Characterization of the C-terminal Propeptide Involved in
Bacterial Wall Spanning of
-Amylase from the Psychrophile
Alteromonas haloplanctis*
Georges
Feller
§,
Salvino
D'Amico
,
Abderrafi M.
Benotmane
,
Fabian
Joly
,
Jozef
Van Beeumen¶, and
Charles
Gerday
From the
Laboratory of Biochemistry, Institute of
Chemistry B6, University of Liege, B-4000 Liege and the
¶ Laboratory of Protein Biochemistry and Protein Engineering,
University of Gent, B-9000 Gent, Belgium
 |
ABSTRACT |
The antarctic psychrophile Alteromonas
haloplanctis secretes a Ca2+- and
Cl
-dependent
-amylase. The nucleotide
sequence of the amy gene and the amino acid sequences of
the gene products indicate that the
-amylase precursor is a
preproenzyme composed by the signal peptide (24 residues), the mature
-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or
secretion helper (192 residues, 21 kDa). In cultures of the wild-type
strain, the 70-kDa precursor is secreted at the mid-exponential phase
and is cleaved by a nonspecific protease into the mature enzyme and the
propeptide. The purified C-terminal propeptide displays several
features common to
-pleated transmembrane proteins. It has no
intramolecular chaperone function because active
-amylase is
expressed by Escherichia coli in the absence of the
propeptide coding region. In E. coli, the 70-kDa precursor
is directed toward the supernatant. When the
-amylase coding region
is excised from the gene, the secretion helper can still promote its
own membrane spanning. It can also accept a foreign passenger, as shown
by the extracellular routing of a
-lactamase-propeptide fusion
protein.
 |
INTRODUCTION |
Most Gram-negative bacteria actively secrete proteins to the
extracellular medium in amounts sometimes comparable to those achieved
by Gram-positive bacteria or by yeast. Polypeptides secreted by
Gram-negative bacteria include biodegradative enzymes, toxins, and
pathogenicity factors that have important industrial or medical applications. As a result, there is now considerable interest in the
elucidation of the molecular mechanisms that allow a polypeptide to
initiate a journey in the cytoplasm and its subsequent routing to a
specific cellular compartment. These studies have highlighted the
remarkable diversity of the targeting processes involved in bacterial
secretion (1-4).
Proteins secreted by the major secretory pathway cross the bacterial
wall in a two-step mechanism via the periplasm. Exoproteins taking this
two-step route possess a N-terminal signal peptide and use the general
sec machinery for inner membrane translocation. Transport
across the outer membrane in the second step requires a secretory
apparatus encoded by large gene clusters, which are either specific or
common to several exoproteins. Polypeptides transported via the
hemolysin-type secretory pathway cross the cell envelope by a
single-step process without periplasmic intermediates. Proteins
targeted through this pathway have no N-terminal signal sequence and
show sec-independent translocation to the medium. However,
they require the assistance of accessory proteins, encoded by genes
contiguous to the exoprotein gene, which are presumed to form a
"pore" or an intermembrane channel. Secretion signals essential for
translocation are located in the C-terminal part of these proteins.
All proteins transported by these pathways do not contain sufficient
internal information to reach the external medium without assistance.
By contrast, the unusual secretion system of gonococcal IgA proteases
employs a two-step route, but after
sec-dependent translocation of the inner
membrane, the periplasmic intermediate is directed to the outer
membrane by a C-terminal propeptide, which is subsequently cleaved by
autolysis of the enzyme precursor (5-7). Propeptides are not uncommon
but are usually found in the N-terminal sequence of proteases; they are
essential for acquisition of the final folding of the active enzyme (8,
9).
-Amylase from the antarctic psychrophile Alteromonas
haloplanctis has been extensively analyzed in the context of
enzyme adaptations to catalysis at low temperatures (10-13). We found that its secretion is assisted by a C-terminal propeptide. Unlike other
propeptides, the C-terminal domain of the
-amylase precursor has no
intramolecular chaperone function but constitutes an autonomous secretion signal that can be purified from culture supernatant after
proteolytic processing. We report here structural and functional analyses of this C-terminal secretion helper.
