Recombinant Schizosaccharomyces pombe Uncharacterized glycosyltransferase C4F11.04c (SPCC4F11.04c)

Q: What structural features might provide insights into SPCC4F11.04c function?

While the three-dimensional structure of SPCC4F11.04c has not been experimentally determined, bioinformatic analysis suggests it likely adopts a GT-A fold typical of many glycosyltransferases. These enzymes typically contain a Rossmann-like nucleotide-binding domain and often possess a DxD motif or similar catalytic residues that coordinate divalent cations (commonly Mn²⁺) essential for the sugar transfer reaction.

A methodological approach to predict structural features would include:

• Multiple sequence alignment with characterized glycosyltransferases

• Homology modeling based on structurally characterized glycosyltransferases

• Secondary structure prediction to identify potential transmembrane regions

• Active site prediction through conservation analysis and structural modeling

• Validation of predictions through targeted mutagenesis studies

Q: What are the optimal methods for recombinant expression and purification of SPCC4F11.04c?

The recombinant expression and purification of SPCC4F11.04c can be achieved through the following methodological approach:

• Expression system: The full-length protein (amino acids 1-345) can be expressed in E. coli as a His-tagged fusion protein.

• Purification protocol:

  • Affinity chromatography using Ni-NTA resin for His-tagged protein

  • Size exclusion chromatography for further purification if needed

  • Final product can be obtained as a lyophilized powder

• Storage conditions:

  • Store in Tris/PBS-based buffer with 6% trehalose at pH 8.0

  • For long-term storage, add glycerol to 50% final concentration

  • Store at -20°C/-80°C in aliquots to avoid repeated freeze-thaw cycles

  • Working aliquots can be kept at 4°C for up to one week

Q: What quality control methods should be employed for recombinant SPCC4F11.04c?

Quality control for recombinant SPCC4F11.04c should include:

• SDS-PAGE analysis to confirm purity (>90% purity is typically achievable)

• Western blot analysis using anti-His antibodies to confirm identity

• Mass spectrometry to verify the intact mass and sequence

• Circular dichroism to assess proper folding

• Dynamic light scattering to evaluate homogeneity and aggregation state

• Activity assays to confirm functional integrity (if the enzymatic function is known)

Q: How can the predicted glycosyltransferase function of SPCC4F11.04c be experimentally verified?

Verification of SPCC4F11.04c's glycosyltransferase function requires a multi-faceted approach:

• Bioinformatic analysis:

  • Protein family multiple sequence alignments to identify conserved catalytic residues

  • Three-dimensional modeling to predict active site architecture

• Mutagenesis studies:

  • Design loss-of-function mutations targeting predicted catalytic residues

  • Express and purify mutant proteins alongside wild-type controls

• Enzymatic assays:

  • Test various nucleotide sugar donors (likely GDP-mannose based on annotation)

  • Screen potential acceptor substrates (possibly inositol phosphoceramides)

  • Develop quantitative assays to measure glycosyltransferase activity

• In vivo functional complementation:

  • Generate SPCC4F11.04c deletion strains in S. pombe

  • Test for phenotypic effects and complementation with wild-type and mutant alleles

The combined results from these approaches would provide strong evidence for the enzymatic function and specificity of SPCC4F11.04c.

Q: What are the likely substrates for SPCC4F11.04c based on its annotation?

Based on its annotation as "Inositol phosphoceramide mannosyltransferase 2," SPCC4F11.04c likely:

• Uses GDP-mannose as the sugar donor, which is one of the nine sugar nucleotide donors used by mammalian glycosyltransferases and is conserved in fungi

• Transfers mannose to inositol phosphoceramide (IPC) acceptors, which are important sphingolipid components in fungal membranes

• May function as either a "retaining" or "inverting" glycosyltransferase, depending on whether the stereochemistry of the glycosidic bond is maintained or inverted during transfer

• Potentially requires divalent cations (commonly Mn²⁺) as cofactors for catalysis

A systematic approach to substrate identification would involve testing activity with various sugar donors and acceptors, followed by product characterization using techniques such as mass spectrometry or NMR.

Q: Is SPCC4F11.04c essential for S. pombe viability?

• Gene deletion study:

  • Create a precise deletion of SPCC4F11.04c using PCR-based gene targeting

  • Analyze the viability of deletion mutants under various growth conditions

• Context from genome-wide studies:

  • Approximately 17.5% of fission yeast genes are essential for vegetative growth

  • About 14% of S. pombe ORFs are found exclusively in this yeast and are absent from S. cerevisiae

• Conditional expression systems:

  • If conventional deletion is lethal, use repressible promoters to control expression

  • Monitor phenotypic consequences of protein depletion

• Functional domain analysis:

  • Create partial deletions or point mutations that affect specific protein domains

  • Determine which domains are essential for function and viability

If SPCC4F11.04c functions in glycolipid biosynthesis or cell wall maintenance, its deletion might affect cell integrity, morphology, or stress resistance, even if not directly essential for viability.

