Paralog of the formylglycine-generating enzyme –
retention in the endoplasmic reticulum by canonical and
noncanonical signals
Santosh Lakshmi Gande
1
, Malaiyalam Mariappan
1
, Bernhard Schmidt
1
, Thomas H. Pringle
2
,
Kurt von Figura
1
and Thomas Dierks
3
1 Zentrum fu
¨
r Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universita
¨
tGo
¨
ttingen, Germany
2 Sperling Foundation, Eugene, OR, USA
3 Fakulta
¨
tfu
¨
r Chemie, Biochemie I, Universita
¨
t Bielefeld, Germany

tsstr. 25,
33615 Bielefeld, Germany
Fax: +49 521 106 6014
Tel: +49 521 106 2092
E-mail:
Website: />chemie/bc1/bc1.htm
(Received 28 October 2007, revised 17
December 2007, accepted 4 January 2008)
doi:10.1111/j.1742-4658.2008.06271.x
Formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sul-
fatases the oxidation of a specific cysteine residue to formylglycine, which
is the catalytic residue required for sulfate ester hydrolysis. This post-trans-
lational modification occurs in the endoplasmic reticulum (ER), and is an
essential step in the biogenesis of this enzyme family. A paralog of FGE
(pFGE) also localizes to the ER. It shares many properties with FGE, but
lacks formylglycine-generating activity. There is evidence that FGE and
pFGE act in concert, possibly by forming complexes with sulfatases and
one another. Here we show that human pFGE, but not FGE, is retained in
the ER through its C-terminal tetrapeptide PGEL, a noncanonical variant
of the classic KDEL ER-retention signal. Surprisingly, PGEL, although
having two nonconsensus residues (PG), confers efficient ER retention
when fused to a secretory protein. Inducible coexpression of pFGE at dif-
ferent levels in FGE-expressing cells did not significantly influence the
kinetics of FGE secretion, suggesting that pFGE is not a retention factor
for FGE in vivo. PGEL is accessible at the surface of the pFGE structure.
It is found in 21 mammalian species with available pFGE sequences. Other
species carry either canonical signals (eight mammals and 26 nonmammals)
or different noncanonical variants (six mammals and six nonmammals).
Among the latter, SGEL was tested and found to also confer ER retention.
Although evolutionarily conserved for mammalian pFGE, the PGEL signal

lacks the enzymatic FGly-generating activity of FGE,
as it lacks the two catalytic cysteines, Cys336 and
Cys341, in the active site of FGE [23–25]. pFGE has a
substrate-binding groove similar to FGE, and shows
weak binding of sulfatase-derived synthetic peptides
in vitro [23–25]. Also in vivo, pFGE seems to contact
nascent sulfatases in the ER. Moreover, pFGE over-
expression interferes with FGly formation, thereby
counteracting FGE function [23,24]. The exact role of
pFGE in this process and how this regulatory effect is
brought about is presently under investigation.
Several observations, including yeast two-hybrid and
biochemical data, are in agreement with heterodimer
formation of FGE and pFGE [23,24], and indeed, Zito
et al. [24] have found multimeric complexes with and
without sulfatases by coimmunoprecipitation. The
structural pFGE dimer found in pFGE crystals and
superposition with the FGE monomer suggests that
heterodimer formation is feasible in a face-to-face
manner with regard to the substrate-binding cleft [25].
Heterodimer formation could be stabilized by an
unfolded sulfatase polypeptide, which might explain
the regulatory function of pFGE. Alternatively, the
inhibitory effect of pFGE observed on FGE function
could be indirect, namely by competing for a common
ER retention mechanism, thereby dislocating FGE
from the ER. In fact, a small fraction of endogenous
pFGE was found to be secreted, and upon overexpres-
sion, pFGE could be detected in other cellular com-
partments of the secretory pathway [23].

ogeneous processing of its N-glycan in the secretory
pathway [23]. Treatment with up to 8 ngÆmL
)1
doxycy-
cline for 28 h led to expression of pFGE ranging from
0.34 to 6.1 lg of pFGE per mg of cell protein (Fig. 1).
Analysis of cells and medium revealed that retention
of pFGE was decreasing with increasing expression
levels, with about 50% retention at the lowest and
12% retention at the highest expression level. This lat-
ter value (about 10% of ‘retained’ protein) is likely to
largely represent newly synthesized material on its way
to the cell surface, because typically no more than
90% of total protein is found in the medium, even in
the case of a native secretory protein (see below). This
indicated that the mechanism used for pFGE retention
is saturable.
The C-terminus is involved in ER retention of
pFGE
In initial experiments, we had expressed human pFGE
carrying a His
6
-tag at the C-terminus to facilitate
detection and purification of pFGE. In these experi-
ments, we noted that about 90% of the tagged pFGE
was secreted [23]. To analyze a possible effect of
the C-terminal His
6
-tag on retention ⁄ secretion of
pFGE, tagged and untagged (wild-type) pFGE were

