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INTRODUCTION |
Almost all sulfatases that have been described are members of an
evolutionary conserved protein family showing extensive homology among
enzymes of prokaryotic, lower eukaryotic, and mammalian origin (1-3).
The three-dimensional fold and, in particular, active site region of
human and bacterial sulfatases are strikingly similar (4-6). The
catalytic residue is a C
-formylglycine
(FGly).1 Its formyl group is
hydrated, leading to two geminal hydroxyls at the
-carbon that both
are required for catalysis (6, 7). During sulfate ester cleavage, one
of the hydroxyls undergoes covalent sulfation with consecutive
desulfation induced by the second hydroxyl (6-8). The importance
of this novel catalytic mechanism is reflected by the fact that failure
to generate the FGly residue leads to synthesis of catalytically
inactive sulfatase polypeptides, as is observed in multiple sulfatase
deficiency, a rare but fatal human lysosomal storage disorder
(9-11).
In eukaryotes, FGly is generated in the endoplasmic reticulum by
oxidation of a conserved cysteine residue (12, 13). This late
cotranslational or early posttranslational protein modification is
directed by a short linear sequence motif comprising a proline and an
arginine as the key residues in +2- and +4-positions
(CXPXR) and, in addition, an adjacent auxiliary
element (LTG; +8 to +10) (2). Replacing the key cysteine by serine or
any other amino acid abolishes FGly formation completely (2, 7, 14).
The motif has to be accessible to the modifying machinery prior to folding of the nascent polypeptide into its native structure (2, 12).
To date, none of the components or cofactors of this machinery have
been identified in eukaryotes. These components are comprised among the
luminal contents of the endoplasmic reticulum, which in
vitro mediate FGly modification independent of protein
translocation and independent of a signal peptide in the sulfatase
substrate (15).
Due to the clear conservation of the FGly modification motif, most of
the sulfatases encoded in various eubacterial genomes are predicted
also to undergo FGly modification by oxidation of a cysteine. This was
shown experimentally for the arylsulfatase of Pseudomonas
aeruginosa (PAS), a member of the cysteine-type sulfatases. Even
after strong overexpression in Escherichia coli, this
cytosolic sulfatase was quantitatively converted to the active FGly-bearing enzyme (16). Hence, the E. coli cytosol
contains the modifying machinery. This machinery is expressed even
under excessive supply with inorganic sulfate. Thus, expression of
E. coli's cysteine-modifying system is independent of the
sulfur status of the cells, in contrast to expression of the sulfatase structural genes, as studied in P. aeruginosa and in
Klebsiella pneumoniae (17-19).
The other well characterized bacterial sulfatase, the arylsulfatase
AtsA of K. pneumoniae, is a serine-type sulfatase that carries an FGly residue generated by oxidation of a serine rather than
a cysteine (20). Generation of FGly (i.e. serine
semialdehyde) from serine most likely is a one-step oxidation process.
In contrast to the cytosolic cysteine-type sulfatases, serine-type
sulfatases are located in the periplasm (18, 21, 22). The key FGly motif (SXPXR) and also the auxiliary
downstream element (LTG) are also conserved in serine-type
sulfatases (2).
Despite these similarities, bacteria have two different pathways for
FGly generation from cysteine and serine, respectively. This is
indicated by two observations. First, substitution of the cysteine to
be modified in PAS by serine totally blocks FGly formation (16).
Second, expression of active, FGly-containing AtsA in E. coli essentially requires coexpression of the K. pneumoniae atsB gene (21), whereas the genomic background of E. coli is sufficient for expression of active and modified PAS
(16).
The atsB gene is located on the same operon as the
structural atsA gene. AtsB acts in trans on AtsA,
since it was fully functional when both genes were co-expressed from
two different plasmids (21). Despite the presence of two serine-type
sulfatase operons in E. coli, each consisting of a sulfatase
gene and an atsB homolog, this species has not been found to
express endogenous sulfatases. The inability of the chromosomal
atsB homologs of E. coli to substitute for the
Klebsiella atsB most likely is explained by repression of
its sulfatase operons.
AtsB is predicted to be a 44-kDa iron-sulfur protein with three
cysteine clusters that are conserved in all AtsB homologs (22).
Iron-sulfur proteins are involved in redox reactions, but only recently
a direct enzymatic oxidoreductase function has been assigned to this
class of proteins (23-25). In this study, we addressed the following
questions. Where in the cell does AtsB fulfill its function? Can it act
on both cytosolic and secretory sulfatases? Does AtsB act specifically
on the Klebsiella sulfatase or on serine-type sulfatases in
general? And finally, does AtsB physically interact with the
modification motif on the sulfatase polypeptide?
