LCN2 Human, Pichia

What is the molecular structure of human LCN2 and how does it differ from mouse LCN2?

Human LCN2 features a characteristic lipocalin fold consisting of an eight-stranded, anti-parallel, symmetrical β-barrel structure. This three-dimensional configuration creates a calyx that is open at one end, enabling the protein to bind and transport small lipophilic molecules . Originally, LCN2 was thought to function primarily as a transporter due to this structure.

The human and mouse LCN2 orthologs share only 62% sequence identity, which is relatively low compared to their putative receptors that can share up to 94% sequence homology . This substantial sequence divergence suggests potential functional differences between species that researchers must consider when translating findings from mouse models to human applications.

What are the primary biological functions of human LCN2?

Human LCN2 demonstrates remarkable functional diversity:

• Bacteriostatic activity: LCN2 plays a key role in innate immunity by sequestering bacterial iron siderophores, thereby limiting bacterial growth

• Immune regulation: Functions as an acute-phase protein during inflammatory responses and potentially acts as an intrinsic "help-me" sensor that promotes neutrophil chemotaxis

• Cell signaling: Involved in controlling cell differentiation, energy expenditure, cell death, and cell migration

• Metabolic regulation: Participates in insulin signaling pathways, though its exact role remains controversial

• Reproductive biology: Originally termed uterocalin, LCN2 is involved in tissue remodeling processes in the uterus during pregnancy

How is LCN2 secreted and what different forms can it take?

Human LCN2 can be secreted in three distinct forms:

• A 25-kDa monomer

• A ~46-kDa homodimer

• A ~135-kDa heterodimer composed of LCN2 and metalloproteinase-9 (MMP-9)

The association with MMP-9 initially suggested that LCN2 might modulate neutrophil gelatinase activity. LCN2 is produced primarily by neutrophils but can also be expressed by hepatocytes as part of the acute-phase response to inflammation. Hepatic expression increases dramatically during turpentine-induced acute phase response and can be significantly stimulated by tumor necrosis factor-α (TNF-α) .

Why choose Pichia pastoris for recombinant human LCN2 expression?

Pichia pastoris offers several advantages for LCN2 expression:

• As a eukaryotic system, it performs post-translational modifications that may be important for proper LCN2 folding and function

• The expression system yields human LCN2 with a molecular weight of approximately 21kDa, which can be purified to >96% purity using chromatographic techniques

• Pichia-expressed LCN2 maintains its capacity to bind small lipophilic substances in its hydrophobic core, preserving its function as a transporter of substances like retinal, biliverdins, and prostaglandins

• The system avoids endotoxin contamination that would be present in bacterial expression systems

What are the optimal conditions for storing recombinant human LCN2?

For maximum stability and activity retention, researchers should follow these storage guidelines:

• Lyophilized LCN2 is stable at room temperature for up to 3 weeks but should be stored desiccated below -18°C for longer periods

• Upon reconstitution, LCN2 should be stored at 4°C if used within 2-7 days

• For future use beyond 7 days, store reconstituted protein below -18°C

• For long-term storage, add a carrier protein (0.1% HSA or BSA)

• Avoid freeze-thaw cycles to prevent protein degradation

What is the recommended protocol for reconstituting lyophilized LCN2?

For optimal reconstitution:

• Use sterile 18MΩ-cm H₂O

• Prepare at concentrations not less than 100 μg/ml

• After initial reconstitution, the solution can be further diluted to other aqueous buffers as needed for specific experimental applications

• LCN2 used in the reconstitution was lyophilized from 0.02M NH₄HCO₃, which should be considered when designing experimental buffers

What are the current understanding and controversies surrounding LCN2 receptors?

Despite substantial research, our understanding of LCN2 receptors remains incomplete:

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

• NGALR (Neutrophil Gelatinase-Associated Lipocalin Receptor) is reported to have the highest affinity for LCN2 among all putative receptors

• Other proposed receptors include LRP2 (Low-density lipoprotein receptor-related protein 2) and MC4R (Melanocortin 4 Receptor)

• The literature contains significant inconsistencies, misinterpretations, and false assumptions regarding these potential LCN2 receptors

How do LCN2-activated signaling pathways influence cellular functions?

