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Applied and Environmental Microbiology, January 2000, p. 363-368, Vol. 66, No. 1
Section Molecular Genetics of Industrial
Microorganisms, Wageningen University, Wageningen, The Netherlands
Received 2 August 1999/Accepted 1 November 1999
Secreted yields of foreign proteins may be enhanced in filamentous
fungi through the use of translational fusions in which the target
protein is fused to an endogenous secreted carrier protein. The fused
proteins are usually separated in vivo by cleavage of an engineered
Kex2 endoprotease recognition site at the fusion junction. We have
cloned the kexin-encoding gene of Aspergillus niger
(kexB). We constructed strains that either overexpressed KexB or lacked a functional kexB gene. Kexin-specific
activity doubled in membrane-protein fractions of the strain
overexpressing KexB. In contrast, no kexin-specific activity was
detected in the similar protein fractions of the kexB
disruptant. Expression in this loss-of-function strain of a
glucoamylase human interleukin-6 fusion protein with an engineered Kex2
dibasic cleavage site at the fusion junction resulted in secretion of
unprocessed fusion protein. The results show that KexB is the
endoproteolytic proprotein processing enzyme responsible for the
processing of (engineered) dibasic cleavage sites in target proteins
that are transported through the secretion pathway of A. niger.
Many secreted eukaryotic proteins
contain a signal peptide and an adjacent propeptide at the amino
terminus. The signal peptide specifies a sequence for translocation
over the endoplasmic reticulum membrane and is normally removed in the
lumen during translocation by a signal peptidase. Propeptides have been
implicated in correct folding and in subcellular sorting of proteases.
They also often function as (auto)inhibitors. The processing of most of
these propeptides occurs at either a monobasic or a dibasic cleavage site (2). Propeptides, which are cleaved after a Lys-Arg or Arg-Arg basic doublet at the P2 and P1 positions (the nomenclature used
is according to reference 27), are specifically
recognized and processed in the trans-Golgi network by the kexin family
of proteases, a subfamily of the subtilase family of proteases
(29). The kexin family consists of the yeast Kex2-like
proteases (EC 3.4.21.61), the mammalian prohormone convertases (PCs)
(EC 3.4.21.93 and EC 3.4.21.94), and the furins (EC 3.4.21.75). All
members of the kexin subfamily are calcium-dependent, neutral, serine proteases that are activated by the removal of the amino-terminal propeptide at a kexin-specific (auto)processing site. The active proteases all contain two additional domains, a subtilisin-like domain
containing the catalytic triad and a conserved P or Homo B domain of
approximately 150 residues. The P domain, which is absent in other
subtilases, is essential for the catalytic activity (21) and
the stability of the protein (18). Kex2-like yeast proteases, furins, and some of the PCs also have a single transmembrane domain (20, 21, 28). In its cytoplasmic tail, yeast Kex2 contains a Golgi retrieval signal, necessary to remain in the trans-Golgi network (33).
In Aspergillus spp., kexin-like activity has been detected
through the cleavage of artificial fusion proteins. Here, artificial fusion proteins are used for the production of foreign proteins, exploiting the efficient production, sorting, and processing of endogenous proteins (reviewed in reference 13). In
these constructs, a consensus Kex2 cleavage site often separates the
foreign protein from the endogenous protein. Studies with such fusion
proteins in Aspergillus niger showed that amino acid
residues directly adjacent to the cleavage site can affect correct
processing of an engineered Kex2 site (30).
Examples of physiological substrates for an A. niger
kexin-like endoprotease are the endopolygalacturonase (Pga) family of proteins (6, 7, 8, 23). They all contain a dibasic cleavage
site at the carboxy-terminal end of their amino-terminal propeptide,
except for PgaII, which has a single arginine residue preceding the
cleavage site (6).
Our objectives in this study were (i) to show that A. niger
expresses a Kex2-like dibasic endoprotease and (ii) to demonstrate that
this endoprotease is responsible for the cleavage of a fusion protein
with an engineered Kex-2 site.
Strains, transformation, and DNA and RNA techniques.