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis, Tagging, and 5'-UTR
Optimization--
atsA-S72C was generated by PCR
mutagenesis of atsA (21) using a coding mutagenic primer
(CGAGCAGGGAATGCGCATGAGCCAGTATTACACCTCGCCGATGTGCGCCCCGGC) that allowed subcloning of the PCR product via its BsmI
site. The generation of pas-C51S was described earlier (16).
The 5'-UTR of the pas gene in pME4055 (16) was substituted
by the short 5'-UTR of atsA (21) by adding an
oligonucleotide (CCCAAGCTTGAACAGGAGAGTCAGTCGTG), with a
HindIII site and an initiator GTG, 5' of pas. The
modified gene was cloned into pBluescriptII SK as a HindIII
fragment downstream of the lac promotor.
The addition of an oligonucleotide coding for a C-terminal
Arg-Gly-Ser-His6 tag to atsA was described
earlier (21). The same tag was fused to AtsB by adding the
oligonucleotide
GGGGGATATCATGCGCTAGTGATGGTGATGGTGATGCGATCCTCT 5' of the
atsB stop codon and subcloning of the obtained PCR product as a BsmI/EcoRV fragment back into the
atsB template vector.
Deletion/Addition of Signal Peptides--
Deletion of
AtsA's signal peptide was achieved by adding an oligonucleotide
(CCCAAGCTTGAACAGGAGAGTCAGTCGTG) with a HindIII site and the indicated initiator GTG, 5' of codon 21 of
atsA, which encodes the first amino acid of mature AtsA. The
PCR product was subcloned as a HindIII/XhoI
fragment back into the atsBA template vector. For
construction of PAS-C51S+SP, the AtsA signal peptide was added to its N
terminus. Using overlapping extension PCR (26), we amplified the 5'-UTR
and signal peptide codons of atsA (internal noncoding
primer: GGGCGTTTGCTCGCGGCGTGCGCGCCACC) and, in a second PCR, the coding region of pas excluding the initiator ATG
(internal coding primer: CACGCCGCGAGCAAACGCCCCAACTTCCTG).
The two PCR products were fused by using them as templates in a third
PCR reaction, due to hybridization of the overlapping complementary
sequences introduced by the two internal primers. From the final PCR
product, a 144-bp EcoRV fragment was subcloned into
pBluescriptII SK-PAS-C51S, thereby replacing the corresponding part in
the 5' region of pas.
Protein Expression in E. coli, Subcellular Fractionation, and
Protein Purification--
E. coli DH5
was transformed
with the following plasmids: pBluescriptII containing either
atsA, atsB, atsBA (atsA and
atsB with or without His tag codons, atsA with or
without signal peptide codons), pas, or pas-C51S
(with or without signal peptide codons). Coexpression of AtsB with PAS
constructs was achieved in double transformants containing also the
pBBR1MCS-atsB plasmid (atsB subcloned from
atsBA as a KpnI/HindIII fragment). The
presence of the two plasmids was maintained in selective medium,
containing ampicillin and chloramphenicol, and was routinely checked by
PCR analysis. Growth conditions, preparation of periplasm, and
purification of hexahistidine-tagged proteins were described earlier
(21).
Generation of Antibodies--
Using purified native PAS protein,
provided by Dr. M. Kertesz (School of Biological Sciences, University
of Manchester, UK) as antigen and specol as adjuvant, polyclonal
antibodies were raised in rabbits injected with 400 µg (first
injection) or 200 µg (two booster injections) of antigen. Anti-AtsB
antibodies were generated similarly, using AtsB-His6
protein as antigen. This was purified from inclusion bodies on
Ni2+-NTA-agarose (Qiagen) in the presence of 8 M urea according to the protocol of the manufacturer. Prior
to rabbit injection, urea was removed by stepwise dialysis (4 M/2 M/1 M urea in PBS). The purity
of AtsB was at least 95% (Fig. 1B). Antibodies were
purified by preadsorption of antisera to immobilized E. coli
protein. Anti-AtsB antibodies furthermore were affinity-purified by
adsorption to SDS-PAGE-purified antigen that was blotted to and excised
from a nitrocellulose membrane. Bound antibodies were eluted with 200 mM glycine (pH 2.8).
Protein and Peptide Analysis--
Expressed AtsB, sulfatase, or
fusion proteins were detected by Western blotting using anti-AtsA (21),
anti-AtsB, anti-PAS (see above), anti-GST (Amersham Biosciences),
anti-hexahistidine (Qiagen), or anti-HA 12C5A (Roche Molecular
Biochemicals) as primary antibodies. ECL signals of corresponding
secondary antibodies were detected by a LAS1000+ imaging system
(Raytest) and quantitated by densitometry of digital images using Aida
3.10 software (Raytest). For SDS-PAGE, see Ref. 27. The activity of
expressed sulfatases was determined in duplicate assays at
saturating substrate concentration, as described earlier for AtsA (20)
and PAS (16, 17).
