Báo cáo khoa học: Mammalian glutaminyl cyclases and their isoenzymes have identical enzymatic characteristics docx

Mammalian glutaminyl cyclases and their isoenzymes
have identical enzymatic characteristics
Anett Stephan
1
, Michael Wermann
1
, Alex von Bohlen
2
, Birgit Koch
1
, Holger Cynis
1
,
Hans-Ulrich Demuth
1
and Stephan Schilling
1
1 Probiodrug AG, Halle ⁄ Saale, Germany
2 Institute for Analytical Sciences, Dortmund, Germany
Introduction
In addition to proteolytic cleavage, glycosylation and
amidation, N-terminal formation of 5-oxoproline (pyro-
glutamate, pGlu) is a common post-translational event
during the biosynthesis of secretory peptides and
proteins, such as thyrotropin-releasing hormone
(TRH), gastrin, fibronectin and neurotensin [1–3]. Glu-
taminyl cyclases (QCs) have been identified in mammals,
invertebrates and plants, catalyzing pGlu formation
from glutaminyl precursors [4–8]. Moreover, compel-
ling evidence suggests an involvement of QCs in
diseases such as osteoporosis and Alzheimer’s disease

(AD) [9,10]. QCs have been shown to catalyze pGlu
formation at the N-terminus of amyloid peptides from
glutamyl precursors, rendering them hydrophobic and
Keywords
Alzheimer’s disease; glutaminyl cyclase
isoenzyme; glycosylation; Golgi apparatus;
Pichia pastoris
Correspondence
S. Schilling, Probiodrug AG, Weinbergweg
22, 06120 Halle ⁄ Saale, Germany
Fax: +49 345 5559901
Tel: +49 345 5559911
E-mail:
(Received 30 June 2009, revised 17 August
2009, accepted 28 August 2009)
doi:10.1111/j.1742-4658.2009.07337.x
Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate resi-
dues at the N-terminus of several peptides and proteins from plants and
animals. Recently, isoenzymes of mammalian QCs have been identified. In
order to gain further insight into the biochemical characteristics of iso-
QCs, the human and murine enzymes were expressed in the secretory
pathway of Pichia pastoris. Replacement of the N-terminal signal anchor
by an a-factor prepropeptide from Saccharomyces cerevisiae resulted in
poor secretion of the protein. Insertion of an N-terminal glycosylation site
and shortening of the N-terminus improved isoQC secretion 100-fold. A
comparison of different recombinant isoQC proteins did not reveal an
influence of mutagenic changes on catalytic activity. An initial character-
ization showed identical modes of substrate conversion of human isoQC
and murine isoQC. Both proteins displayed a broad substrate specificity
and preference for hydrophobic substrates, similar to the related QC.

Likewise, a determination of the zinc content and reactivation of the apo-
isoQC revealed equimolar zinc present in QC and isoQC. Far-UV CD
spectroscopic analysis of murine QC and isoQC indicated virtually identi-
cal structural components. The present investigation provides the first
enzymatic characterization of mammalian isoQCs. QC and isoQC repre-
sent very similar proteins, which are both present in the secretory path-
way of cells. The functions of QCs and isoQC probably complement each
other, suggesting a pivotal role of pyroglutamate modification for protein
and peptide maturation.
Abbreviations
AD, Alzheimer’s disease; BMGY, buffered glycerol complex medium; BMMY, buffered methanol complex medium; GST, glutathione
transferase; IMAC, immobilized metal ion affinity chromatography; pGlu, pyroglutamate; QC, glutaminyl cyclase; TRH, thyrotropin-releasing
hormone; TXRF, total X-ray fluorescence.
6522 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS
more prone to aggregation, probably contributing to
AD pathology [11–14]. A chronic inhibition of QCs
has been shown to attenuate AD-like pathology in
mouse models, introducing QC activity as a target for
drug development [15].
Recently, we have isolated an isoenzyme of human
QC (human isoQC) [16]. Database mining led to the
identification of a protein of 382 amino acids, which
shares a sequence identity with human QC of 45%.
In contrast with QC, a signal anchor was identified in
isoQC, which putatively mediates the retention of the
class II transmembrane protein in the Golgi apparatus.
Thus, the protein shares interesting similarity to glyco-
syltransferases with regard to subcellular localization,
and may act in concert with those in the secretory pro-
tein maturation process. This hypothesis is supported

by the ubiquitous expression pattern of isoQC, which
contrasts with the differential QC expression in glands
and neuronal tissue [16]. Because the differences in
subcellular localization and tissue distribution might
reflect other functions and catalytic specificity, it was
the aim of this study to express heterologously and
characterize the isoQCs of human and murine (murine
isoQC) origin in the methylotrophic yeast Pichia
pastoris. The method required extensive optimization
of expression, which might have implications for other
proteins.
Results
Expression of human isoQC
The isoQC proteins are localized within the Golgi
complex in their native forms. The retention in the
compartment is mediated by the N-terminus, which
includes a membrane anchor directing the nascent pep-
tide chain into the secretory pathway (Fig. 1). In order
to provoke efficient secretory expression in P. pastoris,
the N-terminal region was substituted by the a-factor
prepropeptide from Saccharomyces cerevisiae, which
should result in the secretion of the protein into the
expression medium [17]. The leader sequence thus
functions in a similar manner to the signal peptides
in native QC proteins (Fig. 1). A coding sequence of
six His residues was additionally attached to the 3¢ end
of the human isoQC open reading frame, facilitating
purification. The expression of this construct should
result in a secreted isoQC starting with phenylalanine
48 (isoQC

(F48)
C-His) (Figs 1 and 2). An isoQC expres-
sion construct was generated by applying the vector
backbone of pPICZaA, linearized and used for the
electroporation of P. pastoris. Unexpectedly, a charac-
terization of 50 stably transformed clones for the
presence of isoQC activity in the medium revealed only
very low concentrations of the recombinant protein
(Fig. 3). According to this observation, several
sequence modifications were considered in order to
improve the solubility and, potentially, the secretion
process. In contrast with isoQC, QCs contain one
(mouse, rat) or two (human, bovine) sites of N-glyco-
sylation. One N-terminal site is conserved in all mam-
malian QCs and is also present in a secreted QC from
Drosophila melanogaster, all of which were successfully
expressed in yeast [4,5,18,19]. Hence, in order to
improve the secretion of human isoQC, a potential gly-
cosylation site was introduced at position 73 by the
exchange of isoleucine with asparagine (isoQC
(F48;I73N)
,
Fig. 2), i.e. resembling the glycosylation site of QC as
suggested by a multiple sequence alignment (not
shown). A software-based algorithm predicted a high
probability of derivatization ( />services/NetNGlyc/) of the introduced asparaginyl
residue in the modified isoQC.
The I73N variant of human isoQC was expressed
carrying an N-terminal or C-terminal poly-His fusion
tag (isoQC

(F48;I73N)
N-His, isoQC
(F48;I73N)
C-His). The
comparison of the activity in the expression medium
showed an up to 10-fold higher isoQC secretion of the
now glycosylated variant in comparison with the
unmodified human isoQC
(F48)
C-His (Fig. 3A). No sig-
nificant difference in isoQC activity was observed
between expressed proteins with the different poly-His
fusions, indicating that the tag did not influence the
activity or production process.
In addition to the introduction of the N-terminal
glycosylation site, a cysteine residue present in human
isoQC was exchanged for an alanine. The mutation
appears to be conceivable, because the residue is nei-
ther conserved in murine isoQC nor in other animal
QCs. As the two conserved cysteines have been shown
to form a disulfide bond in QC, the third cysteine in
human isoQC is potentially free and might therefore
interfere with the protein production in yeast as a
result of oxidation. However, further improvement
of enzyme secretion into the medium was observed fol-
lowing expression of the protein (isoQC
(F48;I73N;C369A)
C-His, isoQC
(F48;I73N;C369A)
N-His) (Fig. 2).

