Recombinant Aotus nancymaae Galactoside 2-alpha-L-fucosyltransferase 1 (FUT1), partial

How does the structure of Aotus nancymaae FUT1 inform our understanding of its catalytic mechanism?

While the crystallographic structure of A. nancymaae FUT1 has not been specifically reported in the provided literature, insights can be drawn from structural studies of homologous FUT1 enzymes. Crystallographic studies of recombinant FUT1 from Arabidopsis thaliana have been conducted at high resolution (up to 1.95 Å), which revealed important structural features of this class of enzymes . The crystallization of A. thaliana FUT1 in a monoclinic space group P21 with specific unit-cell parameters (a = 87.6, b = 84.5, c = 150.3 Å, β = 96.3°) provides a framework for understanding the potential structural features of A. nancymaae FUT1 .

The catalytic mechanism of FUT1 likely involves specific binding pockets for the donor molecule (GDP-fucose) and the acceptor substrate (terminal galactosyl residues). Researchers interested in A. nancymaae FUT1 might apply similar crystallization approaches to elucidate its specific structure-function relationships.

What experimental evidence supports the function of FUT1 in Aotus nancymaae?

Current literature provides limited direct experimental evidence specific to A. nancymaae FUT1. Researchers typically rely on comparative genomic approaches and functional predictions based on homology to FUT1 in better-characterized species. The function of FUT1 has been more thoroughly investigated in other organisms, such as Arabidopsis thaliana for plant biology and pigs for resistance to Escherichia coli infections .

To establish experimental evidence for A. nancymaae FUT1 function, researchers should consider:

• Recombinant expression and purification of the enzyme

• In vitro activity assays with various potential substrates

• Generation of knockout or knockdown models to observe phenotypic effects

• Structural studies similar to those performed with A. thaliana FUT1

What expression systems are most effective for producing functional recombinant Aotus nancymaae FUT1?

The optimal expression system for recombinant A. nancymaae FUT1 production depends on research objectives and desired downstream applications. Based on similar enzymes' expression strategies:

When working with A. nancymaae FUT1, consider that strategies similar to those used for A. thaliana FUT1 might be applicable, where researchers successfully produced diffraction-quality crystals from recombinant protein . The expression system choice should align with your specific experimental goals, whether they be structural characterization, activity assays, or interaction studies.

What purification strategies yield the highest purity and activity for recombinant Aotus nancymaae FUT1?

Effective purification of recombinant A. nancymaae FUT1 typically requires a multi-step approach that preserves enzymatic activity while achieving high purity. Based on general principles and related fucosyltransferase purification methods:

• Initial capture: Affinity chromatography using fusion tags (His-tag, GST-tag) provides specific binding and efficient initial purification.

• Intermediate purification: Ion exchange chromatography separates based on charge differences, effectively removing contaminants with different isoelectric points.

• Polishing step: Size exclusion chromatography achieves final purification based on molecular size and shape.

• Activity preservation considerations:

  • Maintain optimal buffer conditions (pH, ionic strength)

  • Include stabilizing agents (glycerol, reducing agents)

  • Minimize freeze-thaw cycles

  • Consider adding cofactors or substrate analogs during purification

For crystallization purposes, additional criteria for purity and homogeneity become critical, as demonstrated in the successful crystallization of A. thaliana FUT1 . Researchers achieved diffracting crystals suitable for structural analysis through careful purification and crystallization condition optimization.

How can researchers optimize crystallization conditions for structural studies of recombinant Aotus nancymaae FUT1?

Crystallization of recombinant A. nancymaae FUT1 would likely benefit from approaches similar to those used for A. thaliana FUT1. The crystallization process requires systematic screening and optimization:

• Initial screening stages:

  • Employ commercial sparse matrix screens covering diverse precipitation agents

  • Test various protein concentrations (typically 5-20 mg/ml)

  • Screen different temperatures (4°C and 20°C are common)

  • Vary drop sizes and ratios in vapor diffusion setups

• Optimization phase:

  • Fine-tune successful condition parameters (pH, precipitant concentration)

  • Introduce additives that promote crystal formation

  • Consider seeding techniques to improve crystal quality

  • Test protein modifications (limited proteolysis, surface mutations)

• Specific considerations for FUT1:

  • Inclusion of stabilizing ligands (GDP-fucose analogs)

  • Detergent screening if membrane-association is present

  • Heavy atom derivatives for phasing (tantalum bromide was successfully used with A. thaliana FUT1)

The crystallization of A. thaliana FUT1 in two different crystal forms, with the best diffracting crystals belonging to the monoclinic space group P21, provides a potential starting point for optimization strategies with A. nancymaae FUT1 .

How do polymorphisms in the FUT1 gene affect enzyme function and what parallels might exist in Aotus nancymaae?

Polymorphisms in FUT1 genes can significantly impact enzyme function and biological outcomes. While specific polymorphisms in A. nancymaae FUT1 have not been extensively documented in the provided literature, insights from other species are informative:

In pigs, a single-nucleotide polymorphism (c.307G>A) in the FUT1 gene has been identified as a key determinant of resistance to enterotoxigenic Escherichia coli F18. Pigs with the AA genotype demonstrate resistance, while those with AG and GG genotypes are susceptible . This polymorphism illustrates how single nucleotide changes can have profound functional consequences.

