GPNMB Human, Sf9

What is the molecular structure of recombinant GPNMB Human produced in Sf9 cells?

GPNMB Human recombinant produced in Sf9 Baculovirus cells is characterized as a single, glycosylated polypeptide chain containing 462 amino acids (spanning positions 22-474 a.a.). It has a molecular mass of 51.8kDa, although its apparent size on SDS-PAGE typically appears between 50-70kDa due to glycosylation. The protein is expressed with a 6 amino acid His tag at the C-Terminus to facilitate purification and detection in experimental settings . The glycosylation of GPNMB is functionally significant, as studies have shown that inhibition of this post-translational modification, such as in SOD1G93A ALS models, can result in increased motoneuron death, suggesting glycosylation is critical for GPNMB's protective functions .

How does the expression system affect GPNMB's functional properties?

The Sf9 baculovirus expression system provides several advantages for producing functional GPNMB that closely resembles the native human protein. Insect cells like Sf9 possess the cellular machinery necessary for complex post-translational modifications, particularly the glycosylation patterns essential for GPNMB function. The recombinant GPNMB is purified using proprietary chromatographic techniques to ensure high purity . When designing experiments with recombinant GPNMB, researchers should consider that while Sf9-produced proteins generally maintain proper folding and functionality, minor differences in glycosylation patterns compared to mammalian systems may impact certain protein-protein interactions. In inflammation studies, researchers have successfully used recombinant GPNMB fragments (rGPNMB) to demonstrate protective effects in models of neurodegeneration, confirming that the Sf9-expressed protein maintains biologically relevant activity .

What are the recommended storage and handling conditions for GPNMB Human, Sf9?

For optimal preservation of GPNMB Human, Sf9 bioactivity, specific storage conditions are recommended based on usage timeframes. For short-term use (within 2-4 weeks), the protein can be stored at 4°C. For longer periods, freezing at -20°C is recommended . To prevent protein degradation during long-term storage, it is advisable to add a carrier protein such as 0.1% Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) . Multiple freeze-thaw cycles should be strictly avoided as they can compromise protein integrity through denaturation and aggregation . When shipping the protein, cool pack conditions are specified to maintain stability during transport . These careful handling procedures are essential for maintaining the structural integrity and biological activity of GPNMB in experimental settings.

What intracellular signaling pathways are activated by GPNMB?

GPNMB activates several key signaling pathways that mediate its diverse cellular functions. After cleavage by ADAM10, soluble GPNMB can interact with various receptors and proteins to trigger downstream signaling cascades. Under basal physiological conditions, GPNMB interacts with Na+/K+-ATPase (NKA), leading to neuroprotection against oxidative stress through activation of the ERK/MEK and AKT/PI3K pathways . These pathways are critical for cell survival and protection against various stressors. In inflammatory contexts, GPNMB exhibits anti-inflammatory properties by suppressing pro-inflammatory cytokine production, including TNFα, IL-6, and IL-12, while enhancing anti-inflammatory IL-10 expression . The ERK1/2 pathway appears to be particularly important for GPNMB's inflammatory modulation, as silencing of GPNMB in BV-2 microglial cells resulted in diminished ERK1/2 phosphorylation following LPS treatment . Additionally, GPNMB can interact with syndecan-4, inhibiting T-cell activation, which contributes to immunosuppression in certain contexts such as cancer .

How does GPNMB influence macrophage and microglia polarization?

GPNMB plays a significant role in regulating macrophage and microglia polarization, particularly in shifting these cells toward an anti-inflammatory phenotype. Research evidence indicates that GPNMB promotes M2-macrophage polarization, which is associated with inflammation resolution and tissue repair . The molecular mechanisms underlying this polarization effect are not fully elucidated but likely involve GPNMB's interactions with multiple receptors and activation of anti-inflammatory signaling pathways. In microglial cells, the role of GPNMB appears contextual. GPNMB expression increases in response to pro-inflammatory stimuli like LPS treatment, with one study showing elevated GPNMB mRNA levels after 6 hours of LPS exposure, persisting up to 24 hours at higher LPS concentrations (100ng/ml) . Interestingly, another study using similar conditions but different culture media (DMEM/F12 versus DMEM) found no change in GPNMB mRNA levels after 24 hours of LPS treatment, highlighting the importance of experimental conditions in studying GPNMB's inflammatory roles . This apparent contradiction underscores the complexity of GPNMB's function in neuroinflammation.

What protein-protein interactions are critical for GPNMB function?

GPNMB engages in multiple protein-protein interactions that mediate its diverse cellular functions. One key interaction is with Na+/K+-ATPase (NKA), which beyond its ion transport function acts as a receptor modulating neuroinflammation. This GPNMB-NKA interaction appears critical for neuroprotection, particularly in models of neurodegenerative diseases like ALS . GPNMB also interacts with syndecan-4, resulting in inhibition of T-cell activation, which may have implications for immune regulation in disease contexts . In addition, GPNMB can interact with CD44 receptors on astrocytes, leading to decreased expression of inflammatory markers like IL-6 and iNOS following pro-inflammatory stimuli . GPNMB also promotes activation of matrix metalloproteinases (MMP-2, MMP-3, and MMP-9), although the precise molecular mechanisms underlying these interactions remain to be fully characterized . The diversity of GPNMB's protein-protein interactions suggests its function may be highly context-dependent, varying based on the cellular environment, disease state, and available binding partners.

