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Contributions of chaperone andContributions of chaperone and

glycosyltransferaseglycosyltransferase activities ofactivities ofOO--fucosyltransferase 1 to Notchfucosyltransferase 1 to Notchsignalingsignaling

Contributions of chaperone andContributions of chaperone and

glycosyltransferaseglycosyltransferase activities ofactivities ofOO--fucosyltransferase 1 to Notchfucosyltransferase 1 to Notchsignalingsignaling

Okajima, T.Okajima, T. et alet al ;; BMC BiologyBMC Biology 2008,2008, 66:1:1

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Notch signaling pathwayNotch signaling pathway

It is a highly conserved cell signaling systemIt is a highly conserved cell signaling systempresent in most multicellular organismspresent in most multicellular organisms

Vertebrates possess four different notchVertebrates possess four different notchreceptors, referred to as Notch1 to Notch4receptors, referred to as Notch1 to Notch4

The Notch receptor is a singleThe Notch receptor is a single--pass transpass trans--membrane receptor proteinmembrane receptor proteinLigand proteins binding to the extraLigand proteins binding to the extra--cellularcellulardomain induce proteolytic cleavage anddomain induce proteolytic cleavage andrelease of the intracellular domain, whichrelease of the intracellular domain, which

enters the cell nucleus to alter geneenters the cell nucleus to alter geneexpressionexpression

InIn Drosophila melanogasterDrosophila melanogaster(the fruit fly)(the fruit fly)there are two ligands named Delta andthere are two ligands named Delta andSerrateSerrate

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Notch signaling pathway

The Notch signaling pathway is importantfor cell-cell communication. It involves gene regulation mechanisms

that control multiple cell differentiation

processes during embryonic and adult life Notch activity needs to be regulatedprecisely

Aberrant Notch activity is associated with

a number of human diseases includingcancers and congenital syndromes.

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The Role of Glucosyltransferases inThe Role of Glucosyltransferases in

Notch signalingNotch signaling

Notch signaling is influenced by two conservedNotch signaling is influenced by two conservedglycosyltransferases,glycosyltransferases, OO--fucosyltransferase1fucosyltransferase1(OFUT1) and Fringe (FNG)(OFUT1) and Fringe (FNG)

OFUT1 transfers fucose from GDPOFUT1 transfers fucose from GDP--fucose ontofucose ontoEGF domains (substrate forFNG)EGF domains (substrate forFNG)

OFUT1 also possesses a chaperone activity thatOFUT1 also possesses a chaperone activity thatpromotes the folding and secretion of Notchpromotes the folding and secretion of Notch

FNG transfersFNG transfers NN--acetylglucosamine (GlcNAc) ontoacetylglucosamine (GlcNAc) ontoOO--linked fucose on EGF domainslinked fucose on EGF domains

FNG modulates Notch signaling during theFNG modulates Notch signaling during thedevelopment of thedevelopment of the DrosophilaDrosophila wingwing

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The fact:

OFUT1 possesses both fucosyltransferaseand chaperone activity for Notch

Question:What will bethe respectivecontributions of thesetwo activitiestothe genetic requirement forOFUT1 in Notch signaling?

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OO--fucosylation ofNotch is not required duringfucosylation ofNotch is not required during

embryonic neurogenesisembryonic neurogenesis

Two complementary approachesTwo complementary approaches

1) Use of a mutant isoform ofOFUT1,1) Use of a mutant isoform ofOFUT1,

OFUT1R245A. This mutation alters an invariantOFUT1R245A. This mutation alters an invariant

arginine within the putative GDP binding site,arginine within the putative GDP binding site,

and it eliminates detectable fucosyltransferaseand it eliminates detectable fucosyltransferase

activity, while retaining chaperone activityactivity, while retaining chaperone activity

2) Use of a second mutant in which wild2) Use of a second mutant in which wild--typetypeOFUT1 is present but unable to fucosylate NotchOFUT1 is present but unable to fucosylate Notch

owing to the absence of its donor substrate,owing to the absence of its donor substrate,

GDPGDP--fucosefucose

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Immunostaining It is the demonstration of a tissueor

cellular constituent (protein) in situ by

detecting specific antibody-antigeninteractions wherethe antibody has beentagged with a visiblelabel.

The visual marker may be a fluorescent

dye, colloidal metal, hapten, radioactivemarker or an enzyme .

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Conclusion

O-fucose modification on Notch receptorsis not absolutely required for theiractivity in fringe-independentdevelopmental processes inDrosophila

successful folding mediated by chaperoneactivityof OFUT1 is sufficienttogenerate functional Notch receptors