studies have identified that zinc transporter genes, including SLC30A4, show higher levels of genetic differentiation between African and non-African populations than would be randomly expected, indicating possible adaptive evolution in response to varying zinc availability in different geographical regions .
Evolutionary Aspects and Genetic Variations
Genetic studies have revealed interesting evolutionary patterns related to SLC30A4 and other zinc transporter genes:
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Zinc transporter genes show signs of positive natural selection in human populations, suggesting adaptation to varying zinc availability in different geographical regions .
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Higher levels of genetic differentiation exist between African and non-African populations for zinc transporter genes compared to random expectations, indicating potential adaptive responses during human migration and settlement .
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Population groups in South Asia show significant enrichment for single nucleotide polymorphisms (SNPs) with unusually extended haplotypes in zinc transporter genes, suggesting recent selective pressures .
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Correlations have been observed between zinc transporter gene variants and environmental factors such as zinc deficiency levels in soil, further supporting adaptive evolution in response to nutritional challenges .
These findings emphasize the evolutionary importance of zinc homeostasis and the critical role of transporters like SLC30A4 in human adaptation to varying nutritional environments.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them when placing your order. We will strive to fulfill your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please contact us in advance as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
- A significant increase in ZnT4 protein levels was observed in the prefrontal cortex of individuals with Major Depressive Disorder compared to control subjects. PMID: 27661418
- TRPML1 collaborates with ZnT4 to regulate zinc translocation between the cytoplasm and lysosomes. PMID: 23368743
- The loss of ZnT4 in airway inflammation contributes to the vulnerability of airway epithelium in diseases such as asthma. PMID: 22254085
- Genomic localization and organization; exclusion in variants of acrodermatitis enteropathica. PMID: 11686514
- ZnT4 is not responsible for acrodermatitis enteropathica in Japanese families. PMID: 11935329
- hZnT4 is constitutively expressed in human breast tissue and may be one of several members of the ZnT family involved in zinc transport into milk. PMID: 11988082
- The hZnT4 protein did not co-localize with intracellular free zinc pools, suggesting that hZnT4 is not involved in the transport of zinc into vesicles destined for secretion into milk. PMID: 12743795
- Decreased intensity of ZnT4 immunoreactivity was observed in the progression from benign to invasive localized prostate cancer and to metastatic disease. PMID: 12955079
- Decreased hZnT-4 expression is associated with prostate tissue abnormalities, independent of total cellular zinc content. PMID: 14968438
- Findings implicate a role for ZnT(4) in mast cell zinc homeostasis and suggest that granule pools of zinc may be distinct from those regulating the activation of procaspase-3 and NF-kappaB. PMID: 15187159
- Zn transporter-4 and Zn transporter-6 are significantly (P<0.05) increased in the hippocampus/parahippocampal gyrus of individuals with early Alzheimer's disease and Alzheimer's disease. PMID: 16580781
- hZnT-4 is found in the plasma membrane. It is most highly expressed in Molt-4 cells, up-regulated after treatment with phytohemagglutinin, and responsible for the measured decrease of intracellular zinc content after high zinc exposure. PMID: 17971500
- Data demonstrate that ZNT4 is extensively present in the Abeta-positive plaques in the cortex of human Alzheimer's disease brains. PMID: 18639746
Q&A
What is the primary cellular function of SLC30A4 (ZnT4)?
SLC30A4 (ZnT4) functions as a zinc transporter that plays a critical role in zinc homeostasis. It specifically transports zinc into the trans-Golgi apparatus for lactose synthesis and across the apical cell membrane for efflux from mammary epithelial cells (MECs) into milk . This function is essential for maintaining proper zinc levels during lactation, as demonstrated by studies in mouse models. Unlike other zinc transporters that may function in various tissues, SLC30A4 appears to have specialized functions in secretory epithelial cells.
How does SLC30A4 deficiency manifest in animal models?