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EXPERIMENTAL PROCEDURES |
Purification of
-Amylase, Propeptide, and Protease--
The
antarctic bacteria A. haloplanctis A23 was grown at 4 °C
for 3-5 days in 1-liter Erlenmeyer flasks containing 400 ml of broth
(16 g/liter Bactotryptone, 16 g/liter yeast extract, 20 g/liter NaCl,
10 g/liter sea salts, 30 g/liter maltose, pH 7.6) run at 250 rpm. After
concentration and diafiltration (10, 11), the culture supernatant was
loaded on a DEAE-agarose column (2.5 × 40 cm) equilibrated in 50 mM Tris-HCl, 1 mM CaCl2, pH 7.5, and eluted with a NaCl linear gradient (500 ml-500 ml, 0-0.8
M NaCl). The propeptide was eluted in front of the
gradient, the protease at 0.3 M NaCl, and the
-amylase
at 0.5 M NaCl. The latter was further purified as described
(11).
Fractions containing the propeptide were brought to 4 M
NaCl and loaded on a Phenyl-Sepharose CL-4B column (1.5 × 20 cm), subsequently washed with a decreasing gradient (40 ml) from 4 to 0 M NaCl in 50 mM Tris-HCl, pH 7.5, at room
temperature. Proteins were eluted with a gradient (40 ml) of 0-10%
(v/v) isopropanol containing 2 mM
PMSF.1 Fractions containing
the propeptide were then loaded on a DEAE-agarose column (1.5 × 40 cm) equilibrated in 20 mM Tris-HCl, 2 mM
PMSF, pH 7.5, and eluted with a gradient of 0-0.2 M NaCl
(500 ml). An Ultrogel AcA 54 column (2.5 × 100 cm) eluted with 20 mM Tris-HCl, pH 7.5, was used as the last chromatographic
step.
The protease recovered from the first fractionation of the crude
supernatant was further purified by Ultrogel AcA 54 and DEAE-agarose chromatography under the above-mentioned conditions, except that PMSF
was omitted. The last purification step was carried out on a Beckman
System Gold chromatograph fitted with an FPLC anion exchange column
(Hydropore-AX, Rainin, 1 × 10 cm) eluted with a gradient of
0-0.2 M NaCl in 5 mM Tris-HCl, 15% (v/v)
isopropanol, pH 7.5. For further experiments, the purified proteins
were conditioned in the appropriate buffers by gel filtration on PD10
columns.
Mutagenesis of the amy Gene--
An expression vector for the
-amylase precursor (p
H12) was constructed by ligating the
HpaI site located 60 nucleotides upstream from the
initiation codon of the amy gene to the SmaI site
of the pUC12 polylinker. This construction was used as template for
subsequent PCR and inverse PCR amplifications by VentR DNA
polymerase (New England Biolabs) using optimized conditions described
elsewhere (14).
The vector p
H12WT* encoding for the recombinant mature
-amylase
was constructed by PCR amplification of the amy gene using a
silent sense primer and the mutating antisense primer
5'-CCTCTAGATTCATGAGGCAGAACTG-3', which introduces a stop codon and a
XbaI site after the mature enzyme coding sequence. The
mutations were returned in the template using a
PvuII-XbaI restriction fragment. Deletion of the
-amylase coding region in pEPCT was carried out by inverse PCR of
p
H12 using silent primers, 24 nucleotides in length, ending at codon GCT for Ala-30 (antisense primer) and starting at codon AAT for Asn-448
(sense primer). The amplification product was purified (QIAquick PCR
purification kit, Qiagen), phosphorylated by T4 polynucleotide kinase,
and circularized by ligation before transformation. Deletion of the
amy gene in plasmid pEPST1 was produced by PstI digestion of p
H12 and recircularization of the vector, retaining only a coding sequence ending at Ala-30.
