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

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Title A lysosomal cysteine proteinase from dictyostelium discoideum contains N-acetylglucosamine-1-phosphate bound to serine but not mannose-6-phosphate on N-linked oligosaccharides.
 
Creator Darshini Mehta, P.
Ichikawa, M.
Salimath, P. V.
Etchison, J. R.
Haak, R.
Manzi, A.
Freeze, H. H.
 
Subject 16 Enzyme Chemistry
 
Description 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.
 
Date 1996
 
Type Article
NonPeerReviewed
 
Format application/pdf
 
Language en
 
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Identifier http://ir.cftri.com/2470/1/THE%20JOURNAL%20OF%20BIOLOGICAL%20CHEMISTRY%20Vol.%20271%2C%20No.%2018%2C%20Issue%20of%20May%203%2C%20pp.%2010897%E2%80%9310903%2C%201996.pdf
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.