SPAC12B10.15c Antibody

What is the SPAC12B10.15c gene and what protein does it encode?

SPAC12B10.15c is a gene in Schizosaccharomyces pombe (fission yeast) that encodes Sup11p, a membrane-associated protein critical for β-1,6-glucan synthesis and cell wall integrity. Structurally, Sup11p is a luminal protein anchored via a signal anchor sequence, with significant homology to Saccharomyces cerevisiae Kre9p. The protein plays an essential role in β-1,6-glucan polymer formation, and its depletion leads to cell wall defects, septum malformation, and the accumulation of aberrant glucan deposits .

Why is Sup11p important for cell wall research?

Sup11p is crucial for research into fungal cell wall formation because it is essential for β-1,6-glucan synthesis, a key component of the yeast cell wall. Experimental evidence demonstrates that Sup11p depletion abolishes β-1,6-glucan detection in the cell wall, confirming its necessity for polymer synthesis. Additionally, genetic interactions with β-1,6-glucanase family members (e.g., gas2+) suggest a regulatory role in glucan remodeling. Understanding Sup11p's function provides insights into fundamental cellular processes and potential antifungal targets, as proper cell wall formation is essential for fungal viability .

How does Sup11p relate to septum formation and cell division?

Sup11p is indispensable for proper septum assembly in fission yeast. In conditional nmt81-sup11 knock-down mutants, severe morphological defects and malformation of the septum occur, with massive accumulation of cell wall material at the center of the closing septum. These depositions consist partially of β-1,3-glucan, which is normally restricted to the primary septum. Analysis of the nmt81-sup11 mutant cell wall revealed that Gas2p, a member of the β-1,3-glucanosyl-transferases GH72 family, plays a crucial role in accumulating the observed septum material depositions .

What expression systems are available for producing SPAC12B10.15c antibody?

The SPAC12B10.15c antibody can be generated using recombinant Sup11p produced in multiple expression systems, offering flexibility for different experimental needs. Available formats include:

These variants enable researchers to select the most appropriate format for their specific experimental design, particularly for structural studies, pull-down assays, and localization experiments.

What methods are recommended for subcellular localization studies of Sup11p?

For subcellular localization of Sup11p, multiple complementary approaches are recommended:

How can SPAC12B10.15c antibody be used to investigate O-mannosylation of Sup11p?

To investigate O-mannosylation of Sup11p using SPAC12B10.15c antibody, researchers can implement the following methodology:

This methodological approach has demonstrated that Sup11p:HA is O-mannosylated and has provided insights into how its expression influences the growth of O-mannosyl transferase mutants .

How can computational approaches like RosettaAntibodyDesign (RAbD) be leveraged to improve SPAC12B10.15c antibody specificity?

RosettaAntibodyDesign (RAbD) offers advanced computational methodology to enhance SPAC12B10.15c antibody specificity through the following approach:

• Initial structure preparation: Begin with the three-dimensional structure of SPAC12B10.15c antibody-antigen complex, either from experimental data or predicted through computational docking.

• Sampling of sequence-structure space: Utilize RAbD's framework to sample diverse sequence, structure, and binding space of the antibody to Sup11p.

• CDR grafting and optimization: The program samples antibody sequences by grafting structures from canonical clusters of CDRs, focusing on optimization for specificity to Sup11p.

• Energy function customization: Employ RAbD's capability to optimize for multiple objectives simultaneously, including binding affinity, stability, and specificity.

• Protocol customization: Design specific protocols via command-line options and input files to focus on enhancing interaction with unique epitopes of Sup11p.

What are the critical controls needed when assessing protein-protein interactions involving Sup11p?

When investigating protein-protein interactions involving Sup11p using SPAC12B10.15c antibody, several critical controls must be implemented:

• Antibody specificity validation: Perform Western blot analysis on wild-type versus sup11-deletion or depletion strains to confirm antibody specificity.

• Negative control pull-downs: Include non-specific IgG of the same isotype as SPAC12B10.15c antibody to identify background binding.

• Competitive inhibition controls: Pre-incubate the antibody with recombinant Sup11p to demonstrate binding specificity.

• Reverse co-immunoprecipitation: Confirm interactions by immunoprecipitating the putative interacting partner and detecting Sup11p.

• Subcellular localization confirmation: Verify that interacting proteins colocalize with Sup11p in cellular compartments using immunofluorescence or fractionation.

• Genetic interaction validation: Corroborate physical interactions with genetic interaction data, such as synthetic lethality or suppressor relationships.

Researchers have used these controls to identify meaningful interactions between Sup11p and β-1,6-glucanase family members, validating the genetic interactions with physical interaction data .

How can high-throughput single-cell sequencing approaches be adapted to study antibody responses to Sup11p?

High-throughput single-cell sequencing technologies can be adapted to study antibody responses to Sup11p following these methodological steps:

• Antigen-specific B cell isolation: Use fluorescently labeled recombinant Sup11p to identify and isolate Sup11p-binding B cells through flow cytometry.