The presence of FGly in AtsA was analyzed at the level of its tryptic
peptides (see Refs. 20 and 21). During reversed-phase HPLC, P2 and P2*
(see "Results"), as detected by mass spectrometry, were recovered
in adjacent fractions. The amounts of these peptides were quantitated
by sequencing on a Procise cLC protein sequencer (Applied Biosystems).
The presence of FGly was verified by mass spectrometry on a
matrix-assisted laser desorption ionization-time of flight Reflex III
instrument (Bruker Daltonics), using a 337-nm nitrogen laser, with a
200-ns extraction delay. Spectra were obtained as averages of 100 laser
shots. 10 mg/ml
-cyano-4-hydroxy-cinnamic acid (Bruker Daltonics) in
50% acetonitrile, 0.1% trifluoroacetic acid served as matrix. Samples
were prepared by the drying droplet method, drying 0.5 µl each of
sample and matrix solution on a stainless steel target. For FGly
identification, 0.5 µl of 2,4-dinitrophenyl hydrazine (Fluka),
saturated in 50% acetonitrile, 0.1% trifluoroacetic acid, was added
to the dried sample/matrix mixture on the target.
Yeast Two-hybrid Experiments--
Fragments of the
Klebsiella atsA gene were cloned as PCR products into the
pAS2 ("bait") vector in frame with the DNA sequence encoding the
HA-tagged Gal4-binding domain (28). For PCR amplification of
atsA fragments, plasmids encoding full-length
atsA or its signal peptide-deleted version (see above) were
used as templates. The forward primer
(CCTGAAGGCCATGGAGGCCACAGGAGAGTCAGTCGTG) introduced the
underlined SfiI site 5' of the atsA fragment, and
the reverse primers (CGGGATCCGGAAGAACGATAGCCGTGGTGG or
CGGGATCCTAGCGGTCGGTCAGCCGCAG) introduced the underlined
BamHI site 3' of the wild type stop codon or of a stop codon
inserted 3' of codon 112 by the primer, respectively. The PCR products
were cloned as SfiI/BamHI fragments into the pAS2
vector, yielding pAS2-AtsA-(1-112), pAS2-AtsA-(21-112), and
pAS2-AtsA-(21-577) (see "Results"). The full-length
atsB gene was cloned into the pACTII ("prey") vector in
frame with the HA-tagged Gal4 activation domain (28) using a 3'
BamHI site present in the multicloning sites of both
originating (pBluescriptII KS) and receiving vector. At the 5'-end, a
pBluescriptII KS KpnI site, blunted with T4 polymerase, was
ligated with a pACTII NcoI site, blunted with Klenow polymerase.
The yeast reporter strain Y190 was transformed with both one bait and
one prey plasmid. As negative controls, transformations with empty pAS2
or pACTII vectors in combination with a hybrid construct were
performed. Cotransformants were selected due to their tryptophan and
leucine prototrophy conveyed by the two plasmids (see Ref. 28).
Expression of the correct fusion proteins by these cotransformants was
routinely controlled by Western blot analysis of cell lysates using
anti-HA or anti-AtsB antibodies (not shown). The
-galactosidase
activity induced in the case of reconstitution of the Gal4p
transcription factor was detected by applying a filter assay with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal) as a substrate (29); the permeabilized cells were incubated with X-gal for 6-8 h at 30 °C. The
-galactosidase activity was quantified in a fluid phase assay of cell lysates using
o-nitrophenyl galactoside as a substrate (30) and calculated
according to Ref. 31.
GST Pull-down Experiments--
Codons 21-112 of atsA
or atsA-S72C (see above) were amplified by PCR using primers
that add a 5' EcoRI site
(GGAATTCTAACAGGAGAGTCAGTCGTG) and a 3' SacI site
(AAGCTTGAGCTCTAGCGGTCGGTCAGCCGCAG). The PCR products were
cloned as EcoRI/SacI fragments into the pGEX-KG vector (32) in frame with its glutathione S-transferase
encoding sequence. The fusion proteins, or GST only, were overexpressed in induced (0.2 mM isopropyl thiogalactoside),
logarithmically growing E. coli DH5
. Bacteria were
disrupted in a French press cell and treated with 5 M urea
in PBS (pH 7.4) for 30 min at room temperature. The soluble material
(75,000 × g supernatant) was subjected to dialysis
against PBS to remove the urea. After dialysis and another
centrifugation (75,000 × g), the supernatants or, as a
control, PBS buffer were loaded on glutathione-agarose (incubation for
30 min at room temperature), which then was washed with 3 × 4 column volumes of PBS. The columns were then loaded at room temperature
with the soluble fraction of an E. coli French press lysate
(in PBS) containing expressed AtsB protein, and the flow-through was
immediately collected without further incubation. After another three
washing steps (as above), the columns were eluted twice with 1.5 column
volumes of 20 mM glutathione in PBS (pH 8.0). The wash and
eluate fractions as well as a final eluate, obtained by boiling the
glutathione-agarose beads in SDS-PAGE sample buffer, were analyzed by
Western blotting.