Q: How can advanced structural biology approaches be applied to SPCC4F11.04c characterization?

Structural characterization of SPCC4F11.04c would benefit from these methodological approaches:

• X-ray crystallography workflow:

  • Optimize protein construct design (remove flexible regions if necessary)

  • Screen crystallization conditions systematically

  • Co-crystallize with donor analogs and/or acceptor substrates

  • Solve structure using molecular replacement with related glycosyltransferases

• Cryo-electron microscopy:

  • Particularly useful if the protein forms larger complexes or is membrane-associated

  • Can potentially capture different conformational states during catalysis

• NMR spectroscopy:

  • For studying protein dynamics and substrate interactions

  • Particularly valuable for identifying flexible regions important for catalysis

• Integrative modeling:

  • Combine low-resolution experimental data with computational predictions

  • Use homology modeling based on related glycosyltransferases with known structures

  • Validate models through targeted mutagenesis and functional assays

• Molecular dynamics simulations:

  • Explore conformational flexibility and substrate binding mechanisms

  • Investigate the role of metal ions in catalysis

These approaches would provide insights into the structural basis of substrate recognition and catalytic mechanism of SPCC4F11.04c.

Q: How can the active site of SPCC4F11.04c be identified and characterized?

The active site of SPCC4F11.04c can be systematically identified and characterized through:

• Multiple sequence alignment:

  • Identify conserved motifs typical of glycosyltransferases (e.g., DxD motif)

  • Compare with characterized enzymes in the same family

• Three-dimensional modeling:

  • Generate homology models based on related glycosyltransferases

  • Identify potential catalytic residues and substrate binding pockets

• Site-directed mutagenesis:

  • Create alanine substitutions of predicted catalytic residues

  • Assess effects on enzyme activity and substrate binding

• Substrate docking and molecular dynamics:

  • Predict binding modes of donor and acceptor substrates

  • Simulate enzyme-substrate interactions

• Biochemical validation:

  • Compare catalytic parameters of wild-type and mutant proteins

  • Analyze effects of metal ions and pH on enzyme activity

This integrated approach would reveal both the identity of catalytic residues and the structural basis for substrate specificity, providing a foundation for more detailed mechanistic studies.

Q: What factors might regulate SPCC4F11.04c activity in vivo, and where is it likely localized?

The regulation and localization of SPCC4F11.04c could be investigated through:

• Localization studies:

  • Many glycosyltransferases are single-pass transmembrane proteins anchored to the Golgi apparatus

  • GFP fusion proteins could reveal the subcellular localization

  • Membrane topology prediction suggests possible transmembrane domains

• Post-translational modifications:

  • Phosphorylation sites could be identified by mass spectrometry

  • Effects of phosphorylation on activity could be tested with phosphomimetic mutations

  • Glycosylation of the enzyme itself might affect folding or stability

• Transcriptional regulation:

  • RNA-seq analysis under various conditions to identify regulatory cues

  • Promoter analysis to identify transcription factor binding sites

• Protein-protein interactions:

  • Identify interaction partners through co-immunoprecipitation or yeast two-hybrid

  • Assess if activity is regulated through complex formation

• Environmental responsiveness:

  • Test enzyme activity under various stress conditions

  • Analyze expression patterns during different growth phases

Understanding these regulatory aspects would provide insights into how SPCC4F11.04c activity is coordinated with broader cellular processes.

Q: How conserved is SPCC4F11.04c across fungal species and what can evolutionary analysis reveal about its function?

A methodological approach to evolutionary analysis of SPCC4F11.04c would include:

• Homology identification:

  • BLAST searches against fungal genomes to identify orthologs

  • Assessment of conservation versus divergence patterns

• Phylogenetic analysis:

  • Construction of phylogenetic trees to trace evolutionary history

  • Correlation with known glycosylation pathways across species

• Conservation mapping:

  • Identify highly conserved regions likely critical for function

  • Map conservation onto structural models to predict functional sites

• Comparative genomic context:

  • Analyze gene neighborhoods across species

  • Identify co-evolved genes that might function in the same pathway

• Functional divergence:

  • Given that 14% of S. pombe ORFs are found exclusively in that yeast and absent from S. cerevisiae, investigate whether SPCC4F11.04c represents a species-specific adaptation

  • Compare substrate specificities of orthologs from different species

Q: What approaches can determine the catalytic mechanism of SPCC4F11.04c, and is it likely a "retaining" or "inverting" glycosyltransferase?

