Glycans that regulate development and notch signaling

>> GOOD AFTERNOON, EVERYBODY IT’S REALLY MY GREAT PLEASURE TO INTRODUCE OUR SPEAKER THIS WEDNESDAY AFTERNOON DR. PAMELA STANLEY PAM CURRENTLY HOLDS THE HORACE W. GOLDSMITHS FOUNDATION CHAIR AND IS PROFESSOR OF CELL BIOLOGY AT THE ALBERT EINSTEIN COLLEGE OF MEDICINE PAM HAS HAD A REMARKABLE CAREER, ACTUALLY WHEN SPEAKING TO MANY OF YOU ABOUT HER PENDING VISIT IT WAS QUITE INTERESTING TO LEARN THE SORT OF DIVERSITY OF WHAT PAM IS KNOWN FOR BECAUSE EACH OF YOU SORT OF CAME AT IT FROM A DIFFERENT ANGLE THIS ISN’T SURPRISING, BECAUSE PAM’S MADE MANY SIGNIFICANT CONTRIBUTIONS TO SEVERAL FIELDS OF SIGH SCIENCE SOME OF YOU KNOW HER FOR SHOW MATTIC CELL GENETICS TO ISOLATE CELL MUTANTS DEFECTS IN ASPECTS OF GLYCOPROTEIN BIOSYNTHESIS, THE LEK MUTANTS THAT COULD NOT BIND BECAUSE OF THE ALTERED SUGARCOAT OTHERS OF YOU KNOW HER WORK ON SCHEMEATOGENESIS, ABLATION RESULTING IN MALE INFERTILITY, BUT MOST OF YOU MENTIONED THE WORK I GUESS PAM IS MOST WELL KNOWN FOR, THAT IS ON NOTCH SIGNALING AS MANY OF OF YOU KNOW THE EXTRACELLULAR DOMAIN OF NOTCH RECEPTORS IS DECORATED WITH CARBOHYDRATES, PAM’S GROUP IS AT THE FOREFRONT OF DECOATING WHICH AND HOW THESE SUGARS MEDIATE NOTCH SIGNALING PAMELA IS THE RECIPIENT OF MANY, MANY AWARDS INCLUDING A MERIT AWARD FROM THE NATIONAL CANCER INSTITUTE AND CARL MEYER AWARD FROM THE SOCIETY FOR GLYCOBIOLOGY FOR HER LECTURE TODAY, GLYCANS THAT REGULATE DEVELOPMENT IN NOTCH SIGNALING SHE WILL DISCUSS EMBRYONIC DEVELOPMENT, AND O-FUCOSE GLYCANS JOIN ME IN WELCOMING DR. PAMELA STANLEY [APPLAUSE] >> OKAY, GOOD AFTERNOON, EVERYBODY I AM VERY, VERY PLEASED TO BE HERE TO SEE ALL MY OLD FRIENDS AND MEET NEW ONES, AND IT IS A GREAT HONOR TO HAVE BEEN SELECTED TO GIVE A WALS LECTURE, SO THANK YOU VERY MUCH TO LARRY AND KELLY FOR NOMINATING ME, AND I HOPE BY THE END OF MY LECTURE THAT YOU WILL ALL FEEL A LITTLE SWEETER AND THAT YOU’LL UNDERSTAND THAT SUGAR IS NOT JUST ICING ON THE CAKE SO THIS IS ALBERT EINSTEIN COLLEGE OF MEDICINE IN THE BEAUTIFUL BRONX, AND GLYCANS WHICH HAVE BEEN THE FOCUS OF MY WORK FOR A LONG TIME BECAME THE FOCUS BECAUSE WE REALIZED THAT THEY FORMED THE FRONTIER OF EVERY CELL THIS IS JUST THE CARTOON FROM SCIENTIFIC AMERICAN, WHERE YOU CAN SEE THAT ANYTHING THAT GETS INTO THE SECRETORY PATHWAY ENDS UP IN THE CELL SURFACE, WHERE THE GLYCANS ARE MOSTLY EXPRESSED IF WE ZERO DOWN AND LOOK MORE AT WHAT THESE GLYCANS ARE, THEY ARE MADE UP OF DIFFERENT SUGARS THAT ARE DEPICTED HERE, AND SO FOR THE TALK I WILL BE USING THE SYMBOLS WHICH WILL BE DEFINED ON EACH SLIDE AND THEY ARE ADDED EITHER DIRECTLY TO PROTEIN AS IN THIS CASE, OR THEY ARE ADDED SUGAR TO SUGAR THEY CAN BE FAIRLY SIMPLE, A SINGLE SHOULD SUGAR OR MORE COMPLICATED THERE’S PROTEOGLYCANS AND SO ON TO APPROACH THE FUNCTIONS OF THESE MODIFICATIONS WE’VE TAKEN A GENETIC APPROACH, AND THE REASON THAT WE REALLY DECIDED TO

STICK WITH IT IS THAT GLYCAN EXPRESSION IS REGULATED THIS IS A TEST SECTION, IT SHOWS GLYCANS THAT ARE EXPRESSED IN SPERM ATID, AND SPERMATAZOA NOT EXPRESSED, AND SINCE THEY ARE REGULATED THEY MUST BE CONTROLLED FOR SOME REASON AND SO OUR APPROACH HAS BEEN TO USE GENETICS, CHO CELLS AS DR TABAK MENTIONED IS WHERE WE BEGAN, AND WE HAVE A NUMBER OF CHO MUTANTS, LOSS AND GAIN OF FUNCTION, AND I LOVE ANYONE WHO WANTS TO USE THE MUTANT TO PLEASE ASK ME BECAUSE OTHERWISE THEY JUST SIT THERE WE’VE USED THEM FOR GLYCOSYLATION ENGINEERING, TRANSFERRATION GENE REGULATION AND TO UNDERSTAND GLYCAN FUNCTIONS SUCH AS LETTIN RECOGNITION, SIGNAL TRANSDUCTION AND FUNCTIONS IN BINDING IN THE ’90s WE MOVED INTO THE MOUSE AND MADE GENE TARGET AND TRANSGENIC MICE TO ZERO IN ON CELL TYPES, SPECIFIC FUNCTIONS OF SUGARS SO LET’S JUST REVIEW WHAT CHO GLYCOSYLATION MUTANT IS LIKE IF WE TAKE ONE, THIS IS A COMPLEX END GLYCAN, LOSS OF FUNCTION MUTANT TO BE RESISTANT, ALL THE SUGARS DISAPPEARED AND WE HAVE A NEW TRUNCATED FORM EXPRESSED ON GLYCOPROTEINS ALTERNATIVELY WE HAVE A SERIES OF GAIN OF FUNCTION MUTANTS, LECTIN FOR EXAMPLE, WHERE WE’VE ACQUIRED A NEW SUGAR THAT’S EXPRESSED ON GLYCOPROTEINS NOW, THE GENE THAT CODES FOR THE ENZYME THAT PUTS ON THIS SUGAR IS CALLED NGAT 3, IT’S HIGHLY REGULATE AND CONSERVED IN MAMMALS SO WE’RE INTERESTED IF WHAT IT DOES IT FIRST CAME ON OUR RADAR WHEN WE ISOLATED A MUTANT RESISTANT TO RICIN, AND THE REASON WAS THE ACQUISITION OF THIS MODIFICATION, THE BISECTING GlcNAc, AND THE ENZYME THAT CODES, THE GENE THAT CODES TO THE ENZYME THAT PUTS THIS ON WAS TURNED ON IN THESE