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RESULTS |
AtsB Is a Cytosolic Protein--
The Klebsiella
arylsulfatase AtsA, when purified from a total K. pneumoniae
cell lysate, was found to be processed by signal peptidase and to carry
FGly72 in 60% of sulfatase polypeptides (20). When
hexahistidine-tagged AtsA, expressed in E. coli together
with AtsB, was purified from the periplasm of the cells, FGly
modification was observed for 48 ± 2% of polypeptides, whereas
in the absence of AtsB no FGly was detected (21). To find out whether
AtsB, like AtsA, has a periplasmic localization, as suggested earlier
(18), we performed a subcellular fractionation of E. coli
cells expressing both AtsA and AtsB. After osmotic shock of the cells
AtsB was found exclusively in the spheroplast pellet and not in the
supernatant containing the periplasmic proteins, among them AtsA (Fig.
1A). The appearance of AtsA
also in the spheroplasts is attributed to incomplete disruption of the
outer membrane. After two-step purification of AtsB, expressed in
hexahistidine-tagged form, by Ni2+-NTA-agarose
chromatography and reversed-phase HPLC (Fig. 1B), the
AtsB-His6 protein was subjected to amino acid sequencing
and found to have an intact N terminus (MLNIAALR). This excludes
processing by the signal peptidase. In conclusion, AtsB is a cytosolic
protein.

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Fig. 1.
Subcellular localization of AtsA and AtsB.
A, E. coli cells expressing AtsA and AtsB were
subjected to osmotic shock, and after sedimentation of spheroplasts,
the periplasmic proteins were recovered from the supernatant.
Equivalent amounts of periplasm and spheroplasts were analyzed by
Western blotting and compared with a total cell lysate. AtsA and AtsB
were simultaneously detected by the corresponding affinity-purified
antibodies. B, hexahistidine-tagged AtsB was purified from
E. coli coexpressing AtsA and AtsB-His6 by
Ni2+-NTA-agarose chromatography (see Coomassie-stained gel
of six consecutive eluate fractions, 0.5 column volumes each) and by
HPLC on a C4 reversed-phase column (see chromatogram). The N-terminal
sequence of the purified AtsB protein, eluting from the C4 column at
16.57 min retention time, is given. It was found to correspond to the N
terminus encoded in the atsB gene.
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AtsB Coexpression Does Not Lead to Activation of PAS-C51S--
The
arylsulfatase PAS of Pseudomonas aeruginosa is a
cysteine-type sulfatase that is quantitatively modified upon expression of its structural gene in E. coli (16). When the critical
cysteine residue 51 was substituted by a serine (PAS-C51S), no FGly
formation was observed (16). Since the sequence motifs
(SXPXR and LTG; see Introduction) determining
AtsB-dependent FGly formation in AtsA are contained also in
PAS-C51S, the C51S form of PAS was expressed with or without AtsB. As
shown in Fig. 2A, PAS-C51S was
expressed as an inactive polypeptide in the absence and presence of
AtsB. Control experiments demonstrated that expression of AtsB does not
affect the expression of active wild type PAS (Fig. 2A). Therefore, we conclude that PAS, when converted to a serine-type sulfatase, is not a substrate for the AtsB-dependent
FGly-generating machinery.

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Fig. 2.
Expression of catalytically active PAS-C51S
requires AtsB and the presence of a signal peptide. The wild type
and C51S forms of PAS were expressed in E. coli in the
absence and presence of AtsB, as indicated. The Western blot shows the
PAS and AtsB polypeptides, as recovered from the soluble fraction of
total cell lysates. The sulfatase activities present in the samples
loaded for SDS-PAGE are given below each lane. PAS and PAS-C51S were
expressed as cytosolic (A) or as secretory proteins
(B) (i.e. without or with the signal peptide of
AtsA, respectively, engineered at the N terminus of PAS (PAS-C51S+SP)).