from platypus, the snail Biomphalaria glabrata, the pla-
narian Schmidtea mediterranea, and the sea anemone
Nematostella vectensis. However, human pFGE and
also pFGE from 20 further mammalian species (from
various primates to squirrel, bat, dolphin, sloth and
wallaby) carry a C-terminal PGEL tetrapeptide, i.e.
lacking the critical basic residue in position 1 but with
an acidic residue and a leucine in positions 3 and 4,
typical for the KDEL retention signal (Fig. 3). More-
over, there are further variants of the PGEL motif in
pFGEs, such as FGEL (guinea pig), MGEL (hyrax),
SGEL (opossum), PEEL (tree shrew, lemur), PREL
(kangaroo rat), and PDEL (lamprey). With the excep-
tion of the latter, these species, like all PGEL species,
are mammals. It should be noted that both proline (or
methionine and phenylalanine) in the first position and
glycine in the second position do not fit with the gen-
eral KDEL-like signal consensus [KRHQSA]-[DENQ]-
E-L deposited in the PROSITE database [26].
The C-terminal PGEL and SGEL tetrapeptides
function as retention signals for pFGE
To look for a potential ER retention function of the
C-terminal PGEL tetrapeptide of human pFGE, three
mutant pFGE proteins were constructed with differ-
ent C-termini. The PGEL tetrapeptide was either
deleted (truncated pFGE) or substituted either by the
canonical KDEL or by SGEL, one of the other non-
canonical tetrapeptide sequences (see above). In sev-
eral independent experiments, one of which is shown
in Fig. 4A, truncated pFGE was mostly secreted,

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Fig. 3. Canonical and noncanonical ER
retention signals in SUMF-encoded proteins.
SUMF2-encoded pFGE and SUMF1-encoded
FGE sequences were recovered from data-
bases (see Experimental procedures; data-
base mining freeze date August 2007) for
67 and 69 species, respectively. Species are
given with their systematic and common

encoded KDEL signals in all species. The
given occurrence of KDELR genes is based
on species for which full-length sequences
could be recovered (those indicated by an
asterisk and other species, not shown).
S. L. Gande et al. ER retention by noncanonical retention signals
FEBS Journal 275 (2008) 1118–1130 ª 2008 The Authors Journal compilation ª 2008 FEBS 1121
effective ER retention. In conclusion, the noncanoni-
cal PGEL and SGEL sequences serve as retention
signals for pFGE.
The data shown above reflect the levels of pFGE
inside and outside the cells 24 h after induction of pFGE
expression. To kinetically analyze retention and secre-
tion of newly synthesized pFGE protein, doxycycline-
induced cells were metabolically labeled for 90 min with
[
35
S]methionine ⁄ cysteine and analyzed by immuno-
precipitation of pFGE from cell lysates and medium
immediately or after 3 and 6 h of chase in unlabeled
growth medium. The data obtained clearly show that
truncated pFGE was significantly secreted already dur-
ing pulse labeling. After 3 h of chase, only 25% of trun-
cated pFGE were retained within the cells (Fig. 4B). On
the contrary, very little of the wild-type and the KDEL
form of pFGE was secreted during the pulse, and most
of these forms were retained intracellularly after 3 h of
chase (88% and 66%, respectively).
The C-terminal PGEL tetrapeptide is
an autonomous ER retention signal

