Project Grant R43GM150367

DESIGN AND SYNTHESIS OF A NEXT GENERATION GLYCOBIOLOGY TOOLBOX FOR CELL SURFACE LABELING - THE CELL MEMBRANE IS THE INTERFACE BY WHICH A CELL INTERACTS AND COMMUNICATES WITH ITS SURROUNDINGS, INCLUDING NEARBY CELLS, PROTEINS, AND ANTIBODIES. MEMBRANES OF HEALTHY CELLS ARE OFTEN MODIFIED IN DISEASED STATES, PROVIDING AN EXTERNAL MEANS TO DISTINGUISH BETWEEN MORPHOLOGICALLY SIMILAR CELLS. OFTEN, DISEASED CELLS DISPLAY MODIFIED CARBOHYDRATES AND GLYCANS ON THEIR CELL MEMBRANES. RECENT DEVELOPMENTS IN ENZYME-MEDIATED CELL SURFACE ENGINEERING PROVIDE PROMISING ROUTES FOR LABELING THESE MODIFICATIONS IN DIFFERENT CELL TYPES. THE APPROACH KNOWN AS CHEMOENZYMATIC GLYCAN LABELING COMBINES GLYCOSYLTRANSFERASES AND UNNATURAL NUCLEOTIDE SUGAR DONORS EQUIPPED WITH CHEMICAL TAGS TO DIRECTLY LABEL SPECIFIC SURFACE GLYCANS WITHIN BIOLOGICAL SAMPLES. RECENTLY, THIS SINGLE STEP LABELLING APPROACH HAS BEEN SUCCESSFULLY USED WITH FUCOSYLTRANSFERASE AND GDP-FUCOSE EQUIPPED WITH BIOTIN TAGS. COMPARED WITH OTHER GENETIC AND METABOLIC TECHNIQUES, THIS METHOD OF LABELING LIVING CELLS IS A MORE DIRECT, SIMPLER PROCEDURE WITH A FASTER TURNOVER CYCLE. IT HAS ALSO BEEN SHOWN TO BE GENERALLY APPLICABLE TO MURINE TUMOR MODELS, AND THUS HAS A HIGH POTENTIAL FOR TRANSFER TO A CLINICAL SETTING. WHILE PUBLISHED REPORTS HAVE FOCUSED ON A LOW NUMBER OF GDP-FUCOSE LABELS, THE HIGH POTENTIAL OF THIS METHOD IS LIMITED ONLY BY THE AVAILABILITY OF NUCLEOTIDE SUGAR LABELS PAIRED WITH ACCEPTABLE GLYCOSYLTRANSFERASES. IN ORDER TO ACCELERATE MEDICAL ADVANCES BASED ON CELLULAR INTERACTIONS, THERE IS AN URGENT NEED TO EXPAND THE AVAILABILITY OF NUCLEOTIDE SUGAR LABELS AND TRANSFERASES THAT CAN BE USED BY THIS METHOD. THE GOAL OF OUR PHASE I PROJECT IS TO MEET THIS NEED AND PROVIDE NON-EXPERT USERS WITH A PLATFORM FOR LABELING GLYCANS ON THE MEMBRANES OF THEIR CHOICE CELLS. MEETING THIS GOAL WILL EXPAND THE AVAILABILITY OF GLYCOENGINEERING REAGENTS AND METHODS BEYOND PUBLISHED STUDIES. IN AIM 1, WE WILL CREATE 12 NEW NUCLEOTIDE SUGAR DERIVATIVES WITH LABELS. DERIVATIVES WILL BE BASED ON SUCCESSFUL STUDIES OF GDP-FUCOSE, AND EXPANDED TO CMP-SIALIC ACID. NUCLEOTIDE SUGARS WILL BE MODIFIED THROUGH CLICK-CHEMISTRY WITH A FLUORESCENT DYE, BIOTIN, OR PHOTOPROBE LABEL. IN AIM 2, WE WILL SCREEN AND OPTIMIZE GLYCOSYLTRANSFERASES CAPABLE OF ATTACHING LABELED NUCLEOTIDE SUGARS TO GLYCAN SUBSTRATES AND CELL SURFACES. WE WILL ADVANCE 10 KITS THAT COMBINE THE MOST EFFECTIVE COMBINATIONS OF LABEL AND TRANSFERASE, ALONG WITH STANDARD OPERATING PROCEDURES AND CONDITIONS THAT ARE ACCESSIBLE TO END USERS. USERS MAY THEN LABEL MEMBRANES WITH FLUORESCENT TAGS FOR CELL SURFACE IMAGING, BIOTIN FOR CELL SELECTION, OR PHOTOPROBES FOR CROSSLINKING. DATA WILL SUPPORT PHASE II EFFORTS TO COMPARE THIS METHOD TO EXISTING TECHNOLOGIES AND PROBE SPECIFIC CELL TYPES.