HMGB1 Human, Sf9

What is HMGB1 and what are its primary biological functions?

HMGB1 (High-Mobility Group Box 1, also known as HMG1, HMG3, SBP-1, or Amphoterin) is an abundant chromatin-binding protein found in eukaryotic cell nuclei that serves dual functions depending on its location. Inside cells, HMGB1 binds to DNA and participates in transcriptional regulation, DNA replication, repair, and chromatin remodeling . It enhances the binding affinity of transcription factors including p53, Rb, and NF-κB to DNA by bending the DNA molecule . In the cytoplasm, HMGB1 regulates apoptosis and autophagy by binding to proteins like Beclin1 and modulating caspase-3 activation . Extracellularly, HMGB1 functions as a pro-inflammatory cytokine with activities resembling tumor necrosis factor (TNF), contributing to various inflammatory and autoimmune conditions .

How does the redox state of HMGB1 affect its biological activity?

The immunological activity of HMGB1 is critically determined by the oxidation states of its three cysteine residues (Cys23, Cys45, and Cys106), creating three distinct forms with different functions :

Researchers must carefully consider HMGB1's redox state when designing experiments, as the biological outcomes can vary dramatically depending on which form predominates.

What post-translational modifications occur in HMGB1 and how do they influence function?

HMGB1 undergoes several important post-translational modifications that regulate its localization and function:

• N-glycosylation: Occurs at residues N37, N134, and N135 . N-glycosylation affects HMGB1's nuclear mobility and reduces its DNA binding affinity through steric hindrance . This has been confirmed through FRAP (Fluorescence Recovery After Photobleaching) analysis showing that non-glycosylated HMGB1 mutants (N37Q/N134Q and N37Q/N135Q) have slower nuclear mobility than wild-type HMGB1 .

• Acetylation: While not directly discussed in the provided materials, acetylation of lysine residues in HMGB1's nuclear localization sequences is known to prevent nuclear re-entry, facilitating cytoplasmic accumulation and subsequent secretion.

• Oxidation: As detailed above, oxidation of cysteine residues creates functionally distinct forms of HMGB1 with different receptor binding capabilities and downstream effects .

When designing studies involving HMGB1, researchers should consider how these modifications might influence experimental outcomes and interpret results accordingly.

What are the technical advantages of expressing human HMGB1 in Sf9 cells?

Expressing human HMGB1 in Sf9 insect cells offers several significant advantages for research applications:

• Post-translational modifications: Sf9 cells can perform important eukaryotic post-translational modifications including N-glycosylation, which has been confirmed for HMGB1 at residues N37, N134, and N135 . This allows researchers to study how these modifications affect HMGB1 function.

• High protein yield: The baculovirus expression system in Sf9 cells typically produces higher protein yields than mammalian expression systems, facilitating structural and functional studies that require substantial amounts of purified protein.

• Proper protein folding: Sf9 cells provide a eukaryotic environment that supports proper folding of complex proteins like HMGB1, preserving their functional domains and activities.

• Simplified purification: The ability to add purification tags such as the 6×His tag allows for efficient purification using affinity chromatography followed by size exclusion chromatography to achieve high protein homogeneity .

• Reduced endotoxin contamination: Unlike bacterial expression systems, insect cell systems produce proteins with minimal endotoxin contamination, which is crucial for immunological studies involving HMGB1.

What is the optimal expression and purification protocol for HMGB1 in Sf9 cells?

Based on the research literature, the following optimized protocol is recommended for HMGB1 expression and purification from Sf9 cells:

• Cloning and baculovirus generation:

  • Clone the HMGB1 gene into a baculovirus transfer vector such as pFastBac HT-B

  • Include an N-terminal 6×His tag for purification

  • Generate recombinant baculovirus according to manufacturer's protocols (e.g., Invitrogen's system)

• Protein expression:

  • Infect Sf9 cells with the recombinant baculovirus

  • Express for approximately 36 hours at 27°C

• Cell lysis and initial purification:

  • Harvest cells and lyse in buffer containing 25 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 30 mM imidazole, and 5 mM β-mercaptoethanol

  • Purify using Ni-NTA affinity chromatography

  • Elute with buffer containing increased imidazole concentration (300 mM)

• Tag removal and final purification:

  • Treat with TEV protease for 16 hours at 4°C to remove the His tag

  • Perform size exclusion chromatography using a Superdex-200 column with buffer containing 25 mM Tris-HCl pH 7.5 and 150 mM NaCl

• Quality control and storage:

  • Verify purity by SDS-PAGE

  • Store in buffer containing 20 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM DTT, and 10% glycerol

  • For long-term storage, aliquot and keep at 4°C (if using within 2-3 weeks) or -80°C for extended periods

How can researchers verify the N-glycosylation status of recombinant HMGB1?

