Recombinant Uncharacterized glycosyltransferase B0361.8 (B0361.8)

What is glycosyltransferase B0361.8 and what organism is it found in?

Glycosyltransferase B0361.8 is an uncharacterized enzyme from the nematode Caenorhabditis elegans. It is a 470-amino acid protein (UniProt accession P53993) that belongs to the glycosyltransferase family. The protein is encoded by the B0361.8 gene in the C. elegans genome. Based on homology studies, B0361.8 appears to be orthologous to human ALG11 (Asparagine-Linked Glycosylation 11), suggesting its involvement in N-glycosylation pathways . The protein contains characteristic glycosyltransferase domains including the conserved DXD motif that is critical for coordinating divalent cations during sugar transfer reactions.

What is the predicted function of B0361.8 based on current research?

B0361.8 likely functions as a mannosyltransferase involved in N-glycan biosynthesis in the endoplasmic reticulum. As an ortholog of human ALG11, it is predicted to catalyze the addition of the fourth and fifth mannose residues to the growing lipid-linked oligosaccharide precursor during N-glycan synthesis . The systematic RNAi screening of glycogenes in C. elegans has demonstrated that knockdown of B0361.8 results in small body size (Sma) phenotype and oocyte morphology variants, with 76% penetrance (n = 50) . This suggests that B0361.8 plays crucial roles in developmental processes and reproductive biology, possibly through its glycosylation activity affecting key cellular proteins involved in these processes.

What approaches can be used to express and purify recombinant B0361.8?

For successful expression and purification of recombinant B0361.8, the following methodological approach is recommended:

• Expression system selection:

  • E. coli: Use for initial attempts, but may result in inclusion bodies due to membrane association

  • S. cerevisiae: Preferable for glycosyltransferases as it provides appropriate ER folding machinery

  • Insect cells (Sf9): Optimal for membrane-associated glycosyltransferases

• Construct design:

  • Clone the full coding sequence (1-470 aa) for complete protein studies

  • Remove N-terminal transmembrane domain (approximately first 30 aa) to improve solubility

  • Add fusion tags: N-terminal MBP tag dramatically improves solubility

  • Include TEV or PreScission protease sites for tag removal

• Purification strategy:

  • Solubilize using mild detergents (0.5% DDM or 1% CHAPS)

  • Employ affinity chromatography (Ni-NTA for His-tagged constructs)

  • Further purify by ion exchange and size exclusion chromatography

  • Maintain glycerol (10-50%) in buffer to preserve stability

Recent studies on similar glycosyltransferases suggest that using MBP fusion tags can significantly improve protein yield and solubility. For instance, using the MBP fusion approach for the steviol glycoside-biosynthesizing enzyme UGT76G1 enabled successful purification and subsequent enzymatic activity assays .

How can researchers assess the enzymatic activity of B0361.8?

To assess the enzymatic activity of B0361.8, researchers should employ these methodologies:

• Substrate preparation:

  • Synthesize or isolate the predicted lipid-linked oligosaccharide substrate (Man₃GlcNAc₂-PP-Dol)

  • Prepare UDP-mannose or GDP-mannose as potential donor substrates

• Reaction conditions:

  • Buffer: 50 mM HEPES pH 7.2-7.5, 10 mM MnCl₂, 5 mM DTT

  • Temperature: Test range from 25-37°C

  • Time course: 1-24 hours with sampling at regular intervals

• Activity detection methods:

  • HPLC analysis of fluorescently-labeled oligosaccharides (similar to methods used for UGT76G1)

  • Mass spectrometry to detect mass shifts after mannose addition

  • Radiometric assay using ¹⁴C-labeled UDP-mannose

• Data analysis:

  • Calculate kinetic parameters (Km, Vmax) using Michaelis-Menten equation

  • Compare activity across different substrates and conditions

Researchers should note that B0361.8, being membrane-associated, may require detergent micelles or liposome incorporation for optimal activity. Controls should include heat-inactivated enzyme and reactions without UDP/GDP-mannose to confirm specificity.

What RNAi approaches are most effective for studying B0361.8 function in C. elegans?

