MANF Human, His

What is the molecular structure of human MANF and why is it significant?

Human MANF is a 20 kDa protein belonging to the ARMET family, synthesized as a 179 amino acid precursor containing a 21 amino acid signal sequence and a 158 amino acid mature chain. The mature MANF protein (Leu25-Leu182) has high evolutionary conservation, with 99%, 98%, and 96% amino acid identity to rat, mouse, and bovine MANF, respectively . MANF and its structural homolog CDNF each contain an N-terminal saposin-like lipid binding domain and a carboxyl-terminal domain that lacks homology to previously characterized protein structures . This unique structure contributes to MANF's dual localization in both the endoplasmic reticulum (ER)/Golgi apparatus and as a secreted protein, enabling its diverse biological functions in neuroprotection and ER stress response .

How does His-tagged human MANF differ from native MANF in experimental applications?

Recombinant human MANF with a polyhistidine tag (typically at the C-terminus) retains the biological properties of native MANF while providing advantages for purification and detection in experimental settings. The His-tagged protein has a calculated molecular weight of 20.0 kDa but migrates as 19-21 kDa under reducing conditions in SDS-PAGE due to glycosylation . When designing experiments, researchers should consider that while the His-tag facilitates purification using metal affinity chromatography, it may potentially influence protein folding or interaction surfaces in some applications. Quality control methods including SDS-PAGE (>95% purity) and SEC-MALS (>98% purity) should be employed to verify protein integrity . Biological activity assays comparing His-tagged MANF to standard MANF preparations are essential, with activity typically measured in cell proliferation assays using rat C6 cells (ED50 < 20 μg/ml, corresponding to >50 IU/mg specific activity) .

What are the optimal storage and reconstitution methods for MANF-His protein?

For maximum stability and activity retention, recombinant MANF-His should be stored as a lyophilized powder at -20°C or -80°C. The protein is typically lyophilized from a 0.22 μm filtered solution in PBS (pH 7.4) with trehalose as a protectant . Upon receipt, aliquoting is necessary to prevent repeated freeze-thaw cycles which can compromise protein integrity. For reconstitution, sterile water should be added to prepare a stock solution of 0.2 μg/μl, followed by centrifugation at 4°C before opening to recover the entire contents . Researchers should validate protein activity after reconstitution, particularly for experiments requiring quantitative measurements of MANF function.

How does MANF protect dopaminergic neurons and what are the implications for Parkinson's disease research?

MANF selectively protects nigral dopaminergic neurons over GABAergic or serotonergic neurons, making it particularly relevant for Parkinson's disease (PD) research . The neuroprotective mechanism involves both intracellular and extracellular pathways. Intracellularly, MANF regulates the unfolded protein response (UPR) by directly binding to IRE1α with high affinity and interacting with PERK and ATF6 with lower affinities . These interactions modulate ER stress, which is a critical factor in dopaminergic neuron degeneration. Extracellularly, MANF acts through autocrine and paracrine signaling to promote neuronal survival.

Research using animal models has demonstrated that MANF promotes the survival of dopamine neurons that degenerate in PD . Importantly, MANF mutants deficient in IRE1α binding lack pro-survival action in superior cervical ganglion (SCG) and dopamine neurons in vitro and show reduced biological activity in PD animal models in vivo . This indicates that the direct interaction with IRE1α is critical for MANF's neuroprotective function. When designing PD-related experiments, researchers should consider both the direct application of recombinant MANF-His and genetic approaches to modulate endogenous MANF expression.

What experimental models are most appropriate for studying MANF's effects on neuronal survival?

Several experimental models have proven effective for investigating MANF's neuroprotective properties:

When using these models, researchers should include appropriate controls, including wild-type MANF and functionally relevant MANF mutants, particularly those affecting IRE1α binding . For quantification, combine multiple readouts such as cell viability assays, apoptosis markers, and functional assessments of neuronal activity.

How does MANF interact with the unfolded protein response pathway?

MANF is a critical regulator of the unfolded protein response (UPR), directly interacting with key UPR sensors. Research has demonstrated that MANF binds directly to IRE1α with high affinity and also interacts with PERK and ATF6 with lower affinities . Through these interactions, MANF competes with BiP (GRP78) for binding to IRE1α, thereby regulating IRE1α phosphorylation, oligomerization, and downstream signaling .

To experimentally demonstrate MANF-IRE1α interactions, multiple complementary approaches should be employed:

• Microscale thermophoresis (MST) to measure binding affinity

• Gel filtration chromatography to co-purify the MANF-IRE1α LD complex

• ELISA-based detection using nickel-coated plates with IRE1α LD-His

These techniques have confirmed that MANF interacts specifically with the monomer of IRE1α LD. For researchers investigating UPR regulation, it's essential to note that MANF is one of the 12 commonly UPR-upregulated genes and is unusual in bypassing general down-regulation of protein synthesis during ER stress .