Verifying the N-glycosylation status of recombinant HMGB1 is crucial for functional studies. The following complementary approaches can be employed:

• Enzymatic deglycosylation:

  • Treat purified HMGB1 with PNGase F, which specifically cleaves N-linked glycans

  • Resuspend the protein in 20 mM ammonium bicarbonate buffer (pH 8.0)

  • Heat at 98°C for 10 minutes to denature the protein

  • Incubate with PNGase F overnight at 37°C

  • Analyze by SDS-PAGE; a downward migration shift indicates the presence of N-glycosylation

• Glycosylation inhibitor treatment:

  • Treat cells expressing HMGB1 with tunicamycin (an inhibitor of N-linked glycosylation)

  • Compare migration patterns of HMGB1 from treated and untreated cells by western blotting

  • A band shift indicates the presence of N-glycosylation in the native protein

• LC-MS/MS analysis:

  • Perform liquid chromatography-tandem mass spectrometry on purified HMGB1

  • After PNGase F treatment, look for a mass increase of 0.98 Da at glycosylation sites, resulting from the conversion of asparagine to aspartic acid

  • This technique can precisely identify the glycosylation sites (N37, N134, and N135 in HMGB1)

• Mutational analysis:

  • Generate HMGB1 mutants where potential glycosylation sites are replaced (e.g., N37Q, N134Q)

  • Compare the molecular weight and migration patterns of wild-type and mutant proteins

  • Differences confirm the presence and location of glycosylation sites

How can researchers assess HMGB1's DNA binding properties and the impact of glycosylation?

HMGB1's DNA binding properties and the effects of glycosylation can be evaluated using several complementary techniques:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified HMGB1 (wild-type or glycosylation mutants) with plasmid DNA

  • Analyze complex formation by gel electrophoresis

  • Research has shown that non-glycosylated HMGB1 (N37Q/N134Q) forms stronger complexes with DNA than wild-type glycosylated HMGB1, as evidenced by retarded migration and some complexes remaining in the wells

• Fluorescence Recovery After Photobleaching (FRAP):

  • Express EGFP-tagged HMGB1 variants in cells

  • Photobleach a nuclear area and monitor fluorescence recovery

  • Non-glycosylated HMGB1 mutants (N37Q/N134Q and N37Q/N135Q) show lower recovery percentages than wild-type HMGB1, indicating reduced mobility due to stronger DNA binding

• Molecular modeling:

  • Model the interactions between N-acetyl-D-glucosamine (GlcNAc)-tagged HMGB1 A or B box and double-stranded DNA

  • This approach has revealed that GlcNAc molecules at N37 and N134 create steric hindrance that interferes with the phosphate backbone or nucleic acids of dsDNA

These techniques provide complementary insights into how glycosylation modulates HMGB1's interaction with DNA, which is essential for understanding its nuclear functions.

What experimental approaches can distinguish between different redox forms of HMGB1?

Distinguishing between HMGB1's redox forms is crucial for understanding its various biological activities. Researchers can employ these approaches:

• Mass spectrometry-based redox proteomics:

  • Treat samples with alkylating agents to trap the existing redox state

  • Digest the protein and analyze peptide fragments by LC-MS/MS

  • Identify the presence of free thiols, disulfide bonds, or oxidized forms at Cys23, Cys45, and Cys106

• Functional bioassays:

  • Test the ability of different HMGB1 preparations to activate TLR4 signaling (characteristic of disulfide HMGB1)

  • Assess chemotactic activity through CXCR4 (characteristic of fully reduced HMGB1)

  • Measure cytokine production in macrophages or dendritic cells

• Redox-specific antibodies:

  • Use antibodies that specifically recognize different redox forms of HMGB1

  • Apply these in western blotting, ELISA, or immunohistochemistry

• Site-directed mutagenesis:

  • Create cysteine-to-serine mutations to mimic the reduced state

  • Create cysteine-to-aspartate mutations to mimic the oxidized state

  • Compare the functional properties of these mutants with wild-type protein

These approaches allow researchers to correlate specific redox forms with biological activities, providing insight into how HMGB1 functions in different physiological and pathological contexts.

How does HMGB1 contribute to cellular processes like autophagy and what methods can assess this function?

HMGB1 plays critical roles in regulating autophagy through several mechanisms :

• Binding to Beclin1: HMGB1 interacts with Beclin1, a key regulator of autophagy, promoting autophagosome formation while limiting apoptosis.

