LCN2 Human, His

What is the molecular structure of human LCN2?

Human Lipocalin 2 features a characteristic three-dimensional fold comprising a single eight-stranded continuously hydrogen-bonded antiparallel β-barrel structure with a calyx that is open at one end . This structural feature allows LCN2 to bind and transport small lipophilic molecules . The protein exists in multiple forms including a 25-kDa monomer, a ~46-kDa homodimer, and in complex with other proteins such as MMP-9 . When designing experimental approaches, researchers should consider which form of LCN2 is most relevant to their biological question, as different forms may exhibit distinct functionalities.

What are the primary biological functions of human LCN2?

LCN2 demonstrates remarkable functional diversity with roles in:

• Innate immunity: Acts as a bacteriostatic agent by sequestering iron-binding bacterial siderophores, thereby blocking bacterial access to iron . This function is particularly important in tissues prone to microorganism insult, such as trachea, lung, and stomach .

• Iron metabolism: Participates in mammalian iron trafficking by binding to iron-loaded siderophores and delivering iron inside cells . This capability influences cellular iron content and subsequent cellular processes.

• Cellular differentiation: Induces the differentiation of mesenchymal progenitor cells into epithelial tubules during kidney development .

• Cell survival regulation: Demonstrates context-dependent effects, either promoting apoptosis by depleting intracellular iron pools or functioning as a survival factor in certain cancer cells .

• Metabolism regulation: Modulates expression of genes involved in β-oxidation, promotes β-cell function, and counteracts obesity-induced glucose intolerance .

How does the histidine tag affect recombinant human LCN2 functionality?

The addition of histidine tags (commonly 6×His) to recombinant human LCN2 facilitates protein purification through metal affinity chromatography but may impact protein functionality in experimental settings. Researchers should implement control experiments comparing tagged versus untagged versions when:

• Studying receptor binding, as the His tag might alter binding affinity or kinetics

• Investigating iron-binding capacity, where the tag could potentially interact with metal ions

• Examining protein-protein interactions, particularly with MMP-9 or other binding partners

• Conducting in vivo studies, where immunogenicity of the tag might become relevant

Consider employing cleavable His tags when absolute native conformations are required for your experimental design.

What receptors are known to bind human LCN2?

Six putative receptors for LCN2 have been proposed, though their relative importance varies by tissue context and experimental system :

The cell-surface receptor SLC22A17 (BOCT) mediates LCN2's apoptotic effects in leukocytic cell lines, while Megalin (LRP2) serves as an alternative receptor in other tissues . When designing receptor-focused experiments, researchers should account for the tissue-specific expression patterns of these receptors.

How can researchers effectively study LCN2 receptor signaling pathways?

To investigate LCN2 receptor signaling:

• Receptor binding assays: Use surface plasmon resonance (SPR) or microscale thermophoresis (MST) with purified receptors and labeled LCN2-His to determine binding kinetics and affinities.

• Signaling cascade analysis: Employ phosphoproteomic approaches to map the temporal activation of downstream effectors following LCN2 treatment.

• Receptor knockdown/knockout models: Utilize siRNA, CRISPR-Cas9, or conditional knockout systems to selectively remove individual receptors and assess the impact on LCN2 response.

• Receptor competition studies: Test whether iron-loaded versus iron-free LCN2 differentially activates specific receptors by pre-loading recombinant LCN2-His with iron siderophores before receptor binding assays.

• Tissue-specific signaling: Recognize that signaling outcomes may differ dramatically between tissue types based on receptor expression profiles and intracellular conditions.

When interpreting results, consider that "there is a fundamental lack in understanding of how these cell-surface receptors transmit and amplify LCN2 to the cell" , suggesting caution in making definitive claims about signaling mechanisms.

What are the optimal approaches for producing recombinant human LCN2-His?

For high-quality recombinant human LCN2-His production:

• Expression system selection: E. coli systems offer high yield but may lack proper post-translational modifications. For glycosylated LCN2, consider mammalian (HEK293 or CHO) or insect cell expression systems.

• Purification strategy:

  • IMAC (Immobilized Metal Affinity Chromatography) using nickel or cobalt resins

  • Size exclusion chromatography as a polishing step to separate monomeric, dimeric, and higher-order forms

  • Consider adding reducing agents during purification to prevent unwanted disulfide formation

• Quality control assessment:

  • SDS-PAGE and Western blotting to confirm purity and identity

  • Circular dichroism to verify proper protein folding

  • Dynamic light scattering to assess homogeneity

  • Endotoxin testing for preparations intended for cell culture or in vivo experiments

• Activity verification: Confirm iron-binding capacity through siderophore-binding assays or bacterial growth inhibition tests.

How can researchers effectively study LCN2's role in iron metabolism?

