(Received for publication, March 14, 1995; and in revised form, July 17, 1995)
From the
A rhamnogalacturonan acetylesterase (RGAE) was purified to
homogeneity from the filamentous fungus Aspergillus aculeatus,
and the NH-terminal amino acid sequence was determined.
Full-length cDNAs encoding the enzyme were isolated from an A.
aculeatus cDNA library using a polymerase chain reaction-generated
product as a probe. The 936-base pair rha1 cDNA encodes a
250-residue precursor protein of 26,350 Da, including a 17-amino acid
signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess
RGAE activity, and the recombinant enzyme was purified and
characterized. Mass spectrometry of the native and recombinant RGAE
revealed that the enzymes are heterogeneously glycosylated. In
addition, the observed differences in their molecular masses, lectin
binding patterns, and monosaccharide compositions indicate that the
glycan moieties on the two enzymes are structurally different. The RGAE
was shown to act in synergy with rhamnogalacturonase A as well as
rhamnogalacturonase B from A. aculeatus in the degradation of
apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that
the expression of rhamnogalacturonan degrading enzymes by A.
aculeatus is regulated at the level of transcription and is
subjected to carbon catabolite repression by glucose.
Pectic polysaccharides are located predominantly in the middle
lamella and primary cell wall of dicotyledonous plants(1) . The
main backbone in pectins can be divided into linear homogalacturonan
(smooth) regions of up to 200 residues
of(1, 4) -linked -D-galacturonic acid
(GalUA)
and highly branched rhamnogalacturonan (hairy)
regions consisting of repeating
-(1,2)-L-Rha-
-(1,4)-D-GalUA disaccharide
units(1, 2, 3) . In general, about half of
the Rha residues in the hairy regions are substituted with neutral
oligosaccharides such as arabinans, galactans, and arabinogalactans.
Most pectic substances are furthermore esterified with acetyl or methyl
groups at some of the GalUA residues in the
backbone(1, 4) .
Many saprophytic and plant pathogenic fungi and bacteria possess an array of extracellular enzymes involved in the degradation of plant cell wall polymers(5, 6) . In the primary cell wall, the cellulose-xyloglucan framework is embedded in a matrix of pectic polysaccharides, which thereby control the access of hydrolytic enzymes to the cellulose and hemicellulose substrates(1, 7) . Thus, pectinases are often the first cell wall degrading enzymes produced by plant pathogens when cultured on purified plant cell walls or during infection(8, 9) . Due to the structural complexity of the pectin matrix a synergistic or sequential action of several different pectinolytic enzymes is required for efficient breakdown(5) . For example, the hydrolysis of smooth regions of pectin by polygalacturonases is highly dependent upon demethylation of the homogalacturonan backbone by pectin methylesterase(5) . Similarly, the degradation of rhamnogalacturonan by rhamnogalacturonases (RGases) depends on the removal of the acetyl esters from the substrate(10, 11) . Thus, the presence of a rhamnogalacturonan acetylesterase (RGAE) in the filamentous fungus Aspergillus aculeatus(12) suggests that this enzyme is essential for the action of RGases in vivo.
With the goal of elucidating the role of rhamnogalacturonan acetylesterase in the enzymatic degradation of plant cell wall rhamnogalacturonan we have undertaken purification of the RGAE from A. aculeatus and isolation and characterization of full-length rha1 cDNAs encoding the enzyme. In addition, we have overexpressed the rha1 cDNA in Aspergillus oryzae, and characterized the purified, recombinant RGAE (rRGAE). In a previous study we have reported the cloning of two structurally and functionally different rhamnogalacturonases, RGase A and RGase B, from A. aculeatus(11) . Here we show that RGAE acts in synergy with RGase A as well as RGase B in the degradation of apple pectin rhamnogalacturonan. As a first step in dissecting the molecular mechanisms that control the regulation of rhamnogalacturonan degrading enzymes in A. aculeatus the expression of their genes is compared at mRNA level.