• Protection of autophagy proteins: HMGB1 binds to Beclin1 and ATG5, suppressing calpain-induced cleavage and modulating the transition from autophagy to apoptosis during inflammation.

• STAT3 signaling inhibition: HMGB1 may inhibit STAT3-mediated signaling, promoting autophagy and providing protection against infection in intestinal epithelial cells.

Methods to assess HMGB1's role in autophagy include:

• Autophagy flux assays:

  • Monitor LC3-II levels with and without lysosomal inhibitors in cells expressing wild-type or mutant HMGB1

  • Use GFP-LC3 puncta formation as an indicator of autophagosome formation

• Protein interaction studies:

  • Perform co-immunoprecipitation experiments to detect HMGB1-Beclin1 interactions

  • Use proximity ligation assays to visualize these interactions in situ

• HMGB1 knockdown/knockout approaches:

  • Use siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate HMGB1 expression

  • Assess the impact on autophagy markers and autophagic flux

• Subcellular localization tracking:

  • Monitor HMGB1 translocation from the nucleus to the cytoplasm during autophagy induction

  • Use fluorescently tagged HMGB1 or immunofluorescence with subcellular markers

These methods can reveal how HMGB1 contributes to autophagy regulation in different physiological and pathological contexts.

How can researchers investigate HMGB1's non-classical secretion mechanisms?

HMGB1 lacks an ER-targeting signal peptide yet undergoes N-glycosylation and is secreted through non-classical pathways. To investigate these mechanisms:

• Secretion pathway inhibitor studies:

  • Treatment with brefeldin A and nocodazole (inhibitors of ER-Golgi transport and microtubule polymerization) does not decrease LPS-stimulated HMGB1 secretion, indicating that the conventional secretion pathway is not involved

  • Use other pathway-specific inhibitors to identify the mechanisms involved

• Live-cell imaging:

  • Create fluorescently tagged HMGB1 constructs

  • Track their movement from the nucleus to the extracellular space in real-time

  • Co-label with markers for different vesicular compartments

• Proteomics approach:

  • Identify HMGB1-interacting proteins during the secretion process using proximity labeling techniques

  • Compare the interactomes of wild-type and glycosylation-mutant HMGB1

• Glycosylation status analysis:

  • Compare the glycosylation patterns of intracellular versus secreted HMGB1

  • Determine if glycosylation status changes during the secretion process

• Secretion kinetics:

  • Use pulse-chase experiments with metabolic labeling to track the time course of HMGB1 synthesis, modification, and secretion

These approaches can help elucidate the unconventional secretion mechanisms of HMGB1 and the role of glycosylation in this process.

What is the relationship between HMGB1 forms and disease pathogenesis?

HMGB1 is implicated in various disease processes, with different forms and modifications playing distinct roles:

• Liver diseases:

  • HMGB1 contributes to alcoholic liver disease (ALD) progression by promoting inflammation and lipid accumulation

  • HMGB1 knockdown reduces SREBP-1 synthesis and lipid accumulation, providing protection against disease progression

  • In liver fibrosis, HMGB1 activates pMEK1/2/pERK1/2/pcJun and PI3K/Akt signaling, enhancing Collagen type I synthesis via RAGE

• Inflammatory conditions:

  • In Sjögren's syndrome, increased extracellular HMGB1 in salivary glands indicates involvement in the inflammatory process

  • In sepsis, HMGB1 catalyzes movement of LPS monomers to CD14, initiating TLR4-mediated proinflammatory responses

• Cancer:

  • HMGB1 plays a role in the relationship between necrosis and malignancy in glioma tumors

  • Overexpression is common in gastrointestinal stromal tumors and related to KIT mutation

Research methods to investigate these relationships include:

• Redox-specific detection in patient samples

• Animal models with redox-locked HMGB1 variants

• Cell-type specific knockout studies

• Therapeutic targeting of specific HMGB1 forms

Understanding which HMGB1 forms predominate in different disease states could guide the development of more targeted therapeutic approaches.

How can researchers develop HMGB1-targeted therapeutic strategies?