To investigate LCN2's iron-binding and transport functions:

• Iron-binding assays:

  • Isothermal titration calorimetry to measure binding affinity of LCN2 to iron-loaded siderophores

  • Fluorescence quenching assays using intrinsic tryptophan fluorescence

  • Absorbance spectroscopy to monitor iron-siderophore complex formation

• Cellular iron trafficking:

  • Use fluorescently labeled LCN2 to track internalization and subcellular localization

  • Employ iron chelators and iron supplementation to modulate intracellular iron pools

  • Measure cellular iron content using inductively coupled plasma mass spectrometry (ICP-MS)

• Functional consequences:

  • Monitor expression of iron-responsive genes (ferritin, transferrin receptor)

  • Assess mitochondrial function and reactive oxygen species production

  • Evaluate cell viability and apoptosis under varying iron conditions

Research has shown that iron-loaded LCN2 delivers iron inside cells, increasing intracellular iron content, while empty LCN2 can deplete intracellular iron pools, potentially inducing apoptosis through upregulation of pro-apoptotic proteins like Bim .

What evidence supports LCN2's role in cardiac hypertrophy?

LCN2 demonstrates significant involvement in cardiac hypertrophy through multiple lines of evidence:

• Temporal expression patterns: In hypertrophic heart rat models, cardiac and circulating LCN2 was "significantly overexpressed before, during, and after development of cardiac hypertrophy and heart failure" , suggesting both predictive and mechanistic roles.

• Genetic manipulation evidence:

  • Lcn2-knockout mice developed smaller hearts

  • Increased LCN2 expression was observed in hypertrophic hearts in models of intrauterine growth restriction

• Cellular mechanisms: In cardiomyocyte cultures, LCN2 activated molecular hypertrophic pathways and increased cell size, while simultaneously reducing proliferation and cell numbers .

• Human correlation: In clinical studies, increased LCN2 was associated with cardiac hypertrophy and diastolic dysfunction in diabetes mellitus patients . The Young Finns Study revealed that LCN2 expression associated with body mass index, cardiac mass, and inflammatory marker levels .

• Genetic determinants: The single-nucleotide polymorphism rs13297295 near the LCN2 gene defined a significant cis-eQTL for LCN2 expression , suggesting genetic control of LCN2 levels may influence cardiac outcomes.

How does LCN2 contribute to cancer progression?

LCN2 demonstrates complex and sometimes contradictory roles in cancer biology:

• EMT and invasion promotion: LCN2 has been shown to induce epithelial to mesenchymal transition (EMT) in breast cancer cells and promote tumor invasion . This involves repression of E-cadherin and upregulation of mesenchymal markers.

• MMP-9 stabilization: LCN2 forms a complex with matrix metalloproteinase-9 (MMP-9) and protects this enzyme from autodegradation, preserving its activity . This may contribute to extracellular matrix remodeling during cancer progression.

• Estrogen receptor interactions: Estrogen receptor α may participate in the pathway leading to LCN2-induced EMT in breast cancer , suggesting hormonal regulation of LCN2's oncogenic effects.

• Context-dependent effects: Intriguingly, LCN2 demonstrates opposing effects depending on the cancer context:

  • In thyroid carcinomas, LCN2 protected cancer cells from apoptosis induced by serum deprivation

  • In 4T1 murine mammary cancer cells expressing constitutively active H-ras, LCN2 overexpression reversed EMT and inhibited invasion and metastasis

  • In esophageal squamous cell carcinoma and Barrett esophagus-associated high-grade dysplasia, LCN2 levels were elevated compared to normal tissues

• Biomarker potential: Preliminary evidence suggests LCN2 may serve as a potential non-invasive urinary biomarker for breast cancer , warranting further investigation into its diagnostic utility.

How do researchers address contradictory findings regarding LCN2 functions?

LCN2 exhibits contradictory functions across different experimental systems, particularly in apoptosis regulation and cancer progression. To address these contradictions:

• Context-specific analysis:

  • Explicitly define cellular context, including cell type, tissue origin, and disease state

  • Document genetic background of experimental models, particularly oncogene status

  • Report iron availability and metabolic state of experimental systems

• Receptor profiling:

  • Characterize the expression pattern of all six putative LCN2 receptors in your experimental system

  • Consider receptor competition or compensatory effects when interpreting results

• LCN2 forms and modifications:

  • Distinguish between effects of monomeric, dimeric, and MMP-9-complexed LCN2

  • Investigate post-translational modifications that might alter functionality

  • Determine iron-binding status of LCN2 in your experimental system

• Standardized reporting:

  • Report concentrations of LCN2 used, ensuring physiological relevance

  • Document exposure time and kinetics of responses

  • Control for potential contaminants in recombinant preparations

The literature highlights these paradoxical functions: "In contrast to its pro-apoptotic activity, Lcn2 has also been reported to be a survival factor" and "Lcn2 is associated with different, even opposite effects, in the presence of activated Ras compared to in the absence of Ras" .

What are the cutting-edge approaches for studying LCN2's role in inflammatory responses?