The A. aculeatus cDNA
library in pYES 2.0 (11) was screened by colony hybridization (15) using a random-primed (16) P-labeled
(>1
10
cpm/µg) PCR product for rha1 as a probe. The hybridizations were carried out in 2
SSC(15) , 5
Denhardt's solution(15) ,
0.5% (w/v) SDS, 100 µg/ml denatured salmon sperm DNA for 20 h at 65
°C followed by washes in 5
SSC at 25 °C (2
15
min), 2
SSC, 0.5% SDS at 65 °C (30 min), 0.2
SSC,
0.5% SDS at 65 °C (30 min) and finally in 5
SSC (2
15 min) at 25 °C.
The pH optimum of rRGAE was
determined by incubation of 1% MHR in citrate/phosphate buffers of
varying pH for 15 min at 30 °C followed by acetate determination.
The temperature optimum was determined from PNP-acetate after
incubation with rRGAE at varying temperatures for 15 min. The specific
activity and Michaelis-Menten constant (K) were
determined by incubating RGAE with different concentrations of MHR in
0.1 M phosphate buffer, pH 6.0, for 15 min at 30 °C. The
substrate concentration (S) ranged from 0.1 to 1.5% and 43 µg of
rRGAE was added to 1.1 ml of substrate. The reaction velocity (V) was calculated and 1 unit is defined as 1 µmol of
acetate released per min. Then V/S was depicted as a function
of S, and K
and V
were
determined by linear regression analysis. The specific activity was
calculated as V
/E, where E is
the amount of enzyme added. To determine the substrate specificity,
RGAE and rRGAE were incubated with acetylated xylan and acetylated
mannan (kindly provided by Dr. Jürgen Puls)
followed by determination of acetate as described.
Figure 1:
The nucleotide sequence of the rha1 cDNA and the deduced primary structure of rhamnogalacturonan
acetylesterase from A. aculeatus. The signal peptide is boxed, the NH-terminal sequence obtained from the
purified, native enzyme is underlined, and the putative N-glycosylation sites are indicated by double
underlines.
The 936-bp cDNA clone pC1RGAE1 contains a
750-bp open reading frame initiating at nucleotide position 39 and
terminating with a TGA stop codon at nucleotide position 789, thus
predicting a 250-residue polypeptide of 26,350 Da (Fig. 1). The
open reading frame is preceded by a 38-bp 5`-noncoding region and
followed by a 116-bp 3`-noncoding region and a poly(A) tail. The
deduced primary structure of RGAE matches the NH-terminal
sequence determined from the purified enzyme (Fig. 1). In
addition, the rha1 cDNA encodes an apparent signal peptide of
17 amino acids(29) , revealed by comparison with the
NH
-terminal sequence of the purified enzyme. Hence, the
mature RGAE has a calculated molecular weight of 24,605. A search of
the Swiss-Prot protein sequence data base with the deduced RGAE amino
acid sequence revealed no significant similarities, implying that RGAE
from A. aculeatus is a novel enzyme representing a new family
of esterases.
The copy number of the rha1 gene in the A. aculeatus genome was determined by Southern blot hybridization. Total DNA isolated from A. aculeatus was digested to completion with BamHI, BglII, EcoRI, or HindIII and hybridized with the rha1 cDNA. The rha1 probe detects only single strongly hybridizing fragments in each digest, indicating that the rha1 gene is present as a single copy in the A. aculeatus genome (Fig. 2).
Figure 2: Southern blot analysis of A. aculeatus. Genomic DNA from A. aculeatus was digested to completion with BamHI (Ba), BglII (Bg), EcoRI (E), and HindIII (H), fractionated on a 0.7% agarose gel, and transferred to a nylon membrane. The blot was hybridized with the radiolabeled 0.9-kb BamHI-XhoI insert of pC1RGAE1 containing the full-length rha1 cDNA from A. aculeatus.
The rha1 cDNA probe hybridized readily to a single 0.9-kb mRNA
species in the 5-day-old mycelium, while the rha1 message did
not accumulate to detectable levels in the fungus when glucose was
present in the growth medium (Fig. 3). This is consistent with
the RGAE activity observed in the culture filtrate from day 5.
Reprobing the same filter with the rhgA and rhgB cDNAs encoding RGase A and RGase B(11) , respectively,
revealed a similar expression pattern for the 1.6-kb rhgA and
1.8-kb rhgB mRNAs, except that the message levels appeared to
be lower compared with rha1 (Fig. 3). In contrast, the
1.5-kb benA encoded -tubulin mRNA accumulated to
comparable levels in both samples irrespective of the presence of
glucose in the medium (Fig. 3).
