From the
The cysteine protease papain is synthesized as a 40-kDa inactive
precursor with a 107-amino-acid N-terminal pro region. Although
sequence conservation in the pro region is lower than in the mature
proteases, a conserved motif
(Gly-Xaa-Asn-Xaa-Phe-Xaa-Asp
The primary translation product of most proteases is an inactive
precursor, or zymogen (Kassell and Kay, 1973; Neurath, 1984). These
precursors include pro regions of various size that are often located
at the N terminus of the protease. The pro region is removed by limited
proteolysis during conversion of the precursor to the mature active
protease, a process called maturation, activation, or proteolytic
processing. Maturation of protease precursors can proceed through
intermolecular or intramolecular non-exclusive pathways. For instance,
exogenous proteases are involved in the activation of the serine
protease precursor trypsinogen (Kunitz and Northrop, 1936) in a
trans reaction. Autocatalytic maturation ( cis reaction) has been documented for all groups of proteases. This
includes the serine proteases subtilisin (Ikemura and Inouye, 1988) and
prohormone convertases (Steiner et al., 1992; Wilcox, et
al., 1991; Germain et al., 1992; Mattews et al.,
1994); the aspartyl protease precursor of proteinase A (van den Hazel
et al., 1992; Woolford et al., 1993), procathepsin D
(Conner and Richo, 1992), and pepsinogen (Al-Janabi et al.,
1972); the precursor of the 72-kDa type IV collagenase of the
metalloprotease family (Stetler-Stevenson et al., 1989), and
precursors of the cysteine proteases, papain (Vernet et al.,
1991), cathepsin S (Brömme et al., 1993), and cathepsin B
(Mach et al., 1994).
As predicted from the observation that
the precursors are catalytically inactive for protein substrates, most
large pro regions are inhibitors of their cognate protease. Indeed,
free pro regions or fragments of pro regions of carboxypeptidase
(SanSegundo, 1982), cathepsin D (Fusek et al., 1991),
subtilisin (Ohta et al., 1991),
Our own interest in the
regulation of cysteine protease activity of the papain superfamily is
based upon the observation that mature and precursor forms of cysteine
proteases have been implicated in a wide variety of human diseases
(Brocklehurst et al., 1987; Mason et al., 1987;
Keppler, 1988). In the absence of three-dimensional structural
information for cysteine protease precursors, we have relied upon
mutational analysis for the study of the precursor of the archetypal
cysteine protease papain.
The primary transcript of the papain
precursor includes a 26-amino-acid signal sequence, a 107-amino-acid
pro region, and a mature domain of 212 amino acids (Vernet et
al., 1990). Conversion of the papain precursor into active mature
papain can proceed in the presence of exogenous proteases
(intermolecular or trans processing) or through a pH-dependent
intramolecular cleavage of the pro region (Vernet et al.,
1991).
In this paper, we identify a conserved amino acid motif
within the pro region of proteases of the papain superfamily. The role
of the motif in the pro region of the papain precursor was assessed by
random mutagenesis followed by functional screening of the mutants.
Based upon this analysis, we show that alteration of the electrostatic
charge of the conserved motif triggers automaturation of the papain
precursor.
Plasmid DNA
prepared from selected yeast colonies (Davis et al., 1980) was
transformed and amplified in E. coli MC1061. All mutants of
interest were sequenced in the region surrounding the conserved motif
(Sanger et al., 1977).
Alignment of 56 cysteine
protease pro regions was performed as a part of an overall
alignment/phylogeny of the papain superfamily.
