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.. Here we show t
Trang 1retention in the endoplasmic reticulum by canonical and noncanonical signals
Santosh Lakshmi Gande1, Malaiyalam Mariappan1, Bernhard Schmidt1, Thomas H Pringle2,
Kurt von Figura1and Thomas Dierks3
1 Zentrum fu¨r Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universita¨t Go¨ttingen, Germany
2 Sperling Foundation, Eugene, OR, USA
3 Fakulta¨t fu¨r Chemie, Biochemie I, Universita¨t Bielefeld, Germany
In the catalytic center of eukaryotic and prokaryotic
sulfatases, a unique amino acid, Ca-formylglycine
(FGly), can be found that is essential for enzymatic
activity [1–5] The FGly participates as an aldehyde
hydrate in the hydrolysis of sulfate esters according to
a novel trans-sulfation⁄ elimination mechanism [5–9]
The FGly in all eukaryotic and in most prokaryotic
sulfatases is post-translationally generated by oxidation
of a specific cysteine residue and, in most cases, this oxidation is catalyzed by the recently discovered form-ylglycine-generating enzyme (FGE), a novel oxygenase with unusual structural and catalytic properties [10–15] The genetic defect of FGE in human leads to multiple sulfatase deficiency, a rare inherited disorder
Keywords
endoplasmic reticulum;
formylglycine-generating enzyme; KDEL receptor; protein
retention; SUMF2
Correspondence
T Dierks, Fakulta¨t fu¨r Chemie, Biochemie I,
Universita¨t Bielefeld, Universita¨tsstr 25,
33615 Bielefeld, Germany
Fax: +49 521 106 6014
Tel: +49 521 106 2092
E-mail: thomas.dierks@uni-bielefeld.de
Website: http://www.uni-bielefeld.de/
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
is found only in one further human protein entering the ER Its conse-quences for KDEL receptor-mediated ER retrieval and benefit for pFGE functionality remain to be fully resolved
Abbreviations
ER, endoplasmic reticulum; FGE, C a -formylglycine-generating enzyme; FGly, C a -formylglycine; PDI, protein disulfide isomerase;
pFGE, paralog of Ca-formylglycine-generating enzyme.
Trang 2of a fatal nature that is characterized by the synthesis
of catalytically inactive sulfatases lacking FGly
[12,13,16,17] All multiple sulfatase deficiency patients
analyzed so far carried mutations in the FGE-encoding
SUMF1gene [12,13,18–20]
In eukaryotes, a paralog gene (SUMF2) encoding a
paralog of FGE (pFGE) can be traced back reliably
via conserved sequence signatures to early
deuterosto-mes [12,13,21–23], and indeed to lophotrochozoans,
prebilaterans and even unicellular eukaryotes, although
it appears to have been lost throughout certain large
clades, such as arthropods (this work; see Results)
Like FGE, pFGE localizes to the endoplasmic
reticu-lum (ER), where FGly formation occurs in newly
syn-thesized sulfatases [23,24] FGE and pFGE show
highly similar tissue-specific expression levels and share
many structural properties [23–25] However, pFGE
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]
Therefore, the question arises of how pFGE and
FGE are retained in the ER Here we show that pFGE
is retained via its C-terminal PGEL tetrapeptide
sequence, which, like the classic KDEL signal, can act
as an autonomous retrieval signal, most likely
engag-ing a KDEL receptor (vertebrates have three
paralo-gous KDELR genes), for retrieving pFGE from the cis-Golgi back to the ER FGE lacks a signal even remotely resembling KDEL in mammals However, pFGE overexpression shows no effect on FGE reten-tion
Results
pFGE retention is mediated by a saturable mechanism
A small fraction of endogenous pFGE can be detected extracellularly, whereas upon overexpression the recombinant pFGE is efficiently secreted [23] To determine whether secretion of human pFGE is due to saturation of the retention⁄ retrieval system, an induc-ible, human-derived expression system was established consisting of a Tet-On HT1080 fibrosarcoma cell line stably expressing the reverse tetracycline-controlled transactivator These cells, upon transient transfection and doxycycline addition, allowed us to trigger pFGE expression at defined levels from a Tet-responsive pro-moter (see Experimental procedures) The transfected HT1080 cells were analyzed for intracellular and extra-cellular pFGE by western blotting using a polyclonal pFGE-specific antibody [23] Extracellular pFGE is detected as a 32.5⁄ 31.5 kDa double band, due to heter-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 His6-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 His6-tag on retention⁄ secretion of pFGE, tagged and untagged (wild-type) pFGE were
