Recombinant Schizosaccharomyces pombe Uncharacterized protein C4C3.09 (SPBC4C3.09)

What is SPBC4C3.09 and why is it significant for research?

SPBC4C3.09 is an uncharacterized protein found in the fission yeast Schizosaccharomyces pombe, with UniProt accession number O43062. Its significance stems from its predicted role in glycosylation processes, specifically as a potential galactosyltransferase. This protein belongs to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database and may be involved in α1,3-galactosylation of S. pombe oligosaccharides .

The protein is particularly interesting because understanding its function could provide insights into eukaryotic glycosylation pathways, which are conserved across species and play crucial roles in cell recognition, protein folding, and cellular communication. As S. pombe shares many features with higher eukaryotes including humans, insights from this protein may have broader implications for understanding similar processes in more complex organisms .

What are the optimal conditions for expression and purification of recombinant SPBC4C3.09?

For optimal expression and purification of recombinant SPBC4C3.09, researchers should consider:

Expression System Selection:

• E. coli-based systems are suitable for basic structural studies but may lack proper post-translational modifications

• Yeast expression systems (particularly S. cerevisiae or native S. pombe) provide more appropriate eukaryotic processing

• Insect or mammalian cell lines may be necessary if specific glycosylation patterns are critical

Purification Strategy:

• Use affinity tags (His, GST, or FLAG) with appropriate cleavage sites for tag removal

• Employ a multi-step purification protocol:

  • Initial capture via affinity chromatography

  • Intermediate purification via ion exchange chromatography

  • Polishing via size exclusion chromatography

Buffer Optimization:

• Maintain pH between 7.0-7.5 with Tris-based buffers

• Include glycerol (25-50%) for stability

• Consider adding specific metal ions or cofactors if enzymatic activity is to be preserved

For long-term storage, the purified protein should be stored at -20°C or -80°C, with working aliquots kept at 4°C for up to one week to avoid repeated freeze-thaw cycles that could compromise protein integrity .

How can I design experiments to assess the putative galactosyltransferase activity of SPBC4C3.09?

To assess the putative galactosyltransferase activity of SPBC4C3.09, a comprehensive experimental design should include:

In vitro enzymatic assays:

• Substrate preparation: Use pyridylaminated oligosaccharides (such as Man₉GlcNAc₂ or Manα1,2-Manα1,2-Man) as potential acceptor substrates

• Donor preparation: Utilize UDP-galactose as the sugar donor

• Reaction conditions: Conduct assays in appropriate buffers with necessary cofactors (typically Mn²⁺ or Mg²⁺)

• Activity detection: Monitor transfer of galactose to acceptor substrates using:

  • HPLC analysis of labeled products

  • Mass spectrometry to confirm molecular weight changes

  • Specific lectins that recognize α1,3-linked galactose residues

Confirmation of linkage specificity:

• Enzyme digestion analysis using α-galactosidase

• ¹H NMR spectroscopy to determine the anomeric configuration

• LC-MS/MS analysis for detailed structural characterization

Control experiments:

• Use known α1,3-galactosyltransferases as positive controls

• Include reactions without enzyme or without donor/acceptor as negative controls

• Compare with related enzymes from the same family (such as those encoded by otg1-otg3 genes)

This experimental approach mirrors that used in previous successful characterizations of related galactosyltransferases in S. pombe, where galactosyltransferase activity was confirmed through multiple complementary techniques .

What are the best approaches for studying protein-protein interactions involving SPBC4C3.09?

For investigating protein-protein interactions involving SPBC4C3.09, researchers should employ a multi-faceted approach:

Affinity-based methods:

• Co-immunoprecipitation (Co-IP): Using antibodies against SPBC4C3.09 or potential interacting partners

• Tandem affinity purification (TAP): By creating fusion proteins with appropriate tags

• Pull-down assays: Using recombinant SPBC4C3.09 as bait to capture interacting proteins

Cross-linking mass spectrometry (XL-MS):
This technique is particularly valuable for capturing transient or weak interactions:

• Apply cross-linkers like DSS (amine-reactive NHS-ester) or DMTMM (carboxyl-to-amine coupling)

• Analyze cross-linked peptides with LC-MS/MS

• Identify interaction interfaces through specialized data analysis

In vivo validation techniques:

• Bimolecular fluorescence complementation (BiFC)

• Förster resonance energy transfer (FRET)

• Yeast two-hybrid system (particularly useful in S. pombe itself)

Computational prediction approaches:

• Phylogenetic profiling to identify potentially functionally linked proteins

• Comparative genomic analysis to detect co-evolved protein pairs

A combination of these approaches provides the most comprehensive understanding of the protein interaction network surrounding SPBC4C3.09, with each method offering complementary strengths and addressing different limitations.