The
A. niger strains used in this study are listed in Table
1. NW219, NW249, and NW266 were used for
the transformation of A. niger, as previously described
(17). Strain NW266 was constructed by the transformation of
NW249 with pIM4003. (The construction of the pIM plasmids is described
below.) MCK-5 was constructed by the cotransformation of NW249 with
pGW635, containing the A. niger pyrA gene, and pIM4002. MCGI
and MCGI
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of the Kexin-Like Maturase of
Aspergillus niger
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
were constructed by the cotransformation of, respectively,
NW219 and NW266 with pGW635 and pFGPDGLAHIL6 (9).
TABLE 1.
A. niger strains used in
this studya
DNA isolation. E. coli DH5
was used for plasmid transformation and propagation. Standard DNA
manipulations were carried out essentially as described by Sambrook et
al. (26). Plasmid pUC19 or phagemid pBluescript SK(+) was
used as a cloning vector for genomic DNA fragments. Cloned hybridizing
fragments and cDNA clones were sequenced with a Thermo Sequenase
fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP
(Amersham Pharmacia Biotech, Uppsala, Sweden) and an ALF automated
sequencer (Amersham Pharmacia Biotech).
For Southern analysis, total DNA from Aspergillus strains
was isolated as previously described (9a). Hybridizations
were done in standard hybridization buffer (SHB) (6× SSC, 5×
Denhardt's solution (27), 0.5% sodium dodecyl sulfate
(SDS), and 100 µg of denatured herring sperm DNA ml
1).
1× SSC contains 0.15 M NaCl and 0.015 M sodium citrate. Washing was
performed at 65°C to a final stringency of 0.1× SSC and 0.1% SDS.
For nonstringent conditions, hybridization was executed at 56°C and
washing was done twice in 4× SSC and 0.1% SDS at the same temperature.
For Northern analysis, strains were grown for 17 h in minimal
medium (24) supplemented with 1% glucose as a carbon source and 0.5% yeast extract in 50-ml cultures in 250-ml Erlenmeyer flasks
in an Innova incubator shaker (New Brunswick Scientific Co., Inc.,
Edison, N.J.) at 250 rpm at 30°C. Mycelium was harvested by
filtration over a nylon membrane (mesh size, 100 µm) and the mycelium
was ground with a Braun II dismembrator (B. Braun Melsungen AG,
Melsungen, Germany). Total RNA was isolated from mycelium samples with
Trizol reagent (Life Technologies, Rockville, Md.). RNA concentrations
were determined spectrophotometrically and equal amounts of RNA were
denatured with glyoxal by standard techniques (26) and
separated on a 1.2% (wt/vol) agarose gel. RNA blots were hybridized at
42°C in SHB to which 10% (wt/vol) dextran sulfate and 50% (vol/vol)
formamide were added. Washing was performed at 65°C to a final
stringency of 0.1× SSC and 0.1% SDS. As a control, Northern blots
were hybridized with A. niger ribosomal protein gene
rpS28.
Cloning of kexB.
A kexB PCR product was
generated by PCR on genomic DNA of A. niger N400 with two
degenerate primers: the forward primer was 5'-CAYGGNACIMGITGYGCNGG-3',
encoding HGTRCAGE, and the reverse primer was 5'-TAYTGNACRTCNCKCCA-3',
encoding WRDVQY (standard IUB-IUPAC symbols are used to indicate the
nucleotide mixes; I indicates inosine). A standard program of 30 thermal cycles was composed of 1 min at 94°C, 1 min at 48°C, and 1 min at 72°C, preceded by an incubation of 4 min at 95°C and
followed by an incubation of 5 min at 72°C. The amplified fragment
was cloned in vector pGEM-T (Promega) and identified by sequencing.
Genomic sequences of the kexB gene were obtained by
screening a EMBL4 genomic library of A. niger N402 by
standard methods (26), with the kexB PCR product
as a probe. Four clones were isolated and from one of these
positive phages, two SalI fragments of about 2 kb were
subcloned in pUC19.
