Báo cáo khoa học: Molecular characterization and gene disruption of mouse lysosomal putative serine carboxypeptidase 1 ppt

Molecular characterization and gene disruption of mouse
lysosomal putative serine carboxypeptidase 1
Katrin Kollmann
1
, Markus Damme
1
, Florian Deuschl
1
,Jo
¨
rg Kahle
2
, Rudi D’Hooge
3
,
Renate Lu
¨
llmann-Rauch
4
and Torben Lu
¨
bke
1
1 Abteilung Biochemie II, Georg-August Universita
¨
tGo
¨
ttingen, Germany
2 Abteilung Molekularbiologie, Georg-August Universita
¨
tGo

¨
ttingen, Germany
3 Laboratory of Biological Psychology, KU Leuven, Belgium
4 Anatomisches Institut, Universita
¨
t Kiel, Germany
The lysosomal compartment plays a pivotal role in the
degradation of macromolecules within the cell. To
date, over 60 soluble lysosomal hydrolases and acces-
sory proteins and 25 lysosomal membrane proteins
have been identified [1–3]. Defects in the lysosomal
proteins mostly result in one of about 50 lysosomal
storage diseases (LSDs) which are characterized by the
accumulation of undigested materials in the lysosomes.
As a result of the clinical relevance of soluble lyso-
somal proteins in LSDs and a notable number of
LSD-like diseases of unknown etiology, there is a com-
mon interest in the identification of the proteome of
the lysosomal compartment and of the soluble luminal
lysosomal mannose 6-phosphate (M6P)-containing
Keywords
gene disruption; lysosomes; processing;
Scpep1; serine carboxypeptidase
Correspondence
T. Lu
¨
bke, Zentrum Biochemie und
Molekulare Zellbiologie, Abteilung
Biochemie II, Georg-August Universita
¨

t
Go
¨
ttingen, Heinrich-Du
¨
ker-Weg 12,
D-37073 Go
¨
ttingen, Germany
Fax: +49 551 395979
Tel: +49 551 395932
E-mail:
(Received 4 July 2008, revised 18
December 2008, accepted 23 December
2008)
doi:10.1111/j.1742-4658.2009.06877.x
The retinoid-inducible serine carboxypeptidase 1 (Scpep1; formerly RISC)
is a lysosomal matrix protein that was initially identified in a screen for
genes induced by retinoic acid. Recently, it has been spotlighted by several
proteome analyses of the lysosomal compartment, but its cellular function
and properties remain unknown to date. In this study, Scpep1 from mice
was analysed with regard to its intracellular processing into a mature dimer
consisting of a 35 kDa N-terminal fragment and a so far unknown 18 kDa
C-terminal fragment and the glycosylation status of the mature Scpep1
fragment. Although Scpep1 shares notable homology and a number of
structural hallmarks with the well-described lysosomal carboxypeptidase
protective protein ⁄ cathepsin A, the purified recombinant 55 kDa precursor
and the homogenates of Scpep1-overexpressing cells do not show proteo-
lytic activity or increased serine carboxypeptidase activity towards artificial
serine carboxypeptidase substrates. Hence, we disrupted the Scpep1 gene in

mice by a gene trap cassette, resulting in a Scpep1 ⁄ b-galactosidase ⁄ neo-
mycin phosphotransferase fusion protein. The fusion protein is devoid of
the C-terminal half of Scpep1, including two amino acids of the assumed
catalytic triad which is indispensable for its predicted serine carboxypepti-
dase activity. However, Scpep1-deficient mice were viable and fertile, and
did not exhibit either lysosomal storage or reduced lysosomal SC activity
under any tested condition.
Abbreviations
AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; CBZ, benzyloxycarbonyl; Cpvl, carboxypeptidase vitellogenic-like; CPY, carboxypeptidase Y;
Ctsa, protective protein ⁄ cathepsin A; FA, furylacryloyl; geo, b-galactosidase ⁄ neomycin phosphotransferase; Lamp1, lysosomal associated
membrane protein 1; LSD, lysosomal storage disease; M6P, mannose 6-phosphate; MEFs, mouse embryonic fibroblasts; MPR, mannose
6-phosphate receptor; PNGase F, peptide N-glycosidase F; RISC, retinoid-inducible serine carboxypeptidase; SC, serine carboxypeptidase;
Scpep1, serine carboxypeptidase 1; Scpep1-gt, Scpep1 gene trap.
1356 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS
proteins in particular. Soluble lysosomal proteins
receive M6P residues on their N-linked oligosaccha-
rides [4], which are recognized in the trans-Golgi net-
work by M6P receptors (MPRs) required for
lysosomal transport [5]. By exploiting the M6P recog-
nition marker of the soluble lysosomal proteins for
MPR-dependent affinity chromatography, followed by
their identification by mass spectrometry, we and
others have identified a novel putative serine carboxy-
peptidase 1 (Scpep1) [6–9]. Originally, Scpep1 was
identified in rat aortic smooth muscle cells by a screen-
ing for retinoid-inducible genes, as reflected by its ini-
tial name, ‘retinoid-inducible serine carboxypeptidase’
(RISC) [10]. Northern blot analyses demonstrated high
transcript levels in kidney and aorta in rat and lower
levels in heart, spleen and lung, whereas the human

