A lysosomal cysteine proteinase from Dictyostelium discoideum contains N-acetylglucosamine-1-phosphate bound to serine but not mannose-6-phosphate on N-linked oligosaccharides.
Previous studies showed that vegetative Dictyostelium discoideum cells make a lysosomal proteinase, proteinase-1, that contains multiple GlcNAc-alpha-1-P residues in phosphodiester linkage to serine. We extended these studies and, in contrast to earlier reports, found that proteinase-1 contains 7.5 mol of Fuc, 8 mol of Man, 2 mol of Xyl, and 30 mol of GlcNAc per calculated mol of protein but no Man-6-P residues. The protein binds to concanavalin A and wheat germ agglutinin lectin affinity columns, and PNGase-F digestion released most of the mannose and xylose but little of the GlcNAc. beta-Elimination under reducing conditions released only GlcNAc-alpha-1-P. There was no evidence for the release of disaccharides or of fucitol. A rabbit antiserum and monoclonal antibodies prepared against proteinase-1 recognize GlcNAc-alpha-1-P residues in immunoblots and are specifically competed by UDP-GlcNAc or GlcNAc-alpha-1-P. Use of other monoclonal antibodies showed the presence of mannose-6-sulfate on N-linked sugar chains, and alpha-fucose residues on the protein. Thus, proteinase-1 has at least two types of modifications: Glc NAc-alpha-1-P-Ser, which we call phosphoglycosylation, and N-linked oligosaccharides. This is the first purified lysosomal enzyme in Dictyostelium that does not contain Man-6-P residues. The GlcNAc-alpha-1-P-specific antibodies also recognize a group of developmentally regulated proteins, especially enriched in vegetative cells. Some of them are also lysosomal cysteine proteinases, and all bind to the GlcNAc-alpha-1-P-specific monoclonal antibody but not to the mammalian CI-Man-6-P receptor. Conversely, lysosomal enzymes that have Man-6-P do not bind to the GlcNAc-alpha-1-P-specific antibody. An exception to this is beta-N-acetylglucosaminidase, where 15% of the activity binds to this antibody. Thus, there appear to be two sets of lysosomal enzymes with distinct post-translational modifications.
Mehta
D P
DP
La Jolla Cancer Research Foundation, California 92037, USA.
Ichikawa
M
M
Salimath
P V
PV
Etchison
J R
JR
Haak
R
R
Manzi
A
A
Freeze
H H
HH
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003429:Cross Reactions; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000378:metabolism*; D004023:Dictyostelium / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008360:Mannosephosphates; D009844:Oligosaccharides; C027693:mannose-6-phosphate; D012694:Serine; C026146:N-acetylglucosamine-1-phosphate; D003546:Cysteine Endopeptidases; D000117:Acetylglucosamine