How can I determine the biological function of SPBC4C3.09 using genetic approaches?

To determine the biological function of SPBC4C3.09 using genetic approaches, implement a systematic strategy:

Gene knockout/deletion studies:

• Generate a clean deletion strain using homologous recombination techniques

• Assess phenotypic changes:

  • Growth rate alterations under various conditions

  • Morphological changes in cell structure

  • Defects in specific cellular processes

Complementation analysis:

• Reintroduce wild-type or mutated versions of SPBC4C3.09

• Test for rescue of knockout phenotypes

• Construct chimeric proteins with related enzymes to identify functional domains

Synthetic genetic interactions:

• Cross SPBC4C3.09 deletion strain with a deletion library

• Identify genetic interactions through:

  • Synthetic lethality

  • Synthetic sickness

  • Epistatic relationships

Conditional expression systems:

• Generate strains with regulated expression (e.g., tetracycline-inducible or repressible)

• Study immediate effects of protein depletion or overexpression

The deletion approach was successfully employed in previous studies where multiple galactosyltransferase genes were knocked out, revealing their roles in glycosylation. Following similar methodology, researchers found that disruption of 10 galactosyltransferase genes, including several previously uncharacterized ones from glycosyltransferase family 8, led to complete loss of galactosylation in S. pombe .

What computational approaches can predict the function of SPBC4C3.09?

For computational prediction of SPBC4C3.09 function, multiple bioinformatic strategies can be employed:

Sequence-based analyses:

• Homology detection through PSI-BLAST and HHpred

• Motif identification using PROSITE, PFAM, and InterPro

• Signal peptide and transmembrane domain prediction via SignalP and TMHMM

Structural prediction and analysis:

• 3D structure prediction using AlphaFold2 or RoseTTAFold

• Active site identification through structural alignment with characterized glycosyltransferases

• Molecular docking with potential substrates to assess binding capabilities

Evolutionary analyses:

• Phylogenetic profiling to identify co-evolved proteins

  • Create profiles encoding presence/absence across genomes

  • Identify proteins with similar evolutionary patterns

  • Cluster proteins with similar phylogenetic profiles

• Ortholog identification across species

• Synteny analysis to identify conserved genomic neighborhood

Integrated approaches:

• Network-based function prediction using protein-protein interaction data

• Machine learning models utilizing bioactivity signatures

  • Generate predictive embeddings from existing annotations

  • Apply deep neural networks to infer bioactivity signatures

These computational predictions should be treated as hypotheses to be experimentally validated. The phylogenetic profiling approach has been particularly successful for functional assignment, with an estimated 18% keyword overlap between proteins with identical profiles, compared to only 4% overlap for random proteins .

What are the most effective proteomics approaches for studying SPBC4C3.09 in different cellular contexts?

Effective proteomics approaches for studying SPBC4C3.09 in various cellular contexts include:

Global proteome analysis:

• Two-dimensional electrophoresis (2-DE) coupled with mass spectrometry

  • Separate proteins based on pI and molecular weight

  • Identify differentially expressed proteins between wild-type and knockout strains

  • Quantify changes in protein abundance under different conditions

Targeted proteomics:

• Selected/Multiple Reaction Monitoring (SRM/MRM) for quantitative analysis

• Parallel Reaction Monitoring (PRM) for high-specificity detection

• Design of specific peptide targets unique to SPBC4C3.09

Protein-centric mass spectrometry techniques:

• Charge detection mass spectrometry (CDMS)

• Native mass photometry (MP)

  • These techniques preserve non-covalent interactions

  • Allow resolution of heterogeneous biomolecular assemblies

  • Particularly useful for large protein complexes

Post-translational modification mapping:

• Phosphoproteomics to identify regulatory phosphorylation sites

• Glycoproteomics to characterize glycosylation patterns

  • Especially relevant given SPBC4C3.09's predicted function

Experimental design considerations:

• Include biological replicates (minimum of three)

• Analyze multiple time points to capture dynamic changes

• Compare different genetic backgrounds or environmental conditions

How does SPBC4C3.09 compare with other uncharacterized proteins in the galactosyltransferase family 8?