The
-lactamase-propeptide fusion in pBLACT was performed by inverse
PCR on a construction made of a propeptide coding region (EcoRV-XhoI) cloned downstream from the
bla gene (XbaI-SphI) of Psychrobacter immobilis (15) in the polylinker of pSP73
(Promega). Amplification used an antisense primer ending at codon AAC
for the C-terminal Asn-362 from
-lactamase and a sense primer
starting at codon AAT for Asn-448 from
-amylase. The hybrid coding
sequence was then introduced in the kanamycin-resistant vector pBGS18+ (16) at restriction sites (XbaI-SphI) identical
to those of the cloned wild-type bla gene. The sequence of
these constructions was checked by double-strand sequencing on an ALF
DNA sequencer (Amersham Pharmacia Biotech).
Production and Purification of the Recombinant
Enzymes--
Recombinant proteins were expressed in Escherichia
coli RR1 under the constitutive lacZ assistance (no
isopropyl-1-thio-
-D-galactopyranoside induction) at
18 °C in a medium containing 16 g/liter Bactotryptone, 16 g/liter
yeast extract, 5 g/liter NaCl, 2.5 g/liter
K2HPO4, 0.1 µM CaCl2,
100 mg/liter ampicillin. The recombinant precursor and mature
-amylase were purified using the protocol developed for the
wild-type enzyme, except that concentration of the supernatant by
ammonium sulfate at 70% saturation was required before the first
chromatographic step.
Enzyme Assays--
-Amylase assay was carried out at 25 °C
using 3.5 mM EPS (Boehringer Mannheim) as substrate and
excess (23 units/ml) of
-glucosidase as coupling enzyme in 100 mM Hepes, 50 mM NaCl, 10 mM
MgCl2, pH 7.1. Activities toward the synthetic substrate
were recorded in a thermostated Uvikon 860 spectrophotometer (Kontron)
and calculated on the basis of an absorption coefficient for
4-nitrophenol of 8,980 M
1 cm
1
at 405 nm (17). The kinetic parameters kcat and
Km were determined by the initial velocity method
using a nonlinear regression computer fit of the saturation curves.
The standard assay of
-lactamase was carried out at 25 °C with
300 µM nitrocefin (Glaxo Group Research) as the substrate in 50 mM phosphate buffer, pH 7.0 (18). Protease assays
using azocasein as the substrate were performed as described previously (19).
Analytical Procedures--
Dissociation constants
Kd for chloride and calcium were determined by
activation kinetics following Cl
or Ca2+
titration of the apo-enzymes and fitting the saturation curves by a
nonlinear regression analysis of the Hill equation, as described previously (11, 12). DTNB titration was carried out in 100 mM Tris, 1 mM EDTA, 1 mM DTNB, 8 M urea, pH 8.0, using an absorption coefficient for
2-nitro-5-thiobenzoate of 13,600 M
1
cm
1 at 412 nm (20).
Circular dichroism spectra were recorded in a 0.2-cm path length cell
under constant nitrogen flush using a Jobin Yvon CD6 dichrograph.
N-terminal amino acid sequences were determined using a
pulsed-liquid-phase protein sequencer (Procise 492, Applied Biosystems,
Perkin-Elmer Division, Foster City, CA) fitted with an on-line
phenylthiohydantoin analyzer. C-terminal amino acid sequences were
obtained on a Procise 494CT sequencer (Applied Biosystems, Perkin-Elmer
Division) equipped for alkylthiohydantoin analysis. Electrospray
ionization mass spectrometry of protein samples was performed on
a BIO-Q electrospray mass spectrometer (Micromass, Altrincham, United
Kingdom).
