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

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

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

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

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

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
∗
∗

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.

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

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.

(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

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

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 +

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-

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
-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.

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

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 (

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)

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

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

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

¨
sser HP, Mann M & Hasilik A (2007)
Integral and associated lysosomal membrane proteins.
Traffic 8, 1676–1686.
3Lu
¨
bke T, Lobel P & Sleat DE (2008) Proteomics of the
lysosome. Biochim Biophys Acta, doi:10.1016/j.bbamcr.
2008.09.018.
4 Pohlmann R, Waheed A, Hasilik A & von Figura K
(1982) Synthesis of phosphorylated recognition marker
in lysosomal enzymes is located in the cis part of Golgi
apparatus. J Biol Chem 257, 5323–5325.
5 Ghosh P, Dahms NM & Kornfeld S (2003) Mannose
6-phosphate receptors: new twists in the tale. Nat Rev
4, 202–212.
6 Sleat DE, Lackland H, Wang Y, Sohar I, Xiao G, Li H
& Lobel P (2005) The human brain mannose 6-phos-
phate glycoproteome: a complex mixture composed of
multiple isoforms of many soluble lysosomal proteins.
Proteomics 5, 1520–1532.
7 Kollmann K, Mutenda KE, Balleininger M, Eckermann
E, von Figura K, Schmidt B & Lu
¨
bke T (2005) Identifi-
cation of novel lysosomal matrix proteins by proteome
analysis. Proteomics 5, 3966–3978.
8 Sleat DE, Zheng H & Lobel P (2007) The human urine
mannose 6-phosphate glycoproteome. Biochim Biophys
Acta 1774, 368–372.

16 Wendland M, Waheed A, Schmidt B, Hille A, Nagel G,
von Figura K & Pohlmann R (1991) Glycosylation of
the Mr 46,000 mannose 6-phosphate receptor. effect on
ligand binding, stability, and conformation. J Biol
Chem 266, 4598–4604.
17 Tsiakas K, Steinfeld R, Storch S, Ezaki J, Lukacs Z,
Kominami E, Kohlschu
¨
tter A, Ullrich K & Braulke T
(2004) Mutation of the glycosylated asparagine residue
286 in human CLN2 protein results in loss of enzymatic
activity. Glycobiology 14, 1C–5C.
18 Remington SJ & Breddam K (1994) Carboxypeptidas-
es C and D. Meth Enzymol 244, 231–248.
19 Galjart NJ, Morreau H, Willemsen R, Gillemans N,
Bonten EJ & d’Azzo A (1991) Human lysosomal pro-
tective protein has cathepsin A-like activity distinct
from its protective function. J Biol Chem 266, 14754–
14762.
20 Odya CE, Marinkovic DV, Hammon KJ, Stewart TA
& Erdos EG (1978) Purification and properties of
prolylcarboxypeptidase (angiotensinase C) from human
kidney. J Biol Chem 253, 5927–5931.
21 Mahoney JA, Ntolosi B, DaSilva RP, Gordon S &
McKnight AJ (2001) Cloning and characterization of
CPVL, a novel serine carboxypeptidase, from human
macrophages. Genomics 72, 243–251.
22 Harris J, Schwinn N, Mahoney JA, Lin HH,
Shaw M, Howard CJ, da Silva RP & Gordon S (2006)
A vitellogenic-like carboxypeptidase expressed by

on lysosomal proteins. Mol Cell Proteomics 5, 686–
701.
30 Dierks T, Schmidt B, Borissenko LV, Peng J, Preusser
A, Mariappan M & von Figura K (2003) Multiple sul-
fatase deficiency is caused by mutations in the gene
encoding the human C(alpha)-formylglycine generating
enzyme. Cell 113, 435–444.
31 Cosma MP, Pepe S, Annunziata I, Newbold RF,
Grompe M, Parenti G & Ballabio A (2003) The multi-
ple sulfatase deficiency gene encodes an essential and
limiting factor for the activity of sulfatases. Cell 113,
445–456.
32 Gopalakrishnan MM, Grosch HW, Locatelli-Hoops S,
Werth N, Smolenova E, Nettersheim M, Sandhoff K &
Hasilik A (2004) Purified recombinant human prosapo-
sin forms oligomers that bind procathepsin D and affect
its autoactivation. Biochem J 383, 507–515.
33 Seyrantepe V, Hinek A, Peng J, Fedjaev M, Ernest S,
Kadota Y, Canuel M, Itoh K, Morales CR, Lavoie J
et al. (2008) Enzymatic activity of lysosomal carboxy-
peptidase (cathepsin) A is required for proper elastic
fiber formation and inactivation of endothelin-1. Circu-
lation 117, 1973–1981.
34 Zhou XY, Morreau H, Rottier R, Davis D, Bonten E,
Gillemans N, Wenger D, Grosveld FG, Doherty P,
Suzuki K et al. (1995) Mouse model for the lysosomal
disorder galactosialidosis and correction of the pheno-
type with overexpressing erythroid precursor cells.
Genes Dev 9, 2623–2634.
35 Pohlmann R, Boeker MW & von Figura K (1995) The

spectrophotometric assay for human plasma carboxy-
peptidase N1. Anal Biochem 108, 348–353.
43 Caeyenberghs K, Balschun D, Roces DP, Schwake M,
Saftig P & D’Hooge R (2006) Multivariate neuro-
cognitive and emotional profile of a mannosidosis
murine model for therapy assessment. Neurobiol Dis 23,
422–432.
44 Ko
¨
ster A, Saftig P, Matzner U, von Figura K, Peters C
& Pohlmann R (1993) Targeted disruption of the M(r)
46,000 mannose 6-phosphate receptor gene in mice
results in misrouting of lysosomal proteins. EMBO J
12, 5219–5223.
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.
Please note: Wiley-Blackwell is not responsible
for the content or functionality of any supplementary