In a final approach to further improve the yield of
the secreted protein, the N-terminus of the recombi-
nant protein was shortened by 59 amino acids in total.
The shortening results in complete deletion of the
transmembrane region, which spans amino acid posi-
tions 35–52, 34–55 or 40–60 according to predictions
of HMMTOP, SUSOI or TMpred [20–22] (all avail-
able at ). The plasmid constructs
were expressed encoding a human protein containing
A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes
FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6523
the glycosylation site and the Cys ⁄ Ala mutation
(isoQC
(E60;I73N;C369A)
N-His), or with only one of
the modifications, either the glycosylation site
(isoQC
(E60;I73N)
N-His) or the mutated cysteine (iso-
QC
(E60;C369A)
N-His) (Fig. 2). The human isoQC con-
structs encoded for an additional poly-His fusion
at the N-terminus. The N-terminal shortening of the
construct resulted in a 10-fold higher protein yield in
comparison with the initial construct (Fig. 3A). As
observed previously, the introduction of the glycosyla-
tion site further improved the amount of isoQC in the
medium. The highest protein yield was finally obtained
after expression of isoQC

(E60;I73N;C369A)
N-His, which
revealed a 100-fold higher isoQC concentration in the
medium compared with the initial expression con-
struct. The broad distribution in activity levels in the
expression medium of some constructs (e.g. iso-
QC
(E60;I73N;C369A)
) are caused by the transformation of
yeast. As a result of the integration of the recombinant
DNA into the genome of the host, large clonal varia-
tions occur with respect to expression, caused, for
instance, by multicopy insertion into the genome
(Fig. 3A).
The results of the determination of the isoQC activ-
ity in the medium were corroborated by western blot
analysis of the expression medium, applying an isoQC-
specific antiserum (Fig. 3A, inset). The most intense
immunostaining was observed following the expression
of isoQC
(E60;I73N;C369A)
N-His.
From all expressed constructs, isoQC
(F48;I73N;C369A)
C-His, isoQC
(E60;I73N;C369A)
N-His and isoQC
(E60;I73N)
N-His were used for a scale-up of expression in shake
Fig. 1. Amino acid sequence alignment of

human and murine isoQCs, human QC
(hQC), mouse QC (mQC) and Drosoph-
ila melanogaster QC (Drome QC). The signal
sequences of QC and the signal anchor of
isoQCs are highlighted in bold italics. The
residues for complexation of zinc ions in the
active site (Asp-Glu-His) (bold) and the core
structure surrounding the active site, con-
taining a conserved disulfide bond (bold and
underlined), are conserved in all enzymes. In
addition, the secreted QC proteins contain
sites of N-glycosylation (bold, italics, under-
lined). The position of the introduced glyco-
sylation site by mutation of an isoleucine
residue in isoQC is shown in bold italics.
The alignments were prepared using
CLUSTALW at EMBnet-CH (http://www.
expasy.ch).
Characterization of glutaminyl cyclase isoenzymes A. Stephan et al.
6524 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS
flasks. The protein was purified to virtual homogeneity
by initial Ni
2+
-immobilized metal ion affinity chroma-
tography (IMAC), followed by hydrophobic interac-
Cytosolic sequence
Membrane anchor
Luminal catalytic domain
Yeast secretion signal
NH

2
COOH
isoQC
(E
60
;I73N)
COOHNH
2
Native isoQC
NH
2
COOH
isoQC
(F
48
)
NH
2
COOH
isoQC
(E
60
; I73N;C369A)
NH
2
COOH
isoQC
(F
48
;I73N)

Ile→ Asn
↓
NH
2
COOH
isoQC
(F
48
;I73N;C369A)
NH
2
COOH
Cys→Ala
↓
isoQC
(F
48
;C369A)
COOH
isoQC
E
61
I74N
NH
2
NH
2
COOH
isoQC
(F

49
)
Human isoQC
Murine isoQC
Fig. 2. Schematic representation of the
different constructs for the secretory
expression of human and murine isoQCs in
Pichia pastoris. The native isoQC with the
cytosolic tail and the membrane anchor is
shown for comparison. This N-terminal
region is exchanged for the a-leader prepro-
sequence of Saccharomyces cerevisiae as a
secretion signal. In some constructs, an
isoleucine is mutated into an asparagine,
generating an N-glycosylation site. Further-
more, a third cysteine at the C-terminus of
human isoQC is mutated into an alanine.
Finally, the constructs differed in terms of
their N-terminus, i.e. the N-terminal amino
acid corresponding to the open reading
frame of isoQC was either a phenylalanine
(Phe48 and Phe49 of human and murine
QC, respectively) or a glutamic acid residue
(position 60 or 61).
C
-
H
i
s
(

F
4
8
)
i
s
o
Q
C
C
-
H
i
s
(
F
4
8
;
C
3
6
9
A
)
i
s
o
Q
C

N
-
H
i
s
(
E
6
0
;
C
3
6
9
A
)
i
s
o
Q
C
N
-
H
is
(
F
4
8
;

I7
3
N
)
i
s
o
Q
C
C
-
H
i
s
(
F
4
8
;
I
7
3
N
)
i
s
o
Q
C
N

-
H
i
s
(
E
6
0
;
I
7
3
N
)
i
s
o
Q
C
N
-
H
i
s
(
F
4
8
;I
7

3
N
;
C
3
6
9
A
)
is
o
Q
C
C
-H
i
s
(F
4
8
;
I
7
3
N
;C
3
6
9
A

)
i
s
o
Q
C
N
-
H
i
s
(
E
6
0
;
I
7
3
N
;
C
3
6
9
A
)
is
o
Q

C
0
1
2
3
4
5
6
QC-activity (µM·min
–1
)
0.001
0.01
0.1
1
10
Log activity
12345678910
36 kDa
A
B
kDa
250
150
100
75
50
37
25
20

15
51
34 6
2
Fig. 3. Characterization of human isoQC expression in Pichia pasto-
ris. (A) Determination of the QC activity in the medium of P. pastoris
expressing the different constructs. At least 50 clones of each con-
struct were checked with regard to QC activity in the culture medium
after small-scale expression. The inset shows western blot analysis
of the expression medium and a logarithmic scatter plot of the acti-
vity determined for each yeast clone investigated. The logarithmic
plot of the QC activity data points to a similar variation of expression
after transformation with the different plasmid constructs, which is
caused by differences in transcriptional efficacy and insertion events
of the expression constructs into the genome of yeast. In western
blot analysis, the proteins were visualized using an isoQC antibody.
A total amount of 4 lg of protein was applied to each lane. Lane 1,
isoQC
(F48)
C-His; lane 2, isoQC
(F48;C369A)
C-His; lane 3, iso-
QC
(E60;C369A)
N-His; lane 4, deglycosylated isoQC
(F48;I73N)
N-His; lane
5, deglycosylated isoQC
(F48;I73N)
C-His; lane 6, deglycosylated iso-