transcript was detected strongly in the kidney and
heart but at a low level in a number of other tissues
[10]. In mice, Scpep1 is expressed in embryonic heart
and vasculature, as well as in a broad range of adult
tissues [10]. The mouse Scpep1 gene (GeneID: 74617;
cDNA Accession No. NM_029023) encodes a product
of 452 amino acids (Protein Accession No.
NP_0832299) that localizes to the lysosomes [11]. Fur-
thermore, it has been demonstrated that, in mice, a
55 kDa Scpep1 precursor is processed into a 35 kDa
form [11]. Although no peptidase activity has been
demonstrated so far, Scpep1 has been assigned to the
serine carboxypeptidase (SC) family S10 because of
reasonable sequence homology to members of this
family, such as the lysosomal protective pro-
tein ⁄ cathepsin A (official gene name Ctsa; 35% simi-
larity) and four conserved domains that are predicted
to constitute the substrate-binding site and three cata-
lytic sites. Each of these catalytic sites accounts for
one amino acid of the catalytic triad Ser-Asp-His
[10,12]. To obtain an insight into the physiological and
cellular function of the putative lysosomal SC Scpep1,
we analysed the molecular properties of Scpep1 and
generated an Scpep1 gene trap (Scpep1-gt) mouse
model.
Results
Molecular forms of Scpep1
In order to generate Scpep1-specific antisera, we
purified a C-terminally His-tagged version of full-
length mouse Scpep1 from secretions of stably

expressing HT1080 cells (HT1080-Scpep1). Coomassie
and silver staining after SDS-PAGE revealed an
apparent molecular size of 55 kDa for the secreted
and purified His-tagged Scpep1 starting with Ile29,
as identified by N-terminal sequencing. We derived
Scpep1-specific antisera from rat and rabbit. Both
antisera were suitable for confirming the lysosomal
localization of endogenous Scpep1 by immunofluores-
cence (see Fig. S1).
Western blot analysis using an antibody directed
against the C-terminal His-tag detected the 55 kDa
Scpep1 in cell extracts and in the medium of HT1080-
Scpep1 cells (Fig. 1A, lanes 3 and 4), but not in
untransfected HT1080 cells (lanes 1 and 2).
Unexpectedly, an additional 18 kDa form of Scpep1
was detectable by the anti-His IgG1 in homogenates of
HT1080-Scpep1 cells (lane 3), thus representing the
C-terminal fragment of processed Scpep1.
Both of our Scpep1-specific antisera detected the
55 kDa precursor in homogenates and secretions of
HT1080-Scpep1 cells (lanes 7, 8 and 11, 12), as well as
a strong signal at 35 kDa in the homogenates (lanes 7
and 11), representing the N-terminal moiety of
processed Scpep1. Rabbit antiserum showed low
B
α
α
1pe
pcS-
tibba

r
Standard
(kDa)
Retention time
(min)
20 25
30
40
35
18
mn082DO
160 67 43 13.7
α
-His
α
-Scpep1
α
-Scpep1
rabbit rat
150
75
50
37
25
20
15
10
C M C M C M C M C M C M
55
35

18
kDa
kDa
0801TH
-
0801TH
siH-1
p
epcS
08
01
T
H
-0801TH
s
i
H-1
p
e
p
cS
0
8
0
1T
H
-080
1
TH
siH-1pepcS

A
Lane 1 2 3 4 5 6 7 8 9 10 11 12
Fig. 1. Molecular forms of Scpep1. (A) Analysis of molecular forms
of Scpep1: 100 lg of cell lysates (C) and 50 lL of medium (M) of
HT1080 and HT1080-Scpep1 were separated by SDS-PAGE, blotted
and probed with the a-His antibody and the a-Scpep1 antisera from
rabbit and rat, respectively. (B) Gel filtration analysis of a lysosome-
enriched fraction (F2): 50 lg of F2 were buffered in 20 m
M Mes
(pH 4.5) containing 150 m
M NaCl, loaded onto a Superdex 75 col-
umn on an analytic SMARTÔ system (Pharmacia) and eluted in
20 lL fractions at a flow rate of 40 lLÆmin
)1
, which were analysed
by western blot using the rabbit a-Scpep1 antibody. A mixture of
molecular mass standard proteins, including IgG (160 kDa), albumin
(67 kDa), ovalbumin (43 kDa) and ribonuclease A (13.7 kDa), was
applied to gel filtration under the same conditions.
K. Kollmann et al. Functional characterization of lysosomal Scpep1
FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1357
cross-reactivity with polypeptides in homogenates of
untransfected human HT1080 (lane 5), whereas no
specific signal could be detected in the medium (lane 6)
or with rat antiserum in HT1080 cells (lanes 9 and 10).
Omitting the reducing agents did not alter the mobility
properties in SDS-PAGE (data not shown). However,
gel filtration chromatography with a lysosome-enriched
fraction from mouse liver showed that the 35 and
18 kDa subunits of Scpep1 co-eluted in the same

fractions with an apparent size between 43 and 67 kDa
(Fig. 1B), suggesting that both fragments are linked to
each other noncovalently.
Processing of Scpep1
To investigate the processing of the endogenous
Scpep1 precursor in mice, mouse embryonic fibroblasts
(MEFs) were pulse labelled with [
35
S]methionine for
1 h and chased for up to 72 h. After immunoprecipita-
tion, Scpep1 was separated by SDS-PAGE and analy-
sed by autoradiography (Fig. 2A). In MEFs which had
been labelled for 1 h (0 h chase), only the  55 kDa
precursor was detected. After 2 h of chase, half of the
55 kDa precursor had been processed into 37 and
20 kDa intermediates. Between 4 and 6 h of chase, the
55 kDa precursor disappeared, whereas the 37 kDa
intermediate and the final 18 kDa fragment which was
derived from the 20 kDa fragment showed constant
signals. At 24 h of chase, the 35 kDa fragment was
finally processed, and both fragments remained detect-
able even after 72 h of chase (Fig. 2A). The medium
did not show any specific Scpep1 signal (data not
shown).
Some lysosomal peptidases, such as cathepsin B and
cathepsin D, undergo autoproteolytic activation
[13,14]. To investigate the autoproteolytic activation of
Scpep1, we incubated 0.16–3.2 lm of recombinant
55 kDa Scpep1 precursor at varying pH (pH 4.5 and
pH 7.5), temperature (4 °C, room temperature, 37 °C)

and incubation time (1–16 h), but could not detect any
processing of the precursor into mature forms by
western blot analysis (data not shown).
In order to further define the Scpep1-processing pro-
tease, MEFs were pulse labelled in the presence or
absence of various protease inhibitors. The conversion
of the 55 kDa Scpep1 precursor into the mature
form was sensitive to the serine protease inhibitor
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)
(Fig. 2B). However, two other serine protease inhibi-
tors, aprotinin and antipain, had no effect, although
the latter interferes with cathepsin A activity [15]. Pep-
statin A, an aspartic protease inhibitor, and the cyste-
ine protease inhibitor E-64, as well as the metal
0 2 4 6 24 48 72
A
B
C
Chase (h)
55
35
150
100
75
50
37
kDa
18
∗
∗