SPBC4C3.09 shares significant similarities with other uncharacterized proteins in glycosyltransferase family 8, particularly those involved in galactosylation processes:

Comparative analysis with related proteins:

Key structural and functional differences:

• Domain organization variations affecting substrate specificity

• Differing transmembrane topologies influencing subcellular localization

• Variation in catalytic residues potentially resulting in different linkage specificity

Research has demonstrated that related proteins in this family, such as those encoded by otg2(+) and otg3(+), exhibit α1,3-galactosyltransferase activity toward different pyridylaminated oligosaccharide substrates. Specifically, Otg2p shows activity toward both Man₉GlcNAc₂ and Manα1,2-Manα1,2-Man oligosaccharides, while Otg3p is active primarily toward Man₉GlcNAc₂ .

The tandem duplication observed between SPBC4C3.09 and related proteins suggests evolutionary expansion of this enzyme family to accommodate diverse glycosylation requirements. This duplication pattern is significant for understanding how enzyme specificity evolves and may provide insights into structure-function relationships within this family.

What role might SPBC4C3.09 play in the broader glycosylation pathways of S. pombe?

SPBC4C3.09 likely plays a specialized role within the complex glycosylation network of S. pombe, contributing to the unique glycan structures that distinguish this organism from other yeasts:

Context within S. pombe glycosylation:

• S. pombe oligosaccharides contain larger amounts of galactose compared to S. cerevisiae

• Galactose residues are attached via α1,2- or α1,3-linkages to both N- and O-linked oligosaccharides

• SPBC4C3.09 likely contributes to this galactosylation pattern, particularly in α1,3-linkage formation

Potential functional roles:

• Modification of specific glycoproteins required for cell wall integrity

• Contribution to protein quality control in the secretory pathway

• Involvement in cell-cell recognition or mating processes

• Potential roles in stress response pathways

Integration with other glycosylation enzymes:
Research on related enzymes has shown that multiple galactosyltransferases work in concert to establish the complete glycosylation pattern. Studies with septuple α-galactosyltransferase disruptants (7GalT∆) revealed residual α1,3-linked galactose residues, indicating the presence of additional enzymes like SPBC4C3.09 that contribute to the complete glycosylation profile .

The identification of novel galactosyltransferases, including those in the same family as SPBC4C3.09, eventually led to the creation of a 10GalT∆ strain that completely lacked galactosylation. This suggests that SPBC4C3.09 functions within a network of partially redundant enzymes that collectively establish the complete glycosylation pattern of S. pombe.

How can contradictory experimental data regarding SPBC4C3.09 function be reconciled?

When faced with contradictory experimental data regarding SPBC4C3.09 function, researchers should implement a systematic approach to reconcile these discrepancies:

Data interpretation framework:

• Evaluate experimental contexts

  • Different expression systems may yield varying post-translational modifications

  • Assay conditions (pH, temperature, cofactors) can drastically affect activity

  • Substrate availability might limit observable functions

• Consider partial or moonlighting functions

  • Enzymes often catalyze secondary reactions at lower efficiency

  • Different domains may have distinct activities

• Assess technical and biological variability

  • Distinguish between technical artifacts and true biological variance

  • Implement appropriate statistical analyses for experimental replication

Resolution strategies:

• Perform independent validation using orthogonal techniques

  • Complement biochemical assays with genetic approaches

  • Verify in vitro findings with in vivo studies

• Conduct structure-function analyses

  • Generate targeted mutations to identify critical residues

  • Create chimeric proteins with related enzymes

• Investigate conditional activities

  • Test function under different physiological conditions

  • Examine activity with diverse substrate panels

This example illustrates how apparent contradictions can lead to deeper understanding when systematically investigated with complementary approaches.

