mfsd5 Antibody

What is MFSD5 and what is its primary function in cellular biology?

MFSD5 (Major Facilitator Superfamily Domain-Containing Protein 5) is a transmembrane protein that functions primarily as a molybdate-anion transporter. It mediates high-affinity intracellular uptake of molybdenum, a rare but essential oligo-element . Structurally, MFSD5 is composed of 450 amino acids with 12 transmembrane regions and displays the typical hydrophobicity pattern of membrane proteins . It belongs to the atypical solute carrier (SLC) superfamily within the Major Facilitator Superfamily (MFS) clan .

Functionally, MFSD5 (also known as HsMOT2) is critical for the cellular uptake of molybdate ions, which are the bioavailable form of molybdenum (MoO4²⁻) . This element is essential for the activity of over 50 pterin-containing enzymes including sulfite oxidase and aldehyde oxidase . MFSD5 knockdown experiments have demonstrated reduced molybdate uptake activity, confirming its role in molybdenum transport .

Where is MFSD5 expressed in mammalian systems?

MFSD5 shows a wide distribution pattern across both central and peripheral tissues. According to detailed histological characterization studies:

• Central Nervous System: MFSD5 protein is abundantly expressed in the adult mouse brain, particularly in both excitatory and inhibitory neurons, but not in astrocytes . Its expression is detected during embryogenesis and continues into adulthood .

• Peripheral Tissues: MFSD5 transcripts have been detected in numerous tissues and cell types, with higher expression levels observed in the cervix, stomach, nerves, and skin . qRT-PCR analysis has confirmed its ubiquitous expression in peripheral organs .

The expression of MFSD5 appears to be regulated by energy homeostasis, as studies have shown that its mRNA levels are significantly down-regulated in specific brain regions during both starvation and high-fat diet feeding, particularly in brain sections containing areas involved in food intake and reward processing .

How is MFSD5 classified within protein families and what are its closest related proteins?

MFSD5 is classified as an atypical solute carrier (SLC) protein within the Major Facilitator Superfamily (MFS) clan . Phylogenetic analyses have revealed several important classification details:

• MFSD5 belongs to the proposed "Atypical MFS Transporter Family 6" (AMTF6) along with MFSD1, with which it shares approximately 20% amino acid sequence identity .

• It is an α-group atypical SLC based on phylogenetic classification .

• MFSD11 has been identified as closely related to MFSD5 in humans, along with other SLC families that have organic substrate profiles .

Protein sequence analysis places MFSD5 within a broader network of transporters with potential functional relationships. The table below outlines key protein identifiers for human MFSD5:

These identifiers facilitate cross-referencing MFSD5 across various protein and genomic databases for comprehensive research .

What experimental applications are validated for MFSD5 antibodies?

MFSD5 antibodies have been validated for multiple experimental applications, with specific performance parameters established for different techniques. The primary validated applications include:

• Enzyme-Linked Immunosorbent Assay (ELISA): MFSD5 antibodies have been validated for detecting the protein in solution-based assays .

• Immunohistochemistry (IHC): Antibodies have been extensively validated for tissue localization studies with recommended dilution ranges of 1:20 to 1:200 .

• Immunofluorescence (IF) and Immunocytochemistry (ICC): MFSD5 antibodies perform well in cellular localization studies with recommended dilution ranges of 1:50 to 1:200 .

• Western Blot (WB): Some antibodies have been verified through western blot analysis, which was used as one of three verification methods alongside co-staining with antibodies from different sources .

The validation of MFSD5 antibodies typically involves multiple verification approaches to ensure specificity, as demonstrated in studies where antibodies were verified through: 1) western blot analysis, 2) co-staining with antibodies from different sources, and 3) additional verification methods .

How should researchers optimize MFSD5 antibody protocols for different experimental techniques?