∗
250
25
20
15
MEF
kDa
∗
MEF
Chase (h)
––
ATDE
46-E
FSBEA
xiM-IP
150
100
75
50
37
kDa
250
25
20
15
kDa
0 4
∗
∗
55

35
18
Chase (h) 0 6 0 6
55
35
NH
4
Cl
–+
150
100
75
50
37
kDa
HT1080-Scpep1
C M C M C M C M
% of t
0
100 – 15 45 100 – 20 55
Antipain
Aprotinin
Pepstatin A
Fig. 2. Processing of Scpep1. (A) Immunoprecipitation of Scpep1
from MEFs using the rat-derived Scpep antiserum after pulse label-
ling with [
35
S]methionine for 1 h. Cells were chased for up to 72 h.
Nonspecific signals are marked with asterisks (*) at chase time 0.
(B) Effects of protease inhibitors on the processing of Scpep1.

MEFs were pulse labelled and chased for 0 or 4 h in the absence
()) or presence of the following inhibitors: EDTA (2 m
M final con-
centration), E-64 (10 l
M), pepstatin A (100 lM), AEBSF (1 mM),
aprotinin (0.3 l
M), antipain (75 lM) and a mixture containing all
inhibitors in the assigned concentrations. Scpep1 was immunopre-
cipitated and visualized by autoradiography. (C) Processing of
Scpep1 in NH
4
Cl-treated HT1080-Scpep1 cells. Intracellular (C) or
secreted (M) Scpep1 was immunoprecipitated after pulse labelling
and 0 or 6 h of chase in the absence ()) and presence (+) of the
lysosomotropic agent NH
4
Cl.
Functional characterization of lysosomal Scpep1 K. Kollmann et al.
1358 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS
chelator EDTA, had no effect on Scpep1 maturation.
These results suggest that, under our test conditions, a
serine protease different from cathepsin A mediates
Scpep1 processing.
In order to investigate whether Scpep1 maturation
occurs in the endosomal–lysosomal compartment,
HT1080-Scpep1 cells were pulse labelled in the absence
or presence of NH
4
Cl (Fig. 2C). In untreated HT1080-
Scpep1 cells, the 55 kDa precursor was processed into

the 35 kDa fragment, but the 18 kDa fragment was
not detected. In addition, HT1080-Scpep1 cells
secreted large amounts of the 55 kDa precursor after
6 h of chase (45% of the total Scpep1 signal at chase
0 h). NH
4
Cl interfered with the intracellular processing
of the precursor to the 35 kDa form, but only moder-
ately enhanced the secretion of the precursor (55% of
the Scpep1 signal at chase 0 h). These results indicate
that Scpep1 matures in late endosomes or lysosomes
and could be targeted in an M6P-dependent manner.
Glycosylation of Scpep1 in MEFs
The amino acid sequence of Scpep1 contains five puta-
tive N-glycosylation sites, four of which are located
within the 35 kDa N-terminal fragment (Asn64,
Asn102, Asn126, Asn192) and one within the 18 kDa
C-terminal fragment (Asn362). MEFs were pulse
labelled for 1 h and chased for 4 h, and immunopre-
cipitated Scpep1 was subjected to peptide N-glycosi-
dase F (PNGase F) treatment for 1 h and separated by
SDS-PAGE (Fig. 3). The major form of the Scpep1
precursor from MEFs migrated at an apparent molec-
ular mass of 55 kDa (lane 1). In addition, a minor
signal was detected with a slightly reduced molecular
mass of  52 kDa that was partially covered by the
55 kDa form and most probably represents a less gly-
cosylated Scpep1. PNGase F treatment of the precur-
sor resulted in a shift towards  40 kDa in size
(lane 2). After 4 h of chase, the  55 kDa precursor

was completely converted into two major mature
subunits of 35 and 18 kDa in size and a minor signal
of  30 kDa that might arise from the 52 kDa precur-
sor form (lane 3). The limited deglycosylation of the
35 kDa subunit led to a deglycosylated  25 kDa frag-
ment via the  30 kDa fragment and an intermediate
of 28 kDa (lane 4). PNGase F treatment of the
18 kDa subunit resulted in a partial shift towards a
lower molecular mass of  16 kDa (lane 4). Consider-
ing an apparent molecular mass of about 2–2.5 kDa
per N-linked oligosaccharide [16,17], the results suggest
that, in MEFs, all N-glycosylation sites are utilized.
SC activity of Scpep1
Like carboxypeptidase Y (CPY) and Ctsa, Scpep1 is
classified as a member of the SC type C family
(S10.013, MEROPS database) and should preferen-
tially exhibit proteolytic activity at acidic pH towards
hydrophobic amino acids in the P1¢ position [18]. Puri-
fied Scpep1 protein, mainly consisting of the 55 kDa
precursor, recombinant CPY as a positive control and
BSA were tested for SC activity towards different
N-terminal blocked peptides, such as CBZ-Phe-Leu
and FA-Phe-Phe (CBZ, benzyloxycarbonyl; FA, furyl-
acryloyl), representing SC type C substrates, FA-Ala-
Lys, as an SC type D substrate, and the non-SC
substrate CBZ-Gly-Leu [19]. Although CPY cleaved
SC substrates such as FA-Phe-Phe in a pH-dependent
manner, neither purified Scpep1 precursor nor BSA
showed proteolytic activity under any test condition
(Fig. 4).