 |
RESULTS |
Sequence of the amy Gene and Characterization of the Gene
Products--
The cloning of the amy gene from A. haloplanctis in E. coli, its nucleotide sequence, and
the deduced amino acid sequence of the native
-amylase have been
reported previously (10). The updated nucleotide sequence of the 3'
region of the gene is shown in Fig. 1
along with the corresponding open reading frame. These new data
indicate that the amy gene encodes an
-amylase precursor
composed of 669 amino acid residues. N- and C-terminal amino acid
sequences of the native
-amylase secreted by A. haloplanctis allow the location of three distinct functional
domains of the precursor: (i) the peptide signal made of 24 residues,
(ii) the mature enzyme composed of 453 residues with a
Mr value of 49,340, and (iii) a large C-terminal
propeptide composed of 192 residues (Fig. 1). Inspection of A. haloplanctis culture supernatants revealed the occurrence of a
21-kDa protein, which was further purified to homogeneity. N- and
C-terminal amino acid sequences identified this component as the
-amylase propeptide and confirmed the unique cleavage site between
Ser-453 and Ser-454 (Fig. 1). Electrospray mass spectrometry yielded an
Mr value of 21,518 ± 1.5, in excellent agreement with that deduced from the gene (21,519). However,
electrophoretic mobility on SDS gels leads to overestimated values (see
Fig. 5). The propeptide is a slightly acidic protein, having a pI = 5.0 determined under non-denaturing conditions. It contains four
cysteine residues. Sulfhydryl titration by DTNB of both the native and the denatured protein in 8 M urea indicates that there is
no free thiol group, demonstrating the occurrence of two disulfide
linkages. There is no significant difference in the amino acid molar
ratios of both native
-amylase and the cleaved C-terminal domain. A search through the GenBank/EMBL data banks failed to reveal any significant homology with known proteins or translated nucleotide sequences, except with the C-terminal region (34% identity in 200 amino acid overlap) of the hypothetical
-amylase from the nematode
Caenorhabditis elegans (Swissprot P91982). To date, only the
nucleotide sequence of C. elegans
-amylase is known, but
multiple sequence alignment with the translated protein indicates that
it also belongs to the Ca2+- and
Cl
-dependent
-amylase family. Its
C-terminal extension (200 residues predicted) is possibly involved in
secretion or it could serve to anchor the enzyme to the eukaryote
membrane.

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Fig. 1.
Nucleotide sequence and derived amino acid
sequence of the C-terminal propeptide from the -amylase
precursor. The propeptide cleavage site is indicated. Amino acids
confirmed by N- and C-terminal sequencing are underlined,
and the four cysteine residues are double-underlined. Amino
acids are numbered, starting at the N-terminal residue of
the native precursor after signal peptide cleavage. Nucleotide
numbering corresponds to the sequenced genomic DNA fragment
(GenBank/EMBL accession number X58627).
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Secondary structure prediction algorithms (GCG 9.0) suggest a high
content of
-sheet-forming residues, with several possible amphipathic
-sheets as indicated by hydrophobic moment analysis. This is further emphasized by the far-UV circular dichroism spectra of
the secretion helper (Fig. 2). Secondary
structure analysis of CD spectra (21) correctly estimated the
-helix
and the
-sheet content (20% and 30%, respectively) of the known
-amylase three-dimensional structure (22) taken as reference. CD
spectra of the propeptide contrasted with those of
-amylase and were
typical of a
-pleated protein (50-60%) with a low
-helical
content (~10%). Addition of urea abolished the CD signals and
confirms the existence of secondary structure organization of the
isolated propeptide.

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Fig. 2.
Far-UV circular dicroism spectra of
-amylase and its propeptide. Spectra of the propeptide
(long dash) and of -amylase (line) were taken
in 5 mM NaH2PO4, pH 7.0. Propeptide
in the presence of 8 M urea (dashed). Data are
expressed in terms of the mean residue ellipticity, .
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Expression of the amy Gene in A. haloplanctis--
A.
haloplanctis is a psychrophilic bacterium efficiently growing at
near-zero temperatures (generation time of 4.0 h at 4 °C) and
reaching cell densities as high as 5 × 1010 cells/ml
after 100 h of growth (Fig. 3).
Addition of maltose up to 3% results in a 200-fold induction of
-amylase expression. Fig. 3 also shows that the amylolytic activity
is sharply produced in the supernatant during the exponential growth
phase. Rabbit antibodies raised against
-amylase and the propeptide
were used to study expression of the amy gene in the
wild-type strain. Western blot analysis of samples taken at all growth
stages (Fig. 4) reveals that
-amylase
is expressed in the culture supernatant as a 70-kDa precursor, which
further dissociates into the mature enzyme and the free propeptide. The
level of extracellular activity correlates with the production of the
precursor rather than with the appearance of the mature enzyme, already
suggesting that the precursor is active. Neither the precursor nor its
two products have been detected in cell pellets.

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Fig. 3.