For A. nancymaae FUT1, potential polymorphisms might influence:

• Substrate specificity or catalytic efficiency

• Protein stability and expression levels

• Interaction with regulatory proteins

• Tissue-specific expression patterns

Researchers interested in A. nancymaae FUT1 polymorphisms should consider:

• Sequencing the FUT1 gene from multiple individuals to identify variants

• Comparative analysis with FUT1 sequences from closely related species

• Functional characterization of identified variants through recombinant expression

• Association studies linking variants to phenotypic differences, if applicable

What techniques are most effective for analyzing FUT1 gene expression patterns in Aotus nancymaae tissues?

Understanding FUT1 gene expression patterns in A. nancymaae tissues requires appropriate molecular techniques tailored to the specific research questions:

When studying A. nancymaae FUT1, researchers should consider comparing expression patterns with those of FUT1 in other primates and model organisms to identify conserved and divergent regulatory mechanisms. The choice of technique should align with specific research questions, available resources, and technical expertise.

How has FUT1 evolved across primate species, and what can we learn from Aotus nancymaae FUT1?

Evolutionary analysis of FUT1 across primate species provides insights into functional conservation and adaptation. While the available literature doesn't directly address FUT1 evolution in A. nancymaae, we can draw parallels from evolutionary patterns observed in other genes within this species.

Phylogenetic analysis of immune-related genes in A. nancymaae has shown interesting evolutionary patterns. For example, MHC-DPB1 exon 2 amino acid sequences in A. nancymaae cluster in a species-specific manner, unlike some other immune genes (DRB and DQB) . This suggests differential evolutionary pressures on various gene families within this species.

For FUT1, comparative genomic approaches might reveal:

• Conserved catalytic domains indicating functional constraints

• Variable regions potentially related to substrate specificity

• Evidence of positive selection in regions involved in pathogen interactions

• Lineage-specific adaptations reflecting environmental pressures

Studying A. nancymaae FUT1 in this evolutionary context may provide insights into primate-specific glycosylation patterns and their biological significance, particularly given this species' importance as a model for malaria research .

What unique features of Aotus nancymaae make its FUT1 particularly valuable for comparative glycobiology research?

A. nancymaae (Nancy Ma's night monkey) possesses several characteristics that make it a valuable model organism, potentially extending to the study of its FUT1 and glycobiology:

• Phylogenetic position: As a New World monkey, A. nancymaae occupies an important evolutionary position for comparative studies with both Old World primates and humans.

• Malaria susceptibility: A. nancymaae serves as an important animal model for malaria research, particularly for pre-clinical evaluation of blood-stage vaccine candidates against Plasmodium falciparum and Plasmodium vivax . This susceptibility may involve surface glycan structures potentially influenced by FUT1 activity.

• Genetic distinctiveness: The species shows interesting patterns of genetic evolution, as demonstrated in studies of its MHC genes, where some sequences cluster in species-specific patterns while others show similarities to human sequences . This mixed pattern of evolutionary conservation might extend to FUT1.

• Research applications: The growing genomic resources for A. nancymaae, including characterized gene sequences on various chromosomes , provide a foundation for comprehensive studies of glycosylation-related genes including FUT1.

Studying A. nancymaae FUT1 may provide unique insights into glycan structure-function relationships in an organism that bridges evolutionary gaps between different primate lineages while offering practical applications in infectious disease research.

How might characterization of Aotus nancymaae FUT1 contribute to malaria vaccine development strategies?

A. nancymaae serves as a critical model for pre-clinical evaluation of blood-stage vaccine candidates against Plasmodium falciparum and Plasmodium vivax . Characterizing FUT1 in this species could contribute to malaria vaccine development in several ways:

• Host-parasite interaction insights: FUT1-mediated fucosylation may influence glycan structures on erythrocyte surfaces, potentially affecting parasite invasion mechanisms. Understanding these modifications could reveal new vaccine targets.

• Comparative glycobiology: Differences in FUT1 activity between humans and A. nancymaae might explain species-specific aspects of malaria susceptibility, informing the design of more effective vaccines that account for these differences.

• Immunological considerations: Fucosylated epitopes can influence immune recognition and response. FUT1 characterization might help explain the immunological differences observed between human and A. nancymaae responses to malaria antigens.

• Translational models: Understanding the similarities and differences between human and A. nancymaae FUT1 would strengthen the validity of this animal model for predicting human responses to glycan-based vaccine formulations.

These applications highlight the importance of basic research on A. nancymaae FUT1 for translational malaria vaccine development efforts.

What analytical techniques best characterize the substrate specificity of recombinant Aotus nancymaae FUT1?

Comprehensive characterization of recombinant A. nancymaae FUT1 substrate specificity requires multiple complementary analytical approaches:

For recombinant A. nancymaae FUT1, researchers might begin with radiochemical assays using GDP-[14C]fucose as donor and various potential acceptor substrates to establish baseline activity profiles. Following identification of preferred substrates, more detailed structural and kinetic analyses would provide deeper insights into specificity determinants.

Drawing from approaches used with A. thaliana FUT1, crystallographic studies with substrate analogs could reveal the structural basis for specificity .