Trang 3transiently expressed in HT1080 cells When analyzed
24 h after transfection, His6-tagged pFGE was nearly
quantitatively secreted (96% of total), whereas
untag-ged pFGE was significantly retained inside the cells
(26% retention) (Fig 2) This cannot be explained by
different expression levels, because the expression of
intracellularly retained pFGE was two-fold higher than
that of pFGE-His6 Thus, the C-terminal His6-tag
impaired retention of pFGE, indicating that the
C-ter-minus of pFGE might be involved in the ER retention
mechanism
pFGE carries canonical or noncanonical ER
retention signals in different species
Inspection of the available pFGE protein sequences
from 67 species (Fig 3) revealed that 34 of these
sequences contain a canonical ER retention signal of
the KDEL type (basic-X-acidic-leucine) at the
C-termi-nus, with the basic residue being lysine (21 species),
arginine (nine) or histidine (four), and the acidic
residue being glutamate (30) or aspartate (four) Mur-ine and rat pFGE carry the canonical KEDL motif, and the prototype KDEL can be found in orthologs 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, whereas the wild-type and the KDEL form were mostly retained Also, the SGEL form showed
Fig 1 Retention of pFGE is mediated by a saturable mechanism HT1080 cells stably expressing the reverse tetracycline-controlled trans-activator (Tet-On cells) were transiently transfected to express pFGE under control of a doxycycline-responsive promoter (see Experimental procedures) Six hours after transfection, pFGE expression was induced with 0.5–8 ngÆmL)1doxycycline, as indicated Twenty-eight hours after induction, pFGE was determined in cell lysates (C) and media (M) by western blotting Note that the aliquots of cells and medium were loaded at a ratio of 10 : 1 The amount of total pFGE in cells and medium and the percentage of intracellular pFGE are given below the lanes Two differentially glycosylated forms of pFGE (arrows) were detected in the medium (see text).
Fig 2 ER retention of pFGE is impaired by a C-terminal tag pFGE
and pFGE-His were transiently expressed in HT1080 cells
Twenty-four hours after transfection, aliquots of cells and medium (at a
ratio of 10 : 1) were analyzed for pFGE by western blotting, using
an antiserum against pFGE The total expression level and the
per-centage of intracellular pFGE are given.
Trang 4—
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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
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
names, and ordered according to the
mod-ern phylogenetic tree (relative to human,
subtrees not uniquely orderable) Sequences
were aligned at their C-terminal regions to
locate conserved pspecific and
FGE-specific anchors, respectively (see
supple-mentary Figs S1 and S2) The last four
encoded residues preceding the stop are
indicated for all 67 pFGE C-termini Species
with canonical pFGE retention signals are
colored in blue, and those with noncanonical
pFGE signals in red (PGEL only) or green
(other noncanonical) For species in black,
no SUMF2 could be recovered; this may be
explained (in some species but not all) by
incomplete coverage of the genome FGE
C-termini are given for those 11 species
having a KDEL-type signal; non-KDEL-like
C-termini are indicated by four periods ( ).
A dash (–) indicates that either no SUMF1
or no SUMF2 sequence could be recovered,
as indicated The presence of the three
KDEL receptor genes is indicated by
num-bers (123) KDELR1 is present in tetrapods
from frog onwards, and KDELR3 in all
tele-ost fish and tetrapods, but apparently not in
chondrichthyans (skates, elephantfish,
shark), agnathans (hagfish, lamprey),
urochordates, or earlier KDELR2 is the only
receptor available to interact with
SUMF-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).
