Glycobiology Research: Disease Biomarkers, Metabolic Disorders, Cancer

1. Department of Glycobiology • Major research areas: a) Identifyingof putative N-, and O-glycanbiomarkersrelated to diseasepathogenesis:aplication ofglyco-proteomics and glyco-genomicsmethodsto gain understanding of aberrantglycosylationassociatedwithvariousphysiologicaland pathophysiological status (InbornMetabolicDisorders and Cancer) * Inherritedmetabolicdisorders of glycoconjugatemetabolism -Lysosomalstoragedisorders (LSD) glycogenosis and Pompe disorder: Caused by defects in degradation of glycoconjugates • >40knownsubtypes • Characteristic by specificmetaboliteaccumulated in tissues/body fluids - Congenitaldisorders of glycosylation (subtypes of CDGs): Caused by defects in glycanbiosynthesis and processing • >70known subtypes • Characteristic by specificaberrantN-, and O-glycanstructures in tissues/body fluids * Thestructure of glycanalternationassociatedwithcolorectalcancer. Wholebloodserumglycoprofiling and structure of O- glycans in pathogenesis.

2. Humanfluids and selected glycoproteins in glycoprofiling and N-, and O- glycanstructureanalysis(cooperation with clinicians). Aplication of high-resolutiontools: LC-MS/MS (CID, ETD, HCD ), LC- ESI, permethylation, fragmentationanalysis, sequentionalanalysisusingexoglycosidases, chemical/enzymaticdesialylation. Dataanalysisusingsubjectrelateddatabases , incl. bioinformaticstools. Multiglyco-gene panel sequencing by thenextgenerationsequencing. - CDGs: N- linkedglycans O- linkedglycans In the CDG patient, an abnormal glycosylation was observed at the whole serum glycoprofiles as well as glycoprofiles of selected human blood biomarkers. Structural analysis led to the selection of number of potential deficient enzymes encoded by glyco-genes, incl. enzyme donor substrate transporter. -LSD/glycogenosisand POMPE disorder: Oligosaccharidemetaboliteproduct For monitoring of Pompe disease esp., in enzyme replacement therapy the MS- and NMR -spectroscopy were applied in determination of clinically relevant glyco-biomarkers. O-glycosylation profile was underlined on the patients suffering from various glycogenosis and hyperlipidemias what resulted in discovery of the new glyco-biomarker in glycogen degradation. - Colorectalcancer: Weappliedglyco-proteomicsapproach to accuratelly profile blood N-, and O-complexglycome by delineatinganaberrantglycosylation.

3. b) Transglycosylases of yeasts and plants: enzymes enabling the restructuralization of energy poor cell walls during the cell development, growth, stress-induced respond / enzymes with expected role in cryptococcal capsule rearrangement and following pathogenesis / stress-induced proteins obtained as side-products of proteomic approach to plant and yeast resources: • - Development of activity assay methods based on fluorescently labelled acceptor oligosaccharides (one-pot method for acceptor labelling and activity assays as dot-blot method, assay after IEF-PAGE, high-throughput methods based on glycochips or in-gel reactions, HPLC, etc.). • - Characterization of yeasts GH 16 and GH 72 transglycosylases - Crh group (Saccharomyces cerevisiae) with focus on their substrate specificities and Phr group (Candida albicans) on importance and cooperation of individual forms in pathogenesis → possible importance as targets of new generation drugs based on inhibition of these enzymes. • - Screening for glycosidases on surface of capsular yeasts, purification and characterization of an alpha-galactosidase form from Cryptococcus flavescens → the ever first lactose-inducible yeast long-chain alpha-galactosidase of GH 36 family.

4. - Localization, purification, characterization and structure-specificity studies of plant transglycosylases mainly unspecific xyloglucan endotransglycosylases (XETs) → „play“ with substrate specificity of these enzymes using bioinformatics, molecular modeling and mutagenesis / found heterotranslycosylation between xyloglucan and oligogalacturonic acids as linear components of pectin. • Purification and structural characterization of Pet c 1.0201, Bet v 1 allergen like PR-10 protein, induced due plant tissue damage and low temperature storage → molecular dynamic simulations of dimer stability of various I. type allergens of Apiaceae family (carrot, celery, parsley) with respect to the relationship between oligomerization and allergenicity.

5. c) Study of antimicrobialsubstances of royaljelly: investigation of the efficacies of major antimicrobial royal jellysubstances -peptide defensin1 and five 8-10 carbonfattyacids, against several collection strains and field isolates of Paenibacillus larvae- bacterial pathogen causing serious honeybee brood disease American foulbrood. American foulbrood - the disease causes economic losses to beekeepers and farmers dependent on pollination Disease-causing agent: Paenibacillus larvae - the pathogen occuring in contaminated food propagates first in midgut and then in bodies of infected larvae what causes their death - infection spreads in colony what leads to its collaps Royal jelly - food of young larvae produced by honeybees - antimicrobial substances present in it could in adequate concentrations hinder the propagation of pathogen bacteria in larval midgut Example of substances: 10-hydroxy-2-decenoic acid 3,10-dihydroxydecanoic acid Determination of minimal inhibitory concentrations of a substance by broth microdilution method