Reagents specific for all N-glycans

PROJECT SUMMARY Glycans have several distinct properties that make them excellent targets for disease biomarkersFirstlytheir location glycans on cell surfaces makes them the first point of contact for cellular interactionsand thus they are crucial in the control of normal metabolic processesSecondlycell surface molecules are also strategically exposed for surveillance by the immune system allowing for the potential of immune recognition of abnormal cellsThirdlyspecific glycan structures that are not presentor are in low amountsin normal states proliferate in disease statessuch as cancerAnd lastlychanges in glycosylation involve many proteinsincluding those that are highly abundantThereforea single change in a cellandapos s glycosylation machinery can affect many different glycoconjugatesTo effectively employ and discover glycan disease markers a wide range of highly specific reagents are urgently neededUsing structurally guided directed evolutionwe will convert the newly identified PNGase F II carbohydrateprocessing enzyme into a pan specific high affinity reagent for peptides and proteins that contain asparaginelinkedN linkedcarbohydrate chainsSuch engineered lectin like reagents derived from enzymes are calledLectenzand have several advantages over lectins and antibodiesThe advantages of Lectenzinclude precise definition of specificityease of recombinant expressionandfor human enzyme homologuesminimal in vivo toxicityenabling their potential use as imaging reagentsA pan specific N glycan Lectenzderived from PNGase F II would directly address the needs of glyco biomarker detection in mass spectrometry based glycomics proteomics analysis by enabling sample enrichmentGlycopeptide sample enrichment aids glycosylation site mapping by eliminating non glycosylated peptideswhich would otherwise lower the signals from glycopeptides that have low ionization efficiencyGlycosylation site mapping is essential in fully characterizing and exploiting glycans as markers of specific disease statesand yet no current reagents exist that can be used to enrich a sample in all constituent N linked glycansThe principle advantages of an engineered Lectenzover an antibody are that the Lectenzis specific to the carbohydrate sequencebutin contrast to antibodieswill recognize that sequence in a broad range of glycansFurtherin contrast to carbohydrate reagents based on plant lectinsengineered Lectenzare derived from enzymes that have exquisite substrate specificities and low toxicities PROJECT NARRATIVE Using structurally guided genetic manipulations we will convert the carbohydrate processing enzyme PNGase F II into a pan specific N glycan affinity reagentcalled a Lectenzfor the detection of glycopeptides and glycoproteins that contain asparagine linked carbohydrate chainsincluding those that are core fucosylated in either theorpositionsThis enzyme presents a unique opportunity to engineer a pan specific N glycan detection reagent that will retain the broad specificity of the parent enzyme for all N linked glycansCurrently no pan specific N glycan affinity reagent exists