Biological significance of N-acetylglucosamine 6-sulfation

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Three species of N-acetylglucosamine-6-sulfotransferases (GlcNAc6ST), namely GlcNAc6ST-1, GlcNAc6ST-2, GlcNAc6ST-3 were expressed and the specificities were compared. GlcNAc6ST-l as well as GlcNAc6ST-2 acted on mucin-type core 2 sugar (GlcNAcβ1-6[Galβ1-3]GalNAc) and there were not significant differences between the two enzymes. Although GlcNAc6ST-2 is known to be involved in lymphocyte homing, it is not clarified whether GlcNAc6ST-1 plays such a role. Cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII induced roling of L-selectin-expressing cells. GlcNAc6ST-3 acted only on core 2 sugars. On the other hand, only GlcNAc6ST-2 acted on core 3 sugars (GlcNAcβ1-3 GalNAc). From this specificity, GlcNAc6ST-2 was considered to be the enzyme, which became newly expressed in mucinous adenocarcinoma, and this point was confined by RT-PCR analysis.We produced knockout mice deficient in the GlcNAc6ST-1 gene. That this mice completely lacked GlcNAc6ST-1 was confirmed by Southern and Northern blot analyses. The mice was born normally, and showed no apparent abnormality. Currently backcross is on the way, so that we are able to analyze possible abnormality in lymphocyte homing.GilcNAc6ST-1 was produced as fasion protein in Tn5 cells using the Baculovirus system. The purified recombinant enzyme was useful for synthesis of sulfated oligosaccharides and sulfation of glycoprotein-bound carbohydrates.