As most lysosomal hydrolases are not active as zym-
ogens, we determined the SC activity in homogenates
of HT1080 cells and HT1080-Scpep1 cells (data not
shown). Although the latter mainly show Scpep1 in its
processed form, we could not detect any differences in
acid SC activity. To exclude cell line and vector-
specific effects of His-tagged Scpep1, we assayed
Chase (h) 0 4
55
PNGase F –+ –+
MEF
35
18
3
2
0
1
0
1
∗
∗
Lane 1 2 3 4
Fig. 3. Glycosylation of Scpep1. MEFs were pulse labelled and
chased for 4 h. Scpep1 was immunoprecipitated from the lysates,
treated with PNGase F and separated by SDS-PAGE. The filled
arrowheads point to the fully glycosylated forms of Scpep1 with
the number of their N-glycans, and the open arrowheads to degly-
cosylated forms of the 35 kDa processed form and the 18 kDa pro-
cessed form. Nonspecific signals are marked with asterisks (*) at
chase time 0.

K. Kollmann et al. Functional characterization of lysosomal Scpep1
FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1359
HT1080 cells that had been transiently transfected with
an untagged variant of Scpep1, His-tagged Scpep1 or
the mock vector (pcDNA3.1-Hygro) (Fig. 5A), as well
as COS-7 cells transfected with Scpep1-His6 and the
pCI-neo mock vector (Fig. 5B). Although large
amounts of processed Scpep1 were detected in the
homogenates, no differences in acid SC activity could be
measured regardless of the cell line and substrate used.
Scpep1-gt mice
In order to obtain an insight into Scpep1 function,
we generated a gene trap mouse model. A blast of
Scpep1 cDNA within the BayGenomics (San Fran-
cisco, CA, USA) database identified the ES cell line
RST426 as a Scpep1-gt cell line. The gene trap vector
used by BayGenomics contains a splice acceptor site
upstream of a b-galactosidase ⁄ neomycin phosphotrans-
ferase (geo) fusion gene (Fig. S2), which inserted into
intron 7 of the Scpep1 gene as confirmed by genomic
sequencing. Hence, the downstream exons 8–13 were
deleted from the gene trap transcript and were
replaced by the promoterless geo cassette. Most
importantly, two amino acids, Asp371 and His431, of
the putative catalytic triad for SC activity were
excluded from the resulting fusion product.
As gene trapping does not consistently result in the
inactivation of a gene, we checked for Scpep1 mRNA
with a 3¢-specific probe by northern blot analyses
(Fig. S3) and for Scpep1 protein by western blotting

(Fig. 5A). In Scpep1-gt mice, the 35 kDa Scpep1 signal
was absent from virtually all tissues tested. However,
an antibody against the geo moiety of the gene trap
fusion product detected a 200 kDa protein in the
tissues of Scpep1-gt mice, corresponding to the 35 kDa
Scpep1 expression pattern in wild-type mice,
confirming the calculated size for the Scpep1-geo
fusion protein of about 200 kDa (Fig. 6A).
Subcellular fractionation of mouse liver after Triton
WR-1339 (tyloxapol) injection, including differential
centrifugation steps followed by a discontinuous
sucrose gradient, enables the isolation of a fraction
(F2) which is  50-fold enriched in lysosomal marker
enzymes such as b-hexosaminidase. Western blot ana-
lysis of each fraction of the lysosomal purification from
wild-type mice showed co-fractionation of the pro-
cessed 35 kDa Scpep1 and 18 kDa Scpep1 with lyso-
somal proteins such as cathepsin D (Ctsd) and
lysosomal associated membrane protein 1 (Lamp1) in
fraction F2 (Fig. 6B). Western blot analyses from sub-
cellular fractions derived from Scpep1-gt mice failed to
detect Scpep1 in fraction F2 (Fig. 6C). In contrast, the
geo antibody revealed a specific  200 kDa signal in
the microsomal fraction P, indicating that the Scpep1-
geo fusion product was retained in the endoplasmic
reticulum and ⁄ or in the Golgi (Fig. 6C).
Phenotype of Scpep1-gt mice
Genotyping of 350 offspring from heterozygous bree-
dings showed the expected Mendelian frequency with
23.6% homozygous Scpep1-gt mice, indicating that

Scpep1 is not essential for correct embryonic develop-
ment. Homozygous Scpep1-gt mice and wild-type mice
showed comparable sizes and weight developments, as
well as fertility and mortality (data not shown). Deter-
minations of blood (full blood count), serum (e.g.
aspartate aminotransferase, c-glutamyl transferase)
and urine parameters showed no pathological findings.
The activities of several lysosomal hydrolases were
normal in various tissues and lysosomes from liver and
kidney of Scpep1-gt mice, and were inconspicuous with
regard to their distribution in a Percoll gradient, indi-
cating that the density and size of the lysosomes were
unaltered (data not shown).
The following organs were regularly examined histo-
logically using semithin sections: liver, lung, kidney,
spleen, pancreas, retina, cornea, and spinal cord; in
some instances, the inner ear (cochlea) and cerebellar
cortex were also investigated. Ultrastructural examina-
tion was performed on liver and spinal cord of two
wild-type and two Scpep-gt mice. We were unable to
find any consistent differences between wild-type and
Scpep-gt mice. In particular, there was no evidence of
lysosomal storage in any of the numerous cell types
inspected.
3.5 4.5 5.5 6.5 7.5 8.5
0.0
2.5
5.0
7.5
10.0