Plasmid construction. The upstream and downstream kexB SalI fragments in pUC19 were each recloned in pBluescript SK(+) digested with SalI and XhoI. The resulting plasmids were selected for having the SalI sites upstream and downstream, respectively, of kexB ligated in the XhoI sites of the pBluescript vectors. Next, the downstream kexB fragment in pBluescript was ligated as a SalI-EcoRI fragment downstream of the upstream kexB fragment, yielding pIM4002 (see Fig. 2). For the construction of pIM4003, a 590-bp ClaI-SalI fragment was first removed from the open reading frame (ORF) of the upstream SalI fragment in pBluescript. The resulting plasmid was digested with HindIII and PstI, and the argB gene was inserted 3' of the truncated kexB fragment. In the resulting plasmid, the kexB downstream fragment was inserted as a PstI-BamHI fragment yielding pIM4003 (see Fig. 3).
Nucleotide and protein sequence analyses. Sequence analysis was performed with the sequence analysis software package PC/Gene (IntelliGenetics, Inc., Geneva, Switzerland). Public databases were searched with the BLAST search tools (1). Multiple alignment was done in CLUSTAL W (31). The SignalP program was used to identify the signal sequence for secretion (22). Putative transmembrane regions were identified with the SOSHUI program (14).
Isolation of KexB containing membrane-protein fractions.
Strains were grown and mycelium was treated as described for the
Northern analysis. Ground mycelium (3 g [wet weight]) was extracted
with 6 ml of 50 mM sodium HEPES (pH 7.6) and 10 mM EDTA. The extract
was clarified by centrifugation (10,000 × g for 15 min
at 4°C) and the clarified supernatant was centrifuged again at
100,000 × g for 90 min at 4°C. The supernatant was
discarded and the pelleted membrane-containing fraction was extracted
with 5 ml of 50 mM sodium HEPES (pH 7.6), 1 mM EDTA, 50 mM NaCl,
and 2% (wt/vol) sodium deoxycholate in 20% glycerol. The extract was clarified again by centrifugation at 100,000 × g for
90 min at 4°C. The supernatant was stored at 
20°C until use.
Kexin enzyme assay.
7-Amino-4-methylcoumarin (AMC) and
tert-butyloxycarbonyl (Boc) methylcoumarinamide (MCA)
derivatives were purchased from Sigma Chemical Co. (St. Louis, Mo.).
The reaction mixture (100 µl) contained 200 mM sodium HEPES (pH 7.0),
1.5 mM CaCl2, and 100 µM MCA derivative (Table
2). Reaction mixtures were incubated at
37°C for 0 to 4 h and the reactions were terminated by the
addition of 1.8 ml of 125 mM ZnSO4 and 0.2 ml of saturated
Ba(OH)2. The precipitate was removed by centrifugation for
3 min at 10,000 × g, and the amount of AMC liberated
was measured with a Hitachi F4500 fluorescence spectrophotometer
calibrated with known amounts of AMC (ex = 370 nm;
em = 445 nm). One unit was defined as 1 pmol of AMC
released per min (12). Protein concentrations were
determined by the bicinchoninic acid method as described by the
manufacturer (Sigma Chemical Co.).
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Western analysis of strains MCGI and MCGI.
Shake flask
cultures were used to express the PgpdA-glaA-hIL-6 fusion
gene from strains MCGI and MCGI, each harboring 10 to 15 copies of
the construct (Table 1). Culture conditions were as described for
Northern analysis except that xylose was used as a carbon source to
suppress the endogenous glucoamylase gene. Culture fluid was
concentrated by deoxycholate-trichloroacetic acid precipitation. Medium
samples were subjected to SDS-polyacrylamide gel electrophoresis and
standard protocols were used for the detection of recombinant human
interleukin-6 fusion protein by Western analysis (26). Mouse
monoclonal human interleukin-6 antibody (R & D Systems, Inc.,
Minneapolis, Minn.) was used for the detection. Recombinant human
interleukin-6 (Sigma-Aldrich, St. Louis, Mo.) was used as a positive control.