Figure 3:
Regulation of rha1, rhgA, and rhgB transcript levels in A. aculeatus. Poly(A) RNAs isolated from A. aculeatus mycelia grown on medium
containing soybean meal plus glucose (+Glc) and soybean
meal without glucose (-Glc) as carbon sources were
subjected to RNA gel blot analysis. The same filter was sequentially
hybridized with a 5.4-kb PstI fragment containing the benA gene of A. nidulans, and cDNA probes for the rha1 encoded RGAE, rhgA for RGase A, and rhgB-encoding RGase B from A.
aculeatus.
Figure 4:
Purification and glycosylation of RGAE
from A. aculeatus. A, Coomassie Brilliant
Blue-stained SDS-PAGE gel showing A. aculeatus culture
supernatant (lane 1), RGAE containing fraction from the
DEAE-Sepharose column (lane 2), RGAE containing fraction from
the gel filtration column (lane 3), purified RGAE (lane
4) (1 µg), and rRGAE (lane 5) (1 µg). B,
Western blot of an SDS-PAGE gel with A. aculeatus culture
supernatant, purified RGAE and rRGAE, probed with a polyclonal rabbit
antiserum raised against authentic RGAE. C, lectin blot of an
identical gel as shown in B, probed with G. nivalis lectin, specific for terminal mannose in glycan structures. D, lectin blot of an identical gel as shown in B,
probed with peanut lectin, specific for Gal1-3GalNAc in some N-linked glycans.
Figure 5:
Mass spectrometric analysis of the native
and recombinant RGAE. Matrix-assisted laser desorption ionization
time-of-flight mass spectrometry of native (A) and recombinant (B) RGAE. The molecular ions carrying one, two, and three
positive charges are indicated as (M + H), (M
+ H)
, and (M +
H)
.
The
recombinant, but not native, RGAE binds Galanthus nivalis lectin specific for a terminal mannose residue in glycan moieties (Fig. 4). A similar pattern was observed with peanut lectin
specific for Gal1-3GalNAc in some N-linked glycans,
supporting the notion that the glycans attached to the enzymes are
different. The polyclonal anti-RGAE antiserum recognized a number of
polypeptides in the A. aculeatus supernatant, suggesting that
they contain identical or similar antigenic epitopes, such as glycan
structures. This is supported by the fact that both G. nivalis and peanut lectins reacted with several extracellular proteins
from A. aculeatus (Fig. 4, C and D).
The differences seen in the amount of mannose (6 pmol/pmol of RGAE, 11
pmol/pmol of rRGAE) and galactose (0 pmol/pmol of RGAE, 1 pmol/pmol of
rRGAE) in the glycan moieties of RGAE and rRGAE confirmed that the two
enzymes are differentially glycosylated. Furthermore, the presence of
glucosamine (2 pmol/pmol of protein) in both RGAE and rRGAE indicates
that at least some of the carbohydrate is N-linked, in
accordance with two potential N-glycosylation sites in the
primary structure of RGAE (Fig. 1).
Figure 6: Synergistic degradation of MHR by RGAE and RGases. The incubations of MHR and MHR-S were performed for 24 h in acetate buffers at optimal pH for RGase A (A) and RGase B (B), and the degradation products were analyzed by HPSEC. The estimated molecular weight (Mw) and degree of polymerization (DP) are indicated on the horizontal axis.
Several independent lines of evidence indicate that the cDNA
clone pC1RGAE1 encodes the rhamnogalacturonan acetylesterase from A. aculeatus. First, the deduced primary structure of the cDNA
encoded protein contains an amino acid sequence with 100% identity to
the NH-terminal sequence determined from the purified,
native RGAE from A. aculeatus. Second, heterologous expression
of the rha1 cDNA in A. oryzae, a filamentous fungus
that does not possess a rhamnogalacturonan acetylesterase activity,
resulted in a RGAE activity in the culture supernatant, similar to that
found in A. aculeatus. Both recombinant and native RGAE were
shown to be active on apple pectin rhamnogalacturonan and PNP-acetate,
but not on acetylated mannan or xylan. Furthermore, the enzyme
characteristics of the purified rRGAE correspond well with the values
reported previously for the native RGAE from A.
aculeatus(12) . Third, a polyclonal rabbit antiserum
raised against the native, purified RGAE recognized the recombinant
enzyme expressed in A. oryzae (Fig. 4B).