pH
ytivitca CS
.ceps
(U·mg
–1
)
Fig. 4. SC activity determination. C-terminally His-tagged 55 kDa
Scpep1 precursor (h) was purified from stably expressing HT1080
cells and incubated at different pH values ranging from 3.5 to 8.5
with FA-Phe-Phe as SC substrate. Yeast CPY (d) served as a posi-
tive control and BSA (D) as a negative control.
Functional characterization of lysosomal Scpep1 K. Kollmann et al.
1360 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS
SC activity in Scpep1-gt mice
Homogenates from various tissues and lysosome-
enriched fractions from liver (F2) of control mice and
Scpep1-gt mice showed equal levels of acid SC activity
(data not shown). We further separated F2 fractions
from control and Scpep1-gt mice by gel filtration and
tested each fraction for Scpep1 and Ctsa by western
blot analysis and acid SC activity. The Ctsa elution
profiles were similar in both F2 fractions ranging from
1.0
2.0
3.0
4.0
CBZ-Phe-Leu CBZ-Leu-Phe CBZ-Gly-Leu
α
1p
e

pcS
-
α
-GAPDH
0801TH
kcom +
1pepcS +
A
Substrate
*
55
35
75
50
37
25
kDa
37
6siH-1pepcS +
0
8
01TH
kcom +
1pepcS +
6siH-
1p
epc
S
+
080

1TH
kcom +
1
pepc
S
+
6siH-1pepcS

+
080
1TH
kcom +
1pepcS +
6s
iH-1pepcS +
0.5
1.0
1.5
2.0
CBZ-Phe-Leu CBZ-Leu-Phe CBZ-Gly-Leu
B
α
1pepcS-
α
-GAPDH
7-SOC
kco
m
+
6siH-1pepcS +

7-S
O
C
kc
om +
6siH-1pepcS

+
7-SOC
kcom +
6siH-1pepcS

+
Substrate
7-
S
O
C
kcom +
6siH-1
p
epc
S
+
*
55
35
75
50
37

25
kDa
37
Specific activity of
acid carboxypeptidases (U·mg
–1
)
Specific activity of
acid carboxypeptidases (U·mg
–1
)
Fig. 5. Expression and acid SC activity of Scpep1 in COS-7 and HT1080 cells. HT1080 (A) and COS (B) cells were transfected with either
the appropriate mock construct (pCI-neo for COS-7; pcDNA3.1-Hgyro for HT1080), Scpep1 or Scpep-His6, as indicated, and assayed for acid
SC activity using various artificial substrates. The columns represent the mean of three technical replicates for each cell line. Scpep1 expres-
sion was monitored by western blot analysis using 100 lg of the cell homogenates, and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) served as loading control. Nonspecific signals are marked with asterisks (*).
K. Kollmann et al. Functional characterization of lysosomal Scpep1
FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1361
fraction 16 to 18 (Fig. 7), whereas Scpep1 signals were
solely detectable in fractions 15–17 from control mice
(Fig. 7). However, the lysosomal SC activity distribu-
tion was roughly identical in both elution profiles,
regardless of the presence or absence of Scpep1
(Fig. 7). Thus, Scpep1 did not show proteolytic activity
towards common lysosomal SC type C and D
substrates.
Discussion
To date, four putative lysosomal SCs have been
identified, but proteolytic activity has only been
proven for Ctsa and the distantly related prolyl-

carboxypeptidase [19,20]. The third putative SC,
carboxypeptidase vitellogenic-like (Cpvl), has been
reported to be a lysosomal SC restricted to
T W
t g
T W
t g
T W
t g
T W
t
g
T W
t g
T W
t g
T W
t g
T W
t g
T W
t g
T W
t g
1 p e p c S
l o r t
n
o c
T W
t g

α
α
1 p e p c S -
α
α
-geo
kDa
25
20
100
75
50
37
15
α
α
-GAPDH
34
250
150
0 8 0 1 T H
F E M
A
α
α
-Scpep1
α
α
-Ctsd
α

α
-Lamp1
kDa
-37
-25
- 150
- 100
-20
-20
-50
-37
-25
α
α
-geo
-150
-50
-75
N E M L P S F1 F2 F3 F4
B
C
Wild-type mice
Fraction
α
α
-Scpep1
α
α
-geo
α

α
-Lamp1
-100
- 150
-37
-25
-20
-50
-75
N E M L P S F1 F2 F3 F4
Scpep1-gt mice
Fraction
Testis
Liver
Kidney
Intestine
Stomach
Bladder
Brain
Lung
Heart
Spleen
Fig. 6. Differential western blot analysis of
Scpep1 expression in tissues from wild-type
(WT) and Scpep1-gt mice. (A) Protein from
various tissue extracts (200 lg per lane) and
cell lysates (HT1080 and MEF, 50 lg per
lane) were separated by SDS-PAGE, blotted
onto poly(vinylidene difluoride) membrane
and probed with the antibodies as indicated.

Tyloxapol-filled lysosomes from mouse liver
of control mice (B) and Scpep1-gt mice (C)
were separated by differential centrifugation
(corresponding to fractions N–S). Fraction L
(light mitochondria) was loaded under a
sucrose gradient (F1–F4), resulting in a
codistribution of Scpep1 with the lysosomal
marker proteins cathepsin D (Ctsd) and
Lamp1 in F2, as shown by western blot
analysis after SDS-PAGE loaded with
250 lg for each fraction of the differential
centrifugation (N–S) and 50 lg of F1–F4 of
the sucrose gradient. The blot membrane
was additionally probed with antibodies
against neomycin phosphotransferase
(a-geo). E, postnuclear fraction; F1–F4,
sucrose gradient fractions 1–4; L, light
mitochondria fraction; M, heavy
mitochondria fraction; N, nuclear fraction;
P, microsomal fraction; S, cytosolic fraction.
Functional characterization of lysosomal Scpep1 K. Kollmann et al.
1362 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS
macrophages [21]. Recently, it has been demonstrated
that Cpvl localizes to the endoplasmic reticulum
rather than to lysosomes, and hence a role in major
histocompatibility complex loading has been sug-
gested [22], but proof for the enzymatic activity of
Cpvl has not yet been served. In this study, we
focused on the characterization of the fourth
putative lysosomal SC, Scpep1, with regard to its

