Chemistry Reference
In-Depth Information
8.4
Insect Glycosylation
As with plants, the
N
-glycans of insects display immunogenic features which
potentially complicate the use of these species as ' glycopharmaceutical factories ' .
Indeed, with regard to
N
-glycosylation, insects are, in terms of core fucosylation,
' half plant/half mammal ', since they display both core
α
1,3 - fucosylation (present
in plants) and core
1,6-fucosylation (a feature of many mammalian glycoproteins;
see Figure 8.4). The analysis of various bee venom glycoproteins (interesting
because they are allergens) also showed the presence of a third form of fucosyl-
ation - the fucosylation of antennal ' LacdiNAc ' (GalNAc
α
1,4GlcNAc - R), a feature
also seen in the trematode parasite
Schistosoma mansoni
and akin to the mamma-
lian Lewis
x
epitope (for the structure of a sulphated derivative of LacdiNAc, see
Figure 1.7b). Generally, though, the recombinant glycoproteins produced in insect
cell lines lack the core
β
1,3-fucose and the fucosylation of LacdiNAc; however,
the natural range of
N
-glycans in High Five (
Trichoplusia ni
) cells, one of the
commonly used baculovirus hosts, includes a signifi cant proportion of core
α
α
1,3 - fucosylated structures [12] .
The presence of core
1,3-fucose is just one reason that re- engineering of insect
cells is also, as with plants and yeasts, necessary to ' humanise ' their glycosylation
[13]. Indeed, insect cells generally lack the potential to produce complex, sialylated
N
-glycans and also have a hexosaminidase in the secretory pathway, which removes
the non-reducing terminal GlcNAc residue transferred by GnT-I and, thus, reduces
their ability to produce larger glycans. Therefore, augmentation of the glycosyl-
ation pattern of insect cells requires their transformation with a number of
mammalian genes as well as identifi cation of the genes encoding the core
α
α
1,3-fucosyltransferase and the secretory pathway hexosaminidase. Major steps in
this direction have been made using
Drosophila melanogaster
as a tool.
It is interesting that, despite the long tradition of using
Drosophila
in genetics,
progress regarding its glycobiology was slow. With the sequencing of its genome,
it was possible to begin identifying homologues of mammalian and plant glyco-
syltransferases of known function. The staining of invertebrate neural tissue with
anti-HRP (see Info Box 1) was often used, but without anyone understanding the
molecular basis for this cross-reaction. However, in 2001, work in our lab demon-
strated the presence of difucosylation of the core GlcNAc (as on bee venom glyco-
proteins) in fl ies for the fi rst time, as well as the activity of a recombinant
Drosophila
core
1,3-fucosyltransferase. Recently, mutations in a fl y hexosaminidase (encoded
by the
fused lobes
gene) and the fl y GnT-I have been shown to affect
N
- glycosylation
as well as neural anatomy (see also Table 8.1 ).
It is, though, in terms of
O
-glycosylation that the glycobiological value of
Drosophila
has become most obvious - whether it be through the use of mutants
of glycosaminoglycan biosynthesis, of Notch signalling, of
O
- mannosylation or of
' traditional ' mucin - type
O
- glycosylation (see Figure 8.4). In all these cases devel-
opmental effects or lethality were associated with mutations in these pathways, for
which the fi rst steps are conserved as compared to mammals (see also Chapter 7 ).
α