How is GPNMB expression altered in neurodegenerative diseases?

GPNMB expression is significantly altered in various neurodegenerative diseases, suggesting its potential role as both a biomarker and disease modifier. In Alzheimer's disease (AD), GPNMB levels are increased in cerebrospinal fluid (CSF) and post-mortem brain tissues of sporadic AD patients . Interestingly, GPNMB expression is particularly elevated in microglia surrounding β-amyloid (Aβ) aggregate plaques, indicating a disease-specific response pattern . In Parkinson's disease (PD), increased GPNMB expression has been detected in the substantia nigra of patients, and the single nucleotide polymorphism (SNP) rs199347, a known risk factor for PD, is located within the GPNMB gene and results in increased GPNMB expression . In amyotrophic lateral sclerosis (ALS), elevated GPNMB levels have been found in CSF, serum, and lumbar spinal cord tissue of sporadic ALS patients . These consistent increases across multiple neurodegenerative conditions have led researchers to propose GPNMB as a marker for disease-associated microglia (DAM) . The specific pattern of GPNMB upregulation in these diseases suggests it may represent a common response mechanism to neurodegeneration, potentially involved in modulating disease progression.

How do lysosomal storage diseases affect GPNMB expression?

Lysosomal storage diseases, particularly those characterized by macrophage dysfunction, demonstrate significant alterations in GPNMB expression patterns. In Niemann-Pick type C disease, which arises from lysosomal dysfunction in macrophages, GPNMB levels are elevated in the liver, brain, and spleen in mouse models, and in the plasma of affected patients . Similarly, in Gaucher disease, another condition characterized by lysosomal dysfunction in macrophages, increased GPNMB levels have been detected in the spleen, plasma, and cerebrospinal fluid of patients . In genetically modified mouse models of type 1 Gaucher Disease, GPNMB levels were also increased and importantly, returned to normal following therapeutic intervention . These observations suggest that GPNMB upregulation may represent a cellular response to lysosomal stress or dysfunction, particularly in macrophage-lineage cells. The consistent elevation of GPNMB across multiple lysosomal storage disorders indicates it may serve as a biomarker for these conditions and potentially play a functional role in the cellular response to lysosomal pathology. The normalization of GPNMB levels following treatment further supports its potential utility as a biomarker for monitoring therapeutic efficacy.

What experimental models are most effective for studying GPNMB function?

Several experimental models have proven effective for investigating GPNMB function across different research contexts. For in vitro studies, BV-2 microglial cell lines have been successfully used to examine GPNMB's role in neuroinflammation, with experimental designs typically involving treatment with pro-inflammatory stimuli such as LPS at concentrations of 10-100ng/ml for time periods ranging from 6-24 hours . Primary astrocyte cultures have also been employed to study GPNMB's effects on cytokine production, particularly through interactions with the CD44 receptor . For in vivo investigations, transgenic mice overexpressing GPNMB have been developed by injecting V5-His-tagged GPNMB cDNA constructs containing a CAG hybrid promoter into fertilized eggs, providing a model system to examine GPNMB's protective effects in conditions such as cerebral ischemia-reperfusion injury . Disease-specific models, such as genetic models of ALS (SOD1G93A), Niemann-Pick type C, and Gaucher disease, have been instrumental in revealing GPNMB's patterns of expression and potential functions in pathological states . When selecting an experimental model, researchers should consider that GPNMB's effects appear to be highly context-dependent, with responses varying based on cell type, disease state, and inflammatory environment.

What techniques are recommended for modulating GPNMB expression in experimental settings?

Several approaches have been successfully employed to modulate GPNMB expression in experimental settings, each with specific applications depending on the research question. For knockdown experiments, siRNA transfection has been effectively used in cell culture models such as BV-2 microglial cells to reduce GPNMB expression and examine its role in inflammatory responses . This approach revealed that silencing GPNMB resulted in decreased levels of pro-inflammatory markers (iNOS, TNF-α, IL-1β) following LPS treatment, suggesting GPNMB's potential role in mediating certain inflammatory responses . For overexpression studies, transgenic mouse models have been created using constructs containing the GPNMB gene under the control of strong promoters such as the CAG hybrid promoter . This approach enabled researchers to demonstrate GPNMB's protective effects in cerebral ischemia-reperfusion injury . An alternative to genetic modulation is the use of recombinant GPNMB fragments (rGPNMB) for treatment studies, which has been valuable in investigating GPNMB's extracellular functions and potential therapeutic applications . This method has shown that administration of rGPNMB reduces motoneuron death in ALS models and lessens cerebral infarct damage in ischemia models . When designing experiments to modulate GPNMB, researchers should consider that its responses may differ depending on the cellular context and the specific domains or fragments being studied.