The most well-characterized model of SLC30A4 deficiency is the "lethal milk" (lm/lm) mouse, which has a truncation mutation in the SLC30A4 gene. These mice exhibit multiple phenotypic manifestations including:
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Low milk zinc concentration
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Smaller mammary glands
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Decreased milk volume
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Lactation failure by lactation day 2
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Defects in mammary gland secretion
This model has been instrumental in understanding the physiological significance of SLC30A4, particularly its role in mammary tissue function and zinc transport mechanisms.
How does SLC30A4 expression differ from other SLC30 family members in cancer?
Unlike most members of the SLC30 family, SLC30A4 exhibits a distinctive expression pattern in cancer tissues. While SLC30A1-3, 5-7, and 9 are significantly upregulated in gastric cancer tissues compared to non-cancerous tissues, SLC30A4 is specifically downregulated . This unique expression profile suggests that SLC30A4 may have a different functional role in cancer progression compared to other SLC30 family members, potentially serving as a tumor suppressor rather than an oncogene.
What is the prognostic significance of SLC30A4 expression in cancer patients?
| Factor | Subgroup | β | SE | Wald | RR (95% CI) | P |
|---|---|---|---|---|---|---|
| TNM stage | T3 | 0.686 | 0.199 | 11.914 | 1.986 (1.345–2.933) | 0.001 |
| N2 | 0.953 | 0.372 | 6.578 | 2.594 (1.252–5.375) | 0.010 | |
| N3 | 1.763 | 0.382 | 21.262 | 5.830 (2.756–12.334) | < 0.001 | |
| M | 1.009 | 0.247 | 16.712 | 2.742 (1.691–4.447) | < 0.001 | |
| SLC30A2 | 0.409 | 0.187 | 4.762 | 1.505 (1.042–2.172) | 0.029 | |
| SLC30A5 | -0.518 | 0.179 | 8.357 | 0.596 (0.419–0.846) | 0.004 | |
| SLC30A7 | -0.472 | 0.180 | 6.863 | 0.624 (0.439–0.888) | 0.009 |
What are the optimal techniques for quantifying SLC30A4 expression in tissue samples?
For reliable quantification of SLC30A4 expression in research contexts, quantitative real-time PCR (qRT-PCR) has been demonstrated as an effective methodology, with the following protocol:
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Extract total RNA from frozen tumor and corresponding non-tumorous tissues using TRIzol reagent
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Determine RNA concentration and purity by ultraviolet absorbance spectroscopy
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Reverse transcribe RNA into cDNA using a suitable First Strand cDNA Synthesis Kit
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Perform qRT-PCR using SYBR Green with the following cycling parameters:
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95°C for 10 min (initial denaturation)
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40 cycles of: 95°C for 15s, 60°C for 30s, and 72°C for 30s
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Run samples in triplicate and calculate mean values
This methodology ensures consistent and reliable quantification of SLC30A4 expression levels for comparative studies.
How can researchers assess the diagnostic potential of SLC30A4 in clinical samples?
Receiver operating characteristic (ROC) curve analysis based on expression data has proven effective for evaluating the diagnostic value of SLC30A4. Studies using TCGA database data have shown that SLC30A4 has high diagnostic value (ROC value of 0.762) for distinguishing gastric cancer patients from healthy individuals . For optimal research outcomes:
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Extract expression data from publicly available databases (e.g., TCGA)
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Perform ROC analysis to determine sensitivity and specificity
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Calculate area under the curve (AUC) values
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Compare with other biomarkers for relative diagnostic efficiency
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Validate findings in independent patient cohorts
This approach provides robust evidence for the potential clinical utility of SLC30A4 as a diagnostic biomarker.
What is known about zinc sensing mechanisms in zinc transporters that might apply to SLC30A4?
While the specific zinc sensing mechanisms of SLC30A4 are not fully characterized in the available literature, insights from related zinc transporters such as ZIP4 provide valuable models. In ZIP4, the transport site in the transmembrane domain acts as the zinc sensor during zinc-dependent endocytosis, with an apparent zinc dissociation constant (KD) of approximately 1.5 μM .