A
B
Fig. 4. Retention of pFGE with and without a C-terminal KDEL,
PGEL or SGEL tetrapeptide. pFGE and C-terminal variants of pFGE
(see text), as indicated, were transiently expressed in stable
HT1080 Tet-On cells (cf. Fig. 1). (A) Six hours after transfection, the
cells were induced with 2 ngÆmL
)1
doxycycline. After induction for
24 h, cells and medium (at a ratio of 10 : 1) were analyzed for
pFGE by western blotting. (B) Twelve hours after induction with
0.5 ngÆmL
)1
doxycycline, cells were starved for 1 h and then meta-
bolically labeled for 90 min with [
35
S]methionine ⁄ cysteine. pFGE
was immunoprecipitated from cell lysates and medium, harvested
after 0, 3 and 6 h of chase. Equal aliquots of precipitates from cells
and medium were analyzed by SDS ⁄ PAGE and phosphorimaging.
Bands were quantified; intracellularly retained pFGE is given as per-
centage of total.
Fig. 5. The PGEL tetrapeptide confers ER retention to lysozyme.
Myc-tagged lysozyme and its C-terminally extended variants (see
text) were transiently expressed in stable HT1080 Tet-On cells. Six
hours after transfection, the cells were induced with 1 lgÆmL
)1
doxycycline. Equal aliquots of cells and medium were analyzed by
western blotting, using c-Myc-specific antibodies. The intracellularly
retained lysozyme is given below the lanes as percentage of total.

tion, and was linear with time for another 10–12 h
(Fig. 7A). To find out whether FGE secretion
is reduced by coexpression of pFGE, FGE was
transiently expressed in Tet-On HT1080 cells stably
expressing pFGE (Fig. 7B). Twelve hours after trans-
fection with the FGE plasmid (the starting point of
A
B
C
D
Fig. 6. PGEL-mediated retention of lyso-
zyme in the ER. HT1080 Tet-On cells tran-
siently expressing (at 2 lgÆmL
)1
doxycycline
induction) pFGE (A) or lysozyme–c-Myc
without (B) or with C-terminal KDEL (C) or
PGEL extension (D) were analyzed by indi-
rect immunofluorescence microscopy (see
Experimental procedures). The merge
reveals colocalization of pFGE and lyso-
zyme–c-Myc with the ER marker PDI medi-
ated by the C-terminal KDEL ⁄ PGEL
extensions. A fraction of lysozyme–c-Myc-
PGEL is detected in Golgi-like structures, as
indicated by the arrows (D).
S. L. Gande et al. ER retention by noncanonical retention signals
FEBS Journal 275 (2008) 1118–1130 ª 2008 The Authors Journal compilation ª 2008 FEBS 1123
linear FGE secretion), pFGE expression was induced
by addition of doxycycline. Measuring intracellular

conferred ER retention. Thus, PGEL is an autono-
mous retention signal. It conferred ER retention with
similar (pFGE) or almost (76%) similar (lysozyme)
efficiency as KDEL itself (Figs 4 and 5). On the one
hand, this is surprising, as Pelham et al. [28] found
that even the canonical HDEL, i.e. the yeast prototype
retention signal with a rather conservative exchange in
the first position, cannot substitute for KDEL in medi-
ating lysozyme retention in COS cells. On the other
hand, in vitro experiments have shown quite efficient
binding of an HDEL tetrapeptide and even weak bind-
ing of a DDEL tetrapeptide [29]. The latter acts as a
low-efficiency retrieval signal when present at the
C-terminus of lysozyme in COS cells coexpressing
either the hERD2.1 or hERD2.2 KDEL receptor [30].
Here, we also studied another noncanonical variant,
SGEL, as a representative of six further PGEL-like
signals found in pFGE, and observed that it also con-
ferred ER retention (Fig. 4A).
The PGEL tetrapeptide is accessible at the
surface of the pFGE molecule
The PGEL C-terminus of pFGE is located on the sur-
face of the molecule as part of an eight amino acid
extension (AGRPPGEL) of a three-stranded b-sheet
opposite to the monomer–monomer interface (Fig. 8)
[25]. The last seven residues including the PGEL could
not be resolved in the crystal structure, suggesting that
they show a high degree of flexibility. As the directly
A
B

a KDEL-type extension at the highly conserved C-ter-
minal region constituting the catalytic site of FGE.
Eight of those 11 species lack pFGE, because – despite
generally high sequencing coverage – the SUMF2 gene
is undetectable (Fig. 3). The presence of a retention
signal on either FGE or pFGE lends support to the
idea that in those species pFGE and FGE mutually
act as retention factors, involving heterodimer forma-
tion. On the other hand, pFGE ⁄ SUMF2 is systemati-
cally absent from the Insecta, whereas all 18 of the
insect species have FGE ⁄ SUMF1, which, however,
lack a KDEL signal (Fig. 3). Thus, there are species in
which a retrieval signal is provided by no, one or both
SUMF-encoded proteins. Importantly, for all species
expressing both FGE and pFGE, heterodimer forma-
tion as a prerequisite for ER retention could apply, as
pFGE always carries a KDEL-related signal.
In fact, there are examples of a retention mechanism
through hetero-oligomer formation with [KRH]-D-E-
L-containing proteins, such as PDI ⁄ prolyl hydroxylase
[31–33] and b-glucuronidase ⁄ egasyn [34]. Similarly,
Ero1 retention occurs through disulfide bridge forma-
tion with RDEL containing ERp44 [35]. pFGE⁄ FGE
heterodimerization and ternary complex formation
with sulfatases was reported by Zito et al. [24] to serve
as a regulatory mechanism for FGE activity. We have
to point out that, although we have several indications
for binding of pFGE to sulfatases as well as to FGE,
we have failed so far to biochemically prove the exis-
tence of pFGE ⁄ FGE heterocomplexes. Our experimen-