PAS-C51S+SP was catalytically active when coexpressed with AtsB. Its
specific activity, as calculated after densitometric quantification of
PAS protein on the Western blot (see "Experimental Procedures"),
corresponded to about 10% of wild type PAS activity.
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AtsB-dependent FGly Formation in AtsA Is Strongly
Reduced after Removal of AtsA's Signal Peptide--
One of the major
differences between AtsA and PAS-C51S is the presence or absence of a
signal peptide, respectively. Therefore, we deleted the signal peptide
of AtsA and investigated whether a cytosolic version of AtsA
(AtsA
SP) is synthesized in active form when coexpressed with AtsB.
It turned out that AtsA
SP showed a very low, albeit significant,
catalytic activity of 0.84 ± 0.14 units/mg (n = 5) (i.e. about 1% of wild type AtsA activity) (Fig. 3A). In the absence of AtsB,
AtsA
SP was expressed as a completely inactive protein. To examine
for the presence of FGly, hexahistidine-tagged versions of wild type
AtsA and AtsA
SP were coexpressed with AtsB. The sulfatases were
purified on Ni2+-NTA-agarose and analyzed for FGly
modification. For this purpose, tryptic peptides were generated and
subjected to HPLC on a reversed-phase column, which allowed us to
separate unmodified and modified peptide 2 (P2 and P2*) comprising
serine or FGly at position 72, respectively. P2 and P2* eluted in
adjacent fractions (20). By amino acid sequencing of these fractions
(Fig. 3, B and C), we found that 56% of the wild
type AtsA polypeptides carried the FGly.

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Fig. 3.
The signal peptide is required for efficient
FGly modification of AtsA. AtsB and AtsA-His6 were
coexpressed in E. coli, the latter as periplasmic wild type,
or as cytosolic (i.e. signal peptide-deleted
(AtsA SP-His6)) proteins. The AtsA proteins, recovered
from the periplasm (AtsA-His6) or from spheroplasts
(AtsA SP-His6), were purified on
Ni2+-NTA-agarose, and their specific catalytic activities
(units/mg of AtsA protein) were determined. A, the Western
blot shows 0.1 and 0.2 µg of periplasmic and cytosolic
AtsA-His6, respectively, having a specific activity of 79 and 0.84 ± 0.14 units/mg (n = 5), respectively.
B-F, the purified proteins were subjected to tryptic
digestion, and their tryptic peptides were separated by reversed-phase
HPLC. The modified and the unmodified forms of peptide 2 (P2* and P2,
respectively) eluted in adjacent fractions (see Ref. 20), as detected
by mass spectrometry. B-C, sequencing of the P2* and P2
fraction demonstrated that 56.2 ± 7.5% of the periplasmic wild
type AtsA contained FGly. For P2*, Edman degradation is blocked at the
position of the FGly (B, cycle 10) and reduced in the
preceding cycle (20, 21). For FGly quantitation, only the amino acid
yields in cycles 3-8 (QYYTSP) were considered, since the first two
cycles showed some background. D, for the cytosolic
AtsA SP-His6, the HPLC fraction corresponding to P2*
mainly contained unmodified P2, as evidenced by sequencing of the
entire peptide showing a serine in position 72 and signals in the
following cycles. E, mass spectrometry detected clearly low
signals for P2* (1587 Da) and high signals for P2 (1589 Da).
F, using 2,4-dinitrophenyl (DNP) hydrazine as a
matrix for matrix-assisted laser desorption ionization mass
spectrometry, P2*, but not P2, was converted into the corresponding
hydrazone (1767 Da). Parts of P2, P2*, and the P2*-hydrazone derivative
contained an oxidized methionine or were desorbed as Na+
adducts, as indicated.
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In the cytosolic AtsA
SP, traces of FGly could be detected. By mass
spectrometry of the HPLC fraction that should contain P2*, we clearly
could detect the FGly-containing peptide. As shown in Fig.
3E, some P2* (1587 Da) was detected that specifically reacted with 2,4-dinitrophenyl hydrazine, used as a matrix for matrix-assisted laser desorption ionization mass spectrometry, to the
corresponding hydrazone (1767 Da; Fig. 3F). This hydrazone formation requires the presence of a formyl group (12). The majority of
peptide in the analyzed HPLC fraction, however, corresponded to the
Ser72-containing P2 (1589 Da; Fig. 3E),
indicating incomplete separation of P2* and P2 by HPLC. This was
confirmed by amino acid sequencing. A serine residue was identified at
the position of the expected FGly (Fig. 3D). Moreover, no
clear decrease of sequencing efficiency at the position of the FGly and
consecutive residues, an obligatory effect of FGly-containing peptides
(Fig. 3, compare B and D) (9, 16, 20), was
observed. As estimated from the yield of amino acids in the sequencing
cycles before and after the position of the FGly in the P2* fraction
(Fig. 3D) and, in comparison, in the P2-fraction (not
shown), P2* contributes less than 10% to the P2/P2* mixture in the
P2*-fraction and less than 2% to total P2/P2* of AtsA
SP. In
conclusion, expression of a signal peptide-deleted AtsA results in a
sulfatase with a 100-fold lower specific enzymatic activity. This
agrees with a similarly reduced FGly content.