QC
(E60;I73N)
N-His; lane 7, deglycosylated isoQC
(F48;I73N;C369A)
N-His;
lane 8, deglycosylated isoQC
(F48;I73N;C369A)
C-His; lane 9, deglyco-
sylated isoQC
(E60;I73N;C369A)
N-His; lane 10, 400 ng of purified and
deglycosylated isoQC
(F48;I73N;C369A)
as a positive control. The degly-
cosylated protein migrates at 37 kDa. (B) SDS-PAGE analysis illus-
trating the purification of human isoQC
(F48;I73N;C369A)
C-His after
expression in shake flasks. Proteins were visualized by Coomassie
staining: lane 1, molecular mass standards (kDa) (Dual Color, Bio-
Rad); lane 2, supernatant after expression; lane 3, isoQC-containing
fractions after initial affinity chromatography on immobilized Ni
2+
ions; lane 4, human isoQC after hydrophobic interaction chromato-
graphy; lane 5, purified protein after desalting. The isoQC protein
corresponds to a band migrating between 50 and 70 kDa. The degly-
cosylation causes a shift to 37 kDa (lane 6). The upper band (75 kDa)
in lane 6 corresponds to the endoglycosidase H
f
.

A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes
FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6525
tion chromatography. A purification process illustrated
by SDS-PAGE analysis is shown in Fig. 3B. As a
result of the competitive inhibition of isoQC and QC
by imidazole, the elution was performed with His,
which is much less inhibitory to QC and isoQC. Inacti-
vation of isoQC, e.g. caused by zinc complexation dur-
ing IMAC, was not observed.
Typical expression in a final culture volume of 4 L
resulted in the isolation of 7 mg of isoQC
(E60;I73N)
N-His and 14 mg of isoQC
(E60;I73N;C369A)
N-His
variant. Expression of isoQC
(F48;I73N;C369A)
C-His was
carried out in a total volume of 8 L, resulting in the
isolation of 7.3 mg of human isoQC protein. The over-
all yield of purification was 60%.
Expression and purification of murine isoQC
On the basis of human isoQC expression in P. pastoris,
three different murine isoQC constructs were gener-
ated. The introduction of a glycosylation site and the
shortening of the N-terminus (isoQC
(E61;I74N)
N-His)
resulted in an increase in secretory expressed murine
isoQC, similar to that observed with human isoQC. In

addition to the poly-His-tagged proteins, an untagged
protein was generated. A comparison of the N-termi-
nally His-tagged and untagged protein did not reveal a
significant influence on isoQC expression (Fig. 4A).
The isoQC
(E61;I74N)
variant was successfully expressed
by fermentation using a 5 L bioreactor, which typically
results in the harvesting of 2 L of isoQC-containing
medium. Homogeneous protein was obtained after
purification, applying two different hydrophobic
interaction chromatographic matrices, followed by
anion-exchange chromatography and size exclusion
chromatography. The purity of murine isoQC was
analyzed by SDS-PAGE (Fig. 4B). A typical purifica-
tion process resulted in the isolation of 8 mg of murine
isoQC protein.
Characterization of the substrate and inhibitor
specificity
In order to rule out an influence of the different modi-
fications of human isoQC on substrate conversion, the
kinetic parameters for the turnover of H-Gln-bNA
(Q-bNA), H-Gln-Glu-OH (QE) and H-Gln-Gln-OH
(QQ) and the competitive isoQC inhibitors benzimid-
azole and benzylimidazole were analyzed. The evalua-
tion of the influence of sequence shortening to
glutamic acid 60, glycosylation of the protein and
mutation of cysteine 369 into alanine did not show a
considerable effect on the kinetic parameters or on the
inhibitory specificity (cf. Tables 1 and 2). Accordingly,

the substrate and inhibitor specificities of the human
isoQC
(F48;I73N;C369A)
C-His variant were analyzed and
compared with the data obtained with a glutathione
transferase (GST)-fusion protein, which was expressed
in Escherichia coli previously [16]. Interestingly, the
substrate specificity of the isoQCs expressed in P. pas-
toris and E. coli was virtually identical for the tested
substrates, i.e. the highest specificity was obtained
for substrates containing hydrophobic residues, e.g.
H-Gln-bNA, H-Gln-Tyr-Ala-OH (QYA) or H-Gln-
Glu-Tyr-Phe-NH
2
(QEYF) (Table 2). A comparison of
N-
His
(
F49
)
C
N
-
His
(
E
61;
I7
4N
)

i
soQ
i
soQ
C
(E
61
;I7
4
N)
i
s
o
Q
C
0. 0
0. 5
1. 0
1. 5
2. A
B
0
QC-activity (µM·min
–1
)
kD a
150
100
75
50

37
25
20
1 5 3 4 6 2
0.001
0.01
0.1
1
10
log activity
Fig. 4. Expression and characterization of murine isoQC in Pichia
pastoris. (A) Determination of QC activity in the medium of P. pas-
toris expressing the indicated constructs. The analysis was per-
formed as described for human isoQC in Fig. 3. (B) SDS-PAGE
illustrating the purification of murine isoQC
(E61;I74N)
after fermenta-
tion. Proteins were visualized by Coomassie staining. Lane 1,
molecular mass standards (kDa) (Dual Color, Bio-Rad); lane 2,
supernatant after expression; lane 3, isoQC-containing fractions
after initial hydrophobic interaction chromatography applying
expanded bed absorption; lane 4, isoQC after hydrophobic inter-
action chromatography; lane 5, isoQC after anion exchange chroma-
tography; lane 6, isoQC after desalting and deglycosylation of the
protein. isoQC corresponds to a protein between 50 and 70 kDa.
The deglycosylated protein migrates at 37 kDa and the endoglycosi-
dase H
f
is at 75 kDa.
Characterization of glutaminyl cyclase isoenzymes A. Stephan et al.

6526 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS
the k
cat
⁄ K
m
values of both human isoQC variants,
however, revealed a prominent difference. The specific-
ity constant obtained with isoQC expressed in P. pas-
toris was two- to three-fold higher for almost every
substrate tested, suggesting a potential influence of the
expression system or of the isoQC fusion construct. As
a result of the very similar Michaelis constants for the
isoQCs expressed in E. coli and P. pastoris, the influ-
ence on substrate specificity was mainly caused by k
cat
.
Similar to K
m
, no significant difference was observed
in the K
i
values between isoQCs expressed in yeast and
E. coli (Table 2). Presumably, the expression and puri-
fication of isoQC in P. pastoris resulted in efficient
recovery of the active protein.
The relative substrate specificity of murine isoQC
was similar to that of the human enzyme (Table 3).
Most substrates, however, were more efficiently con-
verted into products by the murine protein, which con-
trasts with the related QCs [5]. The higher proficiency

was caused by the lower Michaelis constants and
higher turnover numbers.
In order to further characterize the activity of mur-
ine isoQC in comparison with QC, the pH dependence
of catalysis was assessed under first-order conditions,
i.e. at [S] << K
m
. Both enzymes displayed a pH opti-
mum of k
cat
⁄ K
m
of between 7.5 and 8 (Fig. 5). The
kinetic data of the pH dependence were evaluated by
applying a model, which considers three dissociating
groups, one of the substrate and two of the enzyme.
The pK
a
value of the applied substrate H-Gln-AMC
(6.83 ± 0.01) has been determined previously and
matches well with the acidic inflection points of the
pH dependences of isoQC and QC. The nonsymmetric
character of the bell-shaped curve was calculated
assuming two dissociating groups of the enzyme. Eval-
uation of these data resulted in pK
a
values of
9.5 ± 0.3 and 8.2 ± 0.4 for murine isoQC and
9.0 ± 0.2 and 8.3 ± 0.3 for murine QC. Apparently,
all dissociating groups, which influence the catalytic