The first series of four libraries of mutants was amplified in
E. coli and transformed into yeast. Forty-two yeast colonies
from each library were grown into multiwell plates, lysed, and
incubated under conditions which promote auto-conversion of the papain
precursor into mature papain. The activity of papain released was
monitored by spectrofluorometry. One non-functional mutant for each
position (Gly
Each
mutant isolated from each cycle was tested again using another
spectrofluorometric assay which is more sensitive (see
``Experimental Procedures''). By this assay, we compared the
ability of the mutants to undergo processing in a cis and in a
trans reaction. Both activation procedures gave comparable
levels of mature enzyme (Fig. 2). Sixteen mutants were unable to
release significant levels of papain activity (non-functional mutants),
whereas 10 precursor mutants could produce various levels of activity
with respect to wild type propapain (functional mutants). Plasmids from
the two classes of mutants were isolated from yeast and then amplified
in E. coli. All plasmids carried the XmnI restriction
site which is diagnostic of the mutations (). The region
surrounding the sites of the mutations was sequenced. Results for the
two series of mutations are presented in Fig. 3.
As the mutant
Asp
Individual mutants delivered comparable levels of activity following
in vitro conversion to mature papain using either the cis or the trans processing reactions. These observations
indicate that the amino acid replacement did not alter the ability of
the pro region to act as a protease inhibitor or its ability to undergo
autoprocessing.
Three mutant proteins accumulated in yeast partly as
active papain. These are mutations that abolished the negative charge
at position -36 (Asp
Recently, the functional complexity of pro regions of
protease precursors has become apparent. Beyond the initial observation
that they act as disposable inhibitors of their cognate protease,
numerous examples showing their involvement in protein folding and
localization have been reported. However, the molecular mechanisms by
which these functions are accomplished have not been elucidated.
In
previous work we showed that the papain precursor activates itself in a
pH-dependent manner (Vernet et al., 1991). To pursue this
investigation, we initially compared cysteine protease pro region
sequences since sequence conservation is a good indicator of structural
or functional constraints. The presence of a common structural feature,
a conserved heptapeptide, between cysteine protease pro regions and the
cysteine protease inhibitors, mammalian type II cystatins, stimulated
our interest to analyze the function of this conserved motif.
Random
mutagenesis experiments revealed that the set of residues accepted in
the motif is restricted, implying structural and/or functional
constraints within the conserved heptapeptide. Some amino acid
replacements led to non-functional mutants that were not folded
properly, with Gly
The spectrum of amino acids tolerated in the motif
generally matches the one found in the pro region sequence alignment
(compare Figs. 1 and 2). The match is remarkable at position
Phe
Our analysis of
functional mutants revealed that direct or indirect perturbation of the
charge at position Asp
Results
presented in this paper support an electrostatic trigger model of
propapain processing implicating the charge of the conserved amino acid
motif. Two putative pathways for processing can be proposed. The first
pathway implies that the peptide bond between residues
Ala
The pH
dependence of processing would reflect the influence of the charge
state of Asp
A second putative pathway can be proposed which does not
require cleavage within the conserved motif during processing. The
synthetic peptide bearing the consensus sequence can be cleaved at
neutral pH, where the Asp
The occurrence of a
conformation change during processing can be inferred from the
observation that the active site of propapain is accessible to small
organic molecules (Vernet et al., 1991) suggesting that the
pro region is not in constant intimate contact with the active site.
Masking of the active site region by the pro peptide probably prevents
access of macromolecules to the active site, a situation similar to the
one described for procarboxypeptidase B (Coll et al., 1991).
Following the initial cleavage of the pro region, whether within the
conserved motif or not, further removal of the remaining amino acids of
the pro region may result from the proteolytic activity of the putative
intermediate species. It is likely that this step results from an
intermolecular reaction given that the N-terminal residue in mature
papain is located far from the active site.
Further work, including
the determination of the tridimensional structure of a cysteine
protease precursor, should help in distinguishing between the two
proposed processing pathways.
The first two positions of the targeted codons within
the degenerated oligos are synthesized using an equimolar mixture of
the four bases A, T, G, and C (N) and the third position with an
equimolar mixture of G and C (G/C).
We thank L. Laramée for DNA sequencing and D.
