Mining High-Complexity Motifs in Glycans: A New Language To Uncover the Fine Specificities of Lectins and Glycosidases

Abstract

Knowledge of lectin and glycosidase specificities is fundamental to the study of glycobiology. The primary specificities of such molecules can be uncovered using well-established tools, but the complex details of their specificities are difficult to determine and describe. Here we present a language and algorithm for the analysis and description of glycan motifs with high complexity. The language uses human-readable notation and wildcards, modifiers, and logical operators to define motifs of nearly any complexity. By applying the syntax to the analysis of glycan-array data, we found that the lectin AAL had higher binding where fucose groups are displayed on separate branches. The lectin SNA showed gradations in binding based on the length of the extension displaying sialic acid and on characteristics of the opposing branches. A new algorithm to evaluate changes in lectin binding upon treatment with exoglycosidases identified the primary specificities and potential fine specificities of an α1–2-fucosidase and an α2–3,6,8-neuraminidase. The fucosidase had significantly lower action where sialic acid neighbors the fucose, and the neuraminidase showed statistically lower action where α1–2 fucose neighbors the sialic acid or is on the opposing branch. The complex features identified here would have been inaccessible to analysis using previous methods. The new language and algorithms promise to facilitate the precise determination and description of lectin and glycosidase specificities.

Advances in Tools to Determine the Glycan-Binding Specificities of Lectins and Antibodies

“…The first feature accounts for the variability in the binding-site of a protein. We developed a new syntax, or motif language (29), that uses wildcards and logical operators (AND, OR, etc.) to describe variability in monosaccharides or linkages, and that uses other modifiers to allow for gaps of any length.…”

“…These features are the foundations of the MotifFinder software (29) for automated analyses of glycan-array data. The program searches for the individual motifs that best describe specific fine-specificities or primary-specificities, and it searches for the set of motifs and the relationships between them that account for the overall binding pattern of the protein (Fig.…”

“…The flexible motif syntax enables the automated generation of novel motif definitions that can account for unforeseen fine-specificities. This capability could be useful for characterizing protein binding to unusual glycans, as in a previous application of the method (29) to glycans with unequal extensions and substituents on each branch (30). The method identified features that would not have been practically analyzable using manual analyses or previous modes of representing motifs.…”

“…Synthetic strategies address these limitations and are making good progress. New synthetic methods have provided structures that previously were difficult to synthesize, such as asymmetricallybranched N-glycans (29,(71)(72)(73), which were useful for producing arrays of human milk oligosaccharides (30). Several groups have developed arrays for additional classes such as sialylated structures (74 -76), plant cell-wall glycans (77), and microbial glycans (8,78,79).…”

Glycans and glycosaminoglycans in neurobiology: key regulators of neuronal cell function and fate

“…Furthermore, novel high sensitivity, low toxicity alkynyl-fucose substrates have been developed for the visualisation of fucose incorporation into glycopolymers, these alkynyl-fucose substrates are incorporated into N-glycans by a wide range of fucosyl transferases [54] enabling their visualisation in cells using biotin-steptavidin Alexa-488 histochemistry and they may be extracted, separated by SDS PAGE and identified by Western blotting [53]. The complexity of glycans surpasses by several magnitudes that of the other major life biomolecules, proteins, lipids and nucleic acids [9,10,21,55,56] and their analysis has lagged behind due to this complexity however with the improvement in glycan analysis now possible with the methodology outlined above this gap is steadily closing.…”