Nucleotide sequence accession number. The sequence data of the kexB gene has been submitted to the DDBJ, EMBL, and GenBank databases under accession no. Y18127.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cloning of an A. niger kexin homologue.
We
designed degenerate primers based on amino acid sequences shared by
several kexin proteases and amplified a 603-bp PCR product from
A. niger genomic DNA. A genomic SalI fragment of approximately 2 kb hybridized with the probe. We recovered two SalI fragments of similar size from an A. niger
genomic library. The fragments contained adjacent parts of the
complete ORF of the gene we designated kexB (Fig.
1). The ORF encodes a protein of 844 amino acids interrupted by a putative 51-bp intron located 1,763 bp
downstream of the start codon. We identified a partial 1-kb cDNA clone
in a cDNA library of A. niger that confirmed that the intron
was positioned correctly. The cDNA ended in a poly(A) tail 133 bp
downstream of the inferred stop codon. Southern analysis performed
under conditions of low stringency indicated that the kexB
gene is a single copy gene.
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Analysis of the kexB ORF sequence. The encoded protein is highly similar to the kexin subfamily of proteases and also shares significant similarity with several other subtilisins. The highest similarity was with the Kex2-like protease from the yeast Yarrowia lipolytica (10), yielding 42% overall identity.
As has been described for other kexin proteases, five putative domains were identified in the deduced amino acid sequence (Fig. 1). The first 19 amino acid residues of the ORF were identified by the signalP neural network (22) as a signal sequence for translocation over the endoplasmic reticulum membrane. The adjacent propeptide probably encompasses amino acids 20 to 129. At the carboxy-terminal end of this segment, there is a Lys-Arg dibasic (auto)cleavage site. Amino acids 190 to 435 contain a subtilisin-like domain. This domain has 53 to 62% identity with the same domains of the yeast Kex2-like proteases and contains the active site residues of the Asp, His, and Ser catalytic triad and a conserved Asn residue which stabilizes the oxyanion hole in the transitional state (5). The P domain (Fig. 1) is not found in other subtilase subfamilies and its presence therefore strongly suggests that we have indeed cloned a member of the kexin subfamily. There also is a putative single membrane-spanning domain. In the putative cytoplasmic tail, 15 amino acids downstream of the transmembrane domain, we found the peptide sequence YDFEMI. The underlined amino acids residues are identical to the late Golgi retention signal (consensus, YXFXXI) in the cytoplasmic tail of the Saccharomyces cerevisiae protease (33). In a multiple alignment, the A. niger KexB protease was compared to other proteases of the kexin subfamily of proteases and PepC (11) and PepD (15), two A. niger subtilases of the proteinase K subfamily (29). The alignment indicated that the A. niger endoprotease is more similar to the yeast-like kexin proteases than to the furins and PCs and is only weakly related to the A. niger PepC and PepD subtilases.Characterization of kexB disruptants and overproducers. We transformed strain NW249 (Table 2) with plasmid pIM4002 and identified three multicopy transformants with elevated expression of the kexB gene (Fig. 2).
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Characterization of KexB activity. We measured the relative kexin activity of the detergent-solubilized membrane-protein fraction (DSP) for 4 h with Boc-L-K-R-MCA as a fluorogenic substrate in a wild-type strain (NW249), a kexB disruptant (NW266), and a kexB multicopy transformant (MCK-5) (Table 1). The DSP fraction of MCK-5 had more than twice as much hydrolyzing activity towards the MCA substrate as the DSP fraction from the wild-type strain. The DSP fraction of the kexB-disrupted strain had no significant hydrolyzing activity (Fig. 4). The amount of AMC liberated increased linearly with time if DSP fractions of NW249 or MCK-5 were used, indicating that under assay conditions the enzyme activity is stable.
|
), MCK-5 (
), and NW266 (
). The
data represent the means of two independent experiments. Standard
errors are indicated by bars or are within each symbol.
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KexB cleavage of an engineered Kex2 site in a reporter
construct.