Optimization of MFSD5 antibody protocols requires consideration of several key parameters depending on the experimental technique:

For Immunohistochemistry (IHC):

• Recommended dilution range: 1:20 to 1:200

• Buffer composition: 0.01M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol

• Fixation methods should be optimized based on tissue type, with paraformaldehyde fixation commonly used for neural tissues where MFSD5 is highly expressed

• Antigen retrieval methods may be necessary for formalin-fixed tissues

For Immunofluorescence (IF) and Immunocytochemistry (ICC):

• Recommended dilution range: 1:50 to 1:200

• Cell permeabilization is critical for accessing the transmembrane domains of MFSD5

• Co-staining with neuronal markers may be beneficial when studying MFSD5 in neural tissues, as it is expressed in both excitatory and inhibitory neurons

General Protocol Optimization Considerations:

• Sample preparation should account for the membrane-bound nature of MFSD5 with its 12 transmembrane domains

• Storage conditions: Aliquot and store at -20°C or -80°C to avoid repeated freeze-thaw cycles

• Each user should determine optimal dilutions/concentrations empirically for their specific experimental conditions

When adapting protocols for specific research questions, consider that MFSD5 expression may be altered by metabolic conditions such as starvation or high-fat diet, which could affect detection sensitivity in some experimental models .

What controls should be included when using MFSD5 antibodies in research?

Rigorous experimental design for MFSD5 antibody applications should include the following controls:

Positive Controls:

• Human tissue samples known to express MFSD5 at high levels, such as cervix, stomach, nerves, and skin tissues

• Neural tissues, particularly those containing both excitatory and inhibitory neurons where MFSD5 expression has been well-documented

• Cell lines with confirmed MFSD5 expression or overexpression systems (e.g., doxycycline-inducible MFSD5 WT-OE cells)

Negative Controls:

• Primary antibody omission controls to evaluate non-specific binding of secondary detection systems

• Isotype controls using non-specific rabbit IgG at the same concentration as the MFSD5 antibody

• For genetic approaches, MFSD5 knockout models or cells with MFSD5 knockdown can serve as specificity controls

Antibody Validation Controls:

• Western blot verification showing a band at the expected molecular weight

• Peptide competition assays using the immunizing peptide (e.g., recombinant human Molybdate-anion transporter protein amino acids 216-248)

• Co-localization with antibodies from different sources or targeting different epitopes of MFSD5

These controls are particularly important given that MFSD5 belongs to a family of proteins with sequence similarities, which increases the risk of cross-reactivity. For instance, MFSD5 shares approximately 20% sequence identity with MFSD1 , necessitating careful validation of antibody specificity.

How is MFSD5 expression regulated in response to metabolic changes?

MFSD5 expression demonstrates significant responsiveness to metabolic challenges, suggesting a potential role in energy homeostasis regulation. Based on experimental evidence:

• Starvation Response: In mice subjected to food restriction, MFSD5 mRNA expression was significantly down-regulated (P<0.001) in brain regions involved in food intake regulation and reward processing . Specifically, these changes were observed in the cortex and hypothalamus of food-restricted mice .

• High-Fat Diet (HFD) Response: Similarly, MFSD5 mRNA levels were significantly down-regulated in mice subjected to high-fat diet (P<0.001), particularly in brain sections containing areas involved in food intake and reward processing .

This bidirectional regulation (responding to both energy deficit and excess) suggests that MFSD5 may be part of a homeostatic system that responds to metabolic state changes. This is in contrast to MFSD11, which shows an opposite pattern of regulation, being up-regulated by both starvation (P<0.01) and high-fat diet (P<0.001) .

The transcriptional regulation of MFSD5 appears to be tissue-specific, with brain regions involved in energy homeostasis showing the most pronounced changes. These findings support potential involvement of MFSD5 in energy regulation pathways, possibly through its role in molybdenum transport, which affects the activity of multiple metabolic enzymes .

What is known about MFSD5's role in molybdenum transport and its impact on cellular physiology?

MFSD5 (also known as HsMOT2) plays a critical role in cellular molybdenum homeostasis through its function as a high-affinity molybdate transporter. Research has established several key aspects of this function:

• Transport Mechanism: MFSD5 mediates the high-affinity intracellular uptake of molybdenum in the form of molybdate ions (MoO4²⁻), which is the bioavailable form of this essential trace element .

• Functional Verification: Overexpression studies using human MFSD5 (HsMOT2) in Saccharomyces cells have demonstrated that it functions as an ion transporter with molybdate ions as the main substrate . Conversely, MFSD5 knockdown leads to reduced molybdate uptake activity .

• Physiological Significance: Molybdate transported by MFSD5 is essential for the activity of more than 50 pterin-containing enzymes . These enzymes play crucial roles in diverse metabolic processes including:

  • Sulfite oxidation (via sulfite oxidase)

  • Purine metabolism (via xanthine oxidase)

  • Aldehyde detoxification (via aldehyde oxidase)

• Behavioral Impact: Male homozygous MFSD5 knockout mice exhibit more anxious or depressed behavior compared to littermate controls , suggesting that MFSD5-mediated molybdenum transport affects neurological functions potentially through altered enzyme activities.