Trang 5effective 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
[35S]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 labeldur-ing 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 The canonical KDEL signal is known to confer ER retention to any soluble passenger protein that normally traverses this compartment on its way to the cell surface
To test whether this holds true also for the noncanonical PGEL signal, we equipped lysozyme, a typical secretory protein, with either a C-terminal KDEL or PGEL tetra-peptide A c-Myc-tag, also located at the C-terminus, but upstream of the KDEL⁄ PGEL extension, allowed detection through western blotting Upon doxycycline-induced expression, 90% of lysozyme was found in the medium, whereas the form extended with KDEL was quantitatively retained inside the cells (Fig 5) The PGEL-extended form of lysozyme was likewise effec-tively retained (76%)
Using indirect immunofluorescence, we could detect lysozyme–c-Myc intracellularly in Golgi-like structures (Fig 6B), suggesting that this compartment is the bot-tleneck for secretion of overexpressed lysozyme–c-Myc When equipped with a C-terminal KDEL or PGEL extension, lysozyme colocalized with the ER marker protein disulfide isomerase (PDI) (Fig 6C,D) Whereas pFGE and lysozyme–c-Myc-KDEL fully colocalized with PDI (Fig 6A,C), a fraction of lysozyme–c-Myc-PGEL was also detected in the Golgi-like structures (Fig 6D) This became particularly obvious under con-ditions of maximum doxycycline-induced expression,
as chosen in Fig 6 In conclusion, the noncanonical PGEL, like the KDEL tetrapeptide, is a transferable signal conferring ER retention per se Although less efficient than KDEL, PGEL: (a) massively increases
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)1doxycycline 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)1doxycycline, 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.
Trang 6intracellular lysozyme retention (Fig 5); and (b) clearly
shifts this intracellular material from a mainly non-ER
to a mainly ER localization [Fig 6; compare
lyso-zyme–c-Myc staining in (B) and (D)]
Effect of pFGE on FGE retention
Formylglycine-generating enzyme and its paralog
pFGE are both soluble ER-resident proteins [12,13,27]
In contrast to pFGE, FGE lacks a C-terminal
reten-tion signal in vertebrates and most other species
(Fig 3; see Discussion) As there are several
indica-tions that pFGE and FGE interact with each other
(see Introduction), we analyzed whether pFGE confers
ER retention to FGE We constructed a Tet-On
HT1080 cell line stably expressing pFGE under control
of a doxycycline-responsive promoter When the
expression of pFGE was induced by addition of
6 ngÆmL)1 doxycycline, secretion was very low for the first 8 h after induction (not shown) In control
Tet-On HT1080 cells, expressing no pFGE, we studied the expression and secretion kinetics for FGE after transient transfection with a noninducible expression vector FGE is secreted in two forms (37 and 42 kDa) The
42 kDa form represents the full-length FGE, whereas the major 37 kDa form results from N-terminal pro-cessing within the secretory pathway [27] Secretion of FGE by these cells started about 12 h after transfec-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)1doxycycline
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).
Trang 7linear FGE secretion), pFGE expression was induced
by addition of doxycycline Measuring intracellular
and extracellular FGE and pFGE every 2 h, we
observed that pFGE did not interfere with FGE
secre-tion (Fig 7B,C), even though the expression of pFGE
clearly exceeded that of FGE (Fig 7B) In addition,
the data shown in Fig 7A,B may suggest that pFGE
coexpression promotes N-terminal processing of FGE
However, careful quantification of many experiments
(under various conditions) did not provide significant
evidence for this interpretation
Discussion
Human pFGE is retained in the ER through its C-terminal PGEL signal
In an earlier study, we localized pFGE mainly in the
ER, but found smaller amounts also in the Golgi and even in the secretions (13% of endogenous pFGE 16 h after synthesis) [23] In this work, we found that pFGE retention is mediated by a saturable mechanism involv-ing KDEL-like signals at the C-terminus of pFGE In fact, the canonical prototype KDEL can only be found
in pFGE of four species (platypus, planorbid snail, planarian flatworm, and sea anemone; Fig 3) Here we studied retention of human pFGE as a representative
of the most common PGEL-containing pFGEs found
in 21 different species Notably, the first two positions
of the PGEL do not match with any of the deposited consensus KDEL patterns (see below)
We found that deletion of PGEL or positioning a tag C-terminal of PGEL more or less fully impaired retention On the other hand, when added to the C-ter-minus of a secretory protein such as lysozyme, PGEL 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
C
Fig 7 Influence of pFGE coexpression on FGE retention A
Tet-On cell line stably expressing, from a doxycycline-responsive
pro-moter, pFGE (B) or not (A) was transiently transfected with the
noninducible FGE expression plasmid pSB-FGE at time 0 Twelve
hours later, 6 ngÆmL)1doxycycline was added to induce (B)
coex-pression of pFGE at the indicated levels Then, every 2 h, i.e 14–
22 h after transfection, FGE expression and intracellular retention
were quantified, as given below the lanes The relative retention
of FGE as observed in the absence (A) or presence (B) of pFGE is
plotted in (C).