RTEL, KTEL, HQEL). The remaining 50 sequences
differ in either the first position [proline (25 sequences),
methionine (one), phenylalanine (one), or threonine
Fig. 8. The C-termini of the pFGE dimer are exposed at the surface
of the molecule. The ribbon model of the pFGE dimer 3D structure
is shown, as determined through X-ray crystallography [25]. The
three N-terminal residues (27-ATS-29, in red) and C-terminal resi-
dues (292-ADA-294, in yellow) of the resolved structure are shown
in stick representation. Ala27 represents the N-terminus of the
mature form of pFGE. The C-terminal residues 295–301 including
PGEL are not visible in the crystal. The two calcium ions in each of
the monomers are shown as gray spheres.
S. L. Gande et al. ER retention by noncanonical retention signals
FEBS Journal 275 (2008) 1118–1130 ª 2008 The Authors Journal compilation ª 2008 FEBS 1125
(one)], in the second position [glycine (28), alanine
(six), proline (one), methionine (one), arginine (one),
or lysine (one)], or in both positions (24). The PGEL
motif accounts for 21 of the 24 sequences with non-
consensus residues in both positions.
In Fig. 3, the pFGE C-termini of all 67 species are
ordered according to the modern taxonomic tree. It
becomes obvious that pFGEs with canonical retention
signals (colored in blue) originated first, and that the
PGEL signal (red) developed with the mammalian line-
age. PGEL can be found in many different phyloge-
netic groups, from marsupials (wallaby) to primates.
However, within these groups, there is fluctuation
between canonical KDEL-like, PGEL and other non-
canonical retrieval signals (green) in the end leaves of
various subclades. We thus conclude that the invention

retention
The topology of the KDEL receptor-binding pocket
has been probed and found to involve four hydrophilic
residues (Arg5, Asp50, Tyr162 and Asn165 in
KDELR1) located in three different transmembrane
helices, which are highly conserved and found in all
three human KDEL receptor isoforms [29]. These and
other data led to a model in which the KDEL peptide
inserts into a charge-lined pocket formed by the trans-
membrane helices [39]. Asp50 has been suggested to
form an ion pair with the normally positively charged
first residue of the KDEL-type signal. Such ion pairs
are supposed to contribute to the very pH-sensitive
association–dissociation equilibrium, with association
being favored in the slightly acidic environment of the
Golgi, and quantitative dissociation in the neutral ER,
which has a higher pH by roughly 0.5 units [40]. This
view seems to contrast with the finding reported here
that even the nonpolar proline in the PGEL motif con-
fers ER retention. However, in vitro peptide-binding
experiments suggest that this ion pair is not obligatory,
at least not for the association step, and that the
sequence directly upstream of the KDEL-type tetra-
peptide contributes to the interaction with the receptor
[29]. Moreover, mutagenesis of Asp50 did not affect
binding of DDEL-containing ligands in vitro and
in vivo, which suggests that different retrieval signals
make different contacts in the binding pocket.
The variability in the retrieval signature could also
be related to the existence of three paralogous verte-

