A lysosomal cysteine proteinase from dictyostelium discoideum contains N-acetylglucosamine-1-phosphate bound to serine but not mannose-6-phosphate on N-linked oligosaccharides.

Darshini Mehta, P. and Ichikawa, M. and Salimath, P. V. and Etchison, J. R. and Haak, R. and Manzi, A. and Freeze, H. H. (1996) A lysosomal cysteine proteinase from dictyostelium discoideum contains N-acetylglucosamine-1-phosphate bound to serine but not mannose-6-phosphate on N-linked oligosaccharides. Journal of Biological Chemistry, 271 (18). pp. 10897-10903.

PDF THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 18, Issue of May 3, pp. 10897–10903, 1996.pdf - Published Version Restricted to Registered users only Download (339kB)

Abstract

Previous studies showed that vegetative Dictyostelium discoideum cells make a lysosomal proteinase, proteinase- 1, that contains multiple GlcNAc-a-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. b-Elimination under reducing conditions released only GlcNAc-a- 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-a-1-P residues in immunoblots and are specifically competed by UDP-GlcNAc or GlcNAc-a-1-P. Use of other monoclonal antibodies showed the presence of mannose-6-sulfate on N-linked sugar chains, and a-fucose residues on the protein. Thus, proteinase-1 has at least two types of modifications: GlcNAc-a-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-a-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 GlcNAca- 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 GlcNAca- 1-P-specific antibody. An exception to this is b-Nacetylglucosaminidase, where 15% of the activity binds to this antibody. Thus, there appear to be two sets of lysosomal enzymes with distinct post-translational modifications.

Item Type:	Article
Uncontrolled Keywords:	lysosomal proteinase, Dictyostelium discoideum, N-linked oligosaccharides
Subjects:	500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
Divisions:	Dept. of Biochemistry
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	09 Mar 2012 09:39
Last Modified:	09 Mar 2012 09:39
URI:	http://ir.cftri.res.in/id/eprint/2470

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