The critical nature of MFSD5's function is underscored by the wide distribution of the protein across tissues and its evolutionary conservation. As molybdenum is required for the function of enzymes involved in detoxification and metabolic processes, MFSD5's role in molybdenum transport positions it as a key regulator of cellular redox balance and metabolic health.

What is the evidence for MFSD5 interactions with other proteins and cellular pathways?

Research has identified several important protein interactions and pathway connections for MFSD5:

• GLP-1R Interaction: MFSD5 has been reported to interact with the glucagon-like peptide 1 receptor (GLP-1R) in cell-based studies where GLP-1R was overexpressed in CHO cell lines . This interaction suggests that MFSD5 could play a role in glucose homeostasis and pancreatic β-cell proliferation pathways, as GLP-1R is a key mediator of these processes .

• Co-expression Network: Analysis of single-cell transcriptomics has identified genes that are co-expressed with MFSD5, suggesting functional relationships . While MFSD5 itself was not identified as one of the most commonly co-expressed atypical SLCs, the study established methods to identify protein interactions through co-expression analysis .

• Protein Proximity: In situ proximity ligation assays have been used to detect potential interactions at the protein level among related transporters . Though MFSD5-specific interactions were not the focus of the referenced study, the methodology demonstrated that even genes like MFSD9, which was not found to be co-expressed at the RNA level, could be found in proximity to other atypical SLCs at the protein level .

• Glycosylation Pathways: Recent research indicates a potential role for MFSD5 in protein glycosylation, as cell surface binding of an anti-Tn antibody (5F4) increased upon doxycycline induction of MFSD5 WT-OE cells . This suggests MFSD5 may influence post-translational modifications that affect cell surface glycosylation patterns.

These interactions position MFSD5 at the intersection of multiple cellular pathways beyond simple molybdenum transport, including metabolic signaling, post-translational modifications, and potentially broader transporter networks. The GLP-1R interaction is particularly notable as it suggests a potential mechanism through which MFSD5 might influence energy homeostasis and metabolic regulation.

What epitopes are targeted by commonly available MFSD5 antibodies and how does this affect their application?

Commercial MFSD5 antibodies typically target specific regions of the protein that influence their utility in different applications. Based on the search results:

• Common Immunogen: Several commercially available polyclonal antibodies are raised against recombinant human Molybdate-anion transporter protein (amino acids 216-248) . This region appears to be immunogenic and accessible for antibody binding.

• Epitope Considerations: The targeted epitope (aa 216-248) falls within the middle portion of the 450-amino acid MFSD5 protein . Given that MFSD5 has 12 transmembrane domains , this epitope likely corresponds to either an extracellular loop or an intracellular domain, affecting membrane permeabilization requirements for certain applications.

• Application Effects: The epitope location may explain why these antibodies perform well in applications like IHC and IF/ICC (with recommended dilutions of 1:20-1:200 for IHC and 1:50-1:200 for IF/ICC) . The accessibility of this epitope in fixed and permeabilized samples likely contributes to consistent staining patterns in these applications.

What methodological approaches help resolve contradictory findings in MFSD5 localization studies?

Contradictory findings regarding protein localization are common in membrane protein research, including for MFSD5. To resolve such contradictions, researchers can employ several methodological approaches:

• Multi-technique Validation: Combine complementary techniques to verify localization:

  • Cellular fractionation followed by western blotting

  • Immunofluorescence microscopy with co-localization markers

  • Electron microscopy for high-resolution localization

  • Live-cell imaging using fluorescently tagged MFSD5 constructs

• Epitope Tagging Strategies: If contradictory results arise from different antibodies:

  • Generate expression constructs with different epitope tags (HA, FLAG, GFP)

  • Compare localization patterns using tag-specific antibodies

  • Use inducible expression systems to control expression levels

• Controls for Specificity:

  • Include MFSD5 knockout or knockdown samples as negative controls

  • Perform peptide competition assays using the immunizing peptide

  • Use multiple antibodies targeting different epitopes of MFSD5

• Addressing Technical Variables:

  • Systematically compare fixation methods (PFA, methanol, acetone)

  • Test different permeabilization protocols (Triton X-100, saponin, digitonin)

  • Optimize antibody concentrations and incubation conditions

• Functional Verification:

  • Correlate localization with functional assays (e.g., molybdate transport)

  • Use subcellular targeting motif mutations to alter localization and assess functional consequences

The search results note contradictory reports regarding the subcellular location of related proteins, with MFSD1 being found in multiple subcellular locations . Similar challenges may apply to MFSD5, particularly given discrepancies between mRNA and protein levels that can result from different regulatory controls .

