Recombinant Ustilago maydis Dolichol-phosphate mannosyltransferase (DPM1)

What is the primary function of DPM1 in Ustilago maydis?

DPM1 in Ustilago maydis functions as a dolichol phosphate mannosyltransferase (EC 2.4.1.83), catalyzing the synthesis of dolichol phosphate-mannose. This enzyme transfers mannose from GDP-mannose to dolichol phosphate, creating DolP-mannose, an essential mannose donor for various glycosylation processes. The reaction is fundamental for proper N-glycosylation pathways and ultimately affects cell wall integrity, protein function, and potentially pathogenicity. Similar to archaeal DolP-mannose synthases like those in Haloferax volcanii, U. maydis DPM1 likely plays a role in transferring mannose to protein-bound glycans, contributing to the complete glycan structures necessary for proper protein function .

What is the structure and sequence of Ustilago maydis DPM1?

Ustilago maydis DPM1 is a 294-amino acid protein with multiple distinctive domains. The full amino acid sequence is: MSIALDMDASAKMRKQPGSSGWSTSSTPSCSVIVPAFRENLNLRPLVTRLSSAFASQSSS ELANTEIIIVDDNSRDGSVETVSALQSEGYNVRIIVRTSERGLSSAVVRGFREARGQRMI CMDA DLQHPPEAVPSLLALNGQKSFVLGTRYGVGVSMDKDWPLHRIISSGARMLARPL TSASDPMSGFFGITKHSFHTADHHINAQGFKIALDLLVKSGVHSTDIAEVPFSFGLRQEG ESKLDGKVMFKYLQQLVELYRFRFGTVPIVFVLIVLLVLALYIWSHVLAPMLGA .

The protein contains a catalytic domain responsible for the mannosyltransferase activity and multiple predicted membrane-spanning regions that anchor it to the membrane. Drawing parallels from research on archaeal homologs, we can infer that the catalytic domain likely resides on the cytoplasmic side of the membrane, while the transmembrane domains facilitate membrane anchoring and potentially participate in the translocation of DolP-mannose across the membrane .

How does glycosylation mediated by DPM1 affect Ustilago maydis biology?

Glycosylation processes mediated by DPM1 are crucial for multiple aspects of Ustilago maydis biology. The addition of mannose to glycan structures contributes to proper protein folding, stability, and function, particularly for secreted and membrane proteins. In pathogenic fungi like U. maydis, glycosylation affects the cell wall structure, which is essential for withstanding environmental stresses and evading host immune responses during infection.

Studies on U. maydis have demonstrated that proper glycosylation is critical during its dimorphic transition from unicellular haploid cells to pathogenic dikaryotic hyphae when infecting maize plants . This morphological shift is essential for U. maydis virulence and requires precise regulation of cell wall composition and remodeling, processes in which DPM1-mediated glycosylation likely plays a significant role. Additionally, many secreted effector proteins that enable U. maydis to establish a biotrophic interaction with host plants may require proper glycosylation for stability and function .

What expression systems are optimal for producing recombinant Ustilago maydis DPM1?

For optimal expression of recombinant U. maydis DPM1, researchers should consider systems capable of handling membrane proteins with multiple transmembrane domains. Based on current practices for similar proteins, several expression platforms can be recommended:

• Yeast expression systems: Saccharomyces cerevisiae or Pichia pastoris are suitable for expressing fungal membrane proteins like DPM1. These systems provide appropriate post-translational modifications and membrane environments. Vectors containing strong inducible promoters (GAL1 for S. cerevisiae or AOX1 for P. pastoris) with appropriate secretion signals and affinity tags (His, FLAG, or StrepII) facilitate protein purification.

• Bacterial expression systems: For expression of the soluble catalytic domain alone, E. coli systems with vectors like pET or pBAD can be employed. Addition of fusion partners like MBP (maltose-binding protein) can improve solubility.

• Insect cell expression: Baculovirus expression systems offer advantages for membrane proteins requiring complex folding and post-translational modifications.

Regardless of the chosen system, expression should be optimized by testing different growth temperatures (typically 16-30°C), induction conditions, and detergents for extraction if membrane-bound forms are desired .

What methodologies can accurately assess DPM1 enzymatic activity?