process in QC and isoQC, are conserved, supporting
an identical catalytic mechanism.
Characterization of metal dependence and
structural elements
The animal QCs and isoQCs share a structural
relationship to bacterial double-zinc aminopeptidases
[23–25]. Although the coordinating residues of the
aminopeptidases are also conserved in QCs, it has been
shown that only one zinc ion is bound to QC [5,26].
Therefore, murine isoQC without an affinity tag
was analyzed for the presence of transition metal ions
using total X-ray fluorescence (TXRF) spectroscopy.
Table 1. Kinetic parameters of substrate conversion by different human isoQC variants. The substrates are displayed in the three-letter code of amino acids. Reactions were carried out in
0.05
M Tris ⁄ HCl, pH 8.0, at 30 °C. ND, not determined.
Compound
isoQC
(F60;I73N;C369A)
N-His isoQC
(F60;I73N;C369A)
N-His deglycosylated isoQC
(F60;I73N)
N-His
K
m
(mM) k
cat
(s
)1
) K

i
(mM) K
m
(mM) k
cat
(s
)1
) K
i
(mM) K
m
(mM) k
cat
(s
)1
) K
I
(mM)
Substrates
H-Gln-bNA 0.034 ± 0.003 9 ± 2 ND 0.039 ± 0.007 7 ± 1 ND 0.058 ± 0.003 12 ± 1 ND
H-Gln-Gln-OH 0.19 ± 0.01 5.7 ± 0.2 0.16 ± 0.02 4.6 ± 0.3 0.19 ± 0.003 6.2 ± 0.1
H-Gln-Glu-OH 1.10 ± 0.04 4.9 ± 0.2 0.89 ± 0.03 4.3 ± 0.2 0.79 ± 0.02 4.9 ± 0.1
Inhibitors
Benzimidazole 0.24 ± 0.02 0.22 ± 0.03 0.27 ± 0.02
Benzylimidazole 0.0080 ± 0.0002 0.009 ± 0.001 0.010 ± 0.001
A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes
FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6527
A typical spectrum is displayed in Fig. 6A. Three inde-
pendently prepared enzyme samples showed a signifi-
cantly increased zinc concentration. The averaged zinc

content in murine isoQC was 0.99 ± 0.38 moles of
zinc per mole of enzyme, clearly supporting stoichiom-
etric zinc binding of murine isoQCs. Accordingly, full
reactivation of the murine isoQC apo-enzyme was
obtained in the presence of equimolar zinc concentra-
tions (Fig. 6B). In addition to zinc, reactivation was
also obtained with cobalt ions, also suggesting an equi-
molar stoichiometry of metal to protein necessary for
activity. With regard to zinc content and reactivation,
therefore, the isoQCs are very similar to QCs. A cata-
lytic role of the transition metal ion is also suggested
by a comparison of the far-UV CD spectrum of
murine isoQC and its apo-enzyme (Fig. 6C). The CD
Table 3. Kinetic evaluation of peptide substrates by mouse isoQC. Protein was expressed in Pichia pastoris.
a
ND, not determined.
Compound
Murine isoQC Murine QC
K
m
(mM) k
cat
(s
)1
) K
i
(mM) k
cat
⁄ K
m

(mM
)1
Æs
)1
) k
cat
⁄ K
m
(mM
)1
Æs
)1
) K
i
(mM)
Substrates
H-Gln-bNA 0.032 ± 0.003 17.48 ± 0.97 3.55 ± 0.13
b
554 ± 47 550 ± 30
c
1.77 ± 0.18
c
H-Gln-AMC 0.030 ± 0.006 6.98 ± 0.35 ND 311 ± 27 125 ± 4
c
5.9 ± 0.7
c
H-Gln-Gln-OH 0.092 ± 0.005 8.66 ± 0.37 95 ± 6 213 ± 8
c
H-Gln-Glu-OH 0.47 ± 0.04 7.79 ± 0.44 16 ± 2 36 ± 1
c

H-Gln-Gly-OH 0.16 ± 0.01 4.57 ± 0.12 28 ± 2 30 ± 2
c
H-Gln-Gly-Pro-OH 0.102 ± 0.006 11.4 ± 0.4 111 ± 7 ND
H-Gln-Tyr-Ala-NH
2
0.058 ± 0.004 22.9 ± 0.9 394 ± 21 ND
H-Gln-Phe-Ala-NH
2
0.060 ± 0.006 24.1 ± 0.5 403 ± 49 ND
H-Gln-Glu-Tyr-Phe-NH
2
0.029 ± 0.003 11.78 ± 0.61 413 ± 46 ND
H-Gln-Glu-Asp-Leu-NH
2
0.16 ± 0.01 6.4 ± 0.1 104 ± 4 ND
Inhibitors
Imidazole 0.103 ± 0.027 0.16 ± 0.01
c
Benzimidazole 0.124 ± 0.004 0.192 ± 0.003
c
Methylimidazole 0.052 ± 0.005 0.023 ± 0.001
c
Benzylimidazole 0.0039 ± 0.0003 0.0064 ± 0.0007
c
Cysteamine 0.069 ± 0.006 0.042 ± 0.002
c
N-Dimethylcysteamine 0.027 ± 0.003 0.029 ± 0.002
c
a
Reactions were carried out in 0.05 M Tris ⁄ HCl, pH 8.0, at 30 °C.

b
Substrate inhibition.
c
Data from [5].
Table 2. Kinetic parameters of substrate conversion by recombinant human isoQC obtained from different host systems.
a
Compound
isoQC
(F48;I73N;C369A)
C-His GST-isoQC E. coli
K
m
(mM) k
cat
(s
)1
) K
i
(mM)
k
cat
⁄ K
m
(mM
)1
Æs
)1
)
k
cat

⁄ K
m
(mM
)1
Æs
)1
) K
i
(mM)
Substrates
H-Gln-bNA 0.035 ± 0.001 3.4 ± 0.1 1.57 ± 0.09
b
229 ± 22 93 ± 7
c
1.47 ± 0.07
c
H-Gln-AMC 0.030 ± 0.006 1.07 ± 0.03 4.47 ± 0.91
b
103 ± 29 63 ± 6
c
5.73 ± 0.81
c
H-Gln-Gln-OH 0.11 ± 0.01 2.7 ± 0.2 54 ± 5 25 ± 4
c
H-Gln-Glu-OH 0.61 ± 0.06 2.6 ± 0.2 9 ± 1 4 ± 0.6
c
H-Gln-Gly-OH 0.36 ± 0.05 1.65 ± 0.04 9 ± 2 4 ± 0.3
c
H-Gln-Gly-Pro-OH 0.23 ± 0.02 4.0 ± 0.1 38 ± 3 19 ± 1
c