For investigating B0361.8 function in C. elegans through RNAi, several methodological approaches have proven effective:

• Feeding RNAi:

  • This method was successfully employed in the glycogene screening study that identified the B0361.8 phenotype

  • Steps:
    a. Clone B0361.8 cDNA fragment (500-1000 bp) into L4440 vector
    b. Transform into HT115(DE3) E. coli strain
    c. Induce dsRNA production with IPTG on NGM plates
    d. Place L4-stage worms on RNAi plates and analyze F1 progeny

• Microinjection RNAi:

  • For tissue-specific or early embryonic phenotypes

  • Higher concentration of dsRNA delivers more penetrant phenotypes

• Analysis techniques after RNAi:

  • DIC microscopy to observe morphological defects

  • Fluorescent markers to assess specific cellular processes

  • For B0361.8, using the transgenic strain that has fluorescently-labeled membrane probe (mCherry) and GFP-tagged β-tubulin marker allows visualization of germline cells in vivo

• Phenotype assessment protocols:

  • Body size measurement: Use image analysis software to measure length of adult worms

  • Oocyte morphology: Employ DIC or fluorescence microscopy of dissected gonads

  • ER stress: Monitor using hsp-4::GFP reporters to detect activation of unfolded protein response

The study that identified the B0361.8 RNAi phenotype used a feeding RNAi technique applied to wild-type N2 animals and to a strain co-expressing mCherry membrane probe and GFP-tagged β-tubulin marker. This approach effectively revealed the small body size and oocyte morphology variant phenotypes .

What phenotypes are associated with B0361.8 knockdown in C. elegans?

RNAi-mediated knockdown of B0361.8 in C. elegans results in several distinct phenotypes:

• Small body size (Sma):

  • This is a primary phenotype observed in 76% of B0361.8 RNAi-treated animals (n = 50)

  • The small body size likely results from defective N-glycosylation of proteins involved in growth regulation

• Oocyte morphology variant:

  • Abnormalities in oocyte formation and structure were observed in 76% of RNAi-treated animals

  • This suggests a critical role for B0361.8 in germline development and reproduction

• Increased ER stress:

  • B0361.8 RNAi leads to increased ER stress as detected by elevated chaperone expression (C, FM)

  • This is consistent with defects in N-glycosylation, which can trigger the unfolded protein response

The phenotypes observed with B0361.8 RNAi are similar to those seen with other ALG family members, including ALG2 and DPM1, suggesting a conserved role in N-glycan synthesis pathways essential for normal development and reproduction in C. elegans .

What is the relationship between B0361.8 and reproductive biology in C. elegans?

B0361.8 plays a significant role in C. elegans reproductive biology, particularly in oocyte development:

• Oocyte morphology effects:

  • RNAi of B0361.8 results in abnormal oocyte morphology in 76% of treated animals

  • The variant morphology may include irregular shape, size discrepancies, or aberrant distribution of cytoplasmic components

• Mechanistic relationship to glycosylation:

  • Proper N-glycosylation is essential for multiple aspects of oocyte development:

    • Cell surface receptors mediating germline cell signaling

    • Proper folding of secreted proteins involved in oocyte maturation

    • Extracellular matrix components that maintain oocyte structure

  • B0361.8 deficiency likely disrupts these processes through impaired glycosylation

• Comparative analysis with other glycosylation pathways:
  Other glycoconjugate synthesis pathways also affect reproduction in C. elegans:

  • Chondroitin synthesis genes (sqv-5, mig-22) affect cytokinesis and oocyte formation

  • GlcCer synthesis is essential for cell division and oocyte formation

  • GPI-anchor synthesis is critical for oocyte formation and germline development

This evidence collectively suggests that B0361.8-mediated N-glycosylation represents one of several glycosylation pathways essential for proper germline development and reproduction in C. elegans.

How evolutionarily conserved is B0361.8 across species?

B0361.8 shows significant evolutionary conservation across different species, particularly among those with characterized N-glycosylation pathways:

• Cross-species orthology:

  • B0361.8 is orthologous to human ALG11, which encodes alpha-1,2-mannosyltransferase

  • Orthologs are found across diverse eukaryotes, including yeast, flies, and vertebrates

• Domain conservation analysis:
  The table below shows conservation of key functional domains across species:

• Functional conservation:

  • The DXD motif crucial for catalytic activity is highly conserved across all orthologs

  • The glycosyltransferase motifs (M1-M6) identified through sequence alignment are preserved

  • Positive amino acids (K/R) at the N-terminal side of the transmembrane region follow the "positive-inside rule" across species

This high degree of conservation, particularly in the catalytic domain, underscores the fundamental importance of this enzyme in N-glycan biosynthesis across eukaryotic organisms.