• Single-cell RNA and VDJ sequencing: Perform high-throughput sequencing of isolated B cells to capture paired heavy and light chain sequences of antibodies specific to Sup11p.

• Bioinformatic analysis: Identify highly expressed clonal immunoglobulin sequences that bind to Sup11p.

• Recombinant antibody expression: Construct expression vectors containing identified antibody sequences and produce recombinant antibodies.

• Affinity and specificity characterization: Evaluate the binding characteristics of the antibodies using techniques such as ELISA, Biolayer Interferometry, or Surface Plasmon Resonance.

This approach has been successfully implemented for other antigens, such as in the development of antibodies against Staphylococcus aureus protein A, where 676 antigen-binding IgG1+ clonotypes were identified through high-throughput scRNA/VDJ-seq, leading to the identification of potent antibodies with nanomolar affinity .

How should researchers interpret unusual N-glycosylation patterns of Sup11p in O-mannosylation-deficient backgrounds?

When encountering unusual N-glycosylation patterns of Sup11p in O-mannosylation-deficient backgrounds, researchers should consider the following interpretative framework:

• Masked glycosylation sites: In wild-type cells, extensive O-mannosylation in S/T-rich regions of Sup11p may mask potential N-glycosylation sites. In O-mannosylation-deficient mutants (e.g., oma4), these sites become accessible, allowing unusual N-glycosylation on N-X-A sequons.

• Competition between glycosylation types: The data suggests a competition between O-mannosylation and N-glycosylation for substrate access, with O-mannosylation typically taking precedence in wild-type cells.

• Altered protein mobility: The hypo-mannosylated Sup11p will show different electrophoretic mobility, which may be further altered by unusual N-glycosylation. EndoH treatment can help distinguish between these modifications.

• Functional implications: These altered glycosylation patterns may affect Sup11p's stability, localization, or function, potentially explaining growth defects in O-mannosylation mutants.

Research has demonstrated that Sup11p:HA is hypo-mannosylated when expressed in an O-mannosylation mutant background, and this hypo-mannosylated form can be N-glycosylated on an unusual N-X-A sequon located in an S/T-rich region .

What experimental approaches can resolve contradictory immunofluorescence and subcellular fractionation data for Sup11p localization?

When facing contradictions between immunofluorescence and subcellular fractionation data regarding Sup11p localization, researchers should implement the following resolution strategies:

• Super-resolution microscopy: Employ techniques such as STORM or STED to achieve higher resolution of Sup11p localization beyond the diffraction limit of conventional microscopy.

• Live-cell imaging: Use minimally disruptive fluorescent tagging combined with live-cell imaging to track Sup11p dynamics in real-time, avoiding fixation artifacts.

• Immuno-electron microscopy: Apply gold-labeled SPAC12B10.15c antibody for ultrastructural localization at nanometer resolution.

• Organelle-specific markers: Co-localize Sup11p with multiple established markers for different compartments (early Golgi, late Golgi, post-Golgi, plasma membrane) to precisely map its distribution.

• Density gradient optimization: Refine fractionation protocols to achieve better separation of closely related compartments.

• Temporal studies: Examine Sup11p localization throughout the cell cycle to account for potential dynamic relocalization.

Previous research has successfully employed these approaches to establish that Sup11p resides in the late Golgi or post-Golgi compartments, reconciling initial contradictory data from different localization methods .

How might SPAC12B10.15c antibody contribute to developing novel antifungal strategies?

SPAC12B10.15c antibody could significantly contribute to novel antifungal strategies through several research pathways:

• Target validation: The antibody can be used to confirm Sup11p as an essential component for fungal cell wall integrity, validating it as a potential antifungal target.

• High-throughput screening: Develop assays using SPAC12B10.15c antibody to screen compound libraries for molecules that disrupt Sup11p function or localization.

• Structural studies: Utilize the antibody for co-crystallization or cryo-EM studies to determine the precise structure of Sup11p, facilitating structure-based drug design.

• Mechanism elucidation: The antibody can help elucidate Sup11p's enzymatic role in β-1,6-glucan elongation, providing mechanistic insights for inhibitor design.

• In vivo imaging: Develop labeled antibody derivatives to monitor antifungal efficacy in real-time using preclinical imaging techniques.

Current research aims to exploit Sup11p as a target for antifungal drug development, given its essential role in fungal cell wall integrity and the absence of direct homologs in humans, potentially leading to selective antifungal agents.

What methodological approaches could assess Sup11p's interaction with the septum separation pathway?

To investigate Sup11p's interaction with the septum separation pathway, researchers could employ these methodological approaches:

• Transcriptome analysis: Perform RNA-seq or microarray analysis of conditional nmt81-sup11 mutants to identify affected genes in the septum separation pathway. Previous studies have identified significant regulation of several cell wall glucan modifying enzymes in this mutant.