AtsB-dependent Expression of Active Signal
Peptide-containing PAS-C51S--
To find out whether the presence of a
signal peptide would allow for AtsB-dependent FGly
formation in PAS-C51S, PAS-C51S+SP was constructed by fusing PAS-C51S
to the signal peptide of AtsA. When expressed in the absence of AtsB,
PAS-C51S+SP was catalytically inactive. However, when coexpressed with
AtsB a significant sulfatase activity was measured (Fig.
2B), which corresponded to up to 10% of that of wild type
PAS (Fig. 2A). It should be noted that the signal peptide of
most of the expressed PAS-C51S+SP remained unprocessed, as evidenced by
a slightly lower electrophoretic mobility (Fig. 2B). In
fact, less than 5% of PAS-C51S protein and less than 10% of
arylsulfatase activity were recovered in the periplasm of cells coexpressing PAS-C51S+SP and AtsB (data not shown). Independent of this
obvious translocation deficiency, the catalytic activity of PAS-C51S+SP
in contrast to the inactivity of PAS-C51S supports the observation made
for AtsA
SP, namely that AtsB-dependent FGly formation in
serine-type sulfatases requires the presence of a signal peptide.
Furthermore, AtsB activity is not restricted to the sulfatase of its
operon and can even act on a non-Klebsiella sulfatase that
carries a serine-type FGly modification motif and a signal peptide.
AtsB Physically Interacts with the Serine-type FGly Modification
Motif--
As shown above, the signal peptide of a newly synthesized
sulfatase polypeptide, determining its export to the periplasm, is
important for AtsB-dependent conversion of serine to the
active site FGly. Hence, the question arises as to whether the
cytosolic AtsB makes contact to the sulfatase polypeptide and, if so,
whether it interacts directly with the FGly motif or, more indirectly, via the signal peptide.
To address this question, we performed a yeast two-hybrid interaction
assay using as "bait" the N-terminal 112 amino acids of
AtsA with and without signal peptide (AtsA-(1-112) and AtsA-(21-112), respectively) or mature full-length AtsA (AtsA-(21-577)) and as "prey" full-length AtsB. Cell extracts of cotransformants were analyzed by Western blots showing correct expression of the AtsA and
AtsB fused to HA-tagged Gal4-binding and Gal4 activation domain, respectively (not shown). The fusion proteins were detected by anti-HA
antibodies and the Gal4AD-AtsB also by specific anti-AtsB antibodies.
The cotransformants expressed the fusion proteins at similar levels.
Only transformants coexpressing Gal4BD-AtsA-(21-112) and Gal4AD-AtsB
showed induction of
-galactosidase expression, as determined both by
a filter and a fluid phase assay for
-galactosidase activity (Table
I, cotransformants 1-4). Thus, in the
two-hybrid system, AtsA-AtsB interaction is observed only if the signal
peptide and large parts of AtsA, located C-terminal of the modification motif, are deleted. The AtsA-(21-112)/AtsB interaction depended on
AtsB, as the
-galactosidase activity was about 3-fold lower in the
absence of AtsB (Table I, compare cotransformants 4 and 6).
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Table I
Analysis of -galactosidase expression in various yeast two-hybrid
cotransformants
Yeast two-hybrid interaction assays were performed using the indicated
bait and prey hybrids (see "Experimental Procedures"). AtsB was
expressed as full-length protein, whereas AtsA was lacking its signal
peptide (amino acid residues 1-20) and/or its C-terminal residues
113-577. In addition, a S72C mutant of AtsA was investigated. To
perform X-gal filter assays, the indicated cotransformants were grown
on selection plates, transferred to a polyvinylidene difluoride
membrane, permeabilized in liquid nitrogen, and then incubated on a
filter soaked with X-gal. The development of no ( ), weak (+), or
strong (+++) blue color due to X-gal cleavage is given. Quantitation of
-galactosidase activity was performed in a fluid phase assay using
o-nitrophenyl galactoside as a substrate (see
"Experimental Procedures"). -Galactosidase activity is expressed
in units/A600 of cells employed for this assay.
