Phosphorylation of arylsulphatase A occurs through multiple interactions with the UDP-N-acetylglucosamine-1-phosphotransferase proximal and distal toits retrieval site by the KDEL receptor

Phosphorylation of oligosaccharides of the lysosomal enzyme arylsulphatase A (ASA), which accumulate in the secretions of cells that mis-sort most of the newly synthesized lysosomal enzymes due to a deficiency of mannose 6-ph osphate receptors, was found to be site specific. ASA residing within the s ecretory route of these cells contains about one third of the incorporated [2-H-3]mannose in phosphorylated oligosaccharides. Oligosaccharides carryin g two phosphate groups are almost 2-fold less frequent than those with one phosphate group and only a few of the phosphate groups are uncovered. Addit ion of a KDEL (Lys-Asp-Glu-Leu) retention signal prolongs the residence tim e of ASA within the secretory route 6-fold, but does not result in more eff icient phosphorylation. In contrast, more than 90% of the [2-H-3]mannose in corporated into secreted ASA (with or without a KDEL retention signal) is p resent in phosphorylated oligosaccharides. Those with two phosphate groups are almost twice as frequent as those with one phosphate group and most of the phosphate groups are uncovered. Thus, ASA receives N-acetylglucosamine 1-phosphate groups in a sequential manner at two or more sites located with in the secretory route proximal and distal to the site where ASA is retriev ed by the KDEL receptor, i.e. proximal to the trans-Golgi. At each of these sites up to two N-acetylglucosamine 1-phosphate groups can be added to a s ingle oligosaccharide. Of several drugs known to inhibit transit of ASA thr ough the secretory route only the ionophore monensin had a major inhibitory effect on phosphorylation, uncovering and sialylation.