What methodologies can assess MANF's protective effects against oxidative stress?

MANF has been shown to mitigate oxidative stress-induced cellular damage, particularly through enhancement of mitophagy. A comprehensive experimental approach should include:

• Cellular models of oxidative stress: Treat nucleus pulposus (NP) cells or other relevant cell types with tert-butyl hydroperoxide (TBHP) to establish oxidative stress conditions .

• MANF expression modulation: Use both overexpression (plasmid transfection) and knockdown (siRNA) approaches to manipulate MANF levels.

• Functional assessments:

  • Cell viability assays (MTT, CCK-8)

  • Apoptosis detection (flow cytometry with Annexin V/PI)

  • ROS measurement (fluorescent probes)

• Molecular analyses:

  • Western blotting for mitophagy-related proteins (e.g., MFN2, PINK1, Parkin)

  • Quantitative real-time PCR for mRNA expression levels

  • RNA-binding protein immunoprecipitation (RIP) and dual-luciferase reporter assays to identify downstream targets

Research has revealed that MANF overexpression enhances mitophagy by upregulating MFN2 expression, thereby mitigating oxidative stress-induced apoptosis . For validation, knockdown of MFN2 should be performed to determine if it reverses the protective effects of MANF overexpression, confirming the mechanistic pathway.

How does MANF expression change after ischemic injury in different tissues?

MANF expression undergoes significant changes following ischemic injury, with distinct temporal and spatial patterns across tissues. In the liver, MANF protein levels increase significantly at 3 hours following ischemia-reperfusion (I/R) injury, compared to sham operation samples . Immunohistochemical analysis confirms that MANF is significantly elevated in liver tissues at 6 hours post-I/R, with highly expressed MANF mainly appearing around the ischemic zone .

In the brain, a profound shift in MANF expression occurs after ischemic stroke. While MANF is primarily expressed in neurons in uninjured brains, after stroke there is a dramatic transition of expression from neurons to inflammatory cells, particularly phagocytic microglia/macrophages within the ischemic territory . The timing of peak expression differs between species: in humans, peak expression occurs approximately two weeks post-stroke, while in rat ischemic cortex, it is observed one week post-stroke .

To accurately track these expression changes, researchers should employ:

• Temporal analysis (multiple time points)

• Cell-type specific markers in co-localization studies

• Both protein (Western blot, immunohistochemistry) and mRNA (qPCR) quantification methods

What experimental approaches can evaluate MANF's therapeutic potential in tissue injury models?

To assess MANF's therapeutic efficacy in tissue injury models, researchers should implement a multi-faceted experimental design:

In hepatic I/R models, hepatocyte-specific MANF knockout (MANFhep-/-) mice show exacerbated injury, which can be partially rescued by recombinant human MANF (rhMANF) injection . RNA sequencing of primary hepatocytes from wild-type and MANFhep-/- mice has identified differential expression of 652 genes (448 upregulated, 204 downregulated), providing insights into the molecular mechanisms of MANF's protective effects .

How can protein-protein interaction studies enhance our understanding of MANF function?

Elucidating MANF's interactome is crucial for understanding its multifunctional nature. Several complementary techniques should be employed:

• Direct binding assays: Beyond traditional co-immunoprecipitation, advanced methods such as microscale thermophoresis (MST) have demonstrated MANF's direct binding to IRE1α . Similar approaches can identify other interaction partners.

• Competition assays: Studies showing that MANF competes with BiP for interaction with IRE1α provide a model for investigating regulatory mechanisms . Researchers should design experiments with titrated concentrations of potential binding partners.

• Structural biology approaches: The unique structure of MANF, with its N-terminal saposin-like lipid binding domain and novel C-terminal domain, suggests specialized interaction surfaces that can be probed through mutation studies.

• Functional validation: After identifying interaction partners, researchers should verify the biological significance through functional assays. For example, MANF mutants deficient in IRE1α binding lack pro-survival action in neurons, confirming the importance of this interaction .

For novel studies, researchers might investigate whether MANF's interactions with UPR components change under different stress conditions or across cell types, potentially explaining its tissue-specific effects.

What are the methodological considerations for studying MANF in different subcellular compartments?