Finally, the NH
-terminal sequence and amino acid
composition of recombinant RGAE concurred with those of native RGAE,
indicating that the rRGAE secreted by A. oryzae is highly
similar to the native enzyme from A. aculeatus.
Previous work has shown that the production of extracellular polygalacturonases by Aspergillus niger(30) , A. nidulans(31) , and Penicillium frequentans(32) is induced by the presence of pectin and repressed by glucose. In addition, the carbon catabolite repression of the polygalacturonase expression by glucose has been reported to operate at the transcriptional level(30, 33, 34) . The rha1 mRNA accumulates in substantial amounts in A. aculeatus grown on the mixed carbon source after glucose has been depleted from the medium coinciding with the extracellular RGAE activity, whereas the rha1 message is absent in the fungus grown on glucose-containing medium. The comparable expression patterns observed for the rhgA and rhgB mRNAs imply that they are coordinately regulated. Furthermore, the message levels correlate well with the high total RGase activity detected in the 5-day-old culture of A. aculeatus(11) . Taken together, these results indicate that the expression of rhamnogalacturonan degrading enzymes by A. aculeatus is primarily regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.
The heterogeneous size in SDS-PAGE together with the difference of 8-11 kDa in apparent mobility and molecular mass compared with the predicted molecular weight of RGAE imply that the enzyme is modified by glycosylation. By comparison, mass spectrometric analyses resulted in average molecular masses of 26.7 and 28.3 kDa for RGAE and rRGAE, respectively. While the values are significantly lower than those determined by SDS-PAGE, they are consistent with substantial glycosylation of the predicted 24,605-Da RGAE polypeptide. The deduced amino acid sequence of RGAE contains two potential sites for N-linked glycosylation, in good agreement with the presence of glucosamine in both RGAE and rRGAE. Yet, this is probably not sufficient to explain the discrepancy between the observed and calculated molecular weights, suggesting that additional, O-linked glycans could be attached to the enzyme. In addition, the observed differences in the molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the native and recombinant RGAE are structurally different.
The HPSEC chromatograms showed only limited depolymerization of unsaponified MHR when the rRGases were used alone (Fig. 6), although the degradation of MHR by rRGase A was more pronounced than that obtained by Schols et al.(35) . In contrast, the combination of rRGAE with rRGase A and rRGase B revealed a marked synergism between the enzymes, resulting in extensive breakdown of the MHR substrate. Although only 60% of the saponifiable acetyl esters in MHR were hydrolyzed by rRGAE, it enhanced depolymerization of MHR almost to the level seen with MHR-S, implying that acetyl esters are a major hindrance for the action of RGases on rhamnogalacturonan. This is in accordance with previous reports for RGase A (10, 11) and is here shown to be valid for RGase B as well. The remaining acetyl esters seem to inhibit the rRGases mainly in MHR fragments of molecular weight exceeding 30,000 (Fig. 6), indicating that RGAE is sterically hindered by the side chains in heavily substituted regions of the rhamnogalacturonan backbone.
An
expanding inventory of purified microbial pectinolytic enzymes, active
within the smooth regions of pectin, along with a number of cloned
enzymes, has significantly improved our understanding of the structure
and degradation of plant cell wall
pectin(5, 31, 36, 37, 38, 39) .
In contrast, the enzymatic breakdown of the rhamnogalacturonan backbone
in pectic hairy regions has received only limited attention. The
recently reported rhamnogalacturonan
-L-rhamnopyranohydrolase from A. aculeatus(40) together with the previously described RGase A and
RGase B(10, 11) , and the rhamnogalacturonan
acetylesterase (12) cloned and characterized in this study,
imply that analogous to the degradation of the smooth regions, a family
of rhamnogalacturonan degrading enzymes can be found in nature.
Furthermore, the data presented here demonstrate that the deduced
primary structure of RGAE is unique, representing a novel family of
esterases and that the enzyme acts in synergy together with RGase B as
well as RGase A in the degradation of plant cell wall
rhamnogalacturonan.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89714[GenBank].