UDP-GLCNAC-SER-PROTEIN N-ACETYLGLUCOSAMINE-1-PHOSPHOTRANSFERASE FROM DICTYOSTELIUM-DISCOIDEUM RECOGNIZES SERINE-CONTAINING PEPTIDES AND EUKARYOTIC CYSTEINE PROTEINASES

Phosphoglycosylation catalyzed by UDP-GlcNAc:Ser-protein N-acetylgluco samine-1-phosphotransferase (Ser:GlcNAc phosphotransferase) adds GlcNA c alpha-1-P to peptidyl-Ser of selected Dictyostelium discoideum prote ins, Lysosomal cysteine proteinase (CP), proteinase-1(CP7), is the maj or phosphoglycosylated protein in bacterially grown amoebae. GlcNAc-1- P is added within a Ser-rich domain containing SSS, SGSG, or SGSQ repe ated motifs that are not found in other papain-like CPs, We studied th e substrate specificity. of the transferase using peptides containing these motifs and 12 other peptides with one or more Ser residues, Phos phoglycosylation is comparable for all three Dictyostelium CP motifs, but it is not restricted to them, Flanking residues in the other pepti des strongly influence phosphoglyco, sylation efficiency, Dictyosteliu m microsomal membranes also phosphoglycosylate endogenous accepters, a nd some of these accepters occur as an 18 S complex with the transfera se, CP-serine motif peptides inhibit endogenous acceptor phosphoglycos ylation weakly (30-40%) at 800 mu M, whereas catalytically inactive pr oteinase-1(CP7) and other non-phosphoglycosylated eukaryotic CPs, lack ing the serine domain, inhibit transferase activity at 1-4 mu M. SDS d enaturation destroys the inhibitory potential of all CPs showing that transferase recognizes a conformation-dependent feature that is shared by all, Proteinase-1(CP7) expressed in Escherichia coli lacks GlcNAc- 1-P, but it is a substrate for Ser:GlcNAc phosphotransferase, K-m = 5. 6 mu M. Thus, Ser: GlcNAc phosphotransferase recognizes both acceptor peptide sequences and a conformational feature of eukaryotic CPs, This may be physiologically important for establishing or maintaining non- overlapping groups of GlcNAc-1-P- and Man-6-P-modified Dictyostelium p roteins that reside in functionally distinct endo-lysosomal vesicles.