How can researchers effectively assess MFSD5 function in relation to metabolic dysregulation models?

To investigate MFSD5's role in metabolic dysregulation, researchers can implement these methodological approaches:

• Diet Manipulation Studies:

  • Follow established protocols for starvation (food restriction) and high-fat diet experiments that have previously demonstrated MFSD5 regulation

  • Monitor MFSD5 expression changes in key brain regions (cortex, hypothalamus) and peripheral tissues using qRT-PCR and immunohistochemistry

  • Recommended primers for murine studies: MFSD5 forward 5'-tgttgggtgtcatacaagc-3' and reverse 5'-ggtctagcacaggtgtcc-3'

• Cell Culture Models:

  • Utilize N25/2 mouse hypothalamic cells for amino acid starvation studies, following established protocols that have revealed regulation of related transporters

  • Implement complete amino acid starvation for varying time periods (1-16h) to assess temporal dynamics of MFSD5 regulation

  • Apply doxycycline-inducible MFSD5 overexpression systems to study gain-of-function effects

• Functional Assays:

  • Measure intracellular molybdate uptake using radioactive tracers or fluorescent probes in control versus metabolically challenged conditions

  • Assess activity of molybdoenzymes (sulfite oxidase, xanthine oxidase) as functional readouts of MFSD5-mediated molybdate transport

  • Investigate GLP-1R signaling pathways in relation to MFSD5 expression levels, particularly focusing on glucose homeostasis endpoints

• Genetic Approaches:

  • Utilize MFSD5 knockout mice to assess behavioral and metabolic consequences, with particular attention to anxiety/depression phenotypes previously reported

  • Implement tissue-specific conditional knockout models to distinguish central versus peripheral effects

  • Apply CRISPR-Cas9 gene editing to create specific mutations in MFSD5 transport motifs

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics analyses to build comprehensive networks of MFSD5-associated pathways

  • Particular focus should be placed on molybdoenzyme pathways and GLP-1R signaling networks

  • Apply similar analytical approaches to those used in metabolic mapping studies of the SLC superfamily

When designing these studies, researchers should carefully control for sex differences, as male homozygous MFSD5 knockout mice have shown specific behavioral phenotypes , and ensure appropriate reference gene selection for qRT-PCR (e.g., using Gapdh, H3a, and Actb as normalization factors as previously validated) .

How can MFSD5 antibodies be utilized in neuroscience research focusing on energy homeostasis?

MFSD5 antibodies offer valuable tools for investigating neuronal circuits involved in energy homeostasis based on several key findings:

• Brain Region-Specific Expression Analysis:

  • MFSD5 antibodies can be used to map protein expression across brain regions involved in feeding behavior and energy regulation

  • Particularly valuable for immunohistochemical studies of the cortex and hypothalamus, where MFSD5 expression is significantly altered by metabolic challenges

  • Co-staining protocols with markers for specific neuronal populations can identify the precise cellular subsets expressing MFSD5

• Neuronal Subtype Characterization:

  • MFSD5 is expressed in both excitatory and inhibitory neurons but not in astrocytes

  • Researchers can utilize MFSD5 antibodies in combination with markers such as VGLUT1 (excitatory neurons) and VIAAT (inhibitory neurons) to study subtype-specific expression patterns

  • This approach can reveal whether MFSD5 regulation during metabolic challenges is global or restricted to specific neuronal populations

• Circuit-Level Analysis:

  • Implement CLARITY or iDISCO+ tissue clearing methods with MFSD5 immunolabeling to achieve whole-brain mapping of expression

  • Combine with retrograde and anterograde tracing methods to identify MFSD5-expressing neurons within specific circuits

  • Use TRAP (Translating Ribosome Affinity Purification) or single-cell RNA sequencing to profile MFSD5-expressing neurons

• Functional Studies:

  • Use MFSD5 antibodies to validate knockdown or overexpression efficiency in specific brain regions

  • Implement immunohistochemistry following behavioral tests to correlate MFSD5 expression with phenotypic outcomes

  • Combine with physiological recordings to link MFSD5 expression to neuronal activity patterns in feeding circuits

For these applications, researchers should optimize fixation and permeabilization protocols for brain tissue, with recommended antibody dilutions of 1:20-1:200 for IHC and 1:50-1:200 for IF . The demonstrated regulation of MFSD5 in response to both starvation and high-fat diet particularly positions these antibodies as valuable tools for studying neuronal adaptations to metabolic challenges.