Proteau and E. W. Jones for strain BJ7537.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, papain precursor
numbering) was found within the pro region of cysteine proteases of the
papain superfamily. To determinate the function to this conserved
motif, we have mutagenized at random each of the 4 residues
individually within the pro region of the papain precursor. Precursor
mutants were expressed in yeast, screened according to their ability to
be processed through either a cis or trans reaction,
into mature active papain. Three classes of mutants were found.
Non-functional propapain mutants of the first class are completely
degraded by subtilisin indicating that they are not folded into a
native state. Mutants of the second class were neutral with respect to
cis and trans processing. The third class included
mutants that mostly accumulated as mature papain in the yeast vacuole.
They had mutations that had lost the negatively charged
Asp
residues and a mutation that probably
introduces a positive charge, Phe
His. The precursor
of the Phe
His mutant could be recovered by
expression in a vph1 mutant yeast strain which has a vacuolar pH of
about 7. The Phe
His propapain mutant has an optimum
pH of autoactivation about one pH unit higher than the wild type
molecule. These results indicate that the electrostatic status of the
conserved motif participates in the control of intramolecular
processing of the papain precursor.
-lytic protease (Baker
et al., 1992), 72-kDa type IV collagenase (Melchiori et
al., 1992), and cathepsin B (Fox et al., 1992) are potent
specific inhibitors of their related protease. Moreover, pro regions
are strictly required for the expression of native proteases. Direct
demonstration of the role of pro regions in promoting protein folding
either in vitro or in vivo have been reported
(reviewed in Baker et al., 1993). Pro regions have also been
implicated in precursor targeting (Klionsky et al., 1990;
Fabre et al., 1991; Wetmore et al., 1992; Chang
et al., 1994; Fukuda et al., 1994) and membrane
association (McIntyre et al., 1994). In one case, the free pro
region was able to rescue intracellular transport of an alkaline
extracellular protease (Fabre et al., 1992). Thus, pro regions
can play many roles in the folding, transport, and activity of the same
molecule (Winther and S
rensen, 1991; Baker et al., 1992;
Wetmore et al., 1992; Fukuda et al., 1994). Genes
homologue to pro regions of proteases have been found in the mouse
(Denizot et al., 1989) in the absence of sequences encoding
for a mature protease. This provides further evidence that the pro
regions of proteases are not merely disposable appendices that are
eliminated upon activation of the protease.
Yeast and Escherichia coli
Strains
Saccharomyces cerevisiae strains BJ3501
(MAT, pep4::HIS3 prb1-
1.6R his3-
200 ura3-52 can1
gal2) (Jones, 1991) and DT101-2b (MAT
, pep4::HIS3,
vph1::LEU2, his3-
200, ura3-52, leu2-
1,
Gal
) were used in this study. The latter strain was
constructed by crossing strains BJ5447 (MAT
, his3-
200,
pep4::HIS3, ura3-52, leu2-
1, Gal
) with
strain BJ7537 (MATa, his3-
200, ura3-52, leu2-
1 or
leu2-3, 112, lys2-801, ura3-52,
vph1::LEU2).
E. coli strain CJ236 (Bio-Rad) was used for the preparation of
uracil-containing single-stranded DNA. The E. coli strain
MC1061 (Casadaban and Cohen, 1980) was used for the selection of
mutated plasmids and for routine amplification of plasmids.
