Glycobiology, Vol 9, 415-422, Copyright © 1999 by Oxford University Press
DK Sharma, TK Smith, CT Weller, A Crossman, JS Brimacombe and MA Ferguson
De-N-acetylation of N-acetylglucosaminyl-phosphatidylino-sitol (GlcNAc- PI)
is the second step of glycosylphosphatidylino-sitol (GPI) membrane anchor
biosynthesis in eukaryotes. This step is a prerequisite for the subsequent
processing of glucosaminyl-phosphatidylinositol (GlcN-PI) that leads to
mature GPI membrane anchor precursors, which are transferred to certain
proteins in the endoplasmic reticulum. In this article, we used a direct
de-N-acetylase assay, based on the release of [14C]acetate from synthetic
GlcN[14C]Ac-PI and analogues thereof, and an indirect assay, based on the
mannosylation of GlcNAc-PI analogues, to study the substrate specificities
of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and
human (HeLa) cells. The HeLa enzyme was found to be more fastidious than
the trypanosomal enzyme such that, unlike the trypanosomal enzyme, it was
unable to act on a GlcNAc-PI analogue containing 2-O-octyl-d- myo -inositol
or on the GlcNAc-PI diastereoisomer containing l- myo -inositol
(GlcNAc-P(l)I). These results suggest thatselective inhibition of the
trypanosomal de-N- acetylase may be possible and that this enzyme should be
considered as a possible therapeutic target. The lack of strict
stereospecificity of the trypanosomal de-N-acetylase for the d- myo
-inositol component was also seen for the trypanosomal GPI
alpha-manno-syltransferases when GlcNAc-P(l)I was added to the trypanosome
cell-free system, but not when GlcN-P(l)I was used. In an attempt to
rationalize these data, we modeled the structure and dynamics of
d-GlcNAcalpha1-6d- myo -inositol- 1-HPO4-( sn )-3-glycerol and its
diastereoisomer d-GlcNAcalpha1-6l- myo -inositol-1-HPO4-( sn )-3-glycerol.
These studies indicate that the latter compound visits two energy minima,
one of which resembles the low-energy conformer of former compound. Thus,
it is conceivable that the trypanosomal de-N-acetylase acts on GlcNAc-P(l)I
when it occupies a GlcNAc-PI-likeconformation and that GlcN-P(l)I emerging
from the de-N- acetylase may be channeled to the alpha-mannosyltransferases
in this conformation.
ORIGINAL ARTICLES
Differences between the trypanosomal and human GlcNAc-PI de-N- acetylases of glycosylphosphatidylinositol membrane anchor biosynthesis
Division of Molecular Parasitology & Biological Chemistry, Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland.
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