Recombinant Dictyostelium discoideum Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit swp1 (swp1)

FAQs for Researchers on Recombinant Dictyostelium discoideum Dolichyl-diphosphooligosaccharide--protein Glycosyltransferase Subunit Swp1 (Swp1)

What is the functional role of Swp1 in Dictyostelium discoideum N-glycosylation?

Swp1 is a 48 kDa subunit of the oligosaccharyl transferase (OST) complex, which catalyzes the transfer of a preassembled glycan (Glc₃Man₉GlcNAc₂) from dolichol-pyrophosphate to asparagine residues in nascent polypeptides during cotranslational N-glycosylation . This process is critical for protein folding, quality control, and ER-associated degradation.

• Methodological Insight: To study Swp1’s role, researchers often:

  • Use CRISPR/Cas9 to generate swp1 knockout strains and assess glycosylation defects via lectin blotting .

  • Co-express Swp1 with other OST subunits (e.g., Stt3) in heterologous systems (e.g., S. cerevisiae) to reconstitute activity .

What structural features define Swp1’s interaction with the OST complex?

Swp1 is essential for maximal OST activity, though its exact structural role remains under investigation. Key features include:

• A conserved N-terminal domain implicated in binding dolichol-pyrophosphate oligosaccharides .

• A C-terminal region that stabilizes interactions with the Sec61 translocon during cotranslational glycosylation .

• Experimental Design: Crosslinking-mass spectrometry (CL-MS) and cryo-EM are used to map Swp1’s interaction sites within the OST complex .

How is recombinant Swp1 produced for in vitro studies?

Recombinant Swp1 is typically expressed in E. coli or insect cell systems with affinity tags (e.g., His₆) for purification.

• Protocol Example:

  • Clone the swp1 gene (Gene ID: wbp1) into a pET vector.

  • Induce expression with IPTG in BL21(DE3) cells.

  • Purify using Ni-NTA chromatography and validate via SDS-PAGE/Western blot .

How do Swp1 mutations affect OST activity and substrate specificity?

Swp1’s conserved residues (e.g., Asp102 in the catalytic pocket) are critical for glycan transfer. Site-directed mutagenesis reveals:

• Data Contradiction: While Swp1(D102A) abolishes activity in Dictyostelium, partial activity is retained in S. cerevisiae OST, suggesting species-specific functional redundancy .

• Analysis Workflow:

  • Compare glycosylation efficiency of mutant vs. wild-type Swp1 using fluorescently labeled glycopeptide substrates.

  • Quantify kinetic parameters (Km, Vmax) via HPLC-based assays .

What is Swp1’s role in non-canonical cytoplasmic glycosylation pathways?

Swp1 may indirectly regulate cytoplasmic glycosylation by modulating ER stress responses. For example:

• Key Finding: Swp1-deficient cells show elevated ROS levels and impaired Skp1 glycosylation, disrupting developmental signaling .

• Method: Use redox-sensitive dyes (e.g., DCFDA) and GSH/GSSG assays to link Swp1 activity to oxidative stress pathways .

How can Swp1 be used to model neurodegenerative disease mechanisms?

Swp1’s role in protein quality control intersects with pathways implicated in neurodegeneration:

• Case Study: Swp1 interacts with autophagic regulators (e.g., Atg8) to clear aggregation-prone proteins like mutant huntingtin .

• Experimental Approach:

  • Express human amyloid-β in Dictyostelium and assess clearance in swp1⁻ strains via fluorescence microscopy .

Table 1: OST Subunit Conservation Across Eukaryotes

Table 2: Impact of Swp1 Knockout on Glycosylation Efficiency

Key Methodological Challenges

• Heterologous Expression: Co-expressing Swp1 with other OST subunits (e.g., Sec61) is required for functional reconstitution .

• Activity Assays: Use radiolabeled dolichol-linked glycans to bypass challenges in oligosaccharide synthesis .