What emerging applications exist for MFSD5 antibodies in cancer and metabolic disease research?

Recent findings suggest several promising applications for MFSD5 antibodies in cancer and metabolic disease research:

• Cancer Research Applications:

  • MFSD5 expression has been detected in skin cancer tissues according to The Human Protein Atlas data

  • Researchers can use MFSD5 antibodies to evaluate expression patterns across cancer types and correlate with patient survival data

  • Investigation of MFSD5's potential relationship with another protein, MFAP5, which when blocked enhances chemotherapy effectiveness in ovarian and pancreatic cancers

  • Note that while both proteins have similar abbreviations, they are distinct molecules with potentially interesting research connections

• Metabolic Disease Research:

  • MFSD5's interaction with GLP-1R positions it as a potential target in diabetes research

  • Antibodies can be used to investigate co-localization and complex formation between MFSD5 and GLP-1R in pancreatic β-cells

  • Immunoprecipitation with MFSD5 antibodies followed by proteomic analysis can identify additional binding partners in metabolic tissues

  • Evaluation of MFSD5 expression changes in tissue samples from patients with metabolic disorders

• Methodological Advances:

  • Implementation of MFSD5 antibodies in high-throughput tissue microarray analysis

  • Adaptation for flow cytometry to quantify MFSD5 expression in specific cell populations

  • Development of proximity ligation assays to study MFSD5 interactions with GLP-1R and other partners in situ

  • Chromatin immunoprecipitation approaches to identify transcription factors regulating MFSD5 expression

• Therapeutic Development Applications:

  • Utilization of MFSD5 antibodies to screen for compounds that modulate MFSD5 expression or localization

  • Validation of genetic or pharmacological interventions targeting MFSD5 in disease models

  • Evaluation of MFSD5 as a biomarker for metabolic disease progression or treatment response

These emerging applications build upon the established roles of MFSD5 in molybdenum transport and energy homeostasis, extending its relevance to disease contexts where these processes may be dysregulated. The connection to GLP-1R signaling is particularly noteworthy given the importance of this pathway in current diabetes therapies.

How can researchers integrate MFSD5 antibody studies with broader solute carrier (SLC) family research?

Integration of MFSD5 research with the broader SLC superfamily context can provide valuable insights through several methodological approaches:

• Comparative Expression Analysis:

  • Utilize antibody panels against multiple SLC family members including MFSD5 to create comprehensive expression atlases across tissues

  • Implement multiplexed immunofluorescence to simultaneously detect MFSD5 alongside related transporters (e.g., MFSD1, MFSD11) in the same tissue sections

  • Compare expression patterns under various physiological and pathological conditions to identify coordinated regulation

• Network-Based Approaches:

  • Leverage co-expression data showing that MFSD5 shares functional relationships with MFSD11 and SLC families having organic substrate profiles

  • Apply antibodies in proximity ligation assays to verify predicted protein-protein interactions identified through computational approaches

  • Extend the methodology used to study MFSD11 interactions to explore MFSD5's place in transporter networks

• Metabolomic Integration:

  • Connect MFSD5 expression patterns (detected via antibodies) with metabolite profiles measured through targeted metabolomics

  • Apply similar approaches to those used in metabolic mapping of the human SLC superfamily

  • Correlate molybdate levels and molybdoenzyme activities with MFSD5 expression across tissues and experimental conditions

• Evolutionary Considerations:

  • Position MFSD5 research within the evolutionary context of the AMTF6 family to which it belongs along with MFSD1

  • Use antibodies to compare expression patterns of MFSD5 orthologs across species to identify conserved versus divergent functions

  • Connect to phylogenetic analyses showing that MFSD5 and MFSD11 are closely related in humans

• Functional Redundancy Assessment:

  • Apply antibodies to evaluate compensatory expression changes in related transporters when MFSD5 is deleted or overexpressed

  • Investigate whether MFSD5 and MFSD1 (which share 20% sequence identity ) demonstrate functional overlap through co-localization studies

  • Determine if MFSD5 expression patterns change in models where related transporters are disrupted