Abstract
While nucleic acid and protein analysis approaches continue to see significant breakthroughs, analytical strategies for glycan determination have by comparison seen slower technological advances. Here we provide a strategy for glycan probe development using an engineered lectin fusion that can be incorporated into various common pathology lab assay formats including Western blot and agglutination assays. In this proof of concept, we use the natural lectin, Pseudomonas fluorescens agglutinin (PFA), capable of binding core Man alpha(1-3)-Man alpha(1-6)-Man units, where this lectin has previously been shown to bind to the glycans presented by the gp120 coat protein of (HIV) Human Immunodeficiency Virus. In our strategy, we engineered the lectin to possess a fusion of the biotin mimetic tag equence of amino acids V-S-H-P-Q-A-P-F. With the glycan receptive PFA directly linked to the biotin mimic, we could facilitate a probe for various standard clinical assay formats by virtue of coupling to streptavidin-HRP (horseradish peroxidase) or streptavidin beads for Western blot and agglutination assays respectively. We found the PFA fusion retained low nanomolar affinity for gp120 by ELISA (Enzyme Linked Immunosorbent Assay) and microscale thermophoresis. This probe engineering strategy proved effective in the relevant assay formats that may now allow detection for the presence of glycans containing the core Man alpha(1-3)-Man alpha(1-6)-Man units recognized by PFA.
Highlights
Glycans have traditionally been complicated to study as their structural complexity allowing sugar building blocks to be linked at different sites and with different stereochemistries goes beyond the relatively simple linear template-driven synthesis of nucleic acids and proteins [1]
Having purified the lectin Pseudomonas fluorescens agglutinin (Figure 3), we initially examined its ability to bind to the glycans of gp120 as compared to GNA (Galanthus nivalis agglutinin) which is known to bind glycans with (α-1,3)-linked mannose residues
We selected the lectin Pseudomonas fluorescens agglutinin (PFA) as our glycan receptive moiety based on reports of its high yield in bacterial expression along with the extensive literature reporting the ability of PFA to bind the glycans of the HIV coat protein gp120 which we could use as a model target for our proof of concept study [32,33,35]
Summary
Glycans have traditionally been complicated to study as their structural complexity allowing sugar building blocks to be linked at different sites and with different stereochemistries goes beyond the relatively simple linear template-driven synthesis of nucleic acids and proteins [1]. Tools for genetic and proteomic level testing are abundant but we have yet to possess sufficient glycan-related diagnostic tools that may be integrated into standard practice. Because of this bias in attention, there remains a lot to be understood regarding glycans in terms of basic science and from the perspective of clinical diagnostics and is deserving of special consideration [5]. Traditional glycan analysis techniques include chromatographic analysis of metabolically radio-labeled glycans, as well as capture with lectin affinity chromatography, and glycosidase treatment for chemical analysis of the constituent sugars [6,7,8]. Other techniques have included the use of sequential exoglycosidase digestion coupled with methylation analysis utilizing
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