Biomedical Engineering Reference
In-Depth Information
saitoi
(Yamashita et al. 1980, Ichishima et al. 1981, Ichishima et al. 1999),
P. citrinum
(Yoshida et al. 1993) and
T. reesei
(Maras et al. 2000). These
proteins share relatively high similarity at the amino acid level. The
T.
reesei
α-1,2-mannosidase is 51.6% and 51.0% similar to the
A. saitoi
and
P.
citrinum
proteins, while the
A. saitoi
and
P. citrinum
α-1,2-mannosidases
are 70% similar. The crystal structures of these proteins have recently
been determined, leading to detailed knowledge of substrate binding and
cleavage in these enzymes (Van Petegem et al. 2001, Lobsanov et al. 2002).
Three different Class I α-1,2-mannosidase genes were identifi ed and
cloned from
A. nidulans
(Eades and Hintz 2000). The
A. nidulans
mns
IB bore
high similarity to the
A. saitoi
and
P. citrinum
Class I α-1,2-mannosidases
and likely represented its homologue, while the
mns
IA and
mns
IC genes
were more distantly related and likely arose from gene duplication events
.
We therefore expressed and characterized the α-1,2-mannosidase IB (
mns
IB)
and IC (
mns
IC) from
A. nidulans
to determine whether these enzymes
exhibited overlapping functions and to compare their activities with α-1,2-
mannosidases from other fi lamentous fungi. The
Aspergillus
expression
system was selected to allow the proteins, following the addition of secretion
signals, to be secreted into the extracellular media from the organism from
which the gene was originally cloned. Homologous protein expression
is often much more effi cient than heterologous protein expression, a
phenomenon which may be due in part to the sequestration and degradation
of heterologous proteins prior to secretion into the extracellular media
(Gellissen et al. 1992, Archer and Peberdy 1997, van den Hombergh et al.
1997).
Endogenous levels of these mannosidase enzymes are extremely low, as
would be expected for Golgi processing enzymes and their purifi cation is
generally diffi cult (Yoshida et al. 1998). In order to analyze the activity and
specifi city of the α-1,2-mannosidases from
A. nidulans
, it was necessary to
produce and purify relatively large amounts of the proteins. These enzyme
are typically type-II membrane proteins residing in the ER (Moremen et
al. 1994), although a secreted form of α-1,2-mannosidase was purifi ed
from
P. citrinum
(Yoshida et al. 1993). To avoid the diffi culties associated
with purifying an intracellular membrane bound enzyme, we elected
to direct secretion of the enzyme to the extracellular media. Expression
and secretion of the
A. nidulans
α-1,2-mannosidase IB and IC enzymes
(MNSIB and MNSIC) was achieved by replacement of the N-terminal
trans-membrane domains of the proteins with the synthetic secretion signal
MDRFLGRHLGLLRHCLRQ. Since the N-terminal regions of the Class
I α-1,2-mannosidases are not necessary for the catalytic activity of these
proteins (Moremen et al. 1994), removal of this region was not expected to
affect the biochemical properties of the secreted protein. The recombinant
proteins were fused in-frame to the inducible
alcA
promoter for regulated