MUTANTS, IT’S NOT ON IN CHO CELLS THIS HAS A PRETTY DRAMATIC EFFECT IN THAT IF WE LOOK AT RICIN RESISTANCE, LECTIN CELLS ARE TEN-FOLD MORE RESISTANT TO RICIN HOWEVER, THEY ACQUIRE AT THE SAME TIME AN INCREASED HYPER SENSITIVITY TO A DIFFERENT LECTIN, E-PHA, THEY BIND TO GAL RESIDUES HERE YOU CAN SURMISE THE BISECRETARYING GlcNAc CHANGES THE CONFIRMATION SO NOW THEY ARE RECOGNIZED VERY DIFFERENTLY BY THESE DIFFERENT LECTINS AND THEY ALSO ARE RECOGNIZED DIFFERENT BY GALECTINS AND THERE ARE A NUMBER OF GALECTINS WHICH ARE DECREASED IN BINDING TO THESE CELLS SO WHAT IS THE FUNCTION OF THIS VERY INTRIGUING SINGLE SUGAR RESIDUE? ONE OF THE THINGS THAT HAS BEEN KNOWN ABOUT GALECTINS IS THEY PROMOTE GROWTH FACTOR RECEPTOR SIGNALING THIS SHOWS THEM CROSS-LINKED BY GALECTIN 3 IN THIS CASE WE WERE INTERESTED TO SEE IF SOMETHING LIKE THE BISECTING GlcNAc, NOT ON THESE GLYCANS, WOULD AFFECT GROWTH FACTOR SIGNALING TO DO THAT, WE SET UP WILD-TYPE CHO CELLS, AND THEN SOME MUTANTS, INCLUDING LECTIN, AND JUST EXPRESSED AND DID A GROWTH FACTOR SIGNALING ASSAY USING GROWTH FACTOR,ENED HERE WE HAVE REPRESENTATIVE GLYCANS ON THE CELLS, SO IN A WILD-TYPE THERE WILL BE COMPLICATED COMPLEX GLYCANS, WE’RE MISSING ONE BRANCH IN THIS MUTANT IN THIS MUTANT WE HAVE NO GALS IN THE LEC 10 WE HAVE THE BISECTING GlcNAc

WE FOUND IN THERE’S REALLY NO GALS WE HAVE REDUCED PDPS RECEPTOR SIGNALING, BUT IF WE HAVE THE BISECTING GlcNAc, IT’S ALSO SIGNIFICANTLY REDUCED, SAME FOR LOSING A BRANCH SO WHAT HAPPENS IN VIVO, WE KNOCKED IT OUT, AND USED A MAMMARY TUMOR MODEL,MTV/PYMT TO LOOK AT TUMOR BURDEN AND METASTASIS, AND WE FOUND IF THAT ENZYME IS NOT THERE, YOU HAVE AN INCREASED TUMOR BURDEN AND THE SIZE OF THE TUMORS IS ALSO INCREASED AND I’M NOT SHOWING YOU THE DATA, BUT METASTASIS TO THE LUNG IS ALSO INCREASED SO IT SEEMS THAT THE BISECTING GlcNAc PRESENT IN THE CONTROL MICE IS BASICALLY RETARDING TUMOR GROWTH AND LIKE IT DID FOR GROWTH FACTOR SIGNALING SO THE BISECTED N-GLYCANS RETARD GROWTH FACTOR SIGNALING BY AFFECTING THEIR ABILITY TO BIND GALECTINS AND TO BE ACTUALLY ENDOCYTOSED INTO THE CELL THE MODEL HERE IS THAT THE GROWTH FACTOR RECEPTORS ARE ABLE TO SIGNAL LONGER BECAUSE THEY ARE NOT ENDOCYTOSED AS QUICKLY AS NORMAL RELAPSE FREE SURVIVAL IN HUMANS CORRELATES WITH EXPRESSION OF THIS GENE IN THIS COHORT THAT WAS PUBLISHED SO THAT IS SORT AN INTERESTING SIDE EFFECT OF HAVING THIS BISECTED GlcNAc IS MAYBE ACTUALLY RELEVANT TO HUMAN DISEASE SO THERE ARE A LOT OF CHO GLYCOSYLATION MUTANTS I’LL SUMMARIZE THEM HERE THERE’S A NUMBER OF LOSS OF FUNCTION MUTANTS, AND THEN A NUMBER OF GAIN OF FUNCTION MUTANTS, AND THEY HAVE BEEN USEFUL TO STUDY HUMAN DISORDERS OF GLYCOSYLATION BY CORRECTING THE DEFECTS WITH WILD-TYPE GENES AND THEN SHOWING THAT MUTANT GENES CANNOT CORRECT IN THIS CASE LEC13 IS VERY USEFUL BECAUSE ANTIBODIES MADE IN THE CELL LINE ARE A THOUSAND TIMES MORE TOXIC BY ADCC, AND MANY ANTIBODIES ARE IN THE CLINIC THESE DAYS IN THESE LINES WHICH MAKE TRUNCATED GLYCANS ENZYME REPLACEMENT THERAPY HAS BEEN HELPED BECAUSE THEY TARGET GLYCOPROTEINS WITH THOSE SUGARS TO THE RETICULOENDOTHELIAL CELLS AND CAN TARGET TO CELLS EXPRESSING SELECTINS AND AS I MENTIONED LEC10 IS IMPORTANT FOR GROWTH CONTROL AND THEN MUTANTS THAT ARE LACKING — WE USED THE MUTANTS TO AID IN THE DISCOVERY OF NEW ORPHAN-LIKE GLYCOSYLATION ACTIVITIES, SO GENES THAT ARE DISCOVERED IN THE GENOME THAT LOOK LIKE THEY MIGHT BE TRANSFERASE OR A GLYCOCIDASE IF YOU CLEAVE THE N GLYCANS FROM CHO GLYCOPROTEINS YOU FIND A BUNCH OF OLIGO AND GLYCANS AND COMPLEX END GLYCANS FOR EXAMPLE, DOWN HERE THE HIGHEST MOLECULAR WEIGHT ONE HAS A STRUCTURE LIKE THIS NOW, SINCE WE’RE ONLY TALKING ABOUT METH, WE CAN’T ACTUALLY SAY WHAT THE STRUCTURE IS BUT IT IS QUITE LARGE WE DECIDED TO USE THIS APPROACH TO STUDY AN ORPHAN CDNA WHICH HAD BEEN ISOLATED, AND WHEN WE EXPRESSED THAT IN WILD-TYPE CHO CELLS, THIS IS THE PATTERN WE GOT THERE WERE ABSOLUTELY NO COMPLEX N-GLYCANS AND WE OVEREXPRESSED IT IN