Determining the catalytic mechanism would involve:

• Stereochemical analysis:

  • NMR analysis of reaction products to determine if SPCC4F11.04c is "retaining" (preserves anomeric configuration) or "inverting" (reverses configuration)

  • Comparison with characterized glycosyltransferases with known mechanisms

• Kinetic analysis:

  • Steady-state kinetics with varying substrate concentrations

  • Pre-steady-state kinetics to identify rate-limiting steps

  • pH and temperature dependence studies

• Isotope effects:

  • Measure kinetic isotope effects using labeled substrates

  • Distinguish between concerted and stepwise mechanisms

• Mechanistic inhibition studies:

  • Design and test transition state analogs

  • Analyze patterns of inhibition

• Metal ion requirements:

  • Test activity with various divalent cations (Mn²⁺, Mg²⁺, etc.)

  • Determine if metal coordinates substrate, enzyme, or both

The combined results would elucidate whether SPCC4F11.04c follows a direct displacement (inverting) or double displacement (retaining) mechanism, providing fundamental insights into its catalytic strategy.

Q: What role might SPCC4F11.04c play in S. pombe cell wall biogenesis and membrane structure?

If SPCC4F11.04c functions as an inositol phosphoceramide mannosyltransferase, it likely contributes to:

• Glycosphingolipid biosynthesis:

  • Generation of mannosylinositol phosphoceramides (MIPCs)

  • These complex lipids are important components of fungal membranes

• Membrane microdomain organization:

  • Glycosphingolipids contribute to lipid raft formation

  • These microdomains organize membrane proteins and signaling complexes

• Cell wall-membrane interface:

  • Glycolipids may connect membrane components to cell wall polysaccharides

  • This interface is critical for cell integrity and stress response

A comprehensive investigation would include:

• Analysis of membrane lipid composition in wild-type versus mutant strains

• Cell wall integrity testing using agents like Calcofluor White or Congo Red

• Stress response analysis under various conditions

• Electron microscopy to examine cell wall ultrastructure

• Lipidomic profiling to identify specific substrates and products

Q: Could SPCC4F11.04c serve as a potential target for antifungal development?

The potential of SPCC4F11.04c as an antifungal target could be evaluated through:

• Essentiality assessment:

  • Determine if the gene is essential or if its deletion causes significant fitness defects

  • Evaluate growth under various stress conditions relevant to host environments

• Conservation analysis:

  • Compare conservation between pathogenic fungi and humans

  • Identify structural or functional differences that could be exploited for selectivity

• Inhibitor screening:

  • Develop high-throughput assays for enzyme activity

  • Screen chemical libraries for selective inhibitors

• Structure-based drug design:

  • Use structural information to design specific inhibitors

  • Optimize lead compounds for potency and selectivity

• Validation in model systems:

  • Test promising inhibitors in fungal culture

  • Evaluate toxicity in mammalian cell lines

  • Assess efficacy in animal models of fungal infection

If SPCC4F11.04c is involved in fungal-specific glycolipid synthesis with no close homologs in mammals, it could represent an attractive target for selective antifungal development.

Q: What comprehensive experimental strategy would best characterize SPCC4F11.04c function across multiple scales?

A comprehensive strategy for SPCC4F11.04c characterization would integrate:

• Molecular and structural studies:

  • Protein structure determination by X-ray crystallography or cryo-EM

  • Substrate binding studies using isothermal titration calorimetry

  • Site-directed mutagenesis of predicted catalytic residues

• Biochemical characterization:

  • Development of quantitative activity assays

  • Determination of substrate specificity and kinetic parameters

  • Analysis of metal ion and pH dependence

• Cellular studies:

  • Generation and phenotypic characterization of deletion mutants

  • Localization studies using fluorescent protein fusions

  • Lipidomic analysis of wild-type versus mutant strains

• Systems biology approaches:

  • Transcriptomic profiling under various conditions

  • Identification of genetic interactions through synthetic genetic arrays

  • Metabolic flux analysis of relevant biosynthetic pathways

• Integration with glycobiology knowledge:

  • Comparison with characterized glycosyltransferases from model organisms

  • Placement within known glycosylation pathways

This integrated approach would provide a comprehensive understanding of SPCC4F11.04c function from molecular mechanism to cellular significance and evolutionary context.