To investigate LCN2's involvement in inflammation using state-of-the-art approaches:

• Single-cell analysis:

  • Apply single-cell RNA-seq to identify LCN2-responsive cell populations

  • Use CyTOF or spectral flow cytometry to characterize LCN2-expressing cells during inflammatory responses

  • Implement spatial transcriptomics to map LCN2 expression patterns in inflammatory tissues

• In vivo imaging:

  • Develop LCN2-reporter mouse models for real-time monitoring of expression

  • Use intravital microscopy with fluorescently labeled LCN2 to track dynamics during inflammation

  • Apply positron emission tomography with radiolabeled anti-LCN2 antibodies for whole-body imaging

• Mechanistic dissection:

  • Employ CRISPR screens to identify genes that modulate LCN2 response

  • Apply proteomics to characterize the LCN2 interactome during different inflammatory states

  • Utilize conditional knockout models with tissue-specific and temporal control over LCN2 expression

• Translational approaches:

  • Develop organoid models from patient samples to study LCN2 in human inflammatory diseases

  • Explore LCN2 as a biomarker through longitudinal sampling in patient cohorts

  • Investigate LCN2-targeting therapeutics using nanobodies or small molecule inhibitors

What considerations are important when interpreting LCN2 receptor studies?

When evaluating or designing LCN2 receptor studies, researchers should consider:

• Receptor validation challenges:

  • "Although six putative receptors for LCN2 have been proposed, there is a fundamental lack in understanding of how these cell-surface receptors transmit and amplify LCN2 to the cell"

  • The field contains "inconsistencies, misinterpretations and false assumptions in the understanding of these potential LCN2 receptors"

• Structural differences:

  • The putative receptors have markedly different external ligand-binding domains, hydrophobic membrane-spanning regions, and intracellular domains

  • These structural differences likely translate to distinct signaling mechanisms that should be independently validated

• Methodological limitations:

  • Many receptor identifications rely on binding assays that may not reflect functional signaling

  • Co-immunoprecipitation studies (such as those showing mouse LRP6 binding to mouse LCN2 ) require functional validation

  • Over-expression systems may create artificial interactions not present at physiological levels

• Tissue-specific expression patterns:

  • SLC22A17/NGALR belongs to a family of 30 distinct multi-membrane spanning proteins, 13 of which localize to the plasma membrane

  • Different tissues may utilize distinct LCN2 receptor systems, necessitating tissue-specific validation

When designing receptor studies, employ multiple complementary approaches and validate findings across different experimental systems to overcome these limitations.

What are the emerging research areas for LCN2 in metabolic disorders?

LCN2 demonstrates significant potential as a metabolic regulator, with several promising research directions:

• Central nervous system regulation:

  • LCN2 functions as "a pronounced direct appetite suppressor and satiety signal in humans and mice modulating caloric intake as well as fat and lean mass content"

  • Future research should investigate hypothalamic receptor mechanisms and neural circuit integration

• Mitochondrial function:

  • LCN2 "impacts mitochondrial and peroxisomal function and integrity that directly impacts hepatic triglyceride balance, oxidative stress, and apoptosis"

  • Further studies should explore the molecular mechanisms connecting LCN2 signaling to mitochondrial physiology

• β-cell biology:

  • LCN2 "promotes β-cell function, and counteracts obesity-induced glucose intolerance"

  • Additional research on pancreatic islet cells could reveal therapeutic applications for diabetes

• Hepatic metabolism:

  • LCN2 serves as "a key modulator of hepatic lipid homeostasis"

  • Future work should characterize the hepatocyte-specific signaling pathways and potential interactions with other metabolic hormones

• Therapeutic targeting:

  • Developing LCN2 analogs or receptor-specific modulators may offer new approaches for treating metabolic syndrome

  • Personalized medicine approaches could leverage genetic variants such as the rs13297295 SNP to stratify patients for LCN2-targeted therapies

How can researchers develop improved tools for studying LCN2 in complex biological systems?

Advancing LCN2 research requires development of sophisticated tools:

• Receptor-specific reagents:

  • Generate antibodies or nanobodies that selectively block individual LCN2 receptors

  • Develop small molecules that modulate specific receptor interactions

  • Create biosensors for real-time monitoring of receptor activation

• Improved animal models:

  • Design conditional and inducible knockout systems for tissue-specific LCN2 manipulation

  • Create knock-in models expressing tagged LCN2 for tracking endogenous protein

  • Develop humanized mouse models expressing human LCN2 and receptors for translational studies

• Advanced imaging approaches:

  • Implement FRET-based sensors for LCN2-receptor interactions

  • Apply super-resolution microscopy to visualize subcellular LCN2 trafficking

  • Develop PET tracers for non-invasive imaging of LCN2 expression in vivo

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics to build comprehensive models of LCN2 action

  • Apply network analysis to identify key nodes in LCN2 signaling networks

  • Implement machine learning approaches to predict context-specific LCN2 functions

• Translational tools:

  • Develop standardized assays for measuring biologically active LCN2 in clinical samples

  • Create patient-derived organoids for personalized studies of LCN2 function

  • Establish biobanks of samples from patients with LCN2-related conditions for longitudinal analysis