MANF functions in multiple subcellular compartments, including the ER, Golgi, and extracellular space, necessitating compartment-specific experimental approaches:

• Distinguishing intracellular vs. secreted MANF:

  • Cellular fractionation techniques to separate membrane-bound from cytosolic MANF

  • Pulse-chase experiments with labeled MANF to track secretion kinetics

  • Brefeldin A treatment to block secretion and assess accumulation

• ER stress-specific functions:

  • Tunicamycin or thapsigargin treatment to induce ER stress

  • Subcellular co-localization with ER markers during stress response

  • Specific assays for each UPR branch (IRE1α, PERK, ATF6)

• Secreted MANF activities:

  • Conditioned media transfer experiments to isolate paracrine effects

  • Addition of recombinant MANF-His to culture media

  • Blocking antibodies to neutralize extracellular MANF

• Trafficking mechanisms:

  • Signal peptide mutations to alter secretion

  • ER retention signal modifications to force compartmentalization

Research has shown that MANF plays an important role in protecting cells against tunicamycin and thapsigargin-induced cell death . Interestingly, loss of MANF not only renders cells more susceptible to these ER stressors but also increases cell proliferation and decreases cell size, suggesting complex homeostatic functions .

Why might recombinant MANF-His show variable activity across experimental systems?

Several factors can contribute to variability in recombinant MANF-His activity:

When troubleshooting, researchers should employ multiple quality control methods, including SDS-PAGE, Western blot, and biological activity assays. The ED50 in cell proliferation assays using rat C6 cells should be less than 20 μg/ml, corresponding to a specific activity of >50 IU/mg . Additionally, careful reconstitution following manufacturer recommendations (e.g., adding sterile water to prepare a stock solution of 0.2 μg/μl) is essential for maintaining protein activity .

How can researchers address conflicting results between in vitro and in vivo MANF studies?

Reconciling discrepancies between in vitro and in vivo MANF studies requires systematic evaluation of several experimental factors:

• Dosage and delivery considerations:

  • In vitro concentrations may not reflect physiological levels

  • Blood-brain barrier penetration is crucial for CNS studies

  • Tissue distribution after systemic administration affects local concentrations

• Temporal dynamics:

  • MANF expression changes dramatically over time after injury (e.g., 2 weeks post-stroke in humans vs. 1 week in rats)

  • Experimental endpoints should match the relevant biological timescale

• Cell-type specific effects:

  • MANF shifts from neuronal to immune cell expression after stroke

  • Cell culture studies often examine isolated cell types rather than complex interactions

• Mechanistic considerations:

  • MANF mutants deficient in IRE1α binding lack in vivo efficacy in PD models, confirming the importance of this mechanism

  • Evaluate whether key interaction partners are present in both systems

To address these challenges, researchers should design experiments with multiple time points, use cell-type specific MANF knockout or overexpression, and employ comprehensive readouts that reflect both direct cellular effects and systemic responses.

What emerging technologies could advance MANF research in neurodegenerative and inflammatory diseases?

Several cutting-edge technologies hold promise for expanding our understanding of MANF biology:

• Single-cell transcriptomics and proteomics: These technologies can reveal cell-type specific responses to MANF, particularly important given the shift in MANF expression from neurons to immune cells after ischemic injury .

• CRISPR-based approaches: Beyond conventional knockout studies, CRISPR activation or inhibition systems allow temporal control of MANF expression in specific cell populations. This could help dissect the role of MANF in different phases of disease progression.

• Intravital imaging: Real-time visualization of MANF trafficking and function in living tissues could provide insights into its dynamic role during stress responses and injury.

• Protein engineering: Structure-guided modifications of MANF to enhance specific functions (e.g., IRE1α binding, mitophagy promotion) could lead to improved therapeutic variants.

• Biomaterial-based delivery systems: Advanced delivery platforms could overcome challenges in targeting MANF to specific tissues, particularly for crossing the blood-brain barrier in neurodegenerative disease applications.

These approaches, combined with the established understanding of MANF's role in ER stress regulation, neuroprotection, and mitophagy enhancement , could accelerate the development of MANF-based therapeutic strategies for conditions ranging from Parkinson's disease to intervertebral disc degeneration and ischemic injuries.

How might computational approaches enhance understanding of MANF structure-function relationships?

Computational methods offer powerful tools for extending experimental findings and generating new hypotheses about MANF function:

• Molecular dynamics simulations: These can model conformational changes in MANF upon binding to partners like IRE1α, potentially revealing allosteric mechanisms.

• Protein-protein docking: Computational prediction of interaction surfaces between MANF and its binding partners can guide mutagenesis studies to validate key residues.

• Systems biology modeling: Integration of MANF into broader UPR and mitophagy pathway models could reveal emergent properties and feedback mechanisms.

• Machine learning approaches: Analysis of gene expression datasets from MANF knockout studies could identify previously unrecognized patterns and potential biomarkers.

• Evolutionary analysis: Comparative genomics across species, leveraging the high conservation of MANF (99% identity between human and rat) , could identify critical functional domains under selective pressure.

These computational approaches, combined with experimental validation, could help resolve outstanding questions about how MANF's unique structure enables its diverse functions in neuroprotection, ER stress regulation, and mitophagy promotion.