How should researchers address contradictory findings in GPNMB studies?

Addressing contradictory findings in GPNMB research requires careful consideration of several methodological factors. First, researchers must account for cell type-specific effects, as GPNMB's function appears to vary significantly across different cellular contexts. For example, contradictory findings regarding GPNMB's role in inflammation have been observed between different cell types and even within the same cell type under different experimental conditions . Culture conditions can significantly impact results, as demonstrated by the contradictory findings in GPNMB mRNA expression following LPS treatment in BV-2 cells cultured in different media (DMEM supplemented with 10% FBS versus DMEM/F12 with 10% FBS) . When designing experiments, researchers should specify which GPNMB domains or fragments are being studied, as the soluble extracellular domain may have different functions compared to the full-length membrane-bound protein. The timing of measurements is also critical, as GPNMB's expression and effects may change over the course of inflammatory or disease processes . To reconcile contradictory findings, researchers should implement comprehensive experimental approaches that examine GPNMB function across multiple time points, in various cell types, and using complementary techniques for modulating and measuring GPNMB expression and activity. Collaborative efforts to standardize experimental protocols across different laboratories would also help address the current inconsistencies in the literature.

What is the potential of GPNMB as a biomarker for neurodegenerative diseases?

GPNMB shows considerable promise as a biomarker for various neurodegenerative conditions based on its consistent upregulation across multiple disorders. In Alzheimer's disease, increased GPNMB levels have been detected in cerebrospinal fluid (CSF) and post-mortem brain tissues of sporadic AD patients, with particularly high expression in microglia surrounding amyloid plaques . In Parkinson's disease, elevated GPNMB expression has been observed in the substantia nigra, with genetic evidence linking the PD risk SNP rs199347 to increased GPNMB expression . For ALS patients, GPNMB levels are increased in CSF, serum, and lumbar spinal cord tissue . These consistent findings across different neurodegenerative conditions suggest GPNMB may serve as a biomarker for generalized neuroinflammation or microglial activation. The accessibility of GPNMB in biofluids like CSF and serum enhances its potential clinical utility. Future research should focus on establishing standardized detection methods for GPNMB in clinical samples, determining disease-specific expression patterns, and evaluating whether GPNMB levels correlate with disease progression or response to therapy. Longitudinal studies comparing GPNMB levels across disease stages would be particularly valuable in establishing its utility as a prognostic biomarker.

Why have therapeutic approaches targeting GPNMB shown mixed results in clinical trials?

Therapeutic approaches targeting GPNMB have yielded mixed results in clinical trials, particularly in cancer treatment, highlighting the complexity of GPNMB biology. The antibody-drug conjugate glembatumumab vedotin (CDX-011) targeting GPNMB was evaluated in phase I/II trials for advanced breast cancer and melanoma but was discontinued in 2018 after failing to improve survival rates compared to standard therapy in metastatic triple-negative breast cancer patients . Several factors may contribute to these mixed outcomes. First, GPNMB appears to have context-dependent functions, acting in both pro- and anti-inflammatory capacities depending on the cellular environment, disease state, and binding partners . Second, GPNMB's widespread expression across multiple cell types, including those in the central nervous system, may lead to off-target effects when using systemic approaches. Third, the processing of GPNMB by proteases like ADAM10 generates soluble fragments with potentially different biological activities than the membrane-bound form, complicating therapeutic targeting . Future therapeutic approaches might benefit from more targeted delivery systems, development of domain-specific inhibitors or activators, or combinatorial approaches that account for GPNMB's diverse signaling networks. Additionally, better patient stratification based on GPNMB expression patterns may improve clinical outcomes in future trials.

What are the most promising directions for future GPNMB research?

Several promising research directions could advance our understanding of GPNMB biology and its therapeutic potential. First, more detailed characterization of GPNMB's domain-specific functions and binding partners would help clarify its seemingly contradictory roles in different disease contexts. This could involve structural studies of GPNMB-receptor complexes and identification of critical binding motifs. Second, development of conditional and cell-type-specific GPNMB knockout or overexpression models would help delineate its function in specific tissues and cell populations, particularly in complex environments like the CNS. Third, longitudinal studies tracking GPNMB expression throughout disease progression in neurodegenerative conditions could establish its value as a biomarker and identify optimal therapeutic windows. Fourth, development of selective modulators of GPNMB function, rather than global inhibitors, might allow for more nuanced therapeutic approaches that preserve beneficial functions while inhibiting detrimental ones. Finally, integration of GPNMB research with emerging technologies like single-cell transcriptomics and advanced imaging could provide unprecedented insights into its cell-specific functions in complex tissues. These approaches, combined with standardized experimental protocols across research groups, would help resolve current contradictions in the literature and advance GPNMB from a promising research target to a clinically relevant biomarker or therapeutic target.