Functional Analysis of Proteoglycan Galactosyltransferase II RNA Interference Mutant Flies

Heparan sulfate proteoglycan plays an important role in developmental processes by modulating the distribution and stability of the morphogens Wingless, Hedgehog, and Decapentaplegic. Heparan and chondroitin sulfates share a common linkage tetrasaccharide structure, GlcAβ1,3Galβ1,3Galβ1,4Xylβ-O-Ser. In the present study, we identified Drosophila proteoglycan galactosyltransferase II (dβ3GalTII), determined its substrate specificity, and performed its functional analysis by using RNA interference (RNAi) mutant flies. The enzyme transferred a galactose to Galβ1,4Xyl-pMph, confirming that it is the Drosophila ortholog of human proteoglycan galactosyltransferase II. Real-time PCR analyses revealed that dβ3GalTII is expressed in various tissues and throughout development. The dβ3GalTII RNAi mutant flies showed decreased amounts of heparan sulfate proteoglycans. A genetic interaction of dβ3GalTII with Drosophila β1,4-galactoslyltransferase 7 (dβ4GalT7) or with six genes that encode enzymes contributing to the synthesis of glycosaminoglycans indicated that dβ3GalTII is involved in heparan sulfate synthesis for wing and eye development. Moreover, dβ3GalTII knock-down caused a decrease in extracellular Wingless in the wing imaginal disc of the third instar larvae. These results demonstrated that dβ3GalTII contributes to heparan sulfate proteoglycan synthesis in vitro and in vivo and also modulates Wingless distribution.