POFUT2 Antibody

What is POFUT2 and why is it important in cellular biology?

POFUT2 (Protein O-Fucosyltransferase 2) catalyzes the addition of fucose through an O-glycosidic linkage to conserved serine or threonine residues in thrombospondin type 1 repeats (TSRs). This post-translational modification is essential for protein function and stability in numerous biological processes, including:

• Cell adhesion and migration

• Regulation of extracellular matrix interactions

• Embryonic development, particularly during gastrulation

• Protein quality control and secretion

POFUT2 is localized in the endoplasmic reticulum and exists in three isoforms (A, B, and C), each with distinct expression patterns that may contribute to functional diversity . The enzyme has approximately 49-50 kDa molecular weight and plays crucial roles in modifying TSR-containing proteins that regulate diverse cellular processes .

What applications are POFUT2 antibodies validated for?

Most commercially available POFUT2 antibodies have been validated for multiple applications, including:

Researchers should verify specific dilutions and application suitability for their particular antibody, as optimal conditions may vary between manufacturers and lot numbers .

How should I optimize Western blot protocols for POFUT2 detection?

For optimal Western blot detection of POFUT2:

• Sample preparation: Use 20-40 μg of protein lysate from tissues or cells expressing POFUT2 (brain tissue, HEK-293 cells show good expression) .

• Gel electrophoresis: 10% SDS-PAGE gels are generally suitable for resolving the 49-50 kDa POFUT2 protein.

• Transfer conditions: Use standard wet or semi-dry transfer protocols to PVDF or nitrocellulose membranes.

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute POFUT2 antibody to 1:500-1:2000 in blocking buffer. Incubate overnight at 4°C .

• Secondary antibody: HRP-conjugated secondary antibodies should be diluted 1:50,000-1:100,000 for optimal signal-to-noise ratio .

• Detection: Both chemiluminescence and fluorescence detection methods are suitable.

The expected band size is approximately 49-50 kDa, though additional bands may be observed due to isoforms or post-translational modifications .

What controls should be included when using POFUT2 antibodies?

Proper experimental controls are essential for validating POFUT2 antibody specificity:

• Positive controls:

  • Human brain tissue lysate

  • HEK-293 cell lysate

  • Mouse liver or brain tissue for IHC applications

• Negative controls:

  • Primary antibody omission

  • POFUT2 knockout or knockdown samples (when available)

  • Non-expressing tissues/cells (determine empirically)

• Specificity controls:

  • Pre-absorption with immunizing peptide (if available)

  • Use of alternate antibody clones targeting different epitopes

• Loading controls:

  • GAPDH, β-actin, or other housekeeping proteins

These controls help distinguish specific signal from non-specific binding and ensure reliable interpretation of experimental results .

How can I assess the role of POFUT2-mediated O-fucosylation in protein secretion?

O-fucosylation by POFUT2 has been shown to be critical for the secretion of certain TSR-containing proteins, particularly ADAMTS family members. To investigate this function:

• CRISPR/Cas9 knockout approach:

  • Generate POFUT2 knockout cell lines using CRISPR/Cas9 technology

  • Assess secretion of TSR-containing proteins (e.g., ADAMTS9) by analyzing culture media versus cell lysates

  • Research has demonstrated that POFUT2 knockout in HEK293T cells blocks secretion of ADAMTS9

• Site-directed mutagenesis approach:

  • Mutate specific O-fucosylation sites within TSR domains

  • Express wild-type and mutant constructs in cell culture

  • Compare secretion efficiency using Western blot analysis of cell lysate versus media

  • Previous research with ADAMTS13 and Punctin-1 showed reduced secretion when fucosylation sites were mutated

• Rescue experiments:

  • Reintroduce wild-type or enzymatically inactive POFUT2 (e.g., E54A, R294A mutants) into knockout cells

  • Assess recovery of protein secretion

  • This approach can confirm specificity of the observed phenotype

Quantitative analysis should include measurement of intracellular retention, degradation rates, and secretion kinetics to fully characterize the impact of O-fucosylation on protein trafficking.

