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-acetylglucosamine-1-phosphotransferase (Ser:GlcNAc phosphotransferase) adds GlcNAcalpha-1-P to peptidyl-Ser of selected Dictyostelium discoideum proteins. Lysosomal cysteine proteinase (CP), proteinase-1(CP7), is the major phosphoglycosylated protein in bacterially grown amoebae. GlcNAc-1-P is added within a Ser-rich domain containing SSS, SGSG, or SGSQ repeated motifs that are not found in other papain-like CPs. We studied the substrate specificity of the transferase using peptides containing these motifs and 12 other peptides with one or more Ser residues. Phosphoglycosylation is comparable for all three Dictyostelium CP motifs, but it is not restricted to them. Flanking residues in the other peptides strongly influence phosphoglycosylation efficiency. Dictyostelium microsomal membranes also phosphoglycosylate endogenous acceptors, and some of these acceptors occur as an 18 S complex with the transferase. CP-serine motif peptides inhibit endogenous acceptor phosphoglycosylation weakly (30-40%) at 800 microM, whereas catalytically inactive proteinase-1(CP7) and other non-phosphoglycosylated eukaryotic CPs, lacking the serine domain, inhibit transferase activity at 1-4 microM. SDS denaturation 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, Km = 5.6 microM. 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 proteins that reside in functionally distinct endo-lysosomal vesicles.