WILD-TYPE CHO CELLS TO SHOW ALSO THAT THE CELLS BECAME VERY RESISTANT TO THE LECTIN L-PHA THEY ARE NOT MAKING COMPLEX N-GLYCANS, WHY HER RESISTANT TO L-PHA, AND THEY’VE GOT AN INCREASED AMOUNT OF THIS SPECIES WHICH IS MAN-5 SO MAN-5 IS THE SUBSTRATE FOR THIS ENZYME MGAT1, ALSO KNOWN AS GlcNAc T-1, THIS CDNA ENCODES FOR PROTEIN THAT’S A TYPICAL TYPE 2 MEMBRANE BOUND TYPE PROTEIN, AND IT INHIBITS MGAT 1 SO YOU CAN SEE FROM THIS IT CATALYZES GlcNAc TO SUGAR, SUBSTRATES, IF THAT REACTION DOESN’T HAPPEN THE WHOLE PATHWAY IS INHIBITED AND SO THIS SUBSTRATE ACCUMULATES AND THERE ARE NO COMPLEX N-GLYCANS THIS WAS VERY INTERESTING BECAUSE IT’S THE FIRST PHYSIOLOGICAL INHIBITOR OF A GLYCOSYLTRANSFERASE LIKE THIS WHICH PREVIOUSLY OTHER GROUPS HAVE FOUND CHAPERONES OR OTHER ACTIVITIES, BUT NOT AN INHIBITOR SO WE SHOWED IT INHIBITS THE ENZYME ACTIVITY IF YOU MAKE AN EXTRACT OF THE TRANSSECTED CELLS, AND CO–IPs WITH THE ENZYME AND SO WE’VE BEEN LOOKING MORE DIRECTLY AT SPECIFICITY USING A FRET ASSAY IN COLLABORATION FINLAND, THE IDEA WAS TO SEE IF MGAT-1 WAS THE ONLY GlcNAc THAT WOULD INTERACT WITH THIS INHIBITOR SO THE INHIBITOR, GlcNAc T-1 INHIBITORY PROTEIN, L FOR THE LONG FORM, HAS A MONOMERIC VENOUS GSP ATTACHED AT THE C TERMINUS AND IS STABLELY EXPRESSED IN THESE LINES, EACH ONE OF THESE TRANSFERASES WAS CONJUGATED AND THEN TRANSSECTED INTO THE LINE AND WHEN WE DID THAT EXPERIMENT, FRET INTERACTIONS WERE SEEN EITHER IN COS 7 OR LEC1 ONLY WITH MGAT 1, OTHERS IN THE MEDIAL GOLGI DID NOT CAUSE A SIGNIFICANT FRETTED SIGNAL, TRUE IF THEY WERE CO-TRANSSECTED TOGETHER, AND WE CAN SEE THE INHIBITOR ACTUALLY FORMS COMPLEXES AS WELL AS DO EACH OF THOSE ENZYMES AND YOU CAN INHIBIT THIS INTERACTION IF A COMPLEX IS FORMED BY OVEREXPRESSING INDIVIDUAL MGAT 1, CDNA OR THE INHIBITOR BUT NOT ANY OF THE OTHER GlcNAc TRANSFERASES IT SEEMS TO BE SPECIFIC FOR MGAT 1 IN THE GOLGI WHAT IS IT DOING? WELL, WE WERE PRETTY SURPRISED WHEN WE HAD A LOOK AT WHERE IT’S EXPRESSED AND FOUND THAT IT’S VERY HIGHLY EXPRESSED IN TESTERS, MINIMALLY IN OTHER CELL TYPES AND SO WE LOOKED AT THE EXPRESSION SPECIFICALLY OF THE L FORM AND FOUND THAT THAT WAS DEVELOPMENTALLY REGULATED, AT 22 DAYS THE TRANSCRIPTS FOR THIS FORM WERE HIGHER THAN 11 OR 17 AND HIGHER THAN THE ADULT SO THIS WAS INTRIGUING, AND WE HAVE JUST SEEN SOME RECENT DATA ANALYZED FROM RNA-SEQ, WHERE THE MGAT1 GENE IS SHOWN HERE IN RED IN SERTIOLI, AND YOU CAN SEE IT’S EXPRESSED IN ALL OF THEM TO DIFFERING LEVELS, BUT THE INHIBITOR IS EXPRESSED IN SPERMATOCYTES AND SPERMATIDS ONLY IT’S CALLED MGAT 4D, HOWEVER

THERE’S NO EVIDENCE IT’S A TRANSFERASE AT THIS POINT IT COULD BE IT COULD BE AN INHIBITOR AND TRANSFERRATION BUT THAT’S ITS NEW NAME I’M GOING TO KEEP CALLING IT GNTI P THESE ARE MEN WITH TUBULAR ATROPHY, SERTOLI CELL SYNDROME, SPERMAGONIA ONLY, GERM CELLS, MEIOTIC ARREST OF PRIMARY SPERMATOCYTES, AND THEN DECREASED SPERMATIDS OR JUST EVERYTHING BEING NORMAL IN THE NORMAL SITUATION, MGAT1 IS QUITE POORLY EXPRESSED AND THE INHIBITOR IS HIGHLY EXPRESSED, AND SO ON, ALL ALONG SO HERE IN TUBULAR ATROPHY, THERE’S NO GERM CELLS, THE INHIBITOR IS NOT PRESENT AND THE ENZYME TRANSCRIPTS ARE WELL REPRESENTED AND SO THEY ARE COMPLETELY COMPLEMENTARY SO THAT WAS SORT OF AN INTERESTING ASIDE AND OUR MODEL AT THIS POINT IS — OR THE SUMMARY OF THIS DATA THAT IF WE LOOK AT THE TRANSCRIPTS, LOOK AT SPERMATOGONIA, SPERMATOCYTES, ALL GENERATED ON THE SERTOLI CELLS DURING DIFFERENTIATION, A SPERM GOING TO A WOMAN, THE ENZYME IS EXPRESSED HIGHLY IN SPERMATAGONIA, WE EXPECT COMPLEX N-GLYCANS HERE, THEN TRANSCRIPTS FOR THIS GO DOWN A BIT AND THESE ONES COME ON, SO WE EXPECT MUCH MORE REPRESENTATION OF THIS N-GLYCANS, AND SAME HERE, ALTHOUGH PROBABLY A BIT MORE MIXED, AND THEN IN SPERM WE HAVE THE INHIBITOR NOT REPRESENTED AND THE ENZYME THERE SO ARE THESE WORKING IN THE COMPLEMENTARY FASHION AND ARE THEY NEEDED IN A COMPLEMENTARY FASHION DURING SPERMATOGENESIS? COULD THESE HIGH MANNERS IN GLYCANS ENHANCE SERTOLI CELL ADHESION? WE HAVEN’T DONE MUCH ON THIS BUT WE’VE DONE ONE SERIES OF EXPERIMENTS WHERE WE USED TM 4 SERTOLI CELLS WHICH ARE HERE SHOWN ATTACHED TO A DISH, AND THEY ARE ACTUALLY FIXED, AND WE TOOK CELLS EXPRESSING THE INHIBITOR, TWO DIFFERENT FORMS OF IT, AND FOUND THAT IF YOU ALLOWED THEM TO ATTACH AND THEN WASHED FIVE TIMES THAT THESE CELLS REMAINED QUITE STRONGLY ATTACHED TO THE SERTOLI CELLS, WHEREAS THE CONTROL CELLS EXPRESSING COMPLEX N GLYCANS DID NOT, AND A MUTANT THAT’S MISSING THE ENZYME ALTOGETHER HAD THE SAME RESULT AS THE CELLS EXPRESSING THE INHIBITOR OF THE ENZYME AND THESE DATA ARE JUST SHOWN QUANTITATED HERE IT COULD BE THE INHIBITOR IS DESIGNED TO REALLY DECREASE MGAT1 ACTIVITY AFTER THE TRANSCRIPTS HAVE BEEN REDUCED, ANY ENZYME THAT’S REMAINING WILL BE ALSO REDUCED BY THE INHIBITOR AND THAT MAY ENHANCE THE INTERACTION OF THESE GERMS CELLS WITH SERTOLI CELLS, SO THAT’S OUR WORKING MODEL WHAT HAPPENS IN VIVO? WE HAVE DELETED THE ENZYME MGAT1 IN SPERMATAGONIA CRUISING A CRE AT THREE DAYS OF GESTATION, THREE DAYS AFTER BIRTH SO WHAT IS SHOWN HERE IS THE WHOLE TESTERS AND THEN A TESTER’S TUBE YOU’LL, A BLOWUP OF DIFFERENTIATION FROM SPERMATAGONIA THROUGH THE SPERM ON THE SERTOLI CELL F WE DELETE MGAT1 IN THESE CELLS WHAT DO WE FIND? AND WE CAN SHOW THAT IF WE LOOK AT THE EXPRESSION OF COMPLEX N-GLYCANS WITH THE LECTIN HLPA THEY ARE EXPRESSED NICELY HERE IN SPERMATAGONIA AND THERE’S NO REACTIVITY IN THE MUTANT WHICH IS EXPRESSING THE CRE IN