This zinc sensing mechanism involves:
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Direct binding of zinc to the transport site within the transmembrane domain
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Conformational changes induced by zinc binding
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Structural coupling between the transport site and cytosolic regions
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Subsequent initiation of endocytosis or other regulatory events
Similar mechanisms may exist for SLC30A4, where zinc binding could induce conformational changes affecting protein localization or transport activity, thereby regulating zinc homeostasis.
What structural elements might be crucial for SLC30A4 function based on studies of related transporters?
Based on structural studies of related zinc transporters, several key structural elements may be important for SLC30A4 function:
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Transmembrane domains that form the zinc transport pathway
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Cytosolic loops that may serve as regulatory regions or interaction sites with endocytic machinery
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Potential motifs similar to the Leu-Gln-Leu (LQL) motif identified in ZIP4 that is essential for endocytosis
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Histidine-rich regions that may be involved in zinc binding or regulation
The transmembrane domain likely contains the transport site responsible for zinc binding and transport, while cytosolic regions may play roles in regulation, protein-protein interactions, or trafficking signals.
How does SLC30A4 expression correlate with other SLC30 family members in cancer?
SLC30A4 exhibits a distinct expression pattern compared to other SLC30 family genes in cancer. While SLC30A1-3, 5-7, and 9 are significantly upregulated in gastric cancer tissues compared to normal tissues, SLC30A4 is specifically downregulated . This suggests potentially divergent roles in cancer biology, with SLC30A4 possibly functioning as a tumor suppressor.
The correlation extends to clinical parameters as well:
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SLC30A1, 5-7, and 9 show positive associations with nodal metastasis and tumor stage
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SLC30A4 shows negative correlations with these parameters
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SLC30A4, 5-7, 9-10 are significantly related to Helicobacter pylori infection status in gastric cancer patients
These differential expression patterns indicate distinct functional roles for different SLC30 family members in cancer development and progression.
How do SLC30 family members differ in their prognostic implications?
SLC30 family members show opposing prognostic associations in cancer:
These opposing prognostic implications suggest that different SLC30 family members play distinct roles in cancer biology, potentially functioning in different cellular compartments or affecting different aspects of zinc homeostasis relevant to tumor progression.
What experimental approaches would be most effective for studying zinc transport activity of recombinant SLC30A4?
For studying the zinc transport activity of recombinant SLC30A4, several complementary approaches would be effective:
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Cellular zinc measurement assays:
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Express recombinant SLC30A4 in appropriate cell lines
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Utilize zinc-sensitive fluorescent probes (e.g., FluoZin-3) to measure intracellular zinc levels
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Compare zinc levels in cells expressing wild-type vs. mutant SLC30A4 proteins
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Radioactive zinc (65Zn) transport assays:
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Measure zinc uptake or efflux in cells or membrane vesicles expressing SLC30A4
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Determine kinetic parameters (Km, Vmax) of zinc transport
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Vesicular transport assays:
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Prepare membrane vesicles from cells expressing SLC30A4
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Measure zinc transport into vesicles to assess transport activity
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These approaches should be combined with proper controls, including non-transfected cells and cells expressing transport-deficient SLC30A4 mutants.
How should researchers design experiments to study the trafficking and localization of SLC30A4?
To effectively study trafficking and localization of recombinant SLC30A4:
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Fluorescent protein tagging:
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Generate fusion constructs with fluorescent proteins (e.g., GFP) at N- or C-terminus
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Verify that tagged proteins retain transport activity
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Perform live-cell imaging to track localization and trafficking
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Immunofluorescence microscopy:
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Internalization assays:
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Label cell surface SLC30A4 using antibodies against extracellular epitopes
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Monitor internalization over time under different zinc conditions
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Quantify internalization rates using flow cytometry or microscopy-based approaches
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Zinc-dependent trafficking studies:
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Test effects of zinc depletion (using chelators like TPEN) and zinc supplementation
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Compare trafficking of wild-type SLC30A4 with mutant variants
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Establish dose-response relationships between zinc concentration and trafficking
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These experimental approaches would provide comprehensive insights into the regulation and function of recombinant SLC30A4 in cellular contexts.