Growth of A. haloplanctis and
-amylase production. Growth of the psychrophilic bacteria at
4 °C ( ) and -amylase activity in the culture supernatant
( ).
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Fig. 4.
Secretion and maturation of the -amylase
precursor in A. haloplanctis culture supernatant.
Western blots of cell-free supernatant samples corresponding to the
culture shown in Fig. 3. Sampling time and antigen molecular mass are
indicated. Upper panel, antigen detection using rabbit IgG
anti-propeptide. Lower panel, antigen detection by rabbit
IgG anti- -amylase. Secondary antibodies were alkaline
phosphatase-conjugated anti-rabbit IgG.
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Expression of the Recombinant Precursor in E. coli--
In order
to study the maturation mechanism, the precursor was expressed in
E. coli. The amy gene was cloned downstream from the lacZ promoter of pUC12, resulting in the production of
60-100 mg of precursor/liter in the host culture supernatant. N- and C-terminal amino acid sequence determinations indicated that the signal
peptide is correctly cleaved in E. coli and that no
additional post-translational cleavage occurred. This recombinant
precursor is fully active (see below), and a native conformation of the propeptide is expected, taking into account the extracellular location
of the gene product.
When incubated in wild-type conditions (4 °C in sterile broth or in
10 mM imidazole and the medium salts), the purified
precursor is stable for weeks. By contrast, addition of
filter-sterilized aliquots of A. haloplanctis culture
supernatants initiated its cleavage into
-amylase and the
propeptide. This demonstrates the requirement for an extrinsic factor
(i.e. a protease) in the maturation process.
Proteolytic Maturation of the
-Amylase Precursor--
Only one
proteolytic enzyme was detected in A. haloplanctis culture
supernatants. This 45-kDa protease (AHP) is a metalloenzyme (60%
inhibition by excess EDTA) from the serine-protease family (98%
inhibition by 2 mM PMSF). AHP has a broad specificity and readily hydrolyses macromolecular substrates such as precipitated casein or azo-labeled casein. The N-terminal amino acid sequence of AHP
(S-T-P-N-D-P-P-F-D-D-Q-S-Y-Y-E-Q-A-G-) shows strong homology with some
other microbial Ser proteases such as those from Bacillus thuringiensis (accession no. JN0369) and Dichelobacter
nodosus (no. L18984) and, notably, with the protease from a
mesophilic Alteromonas strain (no. D38600).
When the purified AHP and the recombinant precursor are mixed in the
in vivo ratio (
1/10 in sterile broth), cleavage into
-amylase and the propeptide occurs at a rate similar to that recorded in A. haloplanctis cultures (Fig.
5). Furthermore, the N-terminal sequence
of the in vitro processed propeptide is identical to that of
the wild-type propeptide, demonstrating that AHP is the extrinsic
factor required for the maturation of the precursor.

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Fig. 5.
In vitro maturation of the recombinant
-amylase precursor. Upper panel, 12% SDS-PAGE of protein
standards (M, from the top: phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 kDa; carbonic
anhydrase, 31 kDa; and soybean trypsin inhibitor, 21.5 kDa);
Mix, mixture of the purified recombinant precursor and of
wild-type -amylase and its propeptide; Pre, recombinant
-amylase precursor incubated at 4 °C in sterile culture medium
for 1 week; +S, as for Pre with 10% volume of
sterile cell-free supernatant from A. haloplanctis culture;
+AHP, as for Pre with 5% (w/w) purified
serine-protease from A. haloplanctis. Lower
panel, maturation by nonspecific proteases. 12% SDS-PAGE of
M and Mix (see upper panel);
incubation of the precursor at room temperature with 0.1% proteinase K
for 60 min (PrK), 1% Pronase for 160 min (Pro),
or 1% subtilisin for 120 min (Sub). Western blot using IgG
anti-propeptide (data not shown) confirmed the propeptide
release.
