Recombinant Schizosaccharomyces pombe Probable dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ost2 (ost2)

What is the function of Ost2 in Schizosaccharomyces pombe?

Ost2 functions as the epsilon-subunit of the oligosaccharyltransferase (OST) complex in S. pombe. The OST complex catalyzes a critical step in N-linked protein glycosylation, transferring preassembled high-mannose oligosaccharides from dolichol-oligosaccharide donors to consensus glycosylation acceptor sites in newly synthesized proteins within the lumen of the endoplasmic reticulum . Genomic disruption of the OST2 locus is lethal in haploid yeast, demonstrating that expression of the Ost2 protein is essential for viability . Defects in the Ost2 protein cause pleiotropic underglycosylation of both soluble and membrane-bound glycoproteins, highlighting its crucial role in proper protein glycosylation .

How is the OST2 gene conserved across species?

The Ost2 protein shares significant sequence homology with proteins in other organisms, most notably the DAD1 (defender against apoptotic cell death) protein in vertebrates, with which it shares approximately 40% sequence identity . This conservation suggests an ancient evolutionary origin for this component of the N-glycosylation machinery.

The oligosaccharyltransferase complex components, including Ost2, are broadly conserved across eukaryotic species, though with some variations. In Saccharomyces cerevisiae, the oligosaccharyltransferase is an oligomeric complex composed of six non-identical subunits (alpha-zeta), with Ost2 functioning as the epsilon-subunit . This conservation reflects the fundamental importance of N-glycosylation in eukaryotic cell biology.

What phenotypes are associated with OST2 mutations in fission yeast?

Conditional ost2 mutants in S. pombe demonstrate:

• Pleiotropic underglycosylation of soluble and membrane-bound glycoproteins

• Marked reductions in the in vitro transfer of high-mannose oligosaccharide from exogenous lipid-linked oligosaccharide to glycosylation site acceptor tripeptides

• Cell lethality when the gene is completely disrupted, indicating its essential nature

Interestingly, sequence analysis of ost2 mutant alleles has revealed mutations at highly conserved residues in the Ost2p/DAD1 protein sequence, providing insight into critical functional domains of the protein .

What are effective methods for recombinant expression of S. pombe Ost2?

Several expression systems can be used for producing recombinant S. pombe Ost2 protein:

E. coli expression system:
The S. pombe Ost2 protein has been successfully expressed in E. coli as an N-terminal His-tagged fusion protein . This system offers high yield and relative simplicity but lacks eukaryotic post-translational modifications.

S. pombe expression system:
For maintaining native post-translational modifications, S. pombe itself can be used as an expression host. The nmt1 promoter system allows for either constitutive or induced expression of the gene of interest . Specifically:

• Two vectors, pESP-1 and pESP-2, have been developed for protein expression in S. pombe

• These vectors use the nmt1 promoter, which can be regulated by thiamine

• Expressed proteins can be tagged with GST for purification

• Protein yields typically range from 1.0 mg/L to 12.5 mg/L of induced culture

Alternative yeast systems:
S. cerevisiae can also be used, particularly when studying functional conservation between the two yeast species .

How can Ost2 protein be effectively purified?

For His-tagged Ost2 protein expressed in E. coli, the following purification protocol is recommended:

• Express the protein in E. coli and harvest cells

• Lyse cells in appropriate buffer containing protease inhibitors

• Purify using nickel affinity chromatography

• Consider adding 5-50% glycerol to the final purified protein for long-term storage

• Aliquot and store at -20°C/-80°C to avoid repeated freeze-thaw cycles

For GST-tagged proteins expressed in S. pombe:

• Harvest and lyse cells

• Purify using glutathione agarose beads

• The GST tag can be removed using either thrombin or enterokinase protease, depending on the vector used

What assays can be used to evaluate Ost2 function in the OST complex?

Several assays have been developed to study the function of OST components:

In vitro OST activity assay:

• Prepare microsomal membranes from wild-type or mutant strains

• Assay the transfer of high-mannose oligosaccharide from exogenous lipid-linked oligosaccharide to glycosylation site acceptor tripeptides

• Quantify the reduction in activity in mutant strains compared to wild-type

Fluorescent peptide-based assay:
A more refined method uses fluorescent dye-labeled peptides as substrates and SDS-PAGE for separation:

• Incubate purified OST components with fluorescent peptide substrates containing the Asn-X-Thr/Ser sequon

• Include lipid-linked oligosaccharide (LLO) as an oligosaccharide donor

• Separate products by SDS-PAGE and detect fluorescence

• This assay can confirm the necessity of the N-glycosylation sequon

Mass spectrometry analysis:
To confirm glycopeptide products:

• Conduct the OST reaction on a large scale

• Purify the reaction products by HPLC

• Analyze by MALDI-QIT-TOF mass spectrometry

How does Ost2 interact with other components of the oligosaccharyltransferase complex?