SPERMATAGONIA WE FOUND FOR WILD-TYPE, THIS IS TESTER SECTIONS, STAINED WITH HNE, YOU CAN SEE THE GERM CELLS DIFFERENTIALING INTO THE LUMEN OF THE TESTIS WHERE THE SPERM ARE CONGREGATED, IN THE MUTANT MICE WHERE MGAT1 IS KNOCKED OUT IN SPERMATAGONIA THINGS LOOK REASONABLE UP TO SPERMATIDS AND THEN SPERMATIDC FUSE AND FORM THESE AND THIS BLOCKS SPERMATOGENESIS, SEEN CLEARLY IF WE LOOK AT THE EPIDIDIMUS WHICH CONTAINS NEW SPERM COMPARED TO THE CONTROL MOUSE THIS IS A MUCH OLDER MOUSE WHICH NOW HAS ALL SORTS OF THINGS COLLECTING IN THE EPIDIDIMUS BUT NO SPERM AND SO WE’RE TRYING TO WORK OUT THE MECHANISM BEHIND THIS, WE’RE LOOKING AT MICROARRAY DATA AND PROTEOMICS OF GERM CELLS AT 22 DAYS BEFORE THEY SHOW ANY OF THESE PHENOTYPES, TO SEE WHAT PATHWAYS MAY BE MAINLY AFFECTED WE’RE KNOCKING OUT THE INHIBITOR IN THE MOUSE, AND WE’VE ALSO GENERATED A NUMBER OF TRANSGENIC LINES THAT OVEREXPRESS MGAT1 OR INHIBITOR IN SPECIFIC GERM CELLS USING PROMOTERS THAT ARE ACTIVE ONLY IN SPERMATAGONIA OR SPERMATOCYTES OR SPERMATIDS WE HOPE TO BE ABLE TO SHOW THAT THIS INHIBITION OF MGAT1 HAS A FUNCTIONAL ROLE IN SPERMATOGENESIS AND NOW I’D LIKE TO TURN TO THE THIRD PART OF MY TALK WHICH IS ON NOTCH SIGNALING, AND ROLES FOR GLYCANS IN NOTCH SIGNALING THIS IS A FAMILIAR DIAGRAM, I’M SURE, NOTCH RECEPTORS ARE TRANSMEMBRANES, HETERODIMERS, LINKED TOGETHER NON-CO-VEILENTLY OUTSIDE THE CELLS, THE N TERMINAL DOMAIN HAS MANY EGF REPEATS IN THE CASE OF NOTCH F NOTCH 1, IT’S 36 EGF REPEATS IT IS CELL SIGNALING EVENT, INTERACTING WITH NOTCH, LIGAND INTERACTS WITH NOTCH THROUGH THE DSL AND N TERMINAL DOMAIN, AND THAT CAUSES A CONFIRMATIONAL CHANGE THAT ALLOWS THIS PROTEASE TO CLEAVE THE EXTRACELLULAR DOMAIN AND GETS ENDOCYTOSED INTO THIS CELL THEN IT CAN RELEASE THE INTRACELLULAR DOMAIN WHICH GOES INTO THE NUCLEUS AND IN COMPLEX WITH CO-REPRESSORS ACTIVATES MANY DOWNSTREAM TARGETS GENES SO THE MAIN THING IS THAT NOTCH EGF REPEATS REGULATE THIS EVENT AND THEY ARE COATED WITH SUGARS SO THIS IS MOUSE NOTCH1 EXTRACELLULAR DOMAIN WITH 36 EGF REPEATS, AND EACH EGF REPEAT IS ABOUT 40 AMINO ACIDS AND HAS THREE SETS OF DISULFIDE BONDS LINKED THROUGH THESE RESIDUES, AND THE SUGARS ARE ADDED AT A CONSENSUS SITE, SO THE OLD GLUCOSE GLYCANS ARE ADDED BETWEEN CYSTEINE 1 AND 2, THE O FUCOSE ARE ADDED BETWEEN 2 AND 3, AND THE O GlcNAc ARE ADDED BETWEEN 5 AND 6 SO EACH OF THESE IS AN EGF REPEAT AND WE HAVE GLUCOSE, XYLOSE, XYLOSE THERE’S SIMPLE BUT THERE’S MANY AND THREE TIMES SOME EGF REPEATS CAN HAVE THEORETICALLY THREE TYPES OF MODIFICATIONS AND WE HAVE FOCUSED ON THE O-FUCOSE GLYCANS, AND ORIGINALLY WONDERING DO THEY REGULATE NOTCH SIGNALING AND THEN HOW DO THEY REGULATE NOTCH SIGNALING AND THE FIRST EXPERIMENT WE DID AFTER REALIZING THAT FUCOSE WAS

ACTUALLY THE FIRST SUGAR IN THIS GLYCAN WAS TO DELETE THE ENZYME POFUT 1 AND FOUND IF THERE’S IF THERE’S NO POSSIBILITY OF ADDING AN O-FUCOSE TO NOTCH IT’S EMBRYONIC LETHAL, THERE’S A SEVERE VASCULARIZATION DEFECT OF THE EMBRYO THIS IS THE YOLK SAC THERE ARE THE SOMITES ARE NOT PROPERLY FORMED, HERE THEY ARE LUMPY THAT NOTCH TARGET GENE EXPRESSION IS ALTERED, SO HERE DELTA3 EXPRESSION IS REDUCED IN THE PRE-SEMITIC MESODETERMINE BUT ENHANCED IN THE BRAIN AND WE LOOKED AT MANY DIFFERENT NOTCH TARGET GENES, AND THE EMBRYOS ARE SEVERELY RETARDED IN THEIR DEVELOPMENT SO WHAT’S THE MECHANISM? THE PHENOTYPE COPIES GLOBAL LOSS OF NOTCH SIGNALING, BUT IS IT DUE TO AN EFFECT ON NOTCH LIGAND BINDING? AND WE LOOKED AT THAT IN LIGAND DEPENDENED NOTCH SIGNALING ASSAYS WHERE WE USED CHO CELLS THAT HAVE ENDOGENOUS NOTCH RECEPTORS, AND CO-CULTURED THEM EITHER WITH CONTROL CELLS OR WITH THOSE CELLS EXPRESSING THE NOTCH LIGANDS AND THEN TRANSSECTED WITH REPORTERS, LUCIFER ACE HERE WHICH IS DRIVEN BY THE UPSTREAM BINDING SITES WHICH WILL ONLY RESPOND TO R ESPOND TO THE EXTRACELLULAR DOMAIN OF NOTCH RECEPTORS AND WE HAD A MUTANT THAT WAS ABLE TO HELP US UNDERSTAND THIS FUCOSE WAS MORE IMPORTANT OR ENZYME WAS MORE IMPORTANT THIS IS CALLED LEC13, IN THE CELLS THIS IS HOW GDT FUCOSE IS MADE AND THIS JUST SHOWS THAT IN LEC13 CELL THIS