Recombinant Mouse Alpha- (1,3)-fucosyltransferase 11 (Fut11), partial

What is the correct classification of Recombinant Mouse Alpha-(1,3)-fucosyltransferase 11 (Fut11)?

Despite its historical annotation as an α1,3-fucosyltransferase, recent evidence demonstrates that FUT11 actually functions as a protein O-fucosyltransferase (POFUT). FUT11, along with FUT10, has been reclassified based on biochemical studies showing they modify EMI domains by adding O-fucose to specific threonine residues. These enzymes are now considered the third and fourth members of the POFUT family (POFUT3 and POFUT4, respectively), joining the previously characterized POFUT1 and POFUT2 . This reclassification is significant for experimental design and interpretation of results when working with recombinant mouse FUT11.

What are the primary substrates of FUT11?

FUT11 specifically modifies EMI domains found in extracellular matrix proteins. The primary substrate identified in research is the EMI domain of MMRN1 (Multimerin-1), where FUT11 adds O-fucose to threonine residues at positions T216 and T265. Other EMI domain-containing proteins that serve as FUT11 substrates include MMRN2 and EMID1. These proteins contain a conserved fucosylation motif C1XXXX[S/T]X, which is recognized by FUT11. The enzyme only modifies properly folded EMI domains, distinguishing between native and denatured conformations .

How does FUT11 differ from other fucosyltransferases?

FUT11 differs from traditional fucosyltransferases in several key ways:

• Substrate specificity: Unlike α1,3/4-fucosyltransferases that modify glycans, FUT11 directly adds fucose to protein threonine residues within EMI domains

• Structural recognition: FUT11 only recognizes properly folded protein structures, not linear peptides

• Cellular localization: Functions within the ER quality control pathway

• Reaction mechanism: Does not require divalent cations for catalytic activity, suggesting an SN2-like reaction mechanism similar to other fucosyltransferases

• Evolutionary characteristics: Shares polyexonic gene structure with POFUT1/POFUT2, indicating ancient evolutionary origins (>1,000 MYA)

What are the optimal conditions for FUT11 enzymatic assays?

Based on published protocols, optimal conditions for FUT11 enzymatic assays include:

• Temperature: 37°C is the standard incubation temperature

• Reaction duration: 2-4 hours (FUT11 reactions typically reach saturation within 2 hours)

• Substrate concentration: For kinetic analysis, EMI substrate concentrations of 0-40 μM are appropriate

• GDP-fucose concentration: Approximately 5 μM Km for both T216 and T265 sites

• Buffer components: Standard buffer conditions without obligatory requirement for divalent cations (MnCl2 may enhance FUT10 activity, but is not strictly required)

• Analysis method: LC-MS/MS glycoproteomics for precise quantification of fucosylation

Recombinant FUT11 demonstrates higher efficiency than FUT10, showing rapid saturation (within 2 hours) for both T216 and T265 modification sites.

How can recombinant FUT11 be effectively expressed and purified?

For effective expression and purification of recombinant FUT11:

• Expression system: HEK293F cells provide an appropriate mammalian expression system

• Construct design:

  • GFP-tagged FUT11 constructs facilitate purification and detection

  • Consider using secretion-optimized constructs (removal of transmembrane domain)

  • Confirm proper folding using circular dichroism (CD) spectroscopy and nano differential scanning fluorimetry

• Purification approach: Affinity chromatography using the GFP tag

• Quality control:

  • Verify purity by SDS-PAGE with Coomassie blue staining

  • Confirm proper folding using CD spectroscopy

  • Assess thermostability using differential scanning fluorimetry

What methods are effective for studying FUT11 interactions with substrate proteins?

Several complementary approaches have proven effective:

• Computational modeling:

  • AlphaFold2-multimer analysis for predicting enzyme-substrate interactions

  • Examine folding confidence (predicted IDDT) and predicted alignment error (PAE) plots

  • Look for PAE values < 5 Å at the interface between EMI domain and FUT11

• Co-immunoprecipitation:

  • Express tagged versions of both FUT11 and substrate proteins

  • Perform pull-down assays using anti-tag antibodies (e.g., anti-Myc beads)

  • Consider testing substrate variants (e.g., T216A mutant) to validate specific interaction sites

• In vitro binding assays:

  • Use purified recombinant proteins

  • Compare binding to wild-type and mutant substrates

  • Analyze binding kinetics to determine affinity parameters

How does the structure of FUT11 relate to its substrate specificity?

AlphaFold2-multimer and AlphaFold3 analyses have provided valuable insights into FUT11 structure-function relationships:

• GDP-fucose binding pocket: AlphaFold3 predicts a highly conserved pocket in FUT11 for GDP binding, positioned in close proximity to T216 and T265 of the bound MMRN1 EMI domain

• Structural basis for substrate recognition:

  • FUT11 generates high-confidence interaction models with EMI domains (predicted IDDT scores over 90%)

  • The enzyme recognizes the three-dimensional conformation of properly folded EMI domains

  • Denatured (unfolded) EMI domains are poor substrates, suggesting recognition of specific structural elements

• Comparison with FUT10:

  • FUT10 and FUT11 show very high structural similarity

  • This correlates with their approximately 40% protein sequence identity

  • Both enzymes share the ability to recognize and modify EMI domains

  • They differ in kinetic parameters, suggesting subtle differences in active site architecture

What is known about the kinetic parameters of FUT11 compared to FUT10?