H-Gln-Tyr-Ala-NH
2
0.08 ± 0.02 7.7 ± 0.4 207 ± 57 66 ± 13
H-Gln-Phe-Ala-NH
2
0.10 ± 0.02 7.5 ± 0.3 117 ± 34 33 ± 2
H-Gln-Glu-Tyr-Phe-NH
2
0.040 ± 0.004 3.3 ± 0.1 123 ± 6 110 ± 21
H-Gln-Glu-Asp-Leu-NH
2
0.16 ± 0.01 6.4 ± 0.1 55 ± 5 10 ± 1
Inhibitors
Imidazole 0.235 ± 0.013 0.219 ± 0.0009
c
Benzimidazole 0.250 ± 0.005 0.199 ± 0.008
c
Methylimidazole 0.082 ± 0.003 0.079 ± 0.0047
c
Benzylimidazole 0.0062 ± 0.0002 0.0073 ± 0.0005
c
a
Reactions were carried out in 0.05 M Tris ⁄ HCl, pH 8.0, at 30 °C.
b
Substrate inhibition.
c
Data from [16].
Characterization of glutaminyl cyclase isoenzymes A. Stephan et al.
6528 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS
spectra were virtually identical between isoQC, apo-

isoQC and QC. Thus, the loss of the metal ion does
not result in large structural rearrangements. The
virtually identical spectra further support the strong
similarity between QC and isoQC globular domains.
Finally, we characterized whether the conserved
cysteines in human isoQC form a disulfide bond, as
described for human QC [4]. The characterization of
the disulfide status of the protein by SDS-PAGE
clearly suggests disulfide bond formation (Fig. 7).
Therefore, the structurally conserved features of isoQC
and QC proteins not only contain metal complexation
and general fold, but also the formation of a disulfide
bond close to the active center of the protein.
In addition to the two conserved cysteines, human
isoQC contains a third cysteine residue in the C-ter-
minal part of the protein (Fig. 1). The presence of a
third cysteine might imply the formation of dimers in
the secretory pathway because, in an oxidative envi-
ronment, cysteine does not usually appear unbound.
In the case of isoQC, it should be noted that the
cysteine residue is not conserved in murine (Fig. 1),
bovine (UniProt: Q0V8G3) and macaque (UniProt:
Q4R942) isoQCs. Initial expression of murine and
human isoQCs in human HEK293 cells and an
accompanying western blot analysis involving a
reducing or nonreducing sample preparation did not
reveal the formation of homo- or heterodimers (not
shown). The data thus imply that dimerization involv-
ing a covalent interaction does not occur in the Golgi
complex.

Discussion
In a first approach to characterize the recently discov-
ered isoQCs from mouse and humans, we aimed to
C
Wavelength in nm
200 220 240 260
Molar ellipticity
(degrees·cm²·dmol
–1
)
–15 000
–10 000
–5000
0
5000
10 000
15 000
20 000
25 000
m-isoQC apoenzyme
m-isoQC reactivated
mQC
A
Counts
2000
4000
Photon energy, keV
0246810121416
0
m-isoQC

Cl
P
Ca
Cu
Zn
Zn
Br
B
Ratio metal to enzyme
024
Activity (µM·min
–1
)
0
2000
4000
6000
Zinc
Cobalt
Manganese
Nickel
Calcium
Fig. 6. Spectroscopic analysis of murine isoQC. (A) TXRF spectrum
of a murine isoQC preparation. IsoQC was dissolved in 10 m
M
Tris ⁄ HCl, pH 7.6. The evaluation of the measurements revealed
equimolar amounts of zinc bound to the enzyme. (B) Reactivation
of murine apo-isoQC with different divalent metal ions. The enzyme
was inactivated by 1,10-phenanthroline and subjected to dialysis
against Chelex-treated buffer. Reactivation was carried out by the

addition of different concentrations of transition metal ions to the
inactivated protein. The ratios of the concentrations of transition
metal and apo-enzyme are indicated on the x-axis. (C) CD spectro-
scopic analysis of murine isoQC, murine QC and murine apo-isoQC.
The protein was dissolved in 10 m
M potassium phosphate buffer,
pH 6.8. There was virtually no difference in the spectra between
the apo- and holo-enzymes and murine QC, supporting a low
influence of the active site zinc on the structure of isoQC.
pH
68
k
cat
/K
m
(mM
–1
·s
–1
)
0
20
40
60
80
100
120
140
160
180

200
220
240
260
m-isoQC
mQC
Fig. 5. pH dependence of murine isoQC catalysis. Determination
of the specificity constant k
cat
⁄ K
m
for the conversion of H-Gln-AMC
by purified murine isoQC (h) and QC (O), determined under first-
order rate law conditions ([S] << K
m
). The substrate concentration
was 0.005 m
M and the reactions were carried out at 30 °C in a buf-
fer consisting of 0.2
M Tris ⁄ HCl, 0.1 M Mes and 0.1 M acetic acid.
A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes
FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6529
express and characterize the proteins in P. pastoris in
order to compare the catalytic properties with those of
the sister enzyme QC. In contrast with E. coli, P. pas-
toris has been shown to exert many post-translational
modifications, such as N-glycosylation and disulfide
formation [27–31], some of which have been shown to
be present in animal and plant QCs [4,5,19,32] and
may also be important for the expression of isoQCs.

On the basis of these results, we aimed to obtain the
secretory expression of murine and human isoQCs in
P. pastoris. In the native state, isoQC is expressed as a
class II transmembrane protein, which is anchored by
an N-terminal signal peptide in the membrane of the
endoplasmic reticulum and retained in the Golgi appa-
ratus. In order to obtain secreted protein, the short
cytosolic tail, including the major part of the mem-
brane anchor, was deleted and substituted by an a-leader
prepro sequence of yeast, which should direct the
protein efficiently into the secretory pathway.
Although the remaining protein shares 51% sequence
identity with the mature QCs, isoQC expression
resulted in media virtually devoid of QC activity. In
contrast with the QC proteins, human and murine iso-
QCs lack a conserved N-terminal site for N-glycosyla-
tion (Fig. 1). It is known that glycosylation may lead
to an increase in protein solubility [33] and decreased
aggregation propensity [34]. Furthermore, deglycosyla-
tion of human QC resulted in protein precipitation
(results not shown). Therefore, we introduced an
N-glycosylation site into isoQC for heterologous
expression. Glycosylation resulted in a significant
increase in isoQC activity in the medium. Although
the reason for the improvement was not investigated
in detail, an increase in protein solubility and, in turn,
a decrease in hydrophobic interactions between the
protein might be a primary cause. Consistent with this
hypothesis, an enzymatic deglycosylation of the isoQC
protein resulted in a dramatic decrease in solubility to

about 1 mgÆmL
)1
, whereas the glycosylated protein did
not precipitate up to 30 mgÆmL
)1
(not shown). In
addition to glycosylation, the N-terminal truncation to
glutamic acid 60/61 of the proteins further improved
the expression of human and murine isoQCs. Based on
sequence comparisons, the finally deleted region corre-
sponds mainly to unstructured parts of the protein,
which might also exert an influence on efficient protein
production. The sequential optimization of the protein
construct used for expression ended in a 100-fold
improvement of the protein yield, which was secreted
into the medium of the cells and could be purified by
two- to three-step protocols. The secretory expression
in yeast facilitates an efficient purification process,
because yeast – in spite of a fully developed secretory
machinery – secretes only a few proteins into the extra-
cellular space [35]. Because the heterologous protein
reaches a high specific activity in the expression med-
ium, secretion can be regarded as a separation step,
allowing efficient recovery of the protein of interest,
even without an affinity tag, as shown here for murine
isoQC.
Moreover, the expression of proteins in the secretory
pathway facilitates the appropriate formation of post-
translational structural elements, such as disulfide
bonds. As shown in this study, the two cysteine resi-