What experimental approaches can determine if POFUT2 has catalytic-independent functions?

Recent research suggests POFUT2 may have chaperone-like activities independent of its catalytic function. To investigate:

• Enzymatically inactive mutants:

  • Generate catalytically inactive POFUT2 mutants (E54A, R294A, W92A)

  • These mutations abolish enzymatic activity without affecting protein folding

  • Express these mutants in POFUT2-null backgrounds

• Protein interaction studies:

  • Perform co-immunoprecipitation of wild-type and catalytically inactive POFUT2

  • Identify interacting partners using mass spectrometry

  • Compare binding partners between catalytically active and inactive forms

• Structural studies:

  • X-ray crystallography or cryo-EM studies of POFUT2-substrate complexes

  • Focus on the unique loop (residues 265-285) which may be involved in protein-protein interactions

• Subcellular localization analysis:

  • Fluorescence microscopy to track POFUT2 and its substrates

  • Determine if catalytically inactive POFUT2 still colocalizes with substrates

Understanding the dual functions of POFUT2 as both an enzyme and potential chaperone may reveal novel mechanisms in protein quality control within the secretory pathway.

How can POFUT2 antibodies be used to study embryonic development?

POFUT2 plays critical roles during embryonic development, particularly in gastrulation. To study these functions:

• Spatiotemporal expression analysis:

  • Perform immunohistochemistry on embryonic sections at different developmental stages

  • Map expression patterns in specific tissues (trophoblast giant cells, parietal endoderm, visceral endoderm, extraembryonic mesoderm, and anterior primitive streak)

• Conditional knockout phenotyping:

  • Generate tissue-specific POFUT2 conditional knockout models

  • Use POFUT2 antibodies to confirm deletion efficiency

  • Compare phenotypes to global knockout models to identify tissue-specific functions

• Lineage tracing experiments:

  • Combine POFUT2 immunostaining with lineage markers

  • Track changes in epithelial organization, mesoderm formation, and endoderm displacement

  • Analyze effects on signaling pathway components (Nodal, BMP4, Fgf8, Wnt3)

Gene trap and conditional knockout approaches have shown that POFUT2 is essential for maintaining normal epithelial arrangement during early gastrulation, with defects resulting in abnormal mesoderm formation .

What are the methodological considerations when studying POFUT2 in relation to ADAMTS proteins?

ADAMTS (A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeats) family proteins are major targets of POFUT2. For studying their relationship:

• Co-expression analysis:

  • Perform dual immunostaining for POFUT2 and specific ADAMTS proteins

  • Analyze colocalization in tissues where both are expressed

  • ADAMTS9 has been identified as a key POFUT2 target during development

• Secretion assays:

  • Quantify ADAMTS9 secretion in wild-type versus POFUT2-deficient cells

  • Include both intracellular and extracellular fractions in analyses

  • Research has shown that POFUT2 knockout blocks ADAMTS9 secretion

• Functional rescue experiments:

  • In POFUT2 mutant backgrounds, determine if non-fucosylated ADAMTS variants can rescue phenotypes

  • This approach can distinguish between fucosylation-dependent and independent functions

• Extracellular matrix analysis:

  • Examine changes in ECM composition and structure in POFUT2-deficient contexts

  • Since ADAMTS proteins modify ECM, this may reveal mechanisms underlying developmental defects

Research comparing POFUT2 and ADAMTS9 knockout phenotypes has demonstrated similar gastrulation defects, suggesting that disruption of ADAMTS9 function is a primary consequence of POFUT2 deficiency .

How can mass spectrometry be used to confirm POFUT2-mediated O-fucosylation of target proteins?