localization, processing, glycosylation and presumed
SC activity.
Molecular forms of Scpep1
Our antisera raised against the 55 kDa Scpep1
precursor confirmed the lysosomal localization of
endogenous Scpep1 by immunofluorescence and
co-fractionation, as postulated previously by our
group and others [7,11]. Tissue-specific expression
analyses were performed according to a recent study
[11], with highest Scpep1 levels found in visceral
organs such as the liver and kidney. Western blot
analyses of homogenates from HT1080-Scpep1 cells
and from a 50-fold lysosome-enriched fraction
revealed the presence of the expected 35 kDa pro-
cessed fragment and an as yet unknown 18 kDa C-
terminal fragment, in contrast with a recent publica-
tion [11] in which the processing of the Scpep1 pre-
cursor to a C-terminal 35 kDa fragment and a
putative, but undetected, N-terminal 16 kDa peptide
was postulated. The maturation from a zymogen into
a two-chain form bears resemblance to a number of
other SCs, such as barley SC [23] and lysosomal
Ctsa, in particular. Ctsa is synthesized as a 54 kDa
precursor and further processed into N-terminal
32 kDa and C-terminal 20 kDa polypeptides [24].
However, although both subunits of Ctsa are linked
to each other by disulfide bonds to form the 54 kDa
monomer [24], the two-chain form of Scpep1 does
not form disulfide bridges. Moreover, although Ctsa
dimerizes and, together with b-galactosidase and

sialidase, forms a large multienzyme complex [25],
Scpep1 from mouse liver elutes at  50 kDa in gel
filtration assays (Fig. 1B), as predicted for the mono-
mer, and in this regard resembles the yeast SCs
CPY [26] and KEX1 [27], which are also active as
monomers.
The maturation of lysosomal hydrolases from a
zymogen is essential for their functional activation,
and hence must be tightly regulated in terms of pro-
tecting the cell against self-digestion. We could not
mimic autoprocessing, as described in vitro for lyso-
somal cathepsin B, D or L [13,14,28]. The matura-
tion of the Scpep1 precursor into the 35 kDa
fragment is a multistep process (Fig. 2A), which is
prevented by the addition of the serine proteinase
inhibitor AEBSF (Fig. 2B), as well as by the NH
4
Cl-
mediated uncoupling of MPR-dependent lysosomal
transport (Fig. 2C), suggesting that Scpep1 process-
ing is mediated by a lysosomal serine proteinase.
Previously, we have demonstrated that partially puri-
fied mouse Scpep1 derived from BHK cells binds on
immobilized MPR46 and MPR300 and is internalized
by I-cell fibroblasts in an M6P-dependent manner
[7]. Limited deglycosylation by PNGase F digest
demonstrated that all putative N-glycosylation sites
are occupied in MEFs. Sleat et al. [29] identified
M6P sites on 92 MPR-binding proteins derived from
human and mouse brain, which were both of lyso-

somal function or unknown function. Although a
total of 135 M6P sites were identified in 69 proteins,
M6P sites on Scpep1 escaped the analysis [29]. Most
probably, these sites were missed because of the size
of tryptic peptides containing the N -glycosylation
sites, which range from 30 to 62 amino acids, and
thus may exceed the preset mass range of the MS
analysis [29].
13 16 17 18 1914 15
α-Ctsa
α-Ctsa
α-Scpep1
α-Scpep1
F2 WT
F2 gt
B
A
13 14 15 16 17 18 19
0
1
2
3
4
Fraction
Spec. SC activity (mU·mg
–1
)
Fig. 7. SC activity profiling and western blot analyses of Scpep1
and Ctsa after gel filtration of lysosome-enriched fractions. F2 frac-
tions derived from wild-type mice (s) and Scpep1-gt mice (

) were
separated on an FPLC Superdex 200 10 ⁄ 300 GL column in 20 m
M
Mes, pH 4.5, 150 mM NaCl. The collected fractions were assayed
for SC activity (A) and analysed by western blot analyses (B), using
the Scpep1 antiserum to detect the 35 kDa fragment and an anti-
Ctsa rat IgG2B to detect the 32 kDa heavy chain of Ctsa.
K. Kollmann et al. Functional characterization of lysosomal Scpep1
FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1363
The lysosomal localization, processing and conserva-
tion of critical domains are shared features of Scpep1
and Ctsa. However, under conditions adapted to SCs
such as Ctsa or CPY, we failed to demonstrate acid
SC activity of Scpep1 in any approach addressed so
far, regardless of which molecular form of Scpep1 or
substrate was assayed. Because of its zymogen status,
it is reasonable that the purified Scpep1 precursor does
not exhibit SC activity; however, surprisingly, homo-
genates of HT1080 and COS-7 cells, which highly
expressed and subsequently processed mouse Scpep1 in
a tagged or untagged version, did not show any
elevated acid SC activity. The lack of additional SC
activity on Scpep1 overexpression may be ascribed to
an unknown limiting factor of Scpep1 activation. As
an example, lysosomal sulfatases are modified in the
endoplasmic reticulum by the formylglycine-generating
enzyme, which has been shown to be an essential and
limiting factor for sulfatase activity [30,31]. Further-
more, it has been reported that the activation of the
cathepsin D precursor is accelerated when it is com-

