Pan-specific tools and reagents for the enrichment of sialo-glycans

PROJECT SUMMARY Glycans have several distinct properties that make their development as disease biomarkers appealingFirstlytheir location on cell surfaces makes them the first point of contact for cellular interactionsand thus they are crucial in the control of normal metabolic processesand converselythey function as pathogen adhesion receptorsSecondlyspecific glycan structures that are not presentor are in low amounts in normal stateproliferate or alter their sequence in disease statesAndlastlychanges in glycosylation may be found in many proteinsincluding those that are highly abundantThus changes in the normal levels of glycan structuressuch as terminal sialic acidmay be markers of disease statesNew highly specific reagents are required in order to overcome current limitations in the discovery and exploitation of disease related glycansUsing structurally guided genetic manipulationswe are converting the NanB sialidase from Spneumococcus into a high specificity affinity reagent for the detection of all types of sialic acid modifications of glycopeptides and glycoproteinsBecause such a protein has lectin like propertiesbut is derived from an enzymeit is called a andquot Lectenz andquotA NanB Lectenzaddresses a key need in disease glycomarker detectionnamelya robust and easy to produce reagent specific for detecting all types of sialylated glycansThis reagent could be employed in an affinity matrix for sample enrichmentwhich in conjunction with existing MS based methods could provide linkage informationGlycopeptide sample enrichment aids glycomic analyses by eliminating non glycosylated peptideswhich would otherwise attenuate the signals from glycopeptides that have low ionization efficiencyGlycosylation site mapping is essential in fully characterizing and exploiting glycans as markers of specific disease statesbut at presentno reagent exists that can detect sialylated glycansindependent of the type of linkage associated with the sialic acidThusat presenta number of reagents with varying specificities and affinities must be employed to capture or detect all forms of sialylated glycansLectenzoffer numerous advantages over plant lectinsthey are engineered to be high affinity and yet retain the exquisite substrate specificity of the endogenous enzymethey may be efficiently producedand for human homologues have the potential to be employed in vivo with low toxicityWhereas some aspects of Lectenzdevelopment parallel those of antibody evolutionLectenzhave the tremendous benefit of employing a protein na ve template that has the desired specificity PROJECT NARRATIVE Using structurally guided genetic manipulationswe are converting carbohydrate processing enzymes into a high specificity affinity reagentscalled Lectenzfor the detection of disease related modifications of glycopeptides and glycoproteinsHere we are developing a LectenzSia PSspecific for the enrichment of biologically important glycans that contain all forms of sialic acidThe principle advantages of engineered Lectenzover other reagentssuch as antibodies or lectinsis that they have exquisite substrate specificity that is not context dependentthey may be evolved to have desirable binding kineticsand that they may be efficiently produced as monomeric proteins