Trang 8preceding three residues (ADA) stick out at the protein
periphery (Fig 8), the PGEL should be easily
accessi-ble for binding by one of the KDEL receptors It
should be noted, however, that both PGEL and
KDEL are obviously more efficiently bound when
present at the C-terminus of lysozyme, as concluded
from its high retention efficiency
pFGE as a retention factor for FGE?
SUMFgene duplication was a rather ancient event, as
in early unicellular eukaryotes such as Emiliana
hux-leyi, both SUMF1 and SUMF2 genes can be found
(Fig 3) Later, SUMF2 was lost in several clades
Fig-ure 3 shows that KDEL-type signals can also be found
in SUMF1-encoded FGE of several invertebrate
spe-cies In total, 11 of 69 available FGE sequences show
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 retenforma-tion 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-tal data, showing no influence of coexpressed pFGE
on the secretion of FGE (Fig 7), clearly argue against pFGE as a stand-alone retention factor for FGE Ongoing experiments suggest that regulation of retention versus secretion of FGE employs several mechanisms, one of which involves the noncatalytic N-terminal extension of FGE The possibility that pFGE contributes to this regulation cannot be excluded, as the FGE⁄ pFGE coexpression experiments reported here may have missed ternary complex forma-tion with unfolded sulfatase substrates or other inter-acting components in the ER
Bioinformatic and evolutionary considerations
on pFGE and KDEL signals The well-known KDEL ER retrieval signal, discovered
by Munro & Pelham [36], was found to be widely used In fact, many variants have been described, and the pattern [KRHQSA]-[DENQ]-E-L was deposited as
a general consensus in the PROSITE database [26] Informatic inspection of ER proteins deposited in the human ER Aperc¸u (HERA) database led to the suggestion that this pattern should be extended to [KRHQSADEN]-[DENQTFIV]-E-[LF] [37] Even more extensive bioinformatic studies compiled further variations leading to the pattern [KHRDENQAS]-[DENQIYCV]-[DENQ]-L, which shows more flexibil-ity in the penultimate position [38] Of the C-terminal sequences of pFGE from 67 species (Fig 3), only 17
fit with the latter consensus patterns, most of them nearly ideally (KDEL, KEDL, RDEL, RQEL, RNEL, 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.