plexed with prosaposin [32].
The Scpep1-gt mouse did not exhibit an obvious
phenotype and did not show any lysosomal storage,
although we confirmed the loss of the lysosomal
35 kDa mature form of Scpep1. Despite the deletion
of the entire C-terminus, including two critical amino
acids of the putative catalytic triad, we were unable to
show reduced acid SC activity in Scpep1-deficient
mice.
We would like to point out that the computational
modelling of Scpep1 also predicts a Ctsa- (1ivyA)
and CPY-like (1cpy_) folding (http://swissmodel.
expasy.org/SWISS-MODEL.html; Fig. 8). In addition,
the alignment of Scpep1 with several SCs from
mouse, Saccharomyces cerevisiae and Trypanoso-
ma cruzei identifies a highly conserved substrate
binding site (I), as well as three conserved catalytic
regions (II–IV), each embedding one amino acid of
the catalytic triad, and hence strongly favouring
acidic SC activity (Fig. 9).
Consequently, the apparent lack of in vitro SC
activity of Scpep1 in its processed form must be
ascribed either to the selection of an inappropriate
substrate or to a nonproteolytic function of Scpep1.
It is worth mentioning that another study failed to
demonstrate proteolytic activity of Scpep1 for Ctsa
substrates such as endothelin-1 and, in combination
with immunohistological studies, suggests a function
in the homeostasis of the renal and reproductive
Ctsa (1ivyA) Scpep1 (Model: 1ivyA)

CPY (1cpy_) Scpep1 (Model: 1cpy_ )
Fig. 8. Predicted three-dimensional struc-
ture of Scpep1. Scpep1 was homology
modelled with Ctsa and CPY as templates
to predict its three-dimensional structure
using the
SWISSMODEL alignment
mode ( />SWISS-MODEL.html).
Functional characterization of lysosomal Scpep1 K. Kollmann et al.
1364 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS
systems [11]. A recent gene target Ctsa mouse model
(Ctsa
S190A
), in which the catalytic serine residue was
substituted but the protective protein function was
preserved [33], does not develop a secondary galacto-
sialidosis like the ‘classic’ Ctsa-deficient mouse [34].
In Ctsa
S190A
mice, neither Scpep1 nor any other
predicted lysosomal SC efficiently compensates for
the loss of in vitro SC activity, resulting in residual
activities of 5–10% in visceral organs [33], although
Scpep1 and, moreover, Ctsa and Scpep1 are
Fig. 9. Multiple alignment of the primary amino acid sequences of SCs. SCs were aligned according to the CLUSTALW algorithm. Sequences
are highlighted in grey for two or three homologous sequences or in black for amino acids conserved in all four sequences. The substrate
binding site (I) is defined by the line. The catalytic domains embedding the amino acids of the catalytic triad (*) are marked as II–IV. Saccha-
romyces cerevisiae CPY (GI:115901); Trypanosoma cruzei SCP (GI:35181448); mouse Ctsa (GI:84042523); mouse Scpep1 (GI:13436038);
mouse vitellogenic-like carboxypeptidase VLCP (GI:187952735).
K. Kollmann et al. Functional characterization of lysosomal Scpep1

FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1365
expressed in similar cell types [11]. However, instead
of bulk protein storage, Ctsa
S190A
mice manifest
hypertension because of decreased endothelin-1 deg-
radation and, unexpectedly, a significant decrease in
elastic fibres in the skin, elastic arteries or lungs [33],
indicating that bulk proteolysis is compensated for
by other lysosomal proteases, whereas only the loss
of specific proteolytic functions contributes to the
phenotype. In order to reveal Scpep1 function, we
will continue to identify substrates for Scpep1. In an
affinity chromatography approach, we will immobi-
lize mutated Ser167Ala-Scpep1. Thus, substrate
binding should not be affected, but enzymatic activ-
ity should be hampered. With regard to the overall
function of lysosomal SCs, it could be insightful to
crossbreed Scpep1-deficient mice with Ctsa
S190A
mice
to investigate the overlap and distinct functions of
Scpep1 and Ctsa.
Materials and methods
Cell lines and cell culture
If not stated otherwise, the cell lines were grown in com-
plete Dulbecco’s modified Eagle’s medium (GIBCO Life
Technologies, Invitrogen GmbH, Karlsruhe, Germany)
supplemented with 10% fetal bovine serum (PAN Biotech
GmbH, Aidenbach, Germany), 1% penicillin ⁄ streptomycin

(GIBCO Life Technologies, Invitrogen) and 1% l-gluta-
mine (GIBCO Life Technologies, Invitrogen) at 37 °C
with 5% CO
2
.
Antibodies
Antibodies used for this study included monoclonal a-RGS-
His6-Tag mouse IgG1 (Qiagen GmbH, Hilden, Germany),
a-glyceraldehyde-3-phosphate dehydrogenase goat antiserum
(a-GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA,
USA), a-Ctsa rat IgG2B (R&D Systems, Minneapolis, MN,
USA), a-cathepsin D rabbit antiserum [35], a-Lamp1 rat
IgG2A (1D4B, Developmental Studies Hybridoma Bank,
Iowa City, IA, USA) and a-Scpep1 antisera derived from
rabbit and rat. Horseradish peroxidase- and fluorescence-
conjugated secondary antibodies were supplied by Dianova
(Hamburg, Germany) and Invitrogen, respectively.
Cloning, transfection and expression of Scpep1
The Scpep1 cDNA was subcloned into the pcDNA3.1 ⁄
Hygro(+) vector and pCI-neo (Promega GmbH, Mann-
heim, Germany) by add-on PCR, as described previously
[36]. HT1080 cells and COS-7 cells were transfected with
Fugene6 reagent (Roche GmbH, Mannheim, Germany) as
recommended by the manufacturer.
Purification of Scpep1-His6 from stably
expressing HT1080 cells
Scpep1-His6 was essentially purified from HT1080 cells
stably expressing and secreting Scpep1-His6, as described
previously for the 66.3 kDa protein [36].
MS and N-terminal sequencing (Edman digest)

