Biomedical Engineering Reference
In-Depth Information
unit above or below this optimum. The dependence of the activity of these
proteins on calcium was verifi ed by the addition of the ion chelator EDTA,
which signifi cantly reduced enzyme activity. This activity was restored by
the addition of calcium. Addition of non-limiting amounts of the inhibitor
deoxymannojirimycin, a potent inhibitor of Class I α-mannosidases, also
caused activity of the α-1,2-mannosidase IB and α-1,2-mannosidase IC
enzymes to decrease by >80%. This data is consistent with the properties
of other fungal α-1,2-mannosidases, such as those isolated from
T. reesei
,
A. saitoi
and
P. citrinum
. It should be noted that a second mannosidase
which was partially purifi ed from
P. citrinum
and was reported to cleave
Man
9
GlcNAc
2
to Man
8
GlcNAc
2
, had optimal activity at pH 7.0 (Yoshida et
al. 2000).
The Class I α-1,2-mannosidases have been generally classifi ed into
three functional subgroups (Lobsanov et al. 2002). The fi rst subgroup of
enzymes includes the yeast and human α-1,2-mannosidases that reduce
Man
9
GlcNAc
2
to Man
8
GlcNAc
2
isomer B. The second subgroup includes
mammalian Golgi α-1,2-mannosidases, insect α-1,2-mannosidases and
the fungal α-1,2-mannosidases from
T. reesei
,
A. saitoi
and
P. citrinum
.
These enzymes primarily cleave Man
9
GlcNAc
2
down to Man
5
GlcNAc
2
through a Man
8
GlcNAc
2
isomer A or isomer C intermediate. A second
α-1,2-mannosidase which was partially purifi ed from
A. oryzae
appeared to
cleave Man
9
GlcNAc
2
to Man
8
GlcNAc
2
isomer B, which would indicate that
it belongs in the fi rst subgroup of enzymes (Yoshida et al. 2000). The nature
of the substrate binding specifi city and the cleavage products produced
appears to be determined by the shape and size of the substrate binding
pocket. This was determined by comparison of the X-ray crystallography
structures of
P. citrinum
and
S. cerevisiae
structures (Lobsanov et al. 2002)
and was also demonstrated in the
T. reesei
crystal structure (Van Petegem
et al. 2001). The third subgroup of Class I α-1,2-mannosidases involves an
ER mannosidase-like protein which is involved in glycoprotein degradation
and does not hydrolyze Man
9
GlcNAc
2
(Hosokawa et al. 2001, Nakatsukasa
et al. 2001).
The discovery of two different subgroups of enzyme activity in
A. oryzae
would suggest that the multiple enzymes cloned from
A. nidulans
might also
belong to separate subgroups. In order to clarify this, we determined the
substrate specifi city of the
A. nidulans
enzymes by analyzing the cleavage
products of Man
9
GlcNAc
2
by MALDI-TOF mass spectrometry. When the
reactions were allowed to proceed to completion, we found that both the
α-1,2-mannosidase IB and α-1,2-mannosidase IC enzymes were able to cleave
the Man
9
GlcNAc
2
completely and effi ciently to a Man
5
GlcNAc
2
structure.
This supports the classifi cation of both of these enzymes are subgroup 2
enzymes. A more detailed comparison of the amino acid structures of the
A. nidulans
α-1,2-mannosidase IB and α-1,2-mannosidase IC enzymes, to