Trang 9(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
of noncanonical retrieval signals is not coherent with
specific evolutionary developments Nevertheless, it is
interesting to note that the other noncanonical signals
(Fig 3, green) found in mammalian pFGEs are rather
similar to PGEL
Unfortunately, without supporting experimental
data for the many pFGE species listed in Fig 3, we
cannot say whether all the variations of the retention
tetrapeptide are accepted by the in-species KDEL
receptor(s) and the underlying retrieval system, as this
also varies over the vast phylogenetic timescale In the
mammalian system, where experimental data are
avail-able, PGEL is functional as an autonomous retention
signal (Fig 5) Surprisingly, it is commonly found in
mammalian pFGEs and evolutionarily conserved over
a lot of branch lengths; but at the same time, it is
highly specific for this protein, as becomes evident
from the databases, where only very few PGEL ER
proteins can be found In fact, not a single human
PGEL ER protein, apart from pFGE, and only one
human protein with an N-terminal signal peptide and
an SGEL C-terminus (endonuclease domain-containing
protein ENDOD1), could be retrieved from Swissprot
or Ensembl The UCSC browser retrieved in addition
a GDNF receptor-like protein with a PGEL
C-termi-nus, which, even as a membrane protein, may be
sub-jected to KDEL receptor-mediated ER retention The
specific advantage conferred to pFGE by its PGEL
ter-minus needs to be determined
Relevance for KDEL receptor-mediated ER
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-brate KDEL receptor genes All three are found in tet-rapods (mammals, birds, reptiles, amphibians) (Fig 3) KDELR2 is present in all eukaryotes, KDELR3 in all teleost fish and tetrapods, and KDELR1 only in tetra-pods from the frog onwards Human KDEL receptors (ERD2) 1 and 2 are obviously ubiquitously expressed,
as suggested by cDNA libraries from different tissues, but human ERD2.2 is inducible through the ER stress response [30] Both are functionally identical with regard to lysozyme-KDEL and lysozyme-DDEL retrie-val [30] Residues 50–56 of KDEL receptor 1 were sug-gested to determine ligand specificity [29,41] Of these, only positions 54⁄ 55 show minor variations among the three KDEL receptor isoforms (50-DLFTNYI-56⁄ DLFTSFI⁄ DLFTNFI) Unfortunately, no 3D model
of the binding pocket is available Coexpressing the individual receptors with pFGE might show that, indeed, a particular receptor isoform is well adapted for pFGE retrieval
Conclusions
This study on the mechanism of pFGE retention in the ER has uncovered a novel retrieval signal that autonomously confers ER retention to passenger pro-teins Surprisingly, this noncanonical PGEL variant of the classic KDEL signal, although evolutionarily con-served for most mammalian pFGEs, is not being
Trang 10widely used by other mammalian ER proteins Why
the PGEL signal or its other noncanonical variants are
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–
c-Myc with PGEL or KDEL at the C-terminus, plasmid
pCMV2-Lys-cmyc-KDEL [43] served as a template for
add-on PCR, using GTCAGCTAGCCGGCCCGCCAT
GAGGTCTTTGCTAATC as a forward primer, and
reverse primers 5¢-CCGGATATCGATTCACTCACTATC
GATGTTGAGGTC-3¢ (for wild-type lysozyme), 5¢-CC
GGATATCGATTCATAGCTCCCCTGGCTCACTATC-3¢
(lysozyme-PGEL) or 5¢-CCGGATATCGATTCATAGCTC
GTCCTTCTCACT-3¢ (lysozyme-KDEL) Also here, 5¢-end
NheI sites and 3¢-end EcoRV sites facilitated directional
cloning of the PCR product into the pBI Tet vector
Cell culture and transfections
Human HT1080 fibrosarcoma cells were grown in normal
growth medium, i.e in DMEM supplemented with 10%
fetal bovine serum and 1% penicillin⁄ streptomycin
(Invitro-gen, Karlsruhe, Germany) under 5% CO2at 37C HT1080
cells stably expressing the reverse tetracycline-controlled
transactivator rtTA (Tet-On cells) and Tet-On cells stably expressing pFGE were grown in normal growth medium with neomycin or neomycin and puromycin, respectively The stable Tet-On cell line was established by cotransfect-ing HT1080 cells with pUHrT62 (kindly provided by
N Jung, Institute of Chemistry and Biochemistry, Freie Universitaet, Germany), encoding the reverse tetracycline-controlled transactivator [44], and the neomycin-resistant vector pSB4.7pA at a ratio of 10 : 1 Transfectants were selected with increasing concentrations of neomycin from 0.2 to 0.8 mgÆmL)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 primary antibodies Horseradish peroxidase-conjugated goat anti-(rabbit IgG1) sera were used as secondary antibodies ECL signals were quantified using the aida 2.1 software package (Raytest, Straubenhardt, Germany)
Pulse-chase experiments and immunoprecipitation
HT1080 Tet-On cells, grown to 50–60% confluency, were transiently transfected with pBI-pFGE, pBI-pFGEDPGEL
or pBI-pFGE-KDEL plasmids After 6 h, the medium was replaced by medium with 0.5 lgÆmL)1 doxycycline After
12 h of induction, cells were starved in 2 mL of methio-nine⁄ cysteine-free DMEM for 1 h, and pulsed for 90 min with 1 mL of medium containing 100 lCi of 35S-labeled