Biomedical Engineering Reference
In-Depth Information
et al. 2002, Betenbaugh et al. 2004, Tomiya et al. 2004, Viswanathan et al.
2005, Shi and Jarvis 2007); plant cells (Bakker et al. 2001); and mammalian
cell lines (Grabenhorst et al. 1999). Key to all of these expression systems
is the correct production of both the type and complexity of N-glycans
added to glycoproteins.
Protein glycosylation is a post-translational modifi cation of proteins
which involves the attachment of sugar residues to newly synthesized
polypeptides. Protein N-glycosylation occurs primarily in the ER and
Golgi apparatus and involves a series of discrete catalytic steps. A diverse
series of enzymes have evolved to carry out the complex steps of this
pathway. It is becoming increasingly clear that for many of these catalytic
steps, gene families have evolved to generate a number of similar genes to
perform a series of specialized, yet related functions, in effect fi ne tuning the
expressed products. An extensive classifi cation system has been developed
to catalogue the related glycosidases and glycosyltransferases involved in
carbohydrate processing (Henrissat and Davies 2000, Lairson et al. 2008).
Understanding the diverse and specialized functions of the different
glycosylation enzymes in different expression systems is critical for the
engineering of glycosylation-optimized expression systems.
The post-translational processing of glycoproteins has been
characterized to varying degrees in higher eukaryotes (Kornfeld and
Kornfeld 1985, Daniel et al. 1994), yeasts (Dean 1999, Wildt and Gerngross
2005, Jacobs and Callewaert 2009) and fi lamentous fungi (Kalsner et al.
1995, Maras et al. 1997a, Maras et al. 1997b, Maras et al. 1999, Nevalainen
et al. 2005, Fernandez-Alvarez et al. 2010). The early stages of N-glycan
processing in the ER and Golgi are common to all of these systems
(Fig.
1).
Protein N-glycosylation occurs when an oligosaccharide precursor
Glc
3
Man
9
GlcNAc
2
is transferred to an asparagine residue in newly
synthesized proteins on the lumen side of the ER. In all eukaryotes, the
Glc
3
Man
9
GlcNAc
2
is trimmed by α-glucosidase I to remove the three Glc
residues. In higher eukaryotes, up to four mannose residues are then
removed in the ER and Golgi to produce Man
5
GlcNAc
2
, which is the
precursor for complex N-glycan formation. Subsequent processing by
various glycosidases and glycosyltransferases produces complex N-glycans
that may contain mannose, GlcNAc, galactose and sialic acid. In yeast,
however, the Glc
3
Man
9
GlcNAc
2
is not processed to Man
5
GlcNAc
2
, but
rather is fi rst processed to Man
8
GlcNAc
2
. Various mannosyltransferases
then add mannose residues to produce a wide array of mannosylated and
hyper-mannosylated structures. In fi lamentous fungi, less is known about
the ER and Golgi processing of protein N-glycans. Filamentous fungi have
been shown to produce Man
5
GlcNAc
2
(Chiba et al. 1993, Maras et al. 1997a,
Maras et al. 1999), suggesting the presence of glycosylation machinery