dues, which are conserved in murine and human
isoQCs, form a disulfide bond, which is reminiscent of
the disulfide bond formation in human QC. Appar-
ently, the disulfide bridge is an evolutionary conserved
structural element of QC and isoQC (Fig. 1). Accord-
ing to the crystal structure of human QC [25], the
cysteine residues are close to the active site, probably
exerting a stabilizing effect on the flexibility in that
region.
The high degree of sequence similarity between the
isoQCs and their sister enzyme QC was finally mir-
rored by the characterization of the secondary struc-
ture, metal dependence and catalytic activity. A
virtually identical catalytic activity was demonstrated
by the comparison of murine isoQC and murine QC,
which were both expressed in P. pastoris. The data
clearly suggest that the active site of both enzymes has
a very similar structure, forming identical secondary
interactions with the substrate to facilitate binding and
turnover. Apparently, the core features of both pro-
teins, i.e. the active site and the general fold, are iden-
tical, a hypothesis which is also supported by an
alignment of the proteins, which shows a high degree
of conservation of the inner core structures between
QC and isoQC and a weaker similarity in the connect-
ing loops (Fig. 1). The far-UV CD spectra of QC and
isoQC are shown to be identical, confirming the
kDa
1
534 1098762

50
37
Fig. 7. Analysis of disulfide formation in human isoQC expressed
in Pichia pastoris. Lanes 1 and 10, molecular mass standards (kDa);
lanes 2, 3, 8 and 9, sample prepared under reducing conditions
(5% b-mercaptoethanol); lanes 4–7, samples prepared under non-
reducing conditions. Electrophoresis (15% gels) was performed at
a constant voltage of 200 V for 1.5 h, and protein was visualized by
Coomassie staining.
Characterization of glutaminyl cyclase isoenzymes A. Stephan et al.
6530 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS
above-mentioned assumptions. According to the pro-
posed high degree of similarity, isoQCs and QCs con-
tain one zinc ion in the active site, which is responsible
for the catalytic activity of the enzyme. As shown here
by titration experiments, zinc can be replaced by
cobalt, which results in a less active enzyme. Similar
results have been reported previously for human QC
[24]. The results of the titration experiments clearly
suggest that the binding of one transition metal ion is
necessary and sufficient for the exertion of full enzy-
matic activity, and that the metal ion does not exert a
direct effect on the structure of the protein.
Thus, with regard to the recombinant expression
strategy introduced in the present study, it appears
that the various mutagenic changes to the isoQCs did
not exert a relevant influence on the catalytic activity
of the soluble, heterologous proteins. However, a
potential influence of the deletion of the N-terminal
signal anchor in isoQC cannot be fully excluded. In its

native state, the protein is a membrane-bound enzyme
of the Golgi complex, and membrane anchoring might
potentially affect substrate turnover, perhaps by prox-
imity to the membrane or other interacting proteins.
The present results thus mirror well the catalytic
potential of the globular domain, especially in relation
to the sister enzyme QC, but cannot be translated into
the in vivo situation without caution.
The characterization of QC and isoQC revealed that
evolution apparently resulted in globular proteins with
a very similar catalytic power, virtually identical sub-
strate specificity and similar subcellular localization
within the secretory pathway. Most likely, the proteins
had a common ancestor. It still remains unclear,
however, whether the proteins are responsible for the
conversion of the same substrates, i.e. pGlu-modified
proteins and ⁄ or peptide hormones and pGlu-modified
amyloid peptides in neurodegenerative disorders.
The localization of QC and isoQC in the secretory
pathway enables the conversion of secretory proteins
by both enzymes – QC is probably transported within
the regulated pathway [8] and isoQC exerts its function
as a resident enzyme of the Golgi apparatus [16].
Although the localization of both proteins appears to
be virtually identical at first glance, the presence of QC
in vesicles of the regulated secretory pathway might
indicate the responsibility of QC for the conversion
of substrates requiring extensive post-translational
processing, e.g. the neuropeptide TRH [36–38]. The
liberation of the substrate in secretory vesicles requires

the presence of QC in the same compartment, because
N-terminal pGlu formation represents a finishing
reaction in the post-translational maturation of these
hormones. In contrast, many proteins do not require
such processing of the precursor; the N-terminal
glutaminyl residue is directly generated by signal pepti-
dase cleavage in the endoplasmic reticulum, e.g. in
ribonuclease or a-amylase. These proteins are also
likely to be secreted via the constitutive pathway from
the Golgi complex. Therefore, the primary converting
enzyme of these proteins might be isoQC. Taken
together, it appears that the liberation of N-terminal
glutamine in the secretory pathway results inevitably in
N-terminal pGlu formation. The broad and similar
substrate specificity of QC and isoQC might therefore
be important for the conversion of these different
substrates. Finally, the similar cellular distribution of
two proteins with virtually identical specificity, as
shown here, might also point to an overall important
role of pGlu protein formation for physiology. The
elucidation of the physiological function might have
implications for drug development, as a partial
complementation of QC and isoQC might compensate
for the side-effects of potential, isoform-specific drug
candidates. Indeed, it is likely that the protein func-
tions of QC and isoQC complement each other, as QC
knockout mice do not exhibit an apparent phenotype
(S. Schilling et al., unpublished results).
In summary, the first detailed heterologous expres-
sion and characterization study of mammalian isoQCs

was accomplished. Expression was mainly optimized
by the insertion of an artificial glycosylation site into
the isoQC protein, resulting in efficient protein secre-
tion by the yeast P. pastoris, resembling the subcellular
localization in the native tissue of origin. These results
might have implications for the expression of other
mammalian proteins, which display a high tendency to
aggregation and are, therefore, difficult to express. The
isolation of the isoQC protein represents a basis for
structural investigations and drug candidate profiling.
Materials and methods
Materials
The E. coli strain DH5a was applied for all plasmid con-
struction and propagation; P. pastoris strain X-33 (AOX1,
AOX2) was used for the expression of the different isoQC
variants. Yeast was grown, transformed and analyzed
according to the manufacturer’s instructions (Invitrogen,
Karlsruhe, Germany). The glutaminyl peptides were
obtained from Bachem (Bubendorf, Switzerland) or synthe-
sized as described elsewhere [39]. Recombinant pyroglutamyl
aminopeptidase from Bacillus amyloliqefaciens was
purchased from Qiagen (Hilden, Germany) and glutamic
dehydrogenase from Fluka (Seelze, Germany). The imidaz-
ole derivatives were purchased from Sigma-Aldrich
A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes
FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6531
(Taufkirchen, Germany). The low-salt Luria–Bertani
medium, required for the propagation of E. coli, and the
buffered glycerol complex medium (BMGY) and buffered
methanol complex medium (BMMY), required for the