• Temporal protein dynamics: Use time-lapse microscopy with fluorescently tagged Sup11p and key septum separation proteins to track their relative dynamics during cell division.

• Genetic interaction mapping: Create double mutants combining conditional sup11 alleles with mutations in known septum separation pathway components to identify synthetic interactions.

• Phosphorylation state analysis: Use phospho-specific antibodies or mass spectrometry to determine if Sup11p is regulated by cell cycle-dependent phosphorylation, potentially linking it to septum separation timing mechanisms.

• Proximity labeling techniques: Employ BioID or APEX2 fused to Sup11p to identify proteins in close proximity during septum formation and separation phases.

Transcriptome analysis of nmt81-sup11 mutants has already revealed upregulation of genes involved in cell wall organization and septum separation, providing a foundation for more detailed studies of these interactions .

How can in silico developability assessments improve the design of next-generation SPAC12B10.15c antibodies?

In silico developability assessments can significantly enhance the design of next-generation SPAC12B10.15c antibodies through the following methodological approaches:

• Solubility prediction: Employ computational tools like CamSol to design antibody variants with improved solubility profiles. This approach has been validated in creating libraries of humanized mAb variants spanning a broad range of solubility values.

• Aggregation propensity analysis: Use algorithms that identify aggregation-prone regions within the antibody sequence and suggest modifications to reduce this risk.

• Thermal stability prediction: Apply computational methods to estimate the thermal stability of antibody variants, which correlates with shelf-life and manufacturing robustness.

• Post-translational modification prediction: Identify potential sites for unwanted glycosylation, deamidation, or oxidation that could affect antibody performance and stability.

• Developability index calculation: Generate composite scores based on multiple parameters (charge distribution, hydrophobic patches, isoelectric point) to rank candidate antibodies.

• Epitope mapping optimization: Use computational approaches to identify epitopes on Sup11p that are both accessible and conserved, maximizing the utility of the antibody.

These in silico approaches, when combined with experimental validation, can significantly accelerate the development of improved SPAC12B10.15c antibodies with enhanced specificity, stability, and functional properties .

How does the methodology for studying SPAC12B10.15c/Sup11p compare with approaches used for related proteins in other fungal species?

The methodology for studying SPAC12B10.15c/Sup11p can be comparatively analyzed against approaches used for homologous proteins in other fungi:

• Homology identification: Sup11p shows significant homology to Saccharomyces cerevisiae Kre9p, a key protein in β-1,6-glucan synthesis. Comparative studies leverage this homology to develop comprehensive evolutionary models of fungal cell wall synthesis.

• Functional assays: While KRE mutant identification in S. cerevisiae utilized K1 killer toxin resistance screening, S. pombe studies of Sup11p have focused on direct cell wall composition analysis and conditional expression systems due to Sup11p's essential nature.

• Localization techniques: Unlike S. cerevisiae studies that often rely on GFP tagging, Sup11p localization has been more challenging, requiring a combination of immunofluorescence and cellular fractionation due to functional constraints on tagging.

• Genetic interaction mapping: Both Sup11p and Kre9p studies employ synthetic genetic array analysis, but Sup11p research has placed greater emphasis on interactions with O-mannosylation pathways.

• Cell wall analysis: Methods for β-1,6-glucan detection are largely conserved across species, though S. pombe studies of Sup11p have contributed unique insights into septum formation not prominently featured in S. cerevisiae Kre9p research.

The comparative approach reveals that while the fundamental techniques are similar, Sup11p research has revealed unique aspects of fungal cell wall biology not evident in S. cerevisiae models .

What lessons from antibody-drug conjugate (ADC) development could be applied to creating functional SPAC12B10.15c antibody conjugates?

Lessons from antibody-drug conjugate (ADC) development that could enhance the creation of functional SPAC12B10.15c antibody conjugates include:

• Target validation: Employ rigorous validation methods to confirm that in vivo accumulation of the antibody conjugate is Sup11p-specific and not the result of off-target interactions. This could be accomplished using paired preclinical models that differ only in Sup11p expression.

• Genetic manipulation for specificity testing: Utilize CRISPR technology to create matched experimental models with and without Sup11p expression to definitively demonstrate binding specificity.

• Advanced tumor modeling: Apply organoid technology principles to develop more sophisticated fungal colony models that better recapitulate native fungal growth patterns for testing conjugate efficacy.

• Non-invasive imaging integration: Develop methods to track antibody conjugate distribution and efficacy in real-time, allowing for dynamic assessment of antifungal activity.

• Multiple conjugation strategies: Explore various drug conjugation chemistries and linker technologies to optimize the stability, release kinetics, and efficacy of the conjugate.

• Safety profiling: Implement comprehensive tissue cross-reactivity studies to identify potential off-target binding that could lead to toxicity.

These approaches, adapted from ADC development for cancer treatment, could significantly advance the creation of SPAC12B10.15c antibody conjugates as potential diagnostic or therapeutic tools for fungal infections .