Several complementary approaches can be employed to assess the enzymatic activity of U. maydis DPM1:

• In vitro enzymatic assays: Activity can be measured by monitoring the transfer of [14C]- or [3H]-labeled mannose from GDP-mannose to dolichol phosphate. The reaction typically contains purified recombinant DPM1, GDP-[14C]mannose, dolichol phosphate, detergent (e.g., Triton X-100), and appropriate buffers with divalent cations (Mg2+ or Mn2+). Products can be separated using thin-layer chromatography and quantified by radiography.

• LC-MS analysis: A non-radioactive approach involves liquid chromatography coupled with mass spectrometry (LC-MS) to detect and quantify DolP-mannose. This method can identify distinct hexose-modified versions of dolichol phosphate with characteristic retention times, as demonstrated in studies of archaeal DPM synthases. For U. maydis, this would involve extracting lipids followed by targeted LC-MS analysis focusing on the m/z value corresponding to mannose-charged dolichol phosphate species .

• Functional complementation: The activity of DPM1 variants can be assessed by their ability to restore glycosylation in DPM1-deficient yeast strains or U. maydis mutants. The restored glycosylation can be analyzed by techniques such as glycoprotein staining or mass spectrometry of glycopeptides.

How can researchers investigate the membrane topology of DPM1?

Understanding the membrane topology of DPM1 is crucial for elucidating its mechanism. Several experimental approaches can be employed:

• Protease protection assays: Microsomes containing DPM1 can be treated with proteases in the presence or absence of detergents. Protected fragments can be identified by immunoblotting with domain-specific antibodies, revealing which domains are exposed or embedded in the membrane.

• Fluorescence-based approaches: Introducing fluorescent tags at different positions and analyzing their accessibility can provide information about topology. This includes techniques like fluorescence protease protection (FPP) or bimolecular fluorescence complementation (BiFC).

• Cysteine scanning mutagenesis: Systematic replacement of amino acids with cysteines followed by selective labeling with membrane-permeable or -impermeable reagents can map the orientation of specific protein regions.

• Cryo-electron microscopy: For high-resolution structural analysis, cryo-EM can be applied to purified DPM1 reconstituted in nanodiscs or other membrane mimetics.

Drawing from studies of the archaeal homolog AglD, truncation analysis has proven valuable. In Haloferax volcanii, truncated AglD containing only the catalytic domain and two membrane-spanning regions maintained DolP-mannose synthase activity, suggesting that the C-terminal transmembrane domain plays a role in subsequent steps like DolP-mannose translocation rather than mannose charging itself .

What role does DPM1 play in Ustilago maydis pathogenicity?

While no direct studies have specifically examined the role of DPM1 in U. maydis pathogenicity, several lines of evidence suggest its importance:

• Glycosylation in pathogen-host interactions: Proper glycosylation of cell surface and secreted proteins is critical for fungal pathogens to evade host immune responses and establish successful infections. DPM1, as a key enzyme in the mannose transfer pathway, likely contributes to this process.

• Effector protein functionality: U. maydis secretes numerous effector proteins during infection that suppress host immunity and redirect host metabolism. Many of these effectors are likely glycoproteins whose proper function depends on correct glycosylation mediated by pathways involving DPM1 .

• Gene expression patterns: Transcriptomic analyses of U. maydis during infection have revealed differential regulation of genes involved in glycoprotein synthesis. In particular, diploid pathogenic forms show distinct patterns of glycosylation-related gene expression compared to haploid forms, suggesting glycosylation contributes to the biotrophic lifestyle .

Research on other plant pathogens has demonstrated that disruption of DPM1 or other glycosylation enzymes often leads to attenuated virulence due to compromised cell wall integrity, altered recognition by host immune systems, or impaired function of effector proteins.

How is DPM1 expression regulated during different lifecycle stages?

Gene expression analysis suggests that glycosylation pathway genes, potentially including DPM1, are differentially regulated during U. maydis lifecycle transitions. The transcriptomic analysis of U. maydis infecting Arabidopsis reveals distinct expression patterns between haploid and diploid forms:

This differential expression suggests that glycosylation requirements change as U. maydis transitions from saprophytic growth to pathogenic development. The haploid form, which shows necrotrophic-like behavior, appears to broadly upregulate N-glycan synthesis machinery, potentially including DPM1. In contrast, the diploid form, which establishes a biotrophic interaction, shows more selective regulation of glycosylation genes .