The S. pombe oligosaccharyltransferase complex, like that in S. cerevisiae, is composed of multiple subunits. While the exact architecture of the S. pombe complex is not fully characterized in the provided materials, insights can be drawn from the S. cerevisiae system:

• In S. cerevisiae, the OST is an oligomeric complex with six non-identical subunits (alpha-zeta)

• The alpha, beta, gamma, and delta subunits are encoded by OST1, WBP1, OST3, and SWP1 genes, respectively

• Ost2 functions as the epsilon-subunit of this complex

Research approaches to study these interactions include:

• Co-immunoprecipitation experiments

• Two-hybrid assays (as used for studying Rad22A and Rad22B interactions in S. pombe)

• GST pull-down assays to verify direct protein-protein interactions

What is the relationship between S. pombe Ost2 and human DAD1?

The S. pombe Ost2 protein shares approximately 40% sequence identity with the human DAD1 (defender against apoptotic cell death) protein . This relationship has several significant research implications:

• DAD1 was initially identified in vertebrates as a protein involved in preventing apoptotic cell death

• The high degree of conservation suggests fundamental importance in eukaryotic cell biology

• S. pombe can serve as a model system for understanding the function of human DAD1

• Mutations in highly conserved residues in S. pombe Ost2 could provide insights into functionally critical domains of human DAD1

This evolutionary conservation presents opportunities for using S. pombe as a model to investigate aspects of human N-glycosylation disorders.

How can conditional OST2 mutants be generated for functional studies?

Given that OST2 is an essential gene, conditional mutants are valuable for studying its function. Methods for generating such mutants include:

Temperature-sensitive alleles:

• Random mutagenesis of the OST2 gene

• Screening for temperature-sensitive phenotypes

• Characterization of the mutations to identify critical functional residues

Promoter replacement strategies:

• Replace the native OST2 promoter with a regulatable promoter such as nmt1

• The nmt1 promoter is repressed by thiamine, allowing for controlled gene expression

• For faster induction, the urg1 promoter system can be used, which allows induction within 30 minutes (compared to 14-20 hours for full nmt1 induction)

CRISPR-based approaches:
While not specifically mentioned in the search results, CRISPR technologies adapted for S. pombe could potentially be used to create conditional alleles.

What are the differences in glycosylation machinery between S. pombe and other model organisms?

Comparative analysis of glycosylation machinery reveals both conservation and divergence:

Comparison with S. cerevisiae:

• Both yeasts use similar components for N-glycosylation

• S. pombe Ubc13 and Mms2 can function with orthologues of their partner proteins from S. cerevisiae, demonstrating conservation

• Unlike in S. cerevisiae where PCNA is sumoylated in undamaged cells, in S. pombe PCNA is ubiquitinated in S phase

Broader eukaryotic comparison:

• The kinetoplastid parasites have conserved components of the dolichol-cycle and oligosaccharyltransferases of protein N-glycosylation similar to other eukaryotes

• Some organisms show unique glycosyltransferase families, such as the GT67 family in kinetoplastid parasites

• S. pombe has been used as a host for heterologous expression of glycosyltransferases, as it provides eukaryotic post-translational modifications unlike E. coli

What controls should be included when studying recombinant Ost2 function?

For rigorous experimental design when studying Ost2 function, include:

Positive controls:

• Wild-type Ost2 protein for comparison with mutant forms

• Known functional orthologues (e.g., S. cerevisiae Ost2) to demonstrate conservation

• For glycosyltransferase assays, include established enzymes with known activity

Negative controls:

• Ost2 with mutations in the conserved residues identified in temperature-sensitive mutants

• Reactions lacking critical components (e.g., no Mn²⁺/Mg²⁺ ions or no lipid-linked oligosaccharide)

• For peptide substrates, include variants with altered glycosylation motifs:

  • Replacement of Asn with Gln

  • Replacement of Thr/Ser with Ala

  • Replacement of the middle residue with Pro

How can I optimize expression conditions for recombinant S. pombe Ost2?