Peptide mass fingerprint analysis was performed according
to [5] and Edman digest was performed according to [37].
Lysosome (tritosome) isolation
Mice were treated with a single injection of 0.75 mg of
tyloxapol (Sigma-Aldrich, Schnelldorf, Germany) per
gram body weight, 4 days prior to sacrifice. The isolation
of tritosomes, including differential centrifugation and iso-
pycnic centrifugation, was performed according to [38].
Deglycosylation by PNGase F
Cell lysates of HT1080-Scpep1 were subjected to PNGase F
(Roche) treatment as described previously [36].
Pulse labelling of MEFs and HT1080 cells
MEFs or HT1080 and HT1080-Scpep1 cells were plated
onto 10 cm dishes labelled with [
35
S]methionine ⁄ cysteine
(Hartmann Analytic, Braunschweig, Germany), followed by
immunoprecipitation with the rat a-Scpep antiserum, as
described previously for cathepsin D [39]. For some experi-
ments, HT1080-Scpep1 cells were treated with 20 mm
NH
4
Cl throughout the procedure or with protease inhibi-
tors during the chase period.
Carboxypeptidase activity assay of Scpep1
Proteolytic activity was assayed under standard conditions
for SC activity using N-blocked CBZ-Phe-Leu, CBZ-Leu-
Phe or FA-Phe-Phe, as SC type C substrate, FA-Ala-Lys as
an SC type D substrate and CBZ-Gly-Leu as a negative
control for both carboxypeptidase types [40]. All substrates

were supplied by Bachem, Weil am Rhein, Germany. The
released amino acids were determined according to [41] for
CBZ substrates and [42] for FA substrates.
Generation of the Scpep1-gt mouse model
The ES cell line RST426 (129 ⁄ ola) was obtained from Bay-
Genomics. RST426 cells were injected into C57BL ⁄ 6J blast-
ocysts to generate chimeric mice, which were mated with
C57BL ⁄ 6J females to test for germline transmission. The
resulting heterozygous F1 generation was intercrossed to
Functional characterization of lysosomal Scpep1 K. Kollmann et al.
1366 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS
obtain homozygous mutant offspring in a 129 ⁄ ola-
C57BL ⁄ 6J genetic background. All mice were kept in a
conventional animal facility at the Universita
¨
tsmedizin
Go
¨
ttingen.
Genotyping of Scpep1-gt mice
Genotyping on ES cells or tail biopsy was performed on
genomic DNA by either Southern blot analysis after BglII
digestion using a 600 bp PCR-generated probe upstream of
exon 7 or by multiplex-PCR, resulting in a 390 bp fragment
for the wild-type allele and a 200 bp fragment for the gene
trap allele. Primer sequences are shown in Table S1.
Histology
For morphological examination of various tissues, two
wild-type mice and four Scpep1-deficient mice (age,
9–12 months) were used. The animals were deeply anaesthe-

tized with 20 lL of a solution of 10 mgÆmL
)1
Ketavet
(Parke Davis GmbH, Berlin, Germany) and 2 mgÆmL
)1
Rompun (Bayer, Leverkusen, Germany) in 0.15 m NaCl
(intraperitoneal injection), and killed by transcardial perfu-
sion with NaCl ⁄ P
i
followed by glutaraldehyde (6%). Tissue
blocks were post-fixed with osmium tetroxide (2%) and
embedded in araldite. Semithin sections (1 lm) were stained
with toluidine blue and ultrathin sections were stained with
uranyl acetate and lead citrate, and were viewed with a
Zeiss EM 900 electron microscope (Carl Zeiss NTS GmbH,
Oberkochen, Germany).
Behavioural assessment
Fourteen wild-type and 16 gene trap females were examined
using a concise battery of tests. Details of the methods are
given in [43].
Other methods
Lysosomal enzymes were measured using photometric and
fluorometric assays, as described previously [44]. Gel fil-
tration assays with 50 or 1000 lg of a lysosome-enriched
fraction (F2) were performed on a SMARTÔ-HPLC sys-
tem (Pharmacia, GE Healthcare, Freiburg, Germany) and
an A
¨
ktaÔ-Purifier FPLC system (GE Healthcare, Frei-
burg, Germany) with a Superdex 200 PC 3.2 ⁄ 30 column

(Pharmacia) and Superdex 200 10 ⁄ 300 GL column (GE
Healthcare), respectively, in 20 mm Mes, pH 4.5, 150 mm
NaCl.
Acknowledgements
We are grateful to Martina Balleininger, Ellen Ecker-
mann-Felkl, Nicole Eiselt, Klaus Neifer, Leen van
Aerschot and Dagmar Niemeier for excellent technical
assistance, and Bernhard Schmidt for MS expertise
and advice. We thank Professor Kurt von Figura for
stimulating discussions and critical reading of the man-
uscript. This work was supported by the Deutsche
Forschungsgemeinschaft (Grants LU 1173 ⁄ 1-1 and
1-2). The mouse ES cell line RST426 was provided by
Baygenomics, a Program for Genetic Application
(PGA) funded by the National Heart Lung and Blood
Institute (NHLBI).
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Supporting information
The following supplementary material is available:
Fig. S1. Lysosomal localization of endogenous Scpep1
by immunofluorescence.
Fig. S2. Disruption of the Scpep1 gene. (A) Insertion
of the b-geo gene trap cassette into the Scpep1 gene.
(B) Southern blot. (C) Multiplex-PCR analysis of
genomic DNA derived from three F2 mice.
Fig. S3. Multitissue northern blot analysis with RNA
derived from wild-type (+ ⁄ +) mouse tissues, Scpep1-
gt () ⁄ )) tissues and heterozygous tissues (+ ⁄ )).
Table S1. List of primers used in this study.
This supplementary material can be found in the
online version of this article.
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(other than missing material) should be directed to
the corresponding author for the article.
K. Kollmann et al. Functional characterization of lysosomal Scpep1
FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1369