These patterns align with the different cellular demands during these lifecycle stages – the haploid form may require extensive cell wall remodeling during saprophytic growth, while the diploid form needs specific glycosylation patterns to evade host recognition and establish a compatible interaction.

What cellular processes might be affected by DPM1 dysfunction?

DPM1 dysfunction would likely impact multiple cellular processes in U. maydis:

• Protein secretion and stability: Improper glycosylation of secreted proteins could lead to misfolding, degradation, or altered function. This would particularly affect effector proteins needed for host interaction.

• Cell wall integrity: Mannose-containing glycoconjugates are essential components of the fungal cell wall. Disruption of DPM1 would likely compromise cell wall structure, leading to increased susceptibility to environmental stresses and host defense responses.

• Morphological transitions: The dimorphic switch from yeast-like to hyphal growth is crucial for U. maydis pathogenicity. This transition involves extensive cell wall remodeling, which depends on proper glycosylation pathways .

• Host-pathogen interactions: Recent research has shown that U. maydis employs specialized effectors like UmPR-1La to sense plant phenolics and guide hyphal growth in plants, as well as to subvert plant immune responses. The function of such effectors might be compromised by altered glycosylation resulting from DPM1 dysfunction .

• Stress response: Glycoproteins play important roles in stress sensing and response. DPM1 dysfunction might impair adaptation to environmental stresses encountered during infection.

How does Ustilago maydis DPM1 compare to homologs in other fungi?

Comparative analysis of U. maydis DPM1 with homologs in other fungi reveals several important insights:

What insights can be gained from archaeal dolichol phosphate mannose synthases?

Studies of archaeal dolichol phosphate mannose synthases, particularly Haloferax volcanii AglD, provide valuable insights relevant to understanding U. maydis DPM1:

• Catalytic mechanism: The archaeal enzyme AglD has been studied in detail, showing that even truncated versions containing only the catalytic domain and two membrane-spanning regions retain DolP-mannose synthase activity. This suggests that the core catalytic mechanism is highly conserved and requires minimal structural elements .

• Membrane topology: In H. volcanii AglD, the extended C-terminal transmembrane domain is not essential for mannose charging but appears necessary for subsequent steps like DolP-mannose translocation across the membrane. This suggests a dual role for the protein – enzymatic activity and membrane transport facilitation .

• Distinct roles of transmembrane regions: Truncation studies with AglD revealed that truncated proteins containing only up to five membrane-spanning regions failed to transfer mannose to protein-linked tetrasaccharides in vivo, despite maintaining DolP-mannose synthesis activity. This indicates different functional regions within the protein: a catalytic core for mannose charging and additional domains for downstream processing .

• Structural insights: Studies on Pyrococcus furiosus PF0058, an archaeal DolP-mannose synthase with a solved three-dimensional structure, provide a structural template that may inform understanding of the U. maydis enzyme's architecture and mechanism.

What are the key differences between fungal and mammalian DPM systems?

Several important differences distinguish fungal DPM systems, including that of U. maydis, from mammalian counterparts:

• Subunit composition: Mammalian DPM synthase is a complex of three proteins: DPM1 (catalytic subunit), DPM2 (stabilizing subunit), and DPM3 (membrane anchor). In contrast, fungal DPM1 typically functions as a single polypeptide that contains both catalytic and membrane-anchoring domains.

• Membrane topology: Fungal DPM1 contains multiple transmembrane domains that anchor the protein to the ER membrane, with the catalytic domain facing the cytosol. Mammalian DPM1 lacks transmembrane domains and relies on DPM3 for membrane anchoring.

• Regulatory mechanisms: The complex mammalian system allows for sophisticated regulation through assembly/disassembly of the multi-subunit complex. Fungal systems likely employ different regulatory mechanisms focused on expression levels and post-translational modifications of the single DPM1 protein.

• Structural organization: Fungal DPM1 proteins typically have an N-terminal catalytic domain followed by multiple C-terminal transmembrane segments. This arrangement may facilitate coupling of DolP-mannose synthesis with its subsequent utilization in the ER lumen.