CANNOT BIND YOU CAN CORRECT THIS BY ADDING FUCOSE EXTERNALLY, TAKEN UP AND CONVERTED AND SO WE HAVE A MUTANT AND A RESCUE SITUATION AND WE CAN SEE HERE THAT IN A CONTROL CELL STIMULATED WITH L CELLS THERE’S NOT MUCH REPORTER ACTIVITY, STIMULATED WITH DELTA 1 GOOD FOLD STIMULATION AND THAT THIS IS INHIBITED BY A NOTCH INHIBITOR, GAMMA SECRETASE, IT’S DEPENDENT ON NOTCH IN LEC13 WE HAVE VERY LITTLE STIMULATION SO THE FUCOSE ITSELF IS ACTUALLY DOING SOMETHING HERE, BECAUSE AS I CAN SHOW HERE THIS IS JUST THE SAME SORT OF THING WHERE WE’VE RESCUED WITH FUCOSE, OR GALACTOSE AS A DIFFERENT CONTROL SUGAR, AND YOU SEE FUCOSE CAN RESCUE LEC13 FOR DELTA1 STILL LAKES AND JIGGED1 AND GAL CANNOT, CONTROL CELLS DON’T CARE LESS ABOUT FUCOSE BUT MOST IMPORTANTLY THE LEVEL OF THE ENZYME IS THE SAME IN ALL THESE CELL LINES SO THERE’S A LITTLE BIT OF DISCUSSION ABOUT WHETHER IT’S A LACK OF FUCOSE OR LACK OF THE ENZYME, AND IN MAMMALS FUCOSE IS IMPORTANT IN DROSOPHILA IT SEEMS THE ENZYME MAY HAVE SOME FUNCTIONS IN GETTING NOTCH TO THE CELL SURFACE BUT IN OUR CASE, WE COULD LOOK AT BINDING OF O-FUCOSE GLYCANS OF THE LIGANDS TO NOTCH RECEPTORS ON ES CELLS TO ASK THAT QUESTION MUTANT ES CELLS LACKING ENZYME HAVE VERY LITTLE BINDING OF DELTA 1, SOLUBLE DELTA 1, COMPARED TO THE CONTROL ES CELLS THIS IS DEPENDENT ON CALCIUM, A FEATURE OF NOTCH LIGAND BINDING, IT MUST HAVE CALCIUM TO EXHIBIT GOOD BINDING AND IT IS TRUE FOR DELTA AND JAGGED NOTCH LIGANDS AND MOST IMPORTANTLY, IF YOU

LOOK AT THE EXPRESSION OF NOTCH RECEPTORS AT THE SURFACE, THEY ARE THERE IN EQUIVALENT AMOUNTS, IN THE WILD-TYPE ES CELLS VERSUS MUTANT E S CELLS THIS IS NOTCH 3, WE LOOKED AT 1 AND 2 AS CELL THE FUCOSE ITSELF IS IMPORTANT WHAT ABOUT AN INDIVIDUAL SINGLE O-FUCOSE GLYCAN? SO HERE WE’VE GOT THE DIAGRAM AGAIN SHOWING WHERE O-FUCOSE GLYCANS WOULD BE, AND WE MADE A MUTATION JUST IN EGF 12 WHICH IS IN PARTS OF THE LIGAND BINDING DOMAIN OF NOTCH RECEPTORS, SO THIS IS THE ONLY O-FUCOSE HERE, AND THE SURROUNDING EGF REPEATS DON’T HAVE THE CONSENSUS SITES FOR O-FUCOSE AND WE COULD SHOW THAT THIS MUTATION, ELIMINATED FUCOSE IN COOPERATION, USING THIS ASSAY AND SO WHAT HAPPENS IN THE MOUSE? WELL, THE MOUSE WAS FINE, ACTUALLY AND IT ACTUALLY GREW WELL, GREW SLIGHTLY MORE SLOWLY THAN WILD-TYPE, BUT WE DECIDED TO LOOK AT T-CELL DEVELOPMENT BECAUSE NOTCH IS KNOWN TO BE VERY IMPORTANT IN T-CELL DEVELOPMENT AND FOUND THAT THE THYMUS WAS SMALLER IN THE HOMOZYGOUS MUTANTS, THYMUS WEIGHT WAS SMALLER AND THE NUMBER OF THYMUSITES WAS SMALLER LOOKING AT THE SUBSETS DURING DIFFERENTIATION FROM EARLY THYMIC, DOUBLE POSITIVE AND SINGLE POSITIVE T-CELLS, SO NAMED BECAUSE OF EXPRESSION OF CD4 OR CD8, THEN WE FOUND THAT THE — A NUMBER OF THE T-CELL SUBSETS WERE REALLY AFFECTED, DOUBLE POSITIVE T-CELLS WERE REDUCED ABOUT 50%, ENTIRE MEDIATE SINGLE POSITIVE ALSO, AND SINGLE POSITIVES WERE ALSO REDUCED BUT OTHER CELL TYPES IN THE THYMUS WERE NOT ALTERED AND THIS PHENOTYPE WAS CELL-AUTOMONOUS, REDUCED IN T-CELLS OF THE MUTANT, AND IF WE PUT THAT INTO A LETHAL IRRADIATED RECIPIENT, A SINGLE POINT MUTATION IN THE NOTCH RECEPTOR AT THE O-FUCOSE SITE AFFECTS T-CELL DEVELOPMENT AND IT’S IMPORTANT TO HAVE THIS MODIFIED FOR OPTIONAL T-CELL DEVELOPMENT SO THERE’S ANOTHER SUGAR THAT’S ADDED AFTER THE FUCOSE, AND THE NEXT ONE IS GlcNAc, AND ACETYLGLUCOSEAMINE IT FORMED A LITTLE FRINGE ON THE DROSOPHILA WINGS, SO THIS FRINGE GENE OR ONE OF THE FRINGE GENES HAD BEEN MUTATED IN THE MOUSE BEFORE IT WAS EVEN KNOWN WHAT IT DID, AND A GROUP SHOWED YOU GET TREMENDOUSLY DISRUPTED SKELETAL DEVELOPMENT IN THESE MICE SO WE WERE ABLE TO SHOW, AND OTHER GROUPS TOO, THAT FRINGE MODULATES NOTCH LIGAND BINDING HERE WE HAVE CELLS DELTA BINDING, IN THE SAME CELLS BINDING OF JAGGED ONES IS DECREASED SO FRINGE IS A MODULATOR OF NOTCH LIGAND BINDING, AND AN IMPORTANT ONE, AND IN THIS CASE WITH LUNATIC FRINGE IT’S REALLY NEEDED LUNATIC FRINGE WAS ALSO SHOWN TO AFFECT T-CELL DEVELOPMENT, IT’S EXPRESSED DURING THE EARLY PHASE OF T-CELL DEVELOPMENT IT’S TURNED OFF IN DOUBLE POSITIVE CELLS AND COMES BACK ON AGAIN IN THE SINGLE POSITIVE

