Biomedical Engineering Reference
In-Depth Information
residues. Yeasts also produce 'core' oligomannose N-glycans containing
up to 13 mannose residues. In order to manipulate the pathway to produce
complex N-glycans, it is necessary to remove the steps in the pathway
that lead to hypermannosylation (Herscovics and Orlean 1993, Herscovics
1999, De Pourcq et al. 2010) and numerous strategies have been employed
to overcome this challenge (Chiba et al. 1998, Callewaert et al. 2001, Wildt
and Gerngross 2005, Chiba and Jigami 2007, Hamilton and Gerngross 2007,
Oh et al. 2008, Chiba and Akeboshi 2009, Jacobs and Callewaert 2009, De
Pourcq et al. 2010).
Remodeling of the glycosylation pathway of fi lamentous fungi has
focused on two fungal expression systems—
Aspergillus nidulans
and
Trichoderma reesei
. In an early pioneering study, Kalsner et al. (1995) inserted
the mammalian Gnt I gene into the genome of
A. nidulans
. This enzyme
would normally add GlcNAc to the Man
5
GlcNAc
2
precursor as a fi rst step
towards the production of complex mammalian type N-glycans however
expression of the Gnt I alone did not result in the production of N-glycans
containing an additional GlcNAc (GlcNAcMan
5
GlcNAc
2
). This was
conjectured to be due to limiting amounts of the substrate Man
5
GlcNAc
2
.
Maras et al. (1997b) demonstrated the
in vitro
conversion of oligomannose
glycans from cellobiohydrolase I (CBHI) to GlcNAcMan
5
GlcNAc
2
by
treatment with the enzyme Gnt I. Only a small proportion of the N-glycans
were converted, again due to the fact that only a small fraction of the
available N-glycans provided a suitable substrate (Man
5
GlcNAc
2
) for Gnt I.
As a validation of this hypothesis, pre-treatment of the purifi ed CBHI with
α-1,2-mannosidase signifi cantly increased the yield of complex N-glycans,
illustrating the need for effi cient production of suitable substrate. Maras et
al. (1999) also reported the
in vivo
conversion of oligomannose N-glycans
to complex N-glycans by heterologously expressed Gnt I, although the
effi ciency of conversion was low. Again, the conversion process may
have been blocked by a 'bottleneck' preventing production of signifi cant
amounts of substrate for Gnt I. Effi cient removal of mannose in the ER
and Golgi could provide the necessary precursors for the production of
complex N-glycans. More recently, Kainz et al. (2008) demonstrated that
expression of a
Caenorhabditis elegans
α-1,2-mannosidase fused to a
Pichia
pastoris
ER/Golgi targeting signal in
Aspergillus niger
caused an increase
in the relative amount of Man
5
GlcNAc
2
produced on secreted proteins,
however these secreted glycoproteins also contained signifi cant amounts
of higher mannosylated structures. Subsequent expression of Gnt I in
these strains produced detectable amounts of GlcNAcMan
5
GlcNAc
2
. Thus,
endogenous patterns of α-1,2-mannosidase expression are not suffi cient
to drive adequate expression of Man
5
GlcNAc
2
. Further understanding of
these endogenous α-1,2-mannosidase characteristics is important for future
engineering work in these fungal expression hosts.