specific for pFGE remains elusive at present. As long
as the role of pFGE in sulfatase activation through
FGE remains speculative, one can only suppose that
FGE function and possibly trafficking is regulated via
pFGE. If it is true that FGE trafficking out of the cell
eventually reaches even the ER of other cells [42],
anterograde and retrograde transport of this essential
activator of sulfatases definitely need complex regula-
tion.
Experimental procedures
Construction of expression plasmids
C-terminal tetrapeptide variants of pFGE and lysozyme were
constructed by cloning corresponding cDNAs into multiclon-
ing site I of the pBI Tet vector (BD Biosciences, Heidelberg,
Germany), which allows the simultaneous expression of two
genes of interest from one bidirectional tet-responsive pro-
moter. For cloning wild-type pFGE, pFGEDPGEL (‘trun-
cated pFGE’) and pFGE with KDEL or SGEL instead of
PGEL, pFGE cDNA [23] served as a template for an add-on
PCR using 5¢-CTAGCTAGCCACCATGGCCCGGCAT
GGGTTAC-3¢ as a forward primer, and reverse primers
5¢-TCTAGAGATATCTACAGCTCCCCTGGCG-3¢ (for
wild-type pFGE), 5¢-TCTAGAGATATCTACGGCCGGC
CTGCGTC-3¢ (pFGEDPGEL), 5¢-TCTAGAGATATCTA
CAGCTCGTCTTTCGGCCGGCCTG-3¢ (pFGE-KDEL),
or 5¢-TCTAGAGATATCTACAGCTCCCCGGACGGCC-3¢
(pFGE-SGEL). An NheI site was added at the 5¢-end and an
EcoRV site at the 3¢-end, which facilitated directional cloning
of the PCR product into multicloning site I.
For cloning wild-type lysozyme–c-Myc and lysozyme–

)1
. Stable clones were screened first for
doxycycline-dependent fluorescence after transient trans-
fection with a pBI-EGFP plasmid. The best clones were
then rescreened through western blotting for doxycycline-
dependent pFGE production after transient transfection
with pBI-pFGE.
A Tet-On cell-line stably expressing pFGE under control
of a doxycycline-responsive promoter was established by
cotransfecting Tet-On cells with pBI-pFGE and the puro-
mycin resistance vector pSV.pac (10 : 1 ratio). Transfec-
tants were selected as mentioned above and screened for
pFGE expression.
Transient transfections of HT1080 Tet-On cells were per-
formed using Lipofectamine 2000, following the protocol
from Invitrogen. Typically, 2 lg of expression plasmid
DNA (see above) was used for a 3 cm dish. After 6 h of
transfection, medium was replaced by DMEM with various
concentrations of doxycycline ranging between 0 and
1000 ngÆmL
)1
, as indicated for each experiment (see Results
and legends to Figs 1, 4, 5, 6 and 7). Cells and medium
were harvested after 24 h of induction, unless otherwise
specified (see figure legends), and analyzed by western blot-
ting.
Western blotting
For western blot detection of pFGE, FGE and lysozyme–
c-Myc, polyclonal antibodies to pFGE [23], FGE [27]
and c-Myc (Sigma, Taufkirchen, Germany) were used as

tification of pFGE was done using macbas software (Fuji,
Tokyo, Japan).
Immunofluorescence microscopy
HT1080 Tet-on cells were grown on coverslips overnight,
and, the next day, transfected with pBI-based plasmids
allowing transient expression of pFGE or various con-
structs of lysozyme–c-Myc with or without KDEL ⁄ PGEL
extension (see figure legend). After 6 h of transfection, the
cells were induced with 2 lgÆmL
)1
doxycycline. After 24 h
of transfection, the cells were washed twice with NaCl ⁄ P
i
,
fixed with 4% paraformaldehyde for 20 min, and treated
with 50 mm NH
4
Cl for 10 min. The cells were permeabi-
lized with 0.5% saponin in NaCl ⁄ P
i
, incubated with
primary antibodies [rabbit antiserum raised against pFGE
[23], rabbit polyclonal antibodies against c-Myc (Sigma),
and mouse monoclonal antibodies against PDI (Stressgen
Biotechnologies, Ann Arbor, MI, USA)] for 1 h at room
temperature. After being washed three times with NaCl⁄ P
i
containing 0.1% saponin, the primary antibodies were
decorated for 1 h with appropriate secondary antibodies
coupled with either Cy2 or Alexa 546 (Molecular Probes,

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positions 3 and 4. Species with canonical retention
signals are colored in blue, and noncanonical signals
in red (PGEL only) or green (other noncanonical).
Fig. S2. SUMF1-encoded FGEs of some invertebrate
species carry a KDEL-type ER retention signal.
SUMF1 sequences were recovered for the indicated
69 species, which are ordered phylogenetically (see
Fig. 3). C-terminal FGE regions encoded in the last
exon are aligned. Species with an FGE C-terminus
containing a terminal KDEL-type retention signal,
indicated in bold, are colored in blue (canonical signal)
or green (noncanonical signal).
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than missing material) should be directed to the corre-
sponding author for the article.
ER retention by noncanonical retention signals S. L. Gande et al.
1130 FEBS Journal 275 (2008) 1118–1130 ª 2008 The Authors Journal compilation ª 2008 FEBS