CELLS SO TWO GROUPS SHOWED IF LUNATIC FRINGE IS KNOCKED DOWN FEWER DPT CELLS ARE MADE AND COMPETE POORLY WITH WILD-TYPE CELLS IN TRANSFER EXPERIMENTS IF LUNATIC FRINGE IS UPREGULATED IN THESE CELLS, SO MISEXPRESSED IN THESE CELLS, THEY COMPETE WITH THE IMMATURE CELLS AND END UP FORMING B-CELLS IN THE THYMUS SO FRINGE WAS KNOWN TO BE IMPORTANT AND THE QUESTION WAS WHAT ABOUT THE OTHER FRINGES? SO THERE ARE ACTUALLY THREE FRINGES IN THE MAMMALIAN CELLS THEY ARE NOT ALL EXPRESSED IN THE SAME CELLS, AND IF YOU KNOCK OUT MANIC FRINGE OR RADICAL FRINGE, IT DOESN’T AFFECT SKELETAL DEVELOPMENT HOWEVER, IN BOTH T-CELLS AND B CELTS, TRANSCRIPTS ARE PRESENT FOR ALL THREE FRINGES, ALBEIT AT DIFFERENT LEVELS WE DECIDED TO SEE IF WE COULD TEASE OUT FUNCTIONING FOR EACH OF THE FRINGES IN THE DIFFERENT T- AND B-CELL DEVELOPMENT SITUATION AND THE QUESTION WAS ARE THEY ALL FUNCTIONAL? ARE THEY REDUNDANT OR COMPLEMENTARY? WE WANTED TO GENERATE FRINGE TRIPLE KNOCKOUT MICE, AND WE WERE ABLE TO OBTAIN FROM SUSAN COLE FRINGE TRIPLE HEAD MICE WITH ONE COPY OF EACH OF THE FRINGES SO WHEN YOU CROSS THEM TOGETHER WITH THIS ONE, WHICH IS DOUBLE MUTANT FOR MANIC AND RADICAL BUT EXPRESSING LUNATIC, YOU COULD GENERATE MICE THAT ARE MISSING ONE OF THE FRINGES OR TWO OF THE FRINGES OR NONE AND FROM THOSE MICE THE FRINGES THAT REMAIN ARE EITHER TWO OF THEM OR ONE SINGLE FRINGE, OR NO FRINGE SO WE DECIDED TO FOCUS ON THIS GROUP, TO SEE IF THERE WAS A PHENOTYPE WHEN ALL THE FRINGES WERE GONE, AND IF — ALSO IF WE COULD RESCUE WITH THESE SINGLE FRINGE EXPRESSING MICE NOW, ACTUALLY THE — I MENTIONED LUNATIC FRINGE HAS A PHENOTYPE IT DOESN’T HAVE A PHENOTYPE IN ADULT MICE, IT WAS ONLY IF THEY LOOKED AT VERY IMMATURE FETAL CELLS THAT A PHENOTYPE COULD BE OBSERVED SO THE MASS COMPENSATES, IF YOU JUST KNOCK OUT LUNATIC FRINGE BY ITSELF AND NOTHING MUCH WAS KNOWN ABOUT RADICAL AND MANIC AND SO WE COMPARED LIGAND BINDING FOR DOUBLE NEGATIVE T-CELLS, THE IMMATURE T-CELLS IN THE TRIPLE KNOCKOUT, AND FOUND THAT BINDING OF DELTA 4 WAS IS THE LIGAND EXPRESSED ON THE STROMA IS SIGNIFICANTLY REDUCED AND HOWEVER, BINDING OF NOTCH 1 AND NOTCH 2 OR ANTIBODIES AGAINST NOTCH1 AND NOTCH2 SHOW THEY ARE NOT REDUCED, SO THIS IS NOT BECAUSE NOTCH IS NOT GETTING TO THE CELL SURFACE, BUT TO DO WITH PRESUMABLY THE SUGARS ON THESE NOTCH RECEPTORS NOTCH12 IS THE PREDOMINANT NOTCH IN T-CELL DEVELOPMENT NOTCH SIGNALING WAS REDUCED IN THE CELLS LACKING FRINGE THIS IS DELTA1, BOTH OF THESE, AND CD25 SO THIS IS A FAIRLY MINOR EFFECT, BUT IT WAS SIGNIFICANT AND THIS WAS — THIS IS A KNOWN NOTCH TARGET GENE IN THESE T-CELLS AT THAT STAGE OF DEVELOPMENT AND WHEN WE LOOK AT T-CELL SUBSETS IN THE ABSENCE OF FRINGE, WE FOUND NOT ANYTHING DRAMATIC WITH DOUBLE NEGATIVE CELLS BUT DOUBLE POSITIVE CELLS ARE REDUCED, SINGLE POSITIVE CELLS WERE INCREASED THIS IS THE OPPOSITE OF WHAT WE SAW WHEN WE KNOCKED OUT THE O-FUCOSE AND NO SIGNIFICANT DIFFERENCES IN THE OTHER CELL TYPES IN THE THYMUS NOW, IN THE SPLEEN, IT’S BEEN SHOWN THAT LUNATIC AND MANIC FRINGE COOPERATE WITH EACH OTHER, IN FACILITATING THE DIFFERENTIATION OF NEWLY FORMED B-CELLS INTO MARGAL ZONE PRECURSORS AND MARGINAL

ZONE B-CELLS, INTO FALICULARS NOBODY HAS LOOKED AT RADICAL FRINGE IN THIS SITUATION SO IN THE TRIPLE KNOCKOUT MICE, THE WEIGHT OF THE SPLEEN IS REDUCED AND NUMBER — THE FREQUENCY, NOT THE NUMBERS, THE PROPORTION OF CD4 AND CD8 T-CELLS ARE REDUCED IN THE THYMUS THEY WERE INCREASED SO WE’RE LOOKING AT THESE TO SEE IF THEIR ACTIVATION STATE IS ALTERED IN THE GENERATION, ONE CAN IDENTIFY THOSE USING THE DIFFERENT MARKERS, CD21, IGM, FOLLICULAR B-CELLS WERE SLIGHTLY INCREASED, MARGINAL ZONES WERE DECREASED THERE WE ARE WITH THE TRIPLE KNOCK OUT WITH NO DEFECT ON THE OTHER CELL TYPES, MYELOID OR B-CELLS AND NOW WE LOOKED AT THE INDIVIDUAL SINGLELY EXPRESSING FRINGE MICE, SO THIS IS THE TRIPLE HET CONTROL WE CAN SEE THAT IN THE CASES WHERE WE HAVE AN EFFECT IN THE TRIPLE KNOCKOUT, IT HAS BEEN RESCUED BY EACH OF THESE FRINGE ENZYMES BOTH FOR THE DOUBLE POSITIVE T-CELLS AND FOR THE SINGLE POSITIVE T-CELLS IN THE CASE OF THE SPLEEN, IT WAS LARGELY RESCUED BUT IN SOME CASES NOT COMPLETELY RESCUED BY THE SINGLE FRINGES FOR THE T-CELL NUMBERS, FOLLICUL NUMBERS WERE NORMAL AND RADICAL DOES NOT SEEM TO INTERACT VERY MUCH WITH NOTCH PATHWAY IN THE MARGINAL ZONE B-CELLS SO THERE ARE A NUMBER OF KEY QUESTIONS TO ADDRESS FROM THESE DATA AND ONE IS DOES NOTCH1 CARRY ONLY O-FUCOSE IN DOUBLE NEGATIVE T-CELLS LACKING FRINGE? IF THIS IS ALL NOT THERE IS THERE A SUBSTITUTE THERE? AS YOU SAW, THE DELTA4 BINDING WAS VERY LOW, HOWEVER WE DO GET A LOT OF T-CELLS SO POTENTIALLY, THERE’S ANOTHER SUGAR ADDED, OR ANOTHER ENZYME THAT CAN ADD GlcNAc, AND THAT IS SOMETHING WE REALLY WANT TO FIND OUT, WE WANT TO FIND IT OUT IN VIVO BECAUSE NOTCH PATHWAY REGULATION IS VERY SENSITIVE TO CHANGES IN EXPRESSION LEVEL SO WE HAVE THYMUSES FROM THESE MICE, AND WE’LL BE DOING MASS SPEC