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Linker Susceptibility to Proteolysis--
The specific cleavage of
the propeptide by the nonspecific AHP protease prompted us to test the
action pattern of other nonspecific proteases such as Pronase,
proteinase K, and subtilisin. As shown in Fig. 5, these proteases
preferably cleaved the precursor in the linker region between
-amylase and the propeptide. Proteolytic cleavage by E. coli proteases was also noted during expression of the recombinant
precursor. Indeed, about 5% of the produced enzyme is cleaved before
starting the purification procedure. N-terminal sequence of the
propeptide processed in E. coli reveals that the cleavage
site is displaced between Thr-455 and Glu-456 (Fig. 1). A
Mr value of 21,331 for this propeptide is
predicted from the nucleotide sequence, in perfect agreement with the
electrospray ionization mass spectrometric analysis (21,330 ± 4).
It is concluded that the linker region between
-amylase and the
propeptide probably consists of a disordered, solvent-exposed loop,
prone to various proteolytic attacks. This is also supported by the
lack of defined electron density for the last five residues in the
x-ray structure of A. haloplanctis
-amylase (22).
The Propeptide Has No Foldase Activity--
The foldase activity
generally associated with propeptides (8, 9) has been probed by
removing the propeptide sequence from the amy gene and
introducing a stop codon after Ser-453, the last residue of the mature
wild-type enzyme. Properties of the wild-type and recombinant
-amylases as well as those of the recombinant precursor are compared
in Table I. It is shown that both the
kinetic and ion-binding parameters are identical in the three related
enzymes. All the cysteine residues of the native
-amylase and of the
propeptide are engaged in disulfide linkages. Thus, the lack of
significant free sulfhydryl groups, as detected by DTNB titration, also
confirmed the absence of misfolded species in the recombinant
enzymes.
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Table I
Kinetic parameters, dissociation constants, and free thiol groups for
the wild-type and the recombinant -amylases
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Heterologous Secretion in E. coli--
Involvement of the
propeptide in the secretion pathway of A. haloplanctis
-amylase has been analyzed by genetic modification of the
amy gene and its expression in E. coli. Its
function of secretion helper during translocation across the outer
bacterial wall is well illustrated in Fig.
6. When the amy gene (p
H12) is expressed in E. coli, the extracellular targeting of the
precursor follows the bacterial growth curve. At the end of the
exponential growth phase (
30 h), about 80% of the total enzyme
production is found in the cell-free supernatant of E. coli,
15% is found in the periplasmic space, and 5% remains cell-associated
as determined by osmotic shocks (data not shown). The same results were
obtained when the amy gene expression was reduced 100-fold
(in E. coli BL21 expressing the LacI repressor), showing
that the appearance of the precursor in the medium is not the result of
its overexpression. By contrast, removal of the propeptide sequence
(p
H12WT*) leads to periplasmic accumulation of the recombinant
-amylase (followed by its release into the medium during cell
lysis). It is concluded that the propeptide efficiently assists outer
membrane translocation in E. coli. The outer membrane
integrity has been checked by monitoring the
-lactamase activity in
the cell-free supernatants (
-lactamase is a periplasmic enzyme
encoded by the plasmid vector and is responsible for the antibiotic
resistance). Fig. 6 shows that heterologous secretion of the
-amylase precursor (p
H12) induces outer membrane damage in
E. coli as indicated by the release of
-lactamase in the
medium, by the slight growth inhibition and early cell lysis.

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Fig. 6.
Expression of the precursor and of the
recombinant native -amylase in E. coli. Upper
panel, gene constructs encoding the 70-kDa precursor (p H12) and
the native -amylase (p H12WT*) devoid of the propeptide coding
sequence. SP, signal peptide; Ct, C-terminal
propeptide. Lower panel, -amylase and -lactamase
activity in E. coli culture supernatants (activity is
expressed as percent of the maximal activity recorded in the cell-free
supernatant of E. coli (p H12)) and bacterial growth at
18 °C (A550).
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Self-secretion of the Propeptide--
The autonomous outer
membrane insertion of the propeptide was analyzed by removing the
-amylase coding region from the amy gene (pEPCT). In
order to target the modified gene product into the periplasmic space,
the peptide signal and the 30 N-terminal amino acids, which can form
the export initiation domain (23, 24), were not deleted. In addition,
the six last C-terminal residues of the mature
-amylase were also
conserved. Indeed, sequence alignment with other
chloride-dependent
-amylases from insects and
vertebrates shows that the bacterial enzyme is six residues longer at
the C terminus. These residues can therefore belong to the linker
region with the propeptide and were not deleted. The control
construction (pEPST1) is identical but only encodes for the peptide
signal and the 30 first
-amylase residues.