propagation of yeast, were prepared according to the
‘Pichia protocols’ (Invitrogen).
Cloning procedures
The different human and murine isoQC constructs were
inserted into the yeast expression vector pPICZaA (Invitro-
gen) using the EcoRI and NotI restriction sites. The primers
used for cloning are listed in Table 4. The PCR fragment
for the human isoQC
(F48)
C-His was generated using primer
5 (sense) and primer 6 (antisense). Mutagenesis primers 1
and 2 were used for the insertion of the glycosylation site,
and primer 3 (sense) and primer 4 (antisense) were applied
for the mutation of cysteine 369 into alanine. PCR-medi-
ated site-directed mutagenesis was performed according to
standard PCR techniques, followed by digestion of the
parent DNA using DpnI (Quik-Change II site-directed
mutagenesis kit; Stratagene, La Jolla, CA, USA). C- and
N-terminal His-tags were introduced by primer pairs 5 ⁄ 6
and 7 ⁄ 8, respectively. Finally, a human isoQC starting with
glutamic acid 60 and bearing an N-terminal His-tag was
generated by applying the primer pair 8 ⁄ 9. The molecular
cloning of murine isoQC was performed according to
human isoQC. The open reading frame lacking the N-ter-
minal cytosolic tail and the major part of the transmem-
brane region was amplified by PCR using primer 15 (sense)
and primer 11 (antisense). The murine isoQC constructs
starting with codon 61 (Glu) were generated with either an
N-terminal His-tag or without a tag, applying the primer
pairs 14 ⁄ 11 and 10 ⁄ 11, respectively. For the insertion of a

glycosylation site, a mutation was introduced in codon 74
(Ile) using primer 3 as sense and primer 4 as antisense. The
sequences of all expression constructs were verified.
Transformation of P. pastoris and mini-scale
expression
Plasmid DNA was amplified in E. coli DH5a and purified
according to the recommendations of the manufacturer
(Qiagen); 20–30 lg of DNA (in vector pPICZaA) were
linearized with PmeI, precipitated and dissolved in deion-
ized water; 1–5 lg of DNA were applied for the transfor-
mation of competent P. pastoris cells by electroporation
according to the manufacturer’s instructions (BioRad,
Munich, Germany). Selection was carried out on yeast
extract peptone dextrose medium plates containing
100 lgÆmL
)1
of Zeocin. To test yeast clones on expression,
recombinants were grown for 24 h in 10 mL conical tubes
containing 2 mL of BMGY. Afterwards, the cells were
centrifuged and resuspended in 2 mL of BMMY contain-
ing 0.5% methanol. This concentration was maintained
by the addition of methanol every 24 h. isoQC activity
was determined in the supernatant after 72 h. The
expression of human isoQC was confirmed by western blot
analysis applying a polyclonal antibody (rabbit) raised
against human isoQC. Clones displaying the highest QC
activity were chosen for large-scale expression and further
experiments.
Large-scale expression and purification of human
isoQC

The large-scale expression of human isoQC in shake flasks
was performed in a final volume of 4 L, essentially as
described for the mini-scale expression for activity screen-
ing. In addition, the absorbance at 600 nm was adjusted to
unity for the change of BMGY to BMMY. Cells were
removed from the isoQC-containing supernatant by centri-
fugation at 8000 g at 4 °C for 20 min. The pH was adjusted
to 7.0 by the addition of KOH, and the resulting turbid
solution was centrifuged at 16 000 g for 30 min at 4 °C.
Table 4. Oligonucleotides used for the cloning of isoQC constructs.
Oligonucleotide Sequence (5¢-to3¢), restriction sites (italics) Purpose
1 CTGCGGGTCCCATTGAACGGAAGCCTCCCCGAA Introduction of an N-glycosylation site
2 TTCGGGGAGGCTTCCGTTCAATGGGACCCGCAG
3 ACGGTACACAACTTGGCCCGCATTCTCGCTGTG Site-directed mutagenesis C369A
4 CACAGCGAGAATGCGGGCCAAGTTGTGTACCGT
5 ATAT
GAATTCTTCTACACCATTTGGAGC Cloning of isoQC
(F48)
C-His into pPICZaA
6 ATATAT
GCGGCCGCCTAGTGATGGTGATGGTGATGGAGCCCCAGGTATTCAGCCAG
7 ATAT
GAATTCCATCACCATCACCATCACTTCTACACCATTTGGAGCGGC Cloning of isoQC
(F48)
N-His into pPICZaA
8 ATATAT
GCGGCCGCCTAGAGCCCCAGGTATTCAGC
9 ATAT
GAATTCCATCACCATCACCATCACGAGGAGCTGCCGCTGGGCCG Cloning of isoQC
(E60)

N-His
10 ATAT
GAATTCGAGGAGATGTCACGGAGC Cloning of murine isoQC
(E61)
into pPICZaA
11 ATATAT
GCGGCCGCCTAGAGTCCCAGGTACTCGGC
12 GATCTGCGGGTCCCGCTGAACGGAAGCCTTTCAGAAGCC Introduction of an N-glycosylation site
13 GGCTTCTGAAAGGCTTCCGTTCAGCGGGACCCGCAGATC
14 ATAT
GAATTCCATCACCATCACCATCACGAGGAGATGTCACGGAGCCGC Cloning of isoQC
(E60)
N-His into pPICZaA
15 ATAT
GAATTCCATCACCATCACCATCACTTCTATATCGTCTGGAACAGC Cloning of isoQC
(F48)
N-His into pPICZaA
Characterization of glutaminyl cyclase isoenzymes A. Stephan et al.
6532 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS
The purification of His-tagged human isoQC was initi-
ated by IMAC. The culture supernatant was applied to an
Ni
2+
-loaded Chelating Streamline column (2.5 · 22 cm)
(GE Healthcare, Munich, Germany) in expanded bed mode
at a flow rate of 12 mLÆmin
)1
. The column was equilibrated
with 50 mm phosphate buffer, pH 7.0, containing 300 mm
NaCl. After washing with at least 10 column volumes of

equilibration buffer, the bound protein was eluted by a shift
to 50 mm phosphate buffer, pH 7.0, containing 150 mm
NaCl and 100 mm His. Human isoQC-containing fractions
were pooled, and ammonium sulfate was added to a final
concentration of 1 m. The resulting solution was used for
hydrophobic interaction chromatography, employing a
butyl-Sepharose FF column (1.6 · 13 cm) at a flow rate of
4mLÆmin
)1
. The bound protein was washed with 50 mm
phosphate buffer, pH 6.8, containing 1 m ammonium
sulfate for five column volumes, and eluted in a reversed
flow direction with 50 mm phosphate buffer, pH 6.8. Frac-
tions exhibiting isoQC were pooled and applied to a HiPrep
desalting column (2.6 · 10 cm), which was equilibrated
with 50 mm Bis-Tris, pH 6.8, 100 mm NaCl. The purifica-
tion was analyzed by SDS-PAGE and the protein content
was determined according to the methods of Bradford [40]
or Gill and von Hippel [41]. The purified human isoQC
variants were stable for months in 50% glycerol at )20 °C.
Large-scale expression and purification of murine
isoQC
Large-scale expression of murine isoQC was carried out in
a 5 L reactor with 2 L of fermentation medium (Biostat B,
B. Braun Biotech, Melsungen, Germany), essentially as
described elsewhere [4]. Briefly, fermentation was initiated
in basal salt medium supplemented with trace salts at pH
5.5. Biomass was accumulated in a batch and a fed batch
phase with glycerol as the sole carbon source for about
28 h. Expression of isoQC was induced by methanol feed-