Site-directed Saturation Mutagenesis
Each of the
four positions of the conserved motif was subjected to two successive
rounds of mutagenesis (Zoller and Smith, 1982; Kunkel, 1985). The first
round was performed independently at each of the four positions of the
conserved motif using the wild type propapain gene. The template for
these reactions originated from plasmid YpDC222 (Vernet et
al., 1993), an expression vector derived from pVT100-U (Vernet
et al., 1987) in which the propapain gene is under the control
of the -factor promoter. The structure and characteristics of the
synthetic oligonucleotides used to construct the four libraries of
mutants are listed in . The NNG/C of the mutagenic codons
encodes all possible amino acids, although not with equal frequency,
while eliminating two of the three stop codons. The efficiency of
mutagenesis for each of the four libraries of mutants amplified in
E. coli was verified using the restriction enzymes listed in
. The plasmid libraries were amplified in E. coli and then transformed into yeast. Yeast colonies were screened (see
below) for non-functional propapain, i.e. propapain that could
not produce active papain upon activation. For each position of the
motif one non-functional mutant was selected (see below and
) and transformed again into yeast to confirm the phenotype
of the mutation. Those non-functional mutants were then subjected to a
second cycle of mutagenesis/screening using the corresponding initial
oligonucleotide (). Same site suppressors were searched for
within the four new libraries such as to identify amino acids accepted
at each position. The double mutant
Asp
Asn/Ser
Ala was constructed
by site-directed mutagenesis in a separate reaction.
Rapid Screening of Papain Precursor
Mutants
Screening of precursor mutants was done according to a
previously published procedure (Vernet et al., 1993). Briefly,
yeast strain BJ3501 was transformed (Ito et al., 1983) using
plasmid libraries of mutated papain. Clones from the yeast libraries
were grown individually in multiwell plates. The cells were lysed, and
propapain present in the cell lysate was activated at low pH. The
activity of papain produced by the reaction was detected by
spectrofluorometry using the CytoFluor 2300 automatic plate reader
(Millipore). Each plate included yeast cultures expressing wild type
papain (YpDC222), an inactive papain mutant (YpDC228), and the plasmid
vector (YpDC219) as controls (Vernet et al., 1993).
In Vitro cis and trans Processing
Papain precursor
mutants were further characterized using an in vitro assay
(Vernet et al., 1993) more sensitive and accurate than the
plate assay. Briefly, yeast cells were first grown under selective
conditions to ensure plasmid maintenance and then transferred into a
rich medium. The cells were resuspended into 1/10 to 1/30 of the
initial culture volume and lysed using a French Press. The soluble
fraction of the lysate includes propapain. Protein concentrations were
equal for all samples within each experiment. Processing of propapain
was performed using either a cis or a trans reaction
protocol. Complete processing in cis was achieved by
incubating soluble cellular extracts with 50 mM sodium
acetate, pH 4.0, 20 mM cysteine at 60 °C for 30 min. The
trans reaction was complete after incubating soluble cellular
extracts for 2 h at 37 °C in the presence of 0.1 mg/ml of
subtilisin (Nagarse, Sigma). The level of papain activity released by
either the cis or the trans reaction was measured by
spectrofluorometry using the synthetic substrate
carbobenzoxyl-L-phenylalanyl-L-arginine
4-methylcoumarinyl-7-amide hydrochloride (Ménard et
al., 1991).
pH Profile of Activation
Processing in cis was performed using conditions described above except that the pH
of the incubation mixture ranged from 2.7 to 8.2. The rate of
activation at each pH was determined by measuring the level of papain
activity produced over a time course using the spectrofluorometric
assay described above.
Western Blot Analysis and Susceptibility to Protease
Degradation
Yeast-soluble proteins were prepared as described
above. Aliquots were treated with 25 µM of the cysteine
protease inhibitor E-64(
)
(Hanada et
al., 1978) and deglycosylated with endoglycosidase H (Boehringer
Mannheim) according to the supplier's instructions. When
necessary, the extract was treated with 0.1 mg/ml of subtilisin.
Samples were analyzed by Western blot following separation of the
proteins using SDS-polyacrylamide gel electrophoresis (Laemmli, 1970).
Identical quantities of protein in the total extract for each mutants
were loaded on the gel. Immunoreactive species were quantitated using
two rounds of antigen detection
(
)
by counting the
level of I
contained in each selected band using either
an LKB 1282 Compugamma or PhosphorImager (Molecular Dynamics).