Mass spectrometry offers powerful tools for characterizing O-fucosylation:

• Sample preparation:

  • Express and purify putative target proteins from wild-type and POFUT2-deficient cells

  • Perform in-gel digestion using specific proteases (trypsin, chymotrypsin)

  • Enrich for glycopeptides using lectin affinity chromatography (AAL lectin specifically binds fucose)

• LC-MS-based enzyme activity assay:

  • Test wild-type and mutant POFUT2 ability to fucosylate TSR domains in vitro

  • Use synthetic peptides containing the consensus sequon or purified TSR domains

  • This approach has been used to characterize the enzymatic activity of various POFUT2 mutants

• Site-specific glycopeptide analysis:

  • Employ collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods

  • Identify exact sites of O-fucosylation within TSR domains

  • Quantify occupancy rates at each site

• Comparative glycoproteomics:

  • Compare glycopeptide profiles between wild-type and POFUT2-deficient samples

  • Identify all affected proteins and modification sites

This approach allows for confirmation of direct POFUT2 targets and assessment of site occupancy in different biological contexts .

What approaches can assess the functional impact of O-fucosylation on protein-protein interactions?

To determine how O-fucosylation affects protein interactions:

• Surface plasmon resonance (SPR):

  • Compare binding kinetics of fucosylated versus non-fucosylated TSR domains

  • Express and purify TSR domains from wild-type and POFUT2-deficient cells

  • Measure association and dissociation rates with known binding partners

• Proximity labeling approaches:

  • Employ BioID or APEX2 fusion proteins with TSR-containing targets

  • Compare interactomes of fucosylated versus non-fucosylated proteins

  • This approach can identify indirect effects on larger protein complexes

• Structural studies:

  • Crystallographic analysis of fucosylated versus non-fucosylated TSR domains

  • NMR studies to detect conformational changes upon fucosylation

  • Molecular dynamics simulations to predict effects on protein flexibility and interaction surfaces

• Cell-based interaction assays:

  • Co-immunoprecipitation of TSR-containing proteins with binding partners

  • Compare results between wild-type and POFUT2-deficient cells

  • Validate key interactions with purified components in vitro

These approaches can reveal how O-fucosylation modulates interactions that underlie key biological functions of TSR-containing proteins.

How do POFUT2 functions differ between mammalian and parasitic systems?

Recent research has revealed interesting differences in POFUT2 function across species:

• Mammalian systems:

  • POFUT2 is essential for embryonic development in mice

  • Knockout leads to gastrulation defects and embryonic lethality

  • Critical for proper secretion of ADAMTS proteins

• Plasmodium (malaria parasite):

  • POFUT2 O-fucosylates TSR-containing proteins like TRAP and CSP

  • Surprisingly, POFUT2 null mutant P. berghei parasites showed no defects in sporozoite motility

  • Successfully established blood stage infection in mice, contrary to previous studies

• Toxoplasma:

  • POFUT2 modifies thrombospondin repeat proteins

  • The O-fucome (all O-fucosylated proteins) in protists may have unique characteristics

• Experimental approach comparison:

  • Use cross-species antibodies with appropriate validation

  • Consider evolutionary conservation of the epitopes

  • Include species-specific positive controls

This comparison highlights that the importance of O-fucosylation may vary significantly between species, complicating our understanding of glycosylation in different biological systems .

What methodological adaptations are needed when using POFUT2 antibodies across different model organisms?

When applying POFUT2 antibodies to different species:

• Epitope conservation analysis:

  • Perform sequence alignment of the immunogen region across target species

  • Higher sequence identity (>80%) suggests better cross-reactivity

  • Many POFUT2 antibodies target C-terminal regions, which show variable conservation

• Validation strategies:

  • Western blot analysis of lysates from multiple species

  • Include POFUT2 knockout controls when available

  • Optimize antibody dilutions for each species separately

• Application-specific considerations:

  • For IHC: Test multiple antigen retrieval methods (citrate buffer pH 6.0 versus TE buffer pH 9.0)

  • For IF: Optimize fixation protocols for each species and tissue type

  • For WB: Adjust lysis buffers based on tissue type and species

• Known cross-reactivity:

  • Some antibodies are validated for human, mouse, and rat samples

  • Others may have predicted reactivity for additional species (pig, zebrafish, horse, sheep, rabbit, dog)