LCN2 demonstrates extensive pleiotropic effects through multiple mechanisms:

• Overexpression of LCN2 in one cancer cell line resulted in upregulation of 167 genes and downregulation of 96 genes, affecting immunity-related pathways, cellular responses to bacterial molecules, extracellular matrix organization, and cell cycle regulation

• LCN2 may act through several mechanisms simultaneously:

  • As a signal-inducing ligand at specific receptors

  • By masking binding epitopes for other proteins on proposed LCN2 receptors

  • By binding to other biomolecules to prevent their interaction with proteins/receptors

  • By altering general cellular features (osmolarity, iron content, mitochondrial activity)

The biological effect is the cumulative result of interactions with all receptors expressed at a specific time in a specific environment, creating an "extreme high combinatorial diversity of LCN2 activities" .

What contradictory findings exist regarding LCN2's role in metabolic diseases?

The role of LCN2 in metabolic regulation presents significant contradictions:

These contradictions may relate to differences in receptor expression and function. For example:

• LRP2 acts as an endocytic receptor for insulin reabsorption and shows increased expression in early type 2 diabetes

• MC4R deficiency induces insulin resistance and obesity but does not develop hyperglycemia

Additional research is required to understand LCN2's complex roles in metabolic regulation.

How should researchers approach the species-specific differences when translating LCN2 findings?

When translating findings between species, researchers should consider:

• The low sequence identity (62%) between human and mouse LCN2 orthologs

• Systematic studies investigating animal-to-human translational success rates show that translational success is unpredictable

• There have been several reports of functional divergence between mouse and human LCN2 orthologs

• Key differences include post-translational modifications - mouse LCN2 is a PKCδ substrate that needs phosphorylation at T115 for secretion from neutrophils, but equivalent modifications in human LCN2 are not well documented

What methods are most effective for studying LCN2 expression in disease models?

For investigating LCN2 in disease contexts:

• Immunohistochemistry staining with quantitative scoring systems can reveal correlations with prognostic factors

• When analyzing cancer tissues, consider evaluating LCN2 expression in relation to:

  • Necrosis (necrosis-positive cases show statistically higher LCN2 scores)

  • Tumor-infiltrating lymphocytes (TILs ≥5% cases have significantly higher LCN2 scores and intensity)

  • Ki-67 proliferation index (cases with ≥20% index have significantly higher LCN2 scores)

  • Histological grade and molecular subtypes (aggressive subtypes like HER2+ and TNBC show higher LCN2 expression)

How can researchers distinguish between functional effects of different LCN2 forms?

To differentiate between the functional impacts of monomeric, homodimeric, and heterodimeric (with MMP-9) forms of LCN2:

• Use size exclusion chromatography to isolate different molecular weight forms

• Apply non-reducing vs. reducing conditions in SDS-PAGE to preserve or disrupt potential dimeric structures

• Design experiments with recombinant forms of specific LCN2 variants

• Consider co-expression with potential binding partners like MMP-9 to study heterodimeric effects

• Analyze the iron-binding capacity of different forms, as this relates to the bacteriostatic function of LCN2

What are the critical knowledge gaps in LCN2 receptor biology?

Despite extensive research, several fundamental questions remain:

• The precise mechanisms by which the six putative LCN2 receptors transmit signals into cells remain poorly understood

• The relative importance of each receptor in different tissues and disease states needs clarification

• The structural basis for LCN2-receptor interactions requires further investigation

• How species differences in LCN2 sequence affect receptor binding and downstream signaling remains to be fully elucidated

• The interplay between LCN2's iron-binding function and its receptor-mediated signaling requires further study

How might LCN2's role in the gut microbiome influence systemic inflammation?

Emerging evidence suggests important connections between LCN2 and gut microbiota:

• Lcn2 null mice exhibited persistent colonization with segmented filamentous bacteria in the ileum

• This colonization was associated with signs of autolysis in apical villi and detachment of epithelial cells from the basal membrane

• The lack of LCN2 protein provokes expansion of siderophore-dependent bacterial species, resulting in significant changes in intestinal microbiome composition

• These microbiome alterations may increase inflammatory activity in the gastrointestinal tract

• Further research is needed to understand how these local effects in the gut may contribute to systemic inflammatory conditions

What standardized methods should be developed to enhance reproducibility in LCN2 research?

To address contradictions and improve research quality:

• Develop standardized protocols for recombinant LCN2 production in Pichia pastoris with defined quality control parameters

• Establish consistent methods for analyzing different forms of LCN2 (monomer, homodimer, heterodimer)

• Create validated assays for measuring LCN2-receptor interactions with quantifiable parameters

• Design species-comparative studies that account for the 62% sequence identity between mouse and human LCN2

• Implement consistent reporting standards for experimental conditions to facilitate cross-study comparisons