How can studies of SPBC4C3.09 contribute to our understanding of glycosylation in higher eukaryotes?

Studies of SPBC4C3.09 can provide valuable insights into glycosylation processes in higher eukaryotes through several mechanisms:

Evolutionary conservation and divergence:

• S. pombe shares more conserved features with humans than S. cerevisiae, including:

  • Similar gene structures and chromatin dynamics

  • Prevalence of introns and pre-mRNA splicing mechanisms

  • Comparable epigenetic gene silencing pathways

• Galactosyltransferase families show significant conservation across species

• Fundamental catalytic mechanisms are often preserved despite substrate differences

Translational implications:

• Understanding basic mechanisms of glycosyltransferase function

  • Substrate recognition determinants

  • Catalytic mechanisms

  • Regulatory processes

• Insights into glycosylation-related diseases

• Potential applications in glycoengineering

Model system advantages:
S. pombe offers unique advantages as a "micromammal" for studying processes relevant to higher eukaryotes:

• Simpler experimental system with powerful genetic tools

• Well-characterized genome and proteome

• Conservation of key cellular pathways with mammals

The characterization of glycosyltransferases like SPBC4C3.09 contributes to a broader understanding of how these enzymes evolved and diversified across species, potentially revealing fundamental principles that apply to human glycosylation processes and providing targets for therapeutic intervention in glycosylation disorders.

What advanced experimental design approaches can resolve discrepancies in SPBC4C3.09 characterization?

Advanced experimental design approaches to resolve discrepancies in SPBC4C3.09 characterization include:

Randomized controlled experimental design:

• Proper randomization to eliminate selection bias

  • Assign experimental conditions using random number generators

  • Ensure equal group sizes when appropriate6

• Include appropriate controls

  • Positive controls with known galactosyltransferases

  • Negative controls (enzyme-free, substrate-free)

  • Mock-treated samples

Factorial experimental designs:

• Systematically vary multiple parameters

  • Substrate concentrations

  • Buffer conditions

  • Cofactor requirements

• Analyze interaction effects between variables

• Identify optimal conditions for activity

Time-course and kinetic analyses:

• Measure activity at multiple time points

• Determine enzyme kinetics parameters (Km, Vmax)

• Identify potential product inhibition or substrate depletion effects

Multi-method validation approach:

• Combine different detection techniques

  • Radiometric assays

  • Fluorescence-based methods

  • Mass spectrometry

• Cross-validate between in vitro and in vivo systems

• Independent replication in different laboratories

Statistical considerations:

• Conduct power analyses to determine appropriate sample sizes

• Apply appropriate statistical tests based on data distribution

• Account for multiple testing when applicable

• Report effect sizes alongside statistical significance6

By implementing these rigorous experimental design principles, researchers can minimize bias, control for confounding variables, and generate robust, reproducible data that helps resolve contradictions in SPBC4C3.09 characterization.

How might systems biology approaches enhance our understanding of SPBC4C3.09's role in cellular networks?

Systems biology approaches can dramatically enhance our understanding of SPBC4C3.09's role within broader cellular networks through:

Integrative omics analyses:

• Combine multiple data types:

  • Transcriptomics to identify co-regulated genes

  • Proteomics to map protein abundance changes

  • Metabolomics to detect alterations in glycan profiles

  • Interactomics to establish protein-protein interaction networks

• Apply computational integration methods to identify emergent patterns

Network construction and analysis:

• Generate protein-protein interaction networks

• Identify functionally linked proteins through correlated evolution patterns

• Perform pathway enrichment analyses of affected processes in knockout strains

Flux analysis of glycosylation pathways:

• Apply metabolic flux analysis to glycan biosynthesis

• Quantify changes in pathway dynamics in SPBC4C3.09 mutants

• Develop predictive models of glycosylation outcomes

Computational modeling approaches:

• Develop ordinary differential equation models of glycosylation pathways

• Implement agent-based models of glycoprotein processing

• Use machine learning to predict functions from bioactivity signatures

Through these integrative approaches, researchers can move beyond studying SPBC4C3.09 in isolation and understand its role within the complex cellular machinery, potentially revealing unexpected connections and functional relationships that would not be apparent from reductionist approaches alone.