Strains MCGI (wild type) and MCGI (kexB
disruptant) (Table 1) express a fusion gene encoding a
glucoamylase-human interleukin-6 fusion protein separated by a Kex2
site at the fusion junction. Western analysis of the reporter showed
that while the wild-type strain secretes glucoamylase and interleukin-6
separately, the kexB disruptant is unable to hydrolyze the
Kex2 site at the fusion junction and therefore only secretes the intact
fusion protein (Fig. 6).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We used a PCR approach to clone a kexin homologue in A. niger. Kexins have been cloned and characterized from yeasts and mammalian cells, and thus from an evolutionary perspective, the existence of such a function was predictable. The work done with polygalacturonases (6, 7, 8, 23) and with fusion proteins that use engineered Kex2 sites at the fusion junction (reviewed in reference 13) suggested that this function exists in A. niger. The kexB gene we cloned encodes a Kex2-like dibasic endoprotease that has significant similarity to expressed sequence tags (ESTs) of two other filamentous fungi. One EST (GenBank accession no. AA787123) originated from Aspergillus nidulans, and a translation from that EST has 64% identity with KexB. The other EST (GenBank accession no. AIO68899) was from the pyrenomycete Magnaporthe grisea, and a translation from that EST also showed 64% identity with KexB. These findings suggest that this endoprotease function is ubiquitously expressed in filamentous fungi.
Strain MCK-5 has more than 15 copies of the kexB gene, and a high level of overexpression of kexB in that strain was confirmed by Northern analysis. The relatively low increase in kexin activity in the DSP fraction of strain MCK-5 could be due to a regulatory mechanism operative at the posttranscriptional level. When the Kex2 protease was overproduced in the yeast S. cerevisiae, it was transported to the vacuole at an increased rate and degraded (33).
The kexB disruptant grows very poorly on agar plates and its hyphae have an unusual morphology. Under these circumstances, KexB function appears to be essential for normal growth. In shake flask cultures, however, the disruption of kexB has less severe consequences. Biomass is not reduced in liquid culture and there are no indications that secretion of the reporter is hampered in the kexB-disrupted strain. When strains with comparable copy numbers were used, the amount of glucoamylase produced as a separate protein in the control strain was comparable to the amount of fusion protein produced by the KexB disruptant (NW266).
The lack of processing of MCA derivatives by the DSP of the kexB disruptant proves that hydrolysis of the MCA derivatives in the membrane-protein fractions of the wild-type strain and the kexB multicopy strain depends on KexB. In vitro, the specificity of KexB is more comparable to that of the yeast Kex2 maturase (4) than it is to that of mammalian furin (21). However, in vitro Aspergillus KexB appears to process the furin-specific sequence Arg-Val-Arg-Arg better than yeast Kex2. Like Kex2, KexB does not process the tripeptide Glu-Lys-Lys. In yeast, the amino acid at the P4 position is important for the cleavage of a Lys-Lys dibasic cleavage site (25). If a phenylalanine residue is present at P4, then normal processing of the substrate results. In A. niger PgaI, a similar cleavage site (F-A-K-K) is processed in vivo (7).
The results from the in vitro experiments are consistent with the inability of the kexB disruptant to process the glucoamylase-interleukin-6 fusion protein with an engineered Kex2 site at the fusion junction. The yeast monobasic aspartyl protease Mkc7 can also hydrolyze a dibasic cleavage site (16) and monobasic cleavage is operative in A. niger (7). However, such compensation for the loss of kexin activity was not observed with this reporter. Clearly, our results identify the kexB gene as the kexin-encoding gene of A. niger.
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ACKNOWLEDGMENTS |
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We thank R. Contreras for providing plasmid pFGPDGLAHIL6 and Y. Müller for technical assistance.
This work was supported by Dutch Technology Foundation (STW) grant WBI.4100.
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FOOTNOTES |
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* Corresponding author. Mailing address: Section Molecular Genetics of Industrial Microorganisms, Wageningen University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands. Phone: 31 317 485142. Fax: 31 317 484011. E-mail: Peter.Schaap{at}algemeen.mgim.wau.nl.
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Abstract
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Materials and Methods
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Discussion
References
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