Based on HMGB1's roles in disease pathogenesis, several therapeutic strategies can be explored:

• Redox-specific targeting:

  • Develop compounds that selectively bind to and neutralize pro-inflammatory forms of HMGB1

  • Create redox-modulating agents that promote conversion to non-inflammatory forms

• Post-translational modification inhibitors:

  • Design inhibitors of specific enzymes that modify HMGB1 (e.g., glycosylation inhibitors)

  • Target acetylation pathways to prevent nuclear-cytoplasmic translocation and secretion

• Receptor antagonists:

  • Develop antagonists for HMGB1 receptors (TLR4, RAGE) to block downstream signaling

  • Create decoy receptors to capture extracellular HMGB1

• Domain-specific antibodies:

  • Generate antibodies that recognize specific domains or modified forms of HMGB1

  • Use these for both diagnostic purposes and therapeutic neutralization

• Gene therapy approaches:

  • Explore CRISPR-Cas9 or antisense oligonucleotides to modulate HMGB1 expression

  • Develop cell-type specific delivery systems for maximal therapeutic effect

For all these approaches, researchers should:

• Validate targets in relevant disease models

• Assess specificity to minimize off-target effects

• Evaluate effects on both pathological and physiological HMGB1 functions

• Consider combinatorial approaches targeting multiple aspects of HMGB1 biology

What are common challenges in expressing and purifying functional HMGB1 from Sf9 cells?

Researchers frequently encounter these challenges when working with HMGB1 in Sf9 cells:

• Protein aggregation:

  • HMGB1 can form aggregates during expression or purification

  • Solution: Optimize buffer conditions, include stabilizing agents like glycerol (10%), and maintain reducing conditions with DTT (0.5 mM)

• Proteolytic degradation:

  • HMGB1 may be susceptible to proteases

  • Solution: Include protease inhibitors during lysis and purification, work at 4°C, and minimize processing time

• Inconsistent glycosylation:

  • Insect cells may produce heterogeneous glycosylation patterns

  • Solution: Verify glycosylation status using PNGase F treatment and LC-MS/MS analysis

• Maintaining redox state:

  • Different redox forms have distinct functions

  • Solution: Work under controlled redox conditions and verify the redox state using mass spectrometry

• Endotoxin contamination:

  • Even low levels can affect functional assays

  • Solution: Use endotoxin-free reagents and include endotoxin removal steps during purification

• Tag interference:

  • Purification tags may affect protein function

  • Solution: Include TEV protease treatment to remove tags, followed by additional purification steps

Careful attention to these factors will help ensure the production of homogeneous, functional HMGB1 suitable for structural and functional studies.

How can researchers accurately interpret contradictory findings about HMGB1 in the literature?

The HMGB1 literature contains apparent contradictions that can be resolved by considering several key factors:

• Redox state variations:

  • Different laboratories may work with HMGB1 in different redox states

  • Solution: Always characterize and report the redox state of HMGB1 preparations using mass spectrometry or functional assays

• Post-translational modification heterogeneity:

  • Different expression systems and purification methods may yield HMGB1 with varying modifications

  • Solution: Fully characterize modifications and consider their impact on experimental outcomes

• Species differences:

  • HMGB1 from different species may have subtle functional differences

  • Solution: Clearly specify the species origin and consider evolutionary conservation when comparing results

• Experimental context variations:

  • HMGB1 functions differently depending on cell type, disease state, and microenvironment

  • Solution: Carefully document experimental conditions and avoid overgeneralizing findings

• Technical approaches:

  • Different detection methods may have varying sensitivities and specificities

  • Solution: Use multiple complementary techniques to confirm findings

When reviewing literature, researchers should pay particular attention to these factors and design experiments that specifically address potential sources of contradiction.

What techniques can distinguish between intracellular and extracellular functions of HMGB1?

Distinguishing between HMGB1's intracellular and extracellular functions requires specialized approaches:

• Compartment-specific manipulations:

  • Use cell-impermeable neutralizing antibodies to target only extracellular HMGB1

  • Create fusion proteins with compartment-targeting sequences to restrict HMGB1 to specific locations

• Conditional knockout/knockin models:

  • Generate models with tissue-specific or inducible HMGB1 manipulation

  • Create mutants with altered secretion but preserved intracellular functions

• Secretion pathway analysis:

  • Use inhibitors of conventional (brefeldin A) and non-conventional secretion pathways

  • Monitor the impact on HMGB1-dependent processes

• Receptor antagonism:

  • Block extracellular receptors (TLR4, RAGE) without affecting intracellular HMGB1

  • Compare effects with direct HMGB1 inhibition

• Fractionation approaches:

  • Carefully separate nuclear, cytoplasmic, and extracellular fractions

  • Analyze HMGB1 forms and binding partners in each compartment

• Real-time imaging:

  • Track fluorescently tagged HMGB1 movement between compartments

  • Correlate localization changes with functional outcomes

These approaches can help researchers determine whether observed effects are due to intracellular functions of HMGB1 (DNA binding, autophagy regulation) or its extracellular activities (cytokine-like functions, receptor activation).