Chemistry Reference
In-Depth Information
specifi city which generates the
O
-glycan structures. An example for some of the
O
-GalNAc glycan pathways is shown in Figure 7.1. In addition, further modifi ca-
tion of some sugars occurs generating new epitopes. The most important of these
events are sulfation of Gal, GlcNAc and GalNAc and
O
- acetylation of sialic acids
(see Info Box 1).
O
-
-
N
-Acetylglucosamine monosaccharide metabolism on proteins is governed
by GlcNAc modifying enzymes. A specifi c
β
-
O
- GlcNAc transferase ( OGT ) catalyses
the transfer of GlcNAc to the serine and threonine residues on protein acceptors.
The transferase has been isolated from liver and characterized in detail. It is a
heterotrimer of two 110-kDa and one 78-kDa subunits. Some tissue variation of
subunit utilization has been found with the 110-kDa unit present in all cases and
differential expression of the 78-kDa subunit. The larger 110-kDa unit has two
domains linked by a nuclear localization peptide. The C-terminal domain is the
catalytic center of the enzyme while the N-terminus comprises multiple tetratri-
copeptide (TPR) repeats. The TPR domains determine the recognition of protein
substrates. The enzyme is itself a carrier of
β
-
O
- GlcNAc, although the individual
sites are not yet known. It shows several
K
m
values for the donor substrate UDP-
GlcNAc. On the one hand, the enzyme could not be saturated by UDP-GlcNAc at
maximal physiological concentrations while a low apparent
K
m
in the range 500nM
favors OGT utilization of this substrate over the UDP-GlcNAc transporters in the
ER and Golgi which deplete cytosolic levels of the nucleotide sugar. Accordingly,
the sensitivity to UDP-GlcNAc levels implicates a sensor role for OGT and a direct
link with regulation of the many regulation pathways through the action of the
β
-
O
-GlcNAc- proteins associated with this control. Interaction of OGT with the huge
number of protein substrates is mediated through specifi c proteins which bind to
the TPR domains and target OGT to defi ned complexes. OGT knockout mice have
been generated and exhibit embryonic lethality, underlining the need for OGT in
normal cell growth and response to extracellular signals [5] .
The removal of
β
β
-
O
-GlcNAc from proteins is due to the action of a
β
- hexosa-
minidase. This enzyme was show to be specifi c for
β
-
O
- GlcNAc. Protein structure
analysis revealed two catalytic domains, one for the
- hexosaminidase activity and
a second for acetyltransferase, which was also confi rmed enzymatically. A caspase- 3
cleavage site (caspases are a family of proteinases which play an important part in
apoptosis, see Figure 27.2) was reported between these two catalytic domains, but
an explanation for the interaction of these two activities remains speculative.
O
-Man transfer to serine and threonine acceptor sites on proteins is catalyzed
by a family of POMTs. These enzymes are found in the ER and transfer mannose
from a dolicholphosphate mannose donor to the protein acceptor. The isoforms
of POMT vary in their precise substrate specifi cities and appear to be analogous
to the family of ppGaNTases involved in mucin-type glycan biosynthesis. The main
transferases identifi ed in human tissues are POMT1 and 2, and most studies have
focused on the properties and action of these isoforms.
Extension of the
O
-Man glycan occurs through the action of a specifi c transfer-
ase, protein
O
- mannose
N
- acetylglucosaminyl transferase ( POMGnT1 ), located in
the Golgi apparatus, leading to the formation of a Man
β
β
1,2GlcNAc linkage. The