• Evolutionary conservation: While the catalytic mechanism is conserved across kingdoms, the structural organization reflects divergent evolutionary solutions to the common challenge of synthesizing and utilizing DolP-mannose in membrane-associated glycosylation pathways.

How might novel inhibitors of DPM1 affect Ustilago maydis pathogenicity?

Developing and studying selective inhibitors of U. maydis DPM1 could provide valuable insights into both basic biology and potential antifungal strategies:

• Experimental approach: Researchers could employ structure-based drug design utilizing homology models based on archaeal or other fungal DPM structures. High-throughput screening of chemical libraries against recombinant DPM1 could identify lead compounds, which could then be optimized for potency and selectivity.

• Expected outcomes: Selective inhibition of DPM1 would likely disrupt protein glycosylation, potentially resulting in:

  • Compromised cell wall integrity, making the fungus more susceptible to environmental stresses

  • Misfolded or dysfunctional effector proteins, impairing host manipulation

  • Altered surface glycan patterns, potentially increasing recognition by host immune systems

  • Disrupted morphological transitions essential for pathogenicity

• Experimental validation: The effects of identified inhibitors could be assessed through:

  • In vitro enzymatic assays to confirm target engagement

  • Analysis of glycosylation patterns in treated cells using mass spectrometry

  • Microscopic examination of cell morphology and wall integrity

  • Infection assays to determine effects on pathogenicity

  • Transcriptomic analysis to identify compensatory responses

• Potential challenges: Developing highly selective inhibitors might be challenging due to conservation of the catalytic domain across kingdoms. Strategies focusing on fungal-specific regions or regulatory mechanisms might yield more selective compounds.

What are the structural determinants of DPM1 substrate specificity?

Understanding the structural basis of DPM1 substrate specificity would provide fundamental insights into its function:

• Research questions:

  • What structural features determine the preference for dolichol phosphate over other lipid carriers?

  • Which amino acid residues in the catalytic domain are critical for GDP-mannose binding and catalysis?

  • How do the transmembrane domains influence substrate accessibility and product release?

• Methodological approaches:

  • Site-directed mutagenesis of conserved residues in the catalytic domain

  • Chimeric enzymes combining domains from DPMs with different specificities

  • Molecular dynamics simulations to model substrate binding and catalysis

  • X-ray crystallography or cryo-EM of the catalytic domain with bound substrates or substrate analogs

• Comparative analysis: Leveraging knowledge from archaeal DPM synthases like AglD, where truncation studies have provided insights into domain functions. Similar approaches with U. maydis DPM1 could reveal which regions are essential for catalysis versus membrane translocation .

• Structure-function correlations: Correlating structural features with enzymatic parameters (Km, kcat) for various substrates could map the determinants of specificity and provide a rational basis for engineering enzymes with altered properties.

How does DPM1 integrate with other glycosylation enzymes in vivo?

Understanding the integration of DPM1 into the broader glycosylation machinery would provide systems-level insights:

• Protein-protein interactions: Identifying interaction partners of DPM1 using techniques such as:

  • Proximity labeling (BioID, APEX)

  • Co-immunoprecipitation followed by mass spectrometry

  • Fluorescence resonance energy transfer (FRET) with candidate partners

• Spatial organization: Determining the spatial organization of glycosylation enzymes in the ER membrane using:

  • Super-resolution microscopy of fluorescently tagged enzymes

  • Electron microscopy with immunogold labeling

  • Lipid raft association analysis

• Metabolic flux analysis: Tracing the flow of mannose through the glycosylation pathway using:

  • Isotope-labeled mannose precursors

  • Time-course analysis of labeled intermediates

  • Quantitative modeling of pathway kinetics

• Integrative multi-omics approach: Combining data from:

  • Transcriptomics to identify co-regulated genes

  • Proteomics to measure enzyme abundance and modifications

  • Glycomics to assess the impact on final glycan structures

  • Lipidomics to analyze dolichol phosphate pool dynamics

• Temporal regulation: Investigating how DPM1 activity is coordinated with other glycosylation steps during the U. maydis life cycle, particularly during the transition to pathogenic growth, where differential regulation of glycosylation genes has been observed .