When the pEPCT construct is expressed in E. coli, two gene
products recognized by IgG anti-propeptide already appear in the supernatant during the exponential growth phase and further accumulate in the extracellular medium (Fig. 7). As
shown by Western blots, one compound corresponds to the wild-type
propeptide, whereas the second has a slightly higher molecular mass
(±3.5 kDa). It is likely that the propeptide is expressed with the
export initiation domain and that cleavage at the linker by E. coli proteases further occurs in the periplasm, as already noted
for the complete precursor. Autonomous propeptide translocation also
induces outer membrane damage as indicated by
-lactamase leakage,
whereas the control vector encoding for the export initiation domain
alone does not affect the host cells.

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Fig. 7.
Expression of the propeptide in E. coli. Upper panel, gene construct encoding the
propeptide (Ct) preceded by the signal peptide
(SP), the export initiation domain (Ex), and the
linker (L) in pEPCT. The control construct pEPST1 only
encodes signal peptide and export initiation domain. Lower
panel, -lactamase activity in E. coli culture
supernatants (activity is expressed as percent of the maximal activity
recorded in the cell-free supernatant of E. coli (pEPCT))
and bacterial growth at 18 °C (A550).
Inset, Western blot of the purified wild-type propeptide
(A) and of the cell-free supernatant of E. coli
(pEPCT) after 30 h (B) using IgG anti-propeptide for
detection.
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-Lactamase Targeting to the Extracellular Medium--
The
ability of the
-amylase propeptide to export a foreign passenger was
tested by the construction of a
-lactamase-propeptide protein
fusion. The class C
-lactamase from the Gram-negative bacterium
P. immobilis A5 (15) was selected because (i) it is also a
heat-labile psychrophilic enzyme; (ii) like other
-lactamases, the
recombinant enzyme accumulates in the periplasmic space of E. coli; and (iii) it is devoid of disulfide bonds that may impair outer membrane translocation in E. coli as reported
previously (25), although the
-amylase contains four disulfide bonds
but is secreted efficiently.
The coding sequence of the propeptide and of the six C-terminal linker
residues were fused to the bla gene by inverse PCR and
cloned in a kanamycin-resistant vector (pBLACT). The control vector
pBLAC4 is identical but only carries the wild-type bla gene.
The
-lactamase-propeptide fusion remains catalytically active and
provides the usual ampicillin resistance; its specific activity on
nitrocefin in clear periplasmic extracts is similar to that of the
wild-type
-lactamase prepared in the same conditions. Unlike the
native
-lactamase, which remains periplasmic, the
-lactamase-propeptide fusion appears in the cell-free supernatant as
shown in Fig. 8. However, the
extracellular targeting of the fusion is delayed when compared with
-amylase (p
H12), indicating a less efficient translocation
process. No cleavage of the fusion protein was detected in the
supernatant by Western blots using IgG anti-propeptide but mild
proteolytic treatment using proteinase K, Pronase, and A. haloplanctis serine protease allowed to remove the C-terminal
propeptide (data not shown).

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Fig. 8.
Expression of the -lactamase-propeptide
fusion protein in E. coli. Upper panel, gene
constructs encoding the wild-type -lactamase (pBLAC4) and its fusion
with the propeptide (pBLACT). Lower panel, -lactamase
activity in E. coli culture supernatants (activity is
expressed as percent of the maximal activity recorded in the cell-free
supernatant of E. coli (pBLACT)) and bacterial growth at
18 °C (A550).
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DISCUSSION |
In most enzymes possessing an N- or C-terminal propeptide,
truncation of the prosequence precludes correct folding of the associated enzyme leading to inactive polypeptides. A foldase activity
or an intramolecular chaperone function has been therefore attributed
to propeptides (8, 9). We have shown that A. haloplanctis
-amylase produced in the presence or in the absence of the
propeptide has the same catalytic and ion binding properties (Table I).
