Biomedical Engineering Reference
In-Depth Information
Members of the Class I α-1,2-mannosidase gene family have been
identifi ed and characterized in several fi lamentous fungal species, including
Aspergillus saitoi
(Inoue et al. 1995),
A. oryzae
(Yoshida et al. 1998, Yoshida
et al. 2000, Akao et al. 2006),
Penicillium citrinum
(Yoshida and Ichishima
1995),
T. reesei
(Maras et al. 2000),
Magnaportha oryzae
(Zhou et al. 2009)
and
A. nidulans
(Eades and Hintz 2000). The biochemical characterization
of fungal Class I α-1,2-mannosidases has helped elucidate the role of
these genes in protein N-glycosylation in these organisms. Recombinant
expression and purifi cation of the
A. saitoi
,
P. citrinum
and
T. reesei
Class I
α-1,2-mannosidases has allowed refi ned assessment of the specifi c biological
activity of these proteins (Yoshida et al. 1998, Ichishima et al. 1999, Maras
et al. 2000, Van Petegem et al. 2001, Lobsanov et al. 2002, Lobsanov et al.
2008). These enzymes are able to digest the oligosaccharide Man
9
GlcNAc
2
primarily to Man
5
GlcNAc
2
. Digestion of Man
9
GlcNAc
2
to Man
5
GlcNAc
2
typically proceeds very effi ciently from Man
9
GlcNAc
2
to Man
7
GlcNAc
2
,
but much more slowly from Man
7
GlcNAc
2
through Man
6
GlcNAc
2
and
eventually to Man
5
GlcNAc
2
. This fi nding also corroborates the observation
that fi lamentous fungi produce endogenous Man
5
GlcNAc
2
structures for
natural fungal proteins (Chiba et al. 1993, Maras et al. 1997a, Maras et al.
1999).
The sequence characterization of three Class I α-1,2-mannosidase
(
mns
IA,
mns
IB and
mns
IC) genes in the fi lamentous fungus
A. nidulans
was the fi rst report of multiple mannosidases in a single fi lamentous
fungal species (Eades and Hintz 2000). In mammalian systems, multiple
Class I α-1,2-mannosidases have overlapping specifi cities yet display
different tissue expression patterns suggesting tissue-specifi c modifi cation
of N-glycan processing. Two different α-1,2-mannosidase activities of the
lower eukaryote
A. oryzae
also demonstrated distinct substrate specifi cities
(Yoshida et al. 2000), however these simpler organisms are not divided into
specifi c tissues. To determine whether this might be a common pattern
among lower eukaryotes we expressed and compared recombinant forms
of two different Class I α-1,2-mannosidases from
A. nidulans
.
MATERIALS AND METHODS
Strains and Media
The
A. nidulans
expression host T580 (ura
-
), a derivative of strain FGSC4
(Fungal Genetics Stock Center) was used for expression studies.
Cultures
were grown in CYM liquid media described in Kalsner et al. (1995)
supplemented with 10 mM uridine. Protoplasts which integrated the
pyrG
selectable marker (pFB94) were selected for uridine prototrophy on