Detailed kinetic analyses reveal distinct enzymatic properties between FUT11 and FUT10:

These differences suggest that while FUT11 has higher substrate affinity (lower Km), FUT10 has greater catalytic capacity (higher Vmax) when substrate is not limiting .

How can CRISPR-Cas9 be utilized to study FUT11 function?

CRISPR-Cas9 gene editing provides powerful approaches for investigating FUT11 function:

• Knockout generation:

  • CRISPR-Cas9-mediated knockouts of FUT11 can be created in relevant cell lines

  • Single knockouts (FUT11) or double knockouts (FUT10/FUT11) allow assessment of redundancy

  • Confirm successful gene knockout by genomic DNA sequencing

• Functional complementation:

  • Reintroduce wild-type or mutant FUT11 in knockout cells to assess functional rescue

  • Overexpression studies in knockout backgrounds can confirm enzyme sufficiency

• Phenotypic analysis:

  • Assess changes in O-fucosylation patterns by glycoproteomics

  • FUT11 knockout reduces O-fucose stoichiometry at T216 by ~50% and T265 by ~60%

  • Double knockout of FUT10/FUT11 completely eliminates O-fucosylation

  • Either FUT10 or FUT11 overexpression can fully restore modification

What is the significance of FUT11 expression in cancer research?

FUT11 has emerged as a molecule of interest in cancer research, particularly in gastric cancer (GC):

• Gene expression correlations:

  • FUT11 expression shows significant correlations with large gene networks

  • A comprehensive analysis identified 10,801 genes upregulated and 9,424 genes downregulated in co-expression with FUT11 in gastric cancer

  • These patterns may provide insights into dysregulated pathways associated with FUT11 expression

• Pathway associations:

  • GO function and KEGG enrichment analyses of FUT11-associated genes reveal potential involvement in critical cancer-related processes

  • This suggests FUT11 may play roles beyond its enzymatic function in O-fucosylation

• Prognostic relevance:

  • FUT11 expression patterns have demonstrated prognostic significance in gastric cancer

  • This suggests potential utility as a biomarker or therapeutic target

What analytical approaches are recommended for studying FUT11 gene co-expression networks?

For comprehensive analysis of FUT11 co-expression networks:

• Database resources:

  • LinkedOmics (http://www.linkedomics.org) provides a valuable platform for analyzing FUT11 co-expression patterns

  • This database integrates multi-omics data to identify correlations

• Visualization methods:

  • Heat maps displaying the top 50 most significantly up/downregulated genes co-expressed with FUT11

  • These visual representations help identify major patterns and gene clusters

• Enrichment analysis tools:

  • R packages including clusterProfiler and org.Hs.eg.db for conducting GO and KEGG enrichment

  • Set significance threshold (e.g., P < 0.01) for pathway enrichment

  • Visualize results using bubble plots (ggplot2) to display enriched terms

• Interpretation framework:

  • Consider both direct enzymatic functions and potential regulatory roles

  • Analyze how FUT11-mediated O-fucosylation might affect protein function and downstream signaling

  • Integrate findings with known cancer-associated pathways

How do FUT10 and FUT11 function in the cellular quality control system?

FUT10 and FUT11 participate in a non-canonical ER quality control pathway specific to EMI domains:

• Substrate recognition mechanism:

  • Both FUT10 and FUT11 specifically recognize properly folded EMI structures

  • Denatured EMI domains are poor substrates, similar to how POFUT2 only modifies folded TSR domains

  • This property allows these enzymes to function as "folding sensors"

• Comparative specificity:

  • Experiments comparing folded versus reduced/alkylated (unfolded) EMI domains show dramatic differences in modification efficiency

  • This pattern parallels the behavior of other O-glycosyltransferases that modify EGF repeats or TSRs

• Evolutionary implications:

  • FUT10 and FUT11 join POFUT1, POFUT2, POGLUT1, POGLUT2, POGLUT3, and EOGT as structure-specific modifiers

  • This suggests an ancient and conserved role for O-glycosylation in protein quality control

  • All these enzymes operate early in the secretory pathway, adding another layer of regulation to protein folding and secretion

What is the evolutionary history of FUT11 and how does it relate to other POFUTs?

The evolutionary profile of FUT11 provides important context for its functional role:

• Ancient origins:

  • FUT11, like FUT10, POFUT1, and POFUT2, is an ancient enzyme that originated more than 1,000 million years ago

  • This suggests fundamental importance in metazoan biology

• Gene structure conservation:

  • FUT11 shares a polyexonic structure with other POFUTs

  • This structural similarity further supports its reclassification as POFUT4 rather than an α1,3/4-FUT

• Expression patterns:

  • In specialized cells like platelets where MMRN1 is synthesized, FUT11 is one of only four fucosyltransferases expressed (along with POFUT1, POFUT2, and FUT8)

  • This co-expression pattern supports a specific biological role in modifying MMRN1

• Consensus sequence conservation:

  • FUT11 adds fucose to MMRN1 EMI domain within a C1XXXX[S/T]X motif highly conserved across EMI domains

  • This motif conservation suggests selective pressure to maintain this modification site across evolution

  • The presence of potential secondary modification sites in spatial proximity indicates complex evolutionary adaptation