ANALYSIS HOPEFULLY ON NOTCH1 IN COLLABORATION WITH BOB HOLTWANGER CAN A DIFFERENT SUGAR SUBSTITUTE FOR FRINGE? THAT’S ANOTHER QUESTION IN DROSOPHILA IS THERE ANOTHER GlcNAc TRANSFERASE? ALL THESE PUTATIVE SITES, ARE THEY ACTUALLY OCCUPIED AND DOES LOSS OF ONE SUGAR TYPE AFFECT THE EXPRESSION OF ONE OF THE OTHER SUGARS? WE DON’T KNOW AND THEN WHICH NOTCH 1 SITES ARE MODIFIED IN DOUBLE NEGATIVE T-CELLS EXPRESSING ONLY LUNATIC, ONLY MANIC OR ONLY RADICAL? CAN THEY REALLY SUBSTITUTE TOTALLY FOR EACH OTHER OR IS THERE A SUBSET OF SITES NEEDED FOR T-CELL DEVELOPMENT? AND FINALLY, WE HOPE TO TEASE OUT ROLES FOR EACH OF THEM USING THE SINGLE FRINGE MICE IN CO-CULTURE ASSAYS OF T-CELL DEVELOPMENT WHERE YOU CAN USE EACH LIGAND SEPARATELY AND LOOK AT HOW FAR CELLS PROGRESS DURING DEVELOPMENT SO TO SUMMARIZE THEN THE

O-FUCOSE GLYCANS ARE NOTCH SIGNALING, WHAT WE KNOW IS IF WE MISS ONE O-FUCOSE GLYCAN HERE, WE AFFECT T-CELL DEVELOPMENT THERE ARE SIX POTENTIAL ONES THAT COULD MODIFY IT AND MY LAB LOOKED AT GALT1, THEY COULD BE MUCH MORE IMPORTANT THAN THAT THEN THE KNOCKOUT OF ALL FRINGES WOULD GIVE YOU THEORETICALLY THIS, AND WE KNOW THAT LUNATIC IS SKELETAL DEFECTS AND EACH CONTRIBUTE TO T- AND B-CELL DEVELOPMENT AND IF YOU KNOCK OUT POFUT1 YOU END UP WITH NONE OF THESE SUGARS THIS IS A GLOBAL NOTCH SIGNALING BLOCK SO WHICH SIDES ARE MODIFIED AND HOW IN VIVO? A KEY QUESTION AND WHAT ARE ALL THE ESSENTIAL PLAYERS THAT ARE REQUIRED TO GENERATE THESE? BECAUSE EACH OF THESE ENZYMES NEEDS TO BE — NEEDS TO GET ITS SUBSTRATE GDP FUCOSE, THERE ARE TRANSPORTERS AND SO ON, AND OTHER ACTIVITIES THAT CONTRIBUTE TO THE FINAL PRODUCT, WHICH WE ARE ALSO TRYING TO IDENTIFY NOW, THE LAST GLYCAN, THE MOST RECENT GLYCAN ON NOTCH WAS DISCOVERED AND IS AN O-GlcNAc, AND YOU PROBABLY ALL HEARD OF O-GlcNAc, ESPECIALLY HERE, BUT THE O-GlcNAc HA IS WORKED ON MOSTLY AT NIH IS AN O-GlcNAc IN THE CYTOPLASM, THIS IS ADDED WITH A REPEAT THAT HAS TO BE FOLDED PROPERLY IF YOU DENATURE THE EGF REPEATS NONE OF THESE ENZYMES WORK WELL AT ALL SO WE DECIDED TO LOOK AT THIS IN THE FLY BECAUSE THERE ARE MANY TRANSCRIPTS IN THE MOUSE, AND LOOKED AT KNOCKDOWN IN THE DROSOPHILA WING, USING GAL 4 PROMOTER IF WE KNOCKED DOWN THE ENZYME THAT PUTS ON THIS O GlcNAc TRANSFERASE, WE GET WING BLISTERS AND WE SHOWED THAT IF YOU GENETICALLY EXAMINE INTERACTIONS BETWEEN THIS KNOCKDOWN AND NOTCH PATHWAY MUTATIONS, THAT THEY DO INTERACT AND NOTCH MUTATIONS SUPPRESS BLISTERS AND INTERESTINGLY, JUST LAST — TWO YEARS AGO, A HUMAN WHICH HAD COMPLETELY LOST EOGT ACTIVITY WAS REPORTED, AND THE SYNDROME WAS TERMED ADAMS-OLIVER SYNDROME BECAUSE IT’S SIMILAR TO OTHER GENES THAT GIVE RISE TO ADAMS-OLIVER SYNDROME, THE PHENOTYPE IN THIS PERSON, SO ALTERED DIGITS AND CALCIUM DEPOSITS IN THE BRAIN IN THIS PARTICULAR CASE, BUT THERE ARE EFFECTS IN THE SKIN AND SO ON SO THEY ARE QUITE SUGGESTIVE OF NOTCH SIGNALING AND WE’RE INVESTIGATING A MOUSE THAT WILL HOPEFULLY ALLOWS US TO IDENTIFY WHAT THIS EOGT ENZYME DOES SO IN FACT, CONGENITAL DISEASES FROM REDUCE O GLYCANS HAVE BEEN POSSIBLED QUITE A BIT IN THE LAST YEAR OR SO, SO AS WELL AS BEFORE, SO FOR O-FUCOSE THERE ARE AUTOSOMAL DOMINANT MUTATIONS PUBLISHED IN 2013, AND THAT GIVES RISE TO DOWLING-DEGOS DISEASE, A KNOWN SYNDROME BUT THIS IS A NEW CAUSE OF IT I MENTIONED THE MOUSE SITUATION WITH SKELETAL PROBLEMS, THERE’S ALSO HUMAN LUNATIC FRINGE MUTATION GIVING RISE TO SPONDYLOCOSTAL DYSTOSIS AND THIS CAN ADD GLUCOSE AND XYLOSE, EITHER/OR IT GIVES RISE TO THE SAME SORT OF SYNDROME AS DOWLING-DEGOS DISEASE, PUBLISHED LAST YEAR, AND THEN THE ADAMS-OLIVER SYNDROME FOR LOSS OF THIS PARTICULAR O GlcNAc SO IT WOULD — IT’S PRETTY CLEAR

THAT NOTCH NEEDS SUGARS ON ITS EXTRACELLULAR DOMAIN, FOR VARIOUS THINGS INCLUDING LIGAND BINDING, AND IN OTHER CASES FOR THE STABILITY OF NOTCH AT THE CELL SURFACE, AND WE ARE — THOSE OF US WHO ARE INTERESTED IN THIS ARE REALLY TRYING TO NAIL DOWN MECHANISMS BY WHICH THIS ALL WORKS AND BE ABLE TO DEFINE IT IN THE MOLECULAR LEVEL WHAT’S HAPPENING THERE IS VERY NICE CRYSTAL STRUCTURE BY CHRIS GARCIA OF DELTA4 AND NOTCH1 DOMAINS, SHOWING THAT THE O-FUCOSE IS RIGHT IN THERE, AND NEEDS — AND COORDINATES WITH AMINO ACIDS, AND ACTUALLY HE CONCLUDES THAT IT’S LIKE THE 21st AMINO ACID IN THAT SITUATION, BECAUSE IT’S ALLOWING THE INTERACTION WITH DELTA4 TO BE MUCH BETTER THAN IF IT’S NOT THERE SO NOW I WOULD JUST LIKE TO CLOSE BY SAYING THAT IT ALL BEGAN WITH A SIMPLE QUESTION, AND MY QUESTION WAS, IS LECTIN RESISTANCE STABLE AND HERITABLE? NOTHING