Owing to the stringent structural requirements for functional substrate
and ion binding site formation, it can be safely concluded that the
enzyme synthesized without the propeptide is properly folded and,
therefore, that its C-terminal domain is definitely not an
intramolecular chaperone.
By contrast, the propeptide is involved in the translocation across the
outer bacterial wall, as evidenced by its autonomous translocation and
the extracellular targeting of the
-amylase precursor in E. coli (Figs. 6 and 7). In the latter, propeptide-assisted membrane
spanning induces outer membrane alterations, which are likely to be due
to differences in outer wall composition (especially in lipids) between
the mesophilic E. coli and the psychrophilic A. haloplanctis. The purified propeptide has a high
-sheet content (Fig. 2). Additionally, it is interesting that the 15 last residues of
the propeptide can form an amphipathic
-sheet ending with a
C-terminal phenylalanine, which are essential features for the correct
assembly of most bacterial outer membrane proteins such as PhoE
(26).
The following two-step secretion pathway of A. haloplanctis
-amylase can be proposed. The 200-fold induction of
-amylase production suggests the occurrence of a maltose-regulated promoter as
already reported for some other microbial
-amylases (27). After gene
induction and initiation of the translation, the nascent
-amylase
precursor undergoes the classical sec-dependent
inner membrane translocation, as evidenced by the occurrence of a
cleavable signal peptide. The periplasmic intermediate then inserts
into the outer wall via its C-terminal propeptide in a way probably similar to other bacterial outer membrane proteins. However, the next
specific events involve translocation of the
-amylase domain across
the outer wall and the extracellular release of the uncleaved precursor
in a native conformation. The last step requires the assistance of the
nonspecific AHP protease in order to remove the C-terminal secretion
helper by cleavage at the easily accessible linker region. As the
precursor is not detected in A. haloplanctis cell pellets,
the post-transcriptional events leading to secretion seem very fast,
without accumulation of detectable cell-associated intermediates. The
propeptide-assisted mechanism of membrane spanning (through or beside a
possible
-barrel) and the translocation driving force remain
unknown.
To our knowledge, few other bacterial exoenzymes possessing a
C-terminal propeptide have been reported: Neisseria IgA
proteases (5-7), Thermus aquaticus aqualysin I protease
(28), Serratia marcescens SSP protease (29),
Helicobacter pylori VacA cytotoxin (30), Lysobacter
enzymogenes alkaline phosphatase (31), E. coli and
Shigella virulence proteins EspC (32) and VirG (33), and
Bordetella pertussis pertactin (34). According to the
available data, the extracellular routing of these enzymes and of
-amylase should follow the same main steps of the above mentioned
secretion pathway. However, the propeptide from A. haloplanctis
-amylase precursor is unusual because it remains
associated to the precursor in the external medium, it requires
external proteolytic assistance for cleavage, it can be recovered from
supernatants, and it has no intramolecular chaperone function.
 |
ACKNOWLEDGEMENTS |
We are grateful to the Institut
Français de Recherche et de Technologie Polaire for the
support and facilities offered at the Antarctic station Dumont
d'Urville during earlier stages of this work. We also thank B. Samyn
for expertise in the C-terminal amino acid analysis, as well as N. Gerardin-Otthiers and R. Marchand for expert technical assistance.
 |
FOOTNOTES |
*
This work was supported by EU Network Contract ERBCHCT
940521, Concerted Action BIO4-CT95-0017, and Program Grant
BIO4-CT96-0051; by Ministère de l'Education, de la Recherche et
de la Formation, Concerted Action ARC93/98-170; and by Région
Wallonne-Direction Générale des Technologies, Convention
1828.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X58627.
§
To whom correspondence should be addressed. Tel.: 32-4-366-33-43;
Fax: 32-4-366-33-64; E-mail: gfeller{at}ulg.ac.be.
1
The abbreviations used are: PMSF,
phenylmethylsulfonyl fluoride; EPS,
4-nitrophenyl-
-D-maltoheptaoside-4,6-O-ethylidene; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); PCR, polymerase chain reaction; AHP, A. haloplanctis protease.
 |
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