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To test for the dependence of the AtsA-(21-112)/AtsB interaction on
the 72SAPAR FGly modification motif, a 72CAPAR
(S72C) mutant was analyzed. A weak
-galactosidase activity was
induced in AtsA-(21-112)-S72C/AtsB cotransformants (10% of that
observed for AtsA-(21-112)/AtsB) (Table I, cotransformants 4 and 5).
This residual
-galactosidase activity was not dependent on
coexpression of AtsB (Table I, compare cotransformants 5 and 7). We
noted that in the absence of AtsB in the prey, the expression of the
Gal4BD-AtsA-(21-112) and Gal4BD-AtsA-(21-112)-S72C fusion proteins
was considerably increased (not shown). This may contribute to the
relatively high residual
-galactosidase activity observed in these
cotransformants (Table I, cotransformants 6 and 7). Taken together,
these results indicate that interaction of AtsA-(21-112) with AtsB
depends on the serine-type FGly modification motif.
To substantiate the two-hybrid data, we performed biochemical in
vitro interaction experiments using glutathione
S-transferase (GST) pull-down assays. AtsA-(21-112) and its
S72C mutant form were expressed in E. coli as C-terminal
appendices of GST. The fusion proteins were recovered from inclusion
bodies. After solubilization in 5 M urea, dialysis against
PBS and centrifugation the soluble material was bound to
glutathione-agarose columns. After washing, the soluble fraction of a
lysate of E. coli expressing AtsB was applied. After further
washing steps, the GSH-agarose column was eluted with glutathione. The
eluate was analyzed on a Western blot using anti-GST and anti-AtsB
antibodies (Fig. 4, A and
B). GST-AtsA fusion proteins were recovered in the
glutathione eluate as full-length proteins and, in part, as
C-terminally truncated forms (Fig. 4A). Significant amounts
of AtsB (about 600 ng of AtsB/µg of nontruncated GST-AtsA-(21-112)
(i.e. 0.54 mol/mol)) were detected in the eluate of the
GST-AtsA-(21-112) column, whereas only traces of AtsB were present in
the eluate of the GST-AtsA-(21-112)-S72C column (about 0.04 mol of
AtsB/mol of GST-AtsA-(21-112)-S72C; see Fig. 4B). No AtsB
was detected in the eluates of columns loaded with GST or PBS (Fig. 4).
In conclusion, AtsB firmly interacts with AtsA-(21-112) in a
Ser72-dependent manner.

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Fig. 4.
Binding of AtsB to immobilized GST-AtsA
fusion proteins. GST, GST-AtsA-(21-112) and
GST-AtsA-(21-112)-S72C were solubilized in 5 M urea (see
"Experimental Procedures"). After dialysis against PBS and
centrifugation, the soluble material or, as a control, PBS buffer were
loaded on glutathione-agarose, which then was washed thoroughly. All
four columns were then loaded with the soluble fraction of an E. coli lysate (in PBS) containing AtsB protein. After another three
washing steps, the columns were eluted with glutathione and then boiled
with SDS-PAGE sample buffer. The eluates obtained with glutathione and
SDS, were analyzed on a Western blot using goat anti-GST (A)
or rabbit anti-AtsB antibodies (B). The positions of GST (27 kDa), GST-AtsA-(21-112) (39 kDa), and AtsB (44 kDa) are indicated. In
addition to full-length GST-AtsA-(21-112) fusion proteins, the
anti-GST antibody detected C-terminally truncated forms of about 33 and
29 kDa. 200 ng of purified GST (A) and 400 ng of purified
AtsB (B) were used as standards, which allowed us to
quantitate the detected Western blot signals. Controls demonstrated
that the washings efficiently removed unbound GST fusion proteins and
AtsB (not shown). Elution with glutathione was nearly quantitative,
since only minute amounts of GST fusion proteins were extracted from
the glutathione-agarose beads by SDS.
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DISCUSSION |
AtsB Interacts Directly with the Serine-type FGly Modification
Motif--
AtsB is essential for FGly formation in the serine-type
sulfatase AtsA from K. pneumoniae (21). Here we show that
AtsB is a cytosolic protein (Fig. 1). As AtsA is located in the
periplasmic space, a localization predicted for all known serine-type
sulfatases, an interaction of AtsB and AtsA can only occur in the
cytosol (i.e. prior to export of AtsA). In fact, serine
modification is operative in the cytosol, since FGly formation in a
signal peptide-deleted version of AtsA, although extremely inefficient
(Fig. 3), still depended on AtsB. AtsB is not strictly specific for
AtsA, with which it forms an operon at the genomic level. AtsB was able
to activate an artificial serine-type sulfatase that was generated from
the cytosolic cysteine-type sulfatase PAS of P. aeruginosa (Fig. 2). To convert PAS into a substrate of AtsB, the
Cys51 residue had to be replaced by serine and an
N-terminal signal peptide had to be added (see below).