ing according to a three-step profile recommended by Invi-
trogen (‘Pichia fermentation process guidelines’). The
fermentation process was stopped after 65 h. Afterwards,
the cells were separated from the medium by centrifugation
at 8000 g for 20 min.
Ammonium sulfate was added to the supernatant to a
final concentration of 0.8 m and the samples were again
subjected to centrifugation. The supernatant was applied
onto a Butyl Streamline column (2.5 · 22 cm) (GE Health-
care, Munich, Germany) in expanded bed mode at a flow
rate of 15 mLÆmin
)1
. The column was equilibrated with
50 mm phosphate buffer, pH 7.0, containing 0.8 m
(NH
4
)
2
SO
4
. After washing with at least five column vol-
umes of equilibration buffer, the bound protein was eluted
by a shift to 50 mm phosphate buffer, pH 7.0, lacking
(NH
4
)
2
SO
4
. The isoQC-containing fractions were pooled,

and ammonium sulfate was added to a final concentra-
tion of 0.7 m. The resulting solution was applied to a
Butyl-Sepharose FF column (1.6 · 13 cm) at a flow rate of
4mLÆmin
)1
. Bound protein was washed with 50 mm phos-
phate buffer, pH 7.0, containing 0.7 m ammonium sulfate
for four column volumes, and eluted using a gradient from
0.7 to 0.0 m ammonium sulfate over five column volumes.
Fractions exhibiting isoQC were pooled and desalted over-
night by dialysis against 30 mm Bis-Tris, pH 6.8. After-
wards, the protein was applied (4.0 mLÆmin
)1
) to a Uno Q
column (1.2 · 5.3 cm) (BioRad), which was equilibrated
with 30 mm Bis-Tris, pH 6.8. After a washing step using
equilibration buffer, murine isoQC was eluted, applying a
gradient of 0–450 mm NaCl in 10 column volumes. In the
last purification step, size exclusion chromatography was
carried out with a Superdex 75 resin (column 2.6 · 85 cm),
applying a buffer consisting of 30 mm NaH
2
PO
4
, contain-
ing 0.5 m NaCl. The purification steps were analyzed by
SDS-PAGE. The purified murine isoQC was stored
at )20 °C after the addition of 50% glycerol, or without
glycerol at )80 °C.
Enzyme assays and analysis

IsoQC activity was evaluated using H-Gln-bNA substrate
at 30 °C, as described elsewhere [42]. Briefly, the samples
consisted of 0.2 mm fluorogenic substrate, 0.25 units of
pyroglutamyl aminopeptidase in 50 mm Tris ⁄ HCl, pH 8.0,
and an appropriately diluted aliquot of isoQC in a final
volume of 250 lL. For inhibitor testing, the sample compo-
sition was the same as described above, except for the puta-
tive inhibitory compound. Because of the pronounced
substrate inhibition of H-Gln-bNA, inhibitory constants
were determined using H-Gln-AMC at a concentration
range between 0.25 and 4K
m
.
For the investigation of substrate specificity, isoQC activ-
ity was analyzed spectrophotometrically using glutamic
dehydrogenase as the auxiliary enzyme [39,43]. Samples
consisted of varying concentrations of QC substrates (0.25–
10K
m
), 0.3 mm NADH, 14 mm a-ketoglutaric acid and
30 UÆmL
)1
glutamic dehydrogenase in a final volume of
250 lL. The fluorometric assay using H-Gln-AMC was
applied to investigate the pH dependence of the QC-cata-
lyzed substrate turnover, as described elsewhere [5,24]. The
reaction buffer consisted of 0.2 m Tris ⁄ HCl, 0.1 m Mes and
0.1 m acetic acid, adjusted to the desired pH using HCl or
NaOH. This buffer provides a constant ionic strength over
a broad pH range [44]. The resulting kinetic data were eval-

uated by applying a three-inflection-point mathematical
model (bell-shaped curve) using GraFit software (version
5.0.4. for Windows). The QC activity determinations were
carried out under first-order rate law conditions, i.e. at sub-
strate concentrations below 0.25K
m
. Thus, the results repre-
sent the pH dependence of the specificity constants k
cat
⁄ K
m
.
All reactions were carried out at 30 °C, using either the
Polarstar (BMG Labtech, Jena, Germany) or Sunrise
(TECAN, Crailsheim, Germany) reader for microplates.
A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes
FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6533
TXRF and CD spectroscopy
Murine isoQC was desalted by size exclusion chromatogra-
phy using a Sephadex G-25 desalting column (1.0 · 10 cm),
which was equilibrated in 10 mm Tris ⁄ HCl, pH 7.6. The
isoQC-containing fractions were collected and the protein
was concentrated to approximately 3 mgÆmL
)1
by ultracen-
trifugation. Element analysis was performed using TXRF,
as described elsewhere [5,19]. The elution buffer was used
as a background control. Five microliters of undiluted sam-
ple solution or control buffer were applied to the TXRF
quartz glass sample support and dried under IR radiation.

Afterwards, 5 lL of diluted Se aqueous standard solution
(internal standard; Sigma-Aldrich) was added to each sam-
ple and dried again. The X-ray fluorescence signal was col-
lected for 100 s. For all determinations, an Extra II TXRF
module containing molybdenum and tungsten primary
X-ray sources (Seifert, Ahrensburg, Germany), connected
to a Link QX 2000 detector ⁄ analysis device (Oxford Instru-
ments, High Wycombe, UK), was used. The X-ray sources
were operated at 50 kV and 38 mA.
For CD spectroscopic analysis, the proteins were dialyzed
against buffer containing 10 mm NaH
2
PO
4
. CD spectra of
murine QC and murine isoQC were acquired with a Jasco
J-715 spectrapolarimeter using quartz cuvettes of 0.1 cm
path length. The mean of 10 wavelength scans between 190
and 260 nm was calculated, and the spectra were corrected
by subtraction of the buffer spectra. The apo-enzymes and
reactivation of the enzymes were confirmed by QC activity
measurements after spectral analysis.
Inactivation/reactivation and CD spectral analysis
Murine isoQC and mQC were inactivated by dialysis
against 1.0 L of buffer containing 5 mm 1,10-phenanthro-
line, 5 mm EDTA, 500 mm NaCl in 50 mm Bis-Tris, pH
6.8 at 4 °C overnight. The chelating agents were separated
from the apo-enzymes by dialysis against 1 L of either
50 mm Bis-Tris, pH 6.8, 500 mm NaCl, containing 50 gÆL
)1

Chelex-100 (BioRad) or 10 mm NaH
2
PO
4
, pH 6.8, contain-
ing 50 gÆ L
)1
Chelex-100 at 4 °C. The buffer was changed
twice, after 2 and 4 h of dialysis. The final dialysis was per-
formed for 5 h. All buffers were prepared in metal-free
polystyrene containers. Subsequently, the apo-enzyme was
centrifuged at 20 000 g for 1 h at 4 °C, and the protein
concentration was determined at 280 nm.
Reactivation experiments were carried out by incubation
of 20 lL of transition metal solution, prepared in water
(UltraPure, Merck, Darmstadt, Germany), with 20 lLof
apo-enzyme, which was dialyzed against the buffer contain-
ing Bis-Tris at room temperature for 15 min. Finally, the
enzymatic activity was assessed as described above, except
that the reaction buffer contained 2 mm EDTA in order to
avoid rapid reactivation of the enzymes by adventitious
zinc ions present in the buffer.
Acknowledgement
This work was supported by the Investitionsbank
Sachsen-Anhalt, grant number 0904/00007.
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