What functional insights can be gained by comparing B0361.8 with its human ortholog ALG11?

Comparative analysis between C. elegans B0361.8 and human ALG11 provides valuable functional insights:

• Enzymatic activity conservation:

  • Human ALG11 catalyzes two sequential alpha-1,2-mannosylation steps in N-glycan precursor synthesis

  • B0361.8 likely performs the same dual mannosyltransferase function in C. elegans

  • Both enzymes use GDP-mannose as the sugar donor substrate

• Disease relevance:

  • Mutations in human ALG11 cause congenital disorder of glycosylation type Ip (ALG11-CDG)

  • Symptoms include developmental delay, hypotonia, seizures, and abnormal facial features

  • The small body size phenotype in C. elegans B0361.8 knockdown parallels the growth defects in human ALG11 deficiency

• Structural insights through homology:

  • Human ALG11 structure predictions reveal a GT-B fold typical of many glycosyltransferases

  • This architecture consists of two Rossmann-like domains with a catalytic site at their interface

  • B0361.8 likely shares this structural arrangement based on sequence conservation

• Regulatory mechanisms:

  • Both human ALG11 and C. elegans B0361.8 appear to be regulated by ER stress conditions

  • The unfolded protein response (UPR) may modulate expression levels in response to glycosylation demands

This comparative approach enables researchers to use C. elegans B0361.8 as a model to understand human ALG11 function and potentially develop therapeutic approaches for ALG11-CDG.

What methodological approaches can enhance B0361.8 enzyme stability for structural studies?

Enhancing B0361.8 stability for structural studies requires sophisticated protein engineering approaches:

• Computational design strategies:
  Recent work on glycosyltransferase UGT76G1 demonstrated several effective approaches that could be applied to B0361.8:

  • Stabilizing mutation scanning:

    • Use Position-Specific Scoring Matrix (PSSM) analysis to identify conserved positions

    • Apply the Rosetta ddg_monomer method to predict stabilizing mutations

    • Target mutations with ΔΔG values less than -0.5 kcal/mol

  • Structural enhancement mechanisms:

    • Introduce π-π stacking interactions near catalytic residues

    • Improve core packing through strategic hydrophobic substitutions

    • Add proline residues in loop regions to increase rigidity (e.g., G348P, S305P in UGT76G1)

    • Create additional hydrogen bonds through strategic mutations (e.g., S192K in UGT76G1)

• Experimental validation protocols:

  • Circular dichroism (CD) spectroscopy to determine melting temperature (Tm)

  • Differential scanning fluorimetry for high-throughput stability screening

  • Limited proteolysis assays to identify and eliminate flexible regions

• Buffer optimization:

  • Screen additives like glycerol (10-50%), specific detergents, and stabilizing ligands

  • Test thermostabilizing agents such as disaccharides and polyols

Applying these approaches could potentially increase the Tm of B0361.8 by 9-16°C, as observed with UGT76G1 variants, making it amenable to crystallization or cryo-EM structural studies .

How might novel domain assembly strategies be applied to engineer B0361.8 functionality?

Recent advances in glycosyltransferase engineering suggest innovative approaches to modify B0361.8 functionality:

• "Mix and match" auto-assembly strategy:

  • This approach leverages the conserved GT-B structural family architecture of glycosyltransferases

  • The GT-B structure consists of two domains: one binding the sugar donor and one binding the acceptor

  • By creating chimeric glycosyltransferases that combine auto-assembled domains from different GT-B enzymes, broader substrate promiscuity can be achieved

• Implementation methodology for B0361.8:

  • Identify the precise domain boundaries in B0361.8 (donor-binding vs. acceptor-binding)

  • Create expression constructs for each domain with compatible interfaces

  • Test co-expression of B0361.8 donor domain with acceptor domains from other glycosyltransferases

  • Assess activity using various donor-acceptor substrate combinations

• Potential applications:

  • Generate B0361.8-based heterodimeric GTs with expanded substrate range

  • Create glycosyltransferases capable of producing novel glycoconjugates

  • Develop enzymes with increased catalytic efficiency for specific reactions

This domain assembly approach could transform B0361.8 from an uncharacterized enzyme into a versatile biocatalytic tool for glycobiology research and glycan synthesis .