More importantly, AtsB was shown to interact with an N- and
C-terminally truncated AtsA polypeptide (AtsA-(21-112)) encompassing the serine-type FGly modification motif. This interaction depended on
the presence of the serine to be modified, as shown both by yeast
two-hybrid and GST pull-down experiments (Table I, Fig. 4). Hence, we
conclude (i) that AtsB directly recognizes the critical Ser72 and (ii) that AtsA-(21-112), expressed as a
C-terminal appendix of stably folded Gal4BD or GST domains, fulfills
all structural requirements for proper association of its linear FGly
modification motif with AtsB. This allows us to consider the
possibility that AtsB itself is the oxidizing enzyme converting serine
to FGly.
In line with this, enzymatic redox functions have been attributed to
some AtsB-related FeS proteins. Among them are the anaerobic ribonucleotide reductase from E. coli (24) and the HemN
protein catalyzing an oxygen-independent oxidation of
coproporphyrinogen III in anaerobic heme biosynthesis (25). These and
other related proteins generate radical species by reductive cleavage
of S-adenosylmethionine through an unusual FeS center that
is also present in AtsB (23, 33). A possible reaction sequence for
AtsB-mediated FGly formation is outlined in Scheme 1. Transfer of an
electron from the reduced FeS center to S-adenosylmethionine
leads to its reductive cleavage (step 1). The generated deoxyadenosyl
radical abstracts a hydrogen atom from the substrate (i.e.
the Ser72 side chain, under formation of deoxyadenosine and
a substrate radical) (step 2). The single electron of this radical is
then accepted by the FeS center, leading to its rereduction, under formation of FGly (step 3).
SCHEME 1. Proposed mechanism for
AtsB-mediated FGly formation.
According to this mechanism, FGly formation from serine is
a single enzymatic reaction. Hence, AtsB may suffice for FGly formation in serine-type sulfatases, and additional components may merely serve
auxiliary functions (see below). In eukaryotes, serine-type sulfatases
are missing. It is therefore not surprising that only very weak
homologs of AtsB are encoded in mammalian genomes, the best human
homolog being viperin (34), an interferone-inducible antiviral protein
that locates to the endoplasmic reticulum, the site where eukaryotic
FGly formation (from cysteine) occurs. Viperin, however, shows only
26% identity with the N-terminal third of AtsB, and only one of
AtsB's three predicted FeS centers is conserved.
AtsB-dependent FGly Formation Requires a Signal Peptide
in the Sulfatase Substrate--
In GST pull-down experiments, the
interaction of AtsB with the serine-type FGly modification motif was
independent of AtsA's signal peptide. Both in yeast two-hybrid (Table
I) and in GST pull-down experiments (not shown) interaction was even
impaired by the signal peptide. This is in contrast to the in
vivo situation, where FGly formation in AtsA and in the
serine-containing mutant of PAS was clearly dependent on the signal
peptide. In the case of AtsA, deletion of the signal peptide reduced
FGly formation about 100-fold (Fig. 3), and in the case of PAS-C51S,
formation of catalytically active enzyme was totally abolished when a
signal peptide was absent (Fig. 2). The apparent contradiction between the in vitro and the in vivo studies indicates
that the presence of a serine-type FGly modification motif is
sufficient for binding of AtsB but not for FGly formation. In line with
this, we could not detect FGly in the GST-AtsA fusion protein after
incubation for 1 h in the presence of AtsB (data not shown). The
inhibitory effect of the signal peptide in the yeast two-hybrid
experiments can be explained in several ways (e.g. by a
signal peptide-induced misfolding or intracellular mislocalization of
the Gal4BD-AtsA fusion protein).
To be subjected to AtsB-dependent FGly modification, newly
synthesized AtsA obviously has to carry a signal peptide in addition to
the serine-type FGly modification motif. This may be explained in one
of three ways. The hydrophobic signal peptide may exert a direct
intramolecular effect on the sulfatase's folding state and
modification competence. Alternatively, AtsB binds directly, or
indirectly via an adaptor protein, to the signal peptide, and this may
be required for the FGly-forming activity of AtsB. Furthermore, the
signal peptide, after binding of an adaptor, may ferry newly synthesized AtsA to a site where AtsB is active. In the latter two
cases, a chaperone or targeting factor of E. coli's early secretory pathway such as trigger factor, SecB, or SecA may serve as
the putative adaptor protein. A functional in vitro
FGly-generating assay system reconstituted with cytosolic and/or
membrane components of E. coli may help to resolve the
mechanistic role of the signal peptide.