TO DO WITH GLYCANS AND IT STARTED WHEN I PICKED A COLONY AND PUT IT IN A TUBE AND THEN CHARACTERIZED EVERYTHING THAT CAME OUT OF THAT COLONY, WHICH WERE MANY, MANY, MANY THINGS THAT WERE CERTAINLY NOT ON ANYBODY’S MINDS, ESPECIALLY MINE, WHEN I PICKED THE COLONY SO I THINK JUST BASIC SCIENCE UNFORESEEN REWARDS IS HOW I WOULD LIKE TO CONCLUDE AND THANK ALL THE PEOPLE WHO HAVE CONTRIBUTED TO THIS WORK THESE ARE RECENT PEOPLE FROM THE LAB, WHO CONTRIBUTED TO MOST OF THE WORK THAT I MENTIONED TODAY, AND MY FUNDING FROM NIH, BOTH NCI AND NIGMS, FOR MANY, MANY YEARS, AND SO I THANK THEM BECAUSE WITHOUT THEM I WOULDN’T HAVE DONE ANY OF THIS, FROM EINSTEIN AND VARIOUS FOUNDATIONS AND MANY COLLABORATORS OVER THE YEARS, SOME AT EINSTEIN, MANY ELSEWHERE, AND OTHERS IN THE LAB, EACH OF THEM KNOWN FOR THE MUTANTS THAT THEY CHARACTERIZE, AND NOW THERE ARE PEOPLE CARRYING ON WITH NOTCH AND FRINGES AND MGAT 1 AND THAT INHIBITOR I THANK THEM ALL AND THANK YOU FOR YOUR ATTENTION [APPLAUSE] >> THANK YOU SO WE HAVE TIME FOR A FEW QUESTIONS SO THIS IS A SPECIAL TIME AT NIH WE RARELY SERVE COOKIES, BUT FOR THIS LECTURE SHOW, AFTERWARDS THERE’S COOKIES AND BEVERAGES I GUESS OUT THERE THROUGH THAT DOOR, NEAR THE LIBRARY SO THIS IS THE ONLY THING STANDING BETWEEN THE COOKIES >> THE CARBOHYDRATE PEOPLE, YOU KNOW, WERE UNSTOPPABLE PAMELA, VERY NICE A QUICK QUESTION ABOUT THE SPACING YOU KNOW, THE EVOLUTION OF THE EGF REPEATS, DO WE KNOW HOW CONSERVED SPACING IS? >> SOME SITES ARE VERY CONSERVED, LIKE EGF 12 IS HIGHLY CONSERVED, SOME THEM OF THEM ARE NOT SO CONSERVED SO OFF THE TOP OF MY HEAD, I DON’T KNOW I MEAN, PROBABLY ABOUT SIX OR SO OF THE O-FUCOSE THERE COULD BE SITES WHICH DON’T USE EXACTLY THAT CONSENSUS, OR SOME OF THOSE MAY NOT BE USED EVER THAT’S WHY WE REALLY WANT TO DO IT IN VIVO, FROM THE THYMUS, NOTCH1 IN MICE, THAT HAVE NEVER BEEN PERTURBED, EXCEPT FOR HAVING A MUTATION >> IF NO ONE ELSE HAS A QUESTION, A RELATED QUESTION — SORRY, DO YOU THINK BASED ON THE SPERMATOGENES THERE WILL BE

UNIQUE RESULTS FOR THE STEM CELL NICHES? >> OH, I DON’T KNOW I THINK THIS — I DOUBT IT BUT THIS THING WE WERE LOOKING AT IT BECAUSE IT WAS LIKE A TRANSFERASE AND TURNED OUT TO INHIBIT, SO THIS IS AN ARTIFICIAL SITUATION WE’VE OVEREXPRESSED IT IN CELLS BUT THEN YOU LOOK AT WHERE IT’S EXPRESSED, AND IT’S VERY SPECIFIC SORT OF SITE OF EXPRESSION IN THE TESTIS, SO WHATEVER IT IS WE WOULD LIKE TO GET TO THE BOTTOM OF IT, BUT WHEN WHEREVER CELL WE PUT IT IN, EITHER CO-TRANSSECTING WITH MGAT 1 SO THEY ARE EQUALLY EXPRESSED, OR DIFFERENT CELLS THAT INHIBIT MGAT 1 >> FOR SUCH A COMPLEX CODE AND SYSTEM WHAT IS KNOWN ABOUT THE SYNTHESIS AND ADDITION OF THESE DIFFERENT SUGAR MOIETIES IN SPECIFIC THE EDITING BECAUSE IT APPEARS A SIMPLE MISTAKE IN ONE REPEAT COULD HAVE DETRIMENTAL EFFECTS >> YEAH, WELL, THE SYNTHESIS, THE TRANSFERRATIONS ARE ALL KNOWN PRETTY MUCH >> FOR NOTCH AND HOW THEY ARE ADDED, SORRY >> SORRY, WHAT? >> I MEANT TO SAY HOW THESE ARE ADDED TO THE NOTCH, EGF >> FROM GLYCOTRANSFERRATION, THE O-FUCOSE IS ADDED BY GDF FUCOSE, BOTH HAVE TO BE THERE, THAT’S THE SUBSTRATE, SO IF YOU — THERE’S AN ENZYME POQT 1, IN THE FLY THAT COULD LARGELY RESCUE ASPECTS OF IT, ALTHOUGH THERE’S A MORE RECENT PAPER THAT SAYS THERE ARE STILL PROBLEMS, IN OUR SITUATION IN MAMMALS WE COULD SORT OF RESCUE A LITTLE BIT, NOT VERY MUCH, WITH AN ENZYME, AND IF WE DID AN IRRELEVANT ENZYME, OVEREXPRESSED IT, WE GOT THE SAME AMOUNT OF RESCUE IT WAS SO OVEREXPRESSING, WAS NOT GOOD SO IF YOU LOOK IN MICE, WHERE POQT 1 IS KNOCKED OUT IN BONE MARROW, IN HEMOPOIETIC CELLS, THERE’S A LITTLE BIT OF DECREASE IN NOTCH SIGNALING, SO THE ENZYME ITSELF DOES HELP NOTCH GET TO THE SURFACE BUT SO FOR EACH OF THOSE ENZYMES, THAT LAST SLIDE WHICH IS HUMAN DISEASES, THAT’S WHAT’S KNOWN SOMETHING ELSE THAT YOU WERE ASKING? NO? >> ONE FINAL QUESTION >> DOES THE FUCOSE, SAME SITE OR DIFFERENT SITES, IS ANYTHING KNOWN? >> WELL, THAT’S THE QUESTION AND THAT’S WHAT WE WANT TO ADDRESS WITH THE SINGLE FRINGE MICE, AND THE TRIPLE KNOCKOUT MICE BUT IF YOU LOOK AT THEM, LIKE BOB HALTERWANGER HAS DONE EXPERIMENTED REPORTED AT MEETINGS WHERE OVEREXPRESSING EACH FRINGE BY ITSELF WILL GIVE SOME VARIATION SO THEY MAY NOT EQUALLY DO EVERY SINGLE SITE >> SELECTIVE FOR SITES, EACH ONE OF THEM >> AGAIN, YOU KNOW, THIS IS OFTEN WITH PIECES OF NOTCH AND SO ON, OVEREXPRESSION OF THE ENZYMES I THINK IT’S IMPORTANT TO DO IT UNDER PHYSIOLOGICAL CONDITIONS >> WELL, THANK YOU IF YOU COULD JOIN ME IN THANKING OUR SPEAKER [APPLAUSE]