Proteolysis of Synthetic Peptides
The 12-mer
peptide,
H
N-Leu-Gly-Leu-Asn-Val-Phe-Ala-Asp-Met-Ser-Asn-Asp-COO
,
was synthesized by the solid-phase method on an Applied Biosystem 430A
peptide synthesizer using a standard ABI coupling protocol. The peptide
was cleaved from the resin using HF in the presence of anisole (5%
volume) and dimethyl sulfide (5% volume) at -5 °C for 60 min.
After evaporation of the HF, the peptide was washed with ethyl ether
and extracted with 50% acetic acid, followed by water prior to
lyophilization. The peptide was then purified by reverse-phase HPLC on
a Vydac C-18 column (25
0.46 cm) using a linear gradient of
25-50% CH
CN in 25 min all in 0.1% trifluoroacetic
acid. The purified peptide (0.44 mM) was incubated with 3
µM of papain (Sigma) in sodium citrate, pH 4.0, or sodium
phosphate, pH 6.5, buffer, 1 mM EDTA, 20% CH
CN at
25 °C for 10 or 25 min. Hydrolysis was monitored using HPLC under
the conditions described above. Cleavage products were analyzed with a
Beckman model 6300 amino acid analyzer.
Protein Alignments
Numbering of amino acid
residues used the propapain sequence as a reference and corresponds
with the convention defined previously (Vernet et al., 1990),
whereby amino acids in the pro region have negative values starting at
the junction with mature papain
(Asn/Ile
).
(
)
The papain group enzymes have pro regions that have lower
sequence conservation than the sequences of the mature proteins and
regions of no apparent sequence similarity, but could be aligned
between positions -83 and -25 (papain numbering). The pro
regions of the bleomycin hydrolases and cathepsins B and C were not
included since they have non-homologous pro segments. Two sequences
from mouse were included that are homologous with the cysteine protease
pro segments, but do not possess any of the mature protease sequence
(Denizot et al., 1989); their function is not known. The pro
regions were aligned manually based on the alignment/phylogeny of the
mature sequences.
Identification of a Conserved Heptapeptide
Motif
The pro regions of cysteine proteases are more variable
than the mature protease sequences. However, several regions are
relatively well conserved. One such region is a stretch of 7 residues
located between position -42 and -36 (papain numbering).
This heptapeptide contains 4 non-contiguous amino acids that are more
conserved than surrounding residues: Gly,
Asn
, Phe
, and Asp
(Fig. 1). This sequence motif can be written as
Gly-Xaa-Asn-Xaa-Phe-Xaa-Asp (G xN xF xD). It
appeared in all non-cathepsin B and C members of the papain group,
including the kinetoplastids (with Thr in the place of Asn). The
kinetoplastids are protozoa that include the trypanosomes and were
among the first groups to diverge from the ancestors of multicellular
organisms. This indicates that G xN xF xD motif
has an early origin in the evolution of eukaryotes.
Figure 1:
Presence of a conserved amino acid
motif within cysteine protease pro region sequences. Occurrence of
amino acids in cysteine proteases between positions -36 and
-42 (papain pro region numbering) from a set of 56 sequences of
cysteine protease pro regions. Amino acids present in more than 60% of
the sequences are given above each open box. Other amino acids
found at each position are listed using the one-letter code and the number of occurrences within the set of sequences is given
between brackets. Accession numbers for the sequences are:
D90406, D90407, D90408, J02583, J05560, L03212, L08500, M15203, M36320,
M37791, M38422, M64721, M67451, M81341, M84342, M86553, M86659, M90067,
M90696, M94162, M94163, M97695, M97906, S51520, S67655, X02407, X03344,
X03930, X05167, X12451, X13013, X13139, X16465, X16466, X16832, X51900,
X54353, X56753, X62163, X63567, X63568, X66060, X66061, X69877, Y00697,
Z13959, Z13964, Z14028, Z14061 (Genbank/EMBL) and P25249, P25250,
P25251, P25326, P25804 (Swissprot)
Surprisingly,
the same G xN xF xD motif was also observed in
the mammalian type II cystatins (Turk and Bode, 1991), which are
inhibitors of cysteine proteases (not shown). As the
G xN xF xD motif does not occur in
non-mammalian type II cystatins, nor in type I or III cystatins, it has
only appeared recently in evolutionary time. In the SwissProt protein
sequence data base, release 28.0, there are 56 other occurrences of
G xN xF xD in 30 different protein families.