What are the relationships between B0361.8 and membrane trafficking pathways?

The relationship between B0361.8 and membrane trafficking pathways represents an unexplored frontier in glycobiology research:

• Potential interactions with Golgi transport mechanisms:

  • As an ER-resident glycosyltransferase, B0361.8 likely influences protein trafficking through quality control mechanisms

  • The Golgi-associated retrograde protein (GARP) complex, which tethers endosome-derived transport vesicles to the late Golgi , may interact with glycosylation pathways

  • Improperly glycosylated proteins due to B0361.8 deficiency could disrupt normal GARP complex function

• Investigative approaches:

  • Co-immunoprecipitation studies to identify interactions between B0361.8 and trafficking components

  • Live imaging of fluorescently tagged B0361.8 and trafficking markers (RAB-11, RAB-6)

  • Comparative RNAi phenotypic analysis of B0361.8 and trafficking components

• Preliminary evidence from C. elegans studies:

  • Some trafficking regulators affect both apical membrane protein (PEPT-1) localization and recycling endosome (RAB-11) positioning

  • B0361.8 disruption may similarly affect membrane protein trafficking through altered glycosylation

  • The relationship between N-glycosylation quality control and membrane trafficking remains underexplored

This research direction could reveal novel insights into how glycosyltransferase activity influences membrane protein trafficking and cellular compartmentalization.

What are the key unanswered questions about B0361.8 function?

Despite current knowledge, several critical questions about B0361.8 remain unanswered:

• Enzymatic specificity:

  • What is the precise sugar donor preference (GDP-mannose vs. UDP-mannose)?

  • Does B0361.8 catalyze both the fourth and fifth mannose additions like its human ortholog?

  • Are there alternative substrates beyond the canonical N-glycan precursor?

• Structural characterization:

  • What is the three-dimensional structure of B0361.8?

  • How does substrate binding induce conformational changes?

  • What structural features determine dual mannosyltransferase activity?

• Regulatory mechanisms:

  • How is B0361.8 expression regulated during development?

  • What post-translational modifications affect B0361.8 activity?

  • How does B0361.8 respond to ER stress conditions?

• Developmental biology:

  • Which specific glycoproteins affected by B0361.8 deficiency are responsible for the small body phenotype?

  • What molecular mechanisms link B0361.8 to oocyte development?

  • Are there tissue-specific requirements for B0361.8 activity?

• Evolutionary aspects:

  • How has B0361.8 function evolved across nematode species?

  • Are there species-specific differences in substrate specificity?

Addressing these questions would significantly advance our understanding of B0361.8 and N-glycosylation biology in C. elegans.

What interdisciplinary approaches could accelerate B0361.8 characterization?

Accelerating B0361.8 characterization requires innovative interdisciplinary approaches:

• Glycomics-proteomics integration:

  • Combine glycan profiling with proteomics to identify proteins affected by B0361.8 knockdown

  • Use quantitative glycoproteomics to measure site-specific glycosylation changes

  • Implement stable isotope labeling to track glycan flux through the N-glycan pathway

• Systems biology framework:

  • Develop computational models of the N-glycan biosynthesis pathway

  • Integrate transcriptomics, proteomics, and metabolomics data from B0361.8 mutants

  • Use network analysis to identify key nodes connecting glycosylation to developmental processes

• Advanced imaging technologies:

  • Apply super-resolution microscopy to track B0361.8 localization and dynamics

  • Use FRET-based biosensors to monitor enzyme-substrate interactions in vivo

  • Implement correlative light and electron microscopy to study ER morphology changes

• CRISPR-based approaches:

  • Generate precise point mutations in B0361.8 to study structure-function relationships

  • Create conditional alleles to study tissue-specific requirements

  • Implement CRISPRi for temporal control of B0361.8 expression

• Synthetic biology strategies:

  • Reconstitute the N-glycan pathway in vitro with purified components

  • Engineer minimal synthetic cells with defined glycosylation capabilities

  • Create orthogonal glycosylation pathways to probe specificity

These interdisciplinary approaches could transform our understanding of B0361.8 from an uncharacterized glycosyltransferase to a well-defined component of the N-glycosylation machinery with clear connections to development and disease.