To optimize expression of recombinant Ost2:

For E. coli expression:

• Test multiple E. coli strains (BL21, Rosetta, etc.)

• Optimize induction conditions (temperature, IPTG concentration, duration)

• Consider using specialized vectors for membrane proteins

• Test different solubilization conditions to maximize protein recovery

For S. pombe expression:

• The nmt1 promoter is repressed by thiamine; removal of thiamine de-represses the promoter but requires 14-20 hours for full induction

• For faster induction, consider the urg1 promoter system, which allows induction within 30 minutes

• Optimize growth conditions (temperature, media composition)

• For secreted proteins, consider using appropriate signal sequences

Storage conditions:

• Add 5-50% glycerol (final concentration) to purified protein

• Aliquot for long-term storage at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

What approaches can be used to study the role of Ost2 in the broader N-glycosylation pathway?

To investigate Ost2's role in the N-glycosylation pathway:

Genetic approaches:

• Create conditional ost2 mutants and analyze glycoprotein profiles

• Generate epistasis maps by creating double mutants with other glycosylation pathway components

• Use suppressor screens to identify genes that can compensate for ost2 defects

Biochemical approaches:

• Analyze changes in glycoprotein profiles in ost2 mutants using glycan analysis techniques

• Study the effect of Ost2 mutations on the assembly and stability of the OST complex

• Perform in vitro reconstitution of the OST complex with and without Ost2 to determine its precise role

Structural approaches:

• Determine the structure of Ost2 and its interactions within the OST complex

• Use site-directed mutagenesis to test structure-function relationships

• Compare structures across species to identify conserved functional domains

Why might recombinant Ost2 protein show low activity in in vitro assays?

Several factors could contribute to low activity:

• Improper folding: Membrane proteins like Ost2 can be challenging to fold correctly in heterologous systems

  • Solution: Try expression in eukaryotic systems like S. pombe itself

• Absence of cofactors: The OST reaction is stimulated by metal ions

  • Solution: Add exogenous Mn²⁺ or Mg²⁺ ions to reaction mixtures (Mn²⁺ is more effective)

• Incomplete complex formation: Ost2 functions as part of a multi-subunit complex

  • Solution: Consider co-expression with other OST components

• Suboptimal assay conditions: N-glycosylation requires specific conditions

  • Solution: Optimize buffer composition, pH, temperature, and substrate concentrations

• Degraded lipid-linked oligosaccharide (LLO):

  • Solution: Prepare fresh LLO or ensure proper storage conditions

How can I distinguish between direct and indirect effects of OST2 mutations on glycosylation?

To differentiate direct from indirect effects:

• In vitro assays with purified components:

  • Use the fluorescent peptide-based assay with purified Ost2 and minimal OST components

  • Direct effects will be evident even in reconstituted systems

• Structure-function analysis:

  • Create targeted mutations in specific domains

  • Correlate structural changes with functional outcomes

• Temporal analysis:

  • Use rapidly inducible systems (e.g., urg1 promoter) to observe immediate effects of Ost2 depletion

  • Immediate effects are more likely to be direct

• Genetic interaction mapping:

  • Create double mutants with other glycosylation pathway components

  • Synthetic interactions suggest parallel or compensatory pathways

• Biochemical complementation:

  • Test if adding purified wild-type Ost2 can rescue defects in extracts from mutant cells

What are the considerations for using S. pombe as a model for human N-glycosylation disorders?

When using S. pombe to model human disorders:

• Conservation assessment:

  • The 40% identity between Ost2 and human DAD1 suggests conservation of core functions

  • Verify that critical residues affected in human disorders are conserved in S. pombe

• Functional equivalence testing:

  • Determine if human DAD1 can complement S. pombe ost2 mutants

  • Test if mutations corresponding to human disease alleles produce similar phenotypes in S. pombe

• Glycan structure differences:

  • Consider that yeast and humans have different N-glycan processing pathways

  • Focus on early steps of N-glycosylation that are more conserved

• Physiological context:

  • S. pombe lacks the tissue-specific regulation present in humans

  • Some human disease phenotypes may not be recapitulated in unicellular organisms

• Experimental advantages:

  • S. pombe offers genetic tractability and rapid growth

  • Assays developed in S. pombe (e.g., for OST activity) can be adapted for human proteins