Therefore, there remains the possibility that the appearance of the
motif in both cysteine proteases and their inhibitors is fortuitous.
Saturation Mutagenesis and Functional
Screening
The role of the four most conserved positions of the
motif was assessed using two cycles of site-directed mutagenesis
followed by screening of the mutant precursors expressed in S.
cerevisiae (Vernet et al., 1993). This two-step
approach facilitates the discrimination between functional and
non-functional mutants by avoiding the background of non-mutated genes.
Leu, Asn
Stop,
Phe
Ser, and Asp
Met) was chosen
for the second round of mutagenesis. Between 170 and 360 clones from
each new set of libraries were screened for functional mutants.
Figure 2:
Effect of mutations of the conserved motif
upon the function of the papain precursor. Maximum level of papain
activity released following either cis ( light hatched
boxes) or trans ( heavy hatched boxes) in
vitro processing relative to wild type papain precursor papain is
shown by the histogram. Standard deviations of the activities over at
least two independent experiments are presented by the T-shaped
vertical bars. Mutants of the four positions of the motif are
presented in separate quadrants. Mutants for each position, with the
exception of the double mutant
AspAsn/Ser
Ala, originate from
two series of random mutagenesis.
Figure 3:
Compilation of the results of eight
independent random mutagenesis experiments. Amino acid replacements
that prevent the production of active papain upon in vitro activation of the precursor are given above the conserved amino
acid motif ( boxed). Amino acid replacements that are
tolerated, other than amino acids found in the wild type sequence, are
given below the motif. Mutation AspAsn was
constructed by site-directed mutagenesis together with the mutation
Ser
Ala such as to avoid the creation of a potential
N-linked glycosylation site. Codons and the number of
independent isolates are given between
brackets.
Among the
seven different mutants isolated at position Gly,
Ala was the only side chain accepted. Asn
could be
replaced by amino acids with small polar side chains (Gly, Thr, and
Ser) but not by bulkier hydrophobic side chains (Ile and Phe). The only
stop codon of the whole random mutation search was found at position
40. Neither Pro nor Gly nor Ser were accepted at position
Phe
but replacement with two aromatic side chains
(Trp or His) or with the hydrophobic side chains Leu or Met led to a
functional precursor. The negatively charged Asp
can be replaced by amino acids with small uncharged side chains
(Gly or Cys) but not by those with bulkier side chains, even when the
negative charge is maintained (Glu).
Asn did not appear in our screen, we decided to
construct this analogous mutation in order to evaluate the relative
importance of the charge and the size of the side chain at position
-36. Position -34 is occupied by a Ser, precluding the
direct construction of the desired mutant due to the creation of a
potential N-linked glycosylation site (Asn-Xaa-Ser/Thr).
Therefore, we first replaced Ser
with Ala and
verified that this mutant of the precursor is functional (data not
shown). The double mutant
Asp
Asn/Ser
Ala is not functional
(Figs. 2 and 3).
Analysis of Non-functional Mutants
One
non-functional propapain mutant for each of the positions of the motif
was selected and analyzed by quantitative Western blot before and after
treatment with subtilisin (Fig. 4 A). Wild type papain
precursor is fully converted into mature active papain upon treatment
with subtilisin whereas mutants that are not folded properly are
degraded by the protease. For mutants
Gly
Thr, the level of propapain accumulated in the
cell is comparable to the level of the wild type protein
(Fig. 4 A). However, following incubation with subtilisin
the protein is almost completely degraded while the wild type propapain
is fully converted to mature papain ( trans processing). The
effect of the Asn
Ile and Phe
Pro
mutations is even more severe with little accumulation of propapain in
the cell, which indicates that the protein is very unstable. The
Asp
Asn/Ser
Ala double mutant has
an intermediate behavior; it has a level of propapain expression 10%
that of the wild type which is completely degraded upon incubation with
subtilisin. Proteolytic sensitivity and reduced expression in yeast of
the non-functional mutants indicates that the proteins are not folded
properly.
Figure 4:
Western blot analysis of papain precursor
mutants. Autoradiogram of Western blots of total cellular extracts
before (-) and after (+) treatment with subtilisin. The
level of propapain and papain relative (%) to wild type ( WT)
propapain and papain, respectively, are given below each sample. The
source of the sample is indicated above each series of samples.
A, non-functional mutants. The autoradiograms were exposed for
41 h with the exception of the one for the AsnIle
and Phe
Pro mutants (83 h of exposure). B,
functional mutants. The autoradiograms were exposed for 48 h. Molecular
masses of standards (kDa) and the location of propapain and mature
papain are indicated in the left and right margins,
respectively.
Analysis of Functional Mutants
The 10 functional
mutants were also characterized by quantitative Western blot analysis.
Most mutant proteins accumulate in the yeast cell as inactive
precursors at levels ranging from less than 5 to 100% of the level of
wild type precursor. Crude preparations of these mutants were incubated
in conditions known to promote auto processing of the papain precursor
(Vernet et al., 1991). Under these conditions neither the wild
type papain precursor treated with E-64 nor an active site
CysSer precursor mutant were processed. Unstable
non-functional mutants were not degraded when incubated under similar
conditions (data not shown). This shows the absence of interfering
proteases in the crude extract and the requirement of the active site
for processing of the precursors ( cis processing).
Gly and
Asp
Cys) and mutation Phe
His
(Figs. 4 B and 5). Attempts at further activation in cis or trans of the mutants led only to a marginal increase
in papain activity (Fig. 5). At the pH of the yeast vacuole of
about 6, there would be a significant positive charge on
His
. It appears that alteration of the charge of
the conserved motif triggers processing of the papain precursor. Rescue of the Phe
His Precursor Mutant in a vph1
Yeast Strain-If the presence of a positive charge within the
conserved motif is responsible for the premature processing of
propapain, it should be possible to stabilize the precursor
Phe
His at higher pH where the His side chain would
not be protonated. The vacuolar pH of the vph1 yeast mutant
(Preston et al., 1989) is maintained at pH 6.9 and was used
for our experiments. Yeast strain BJ3501 accumulates wild type
propapain that has the potential to be fully converted into mature
active papain in a cis or a trans reaction
(Fig. 6). As shown above, the same strain accumulates mostly
mature Phe
His mutant, and complete conversion to
mature papain can be obtained by cis or trans processing. However, the Phe
His mutant,
accumulates in the vph1 yeast mutant as the papain precursor.
The Phe
His precursor can then be processed into
mature papain by incubation at low pH ( cis processing) or in
the presence of subtilisin ( trans processing) (Fig. 6). pH Profile of Activation of Phe
His Mutant-The
availability of the mutant Phe
His papain precursor
now made it possible to measure the rate of autoprocessing as a
function of pH. The rate of activation of Phe
His
propapain expressed in the vph1 yeast strain was determined at
pH 2.5-8 and compared to the activation profile for the wild type
molecule (Fig. 7). The maximal rate of activation for the wild
type precursor was obtained at a pH 3.3, which is close to that
reported previously (Vernet et al., 1991). The corresponding
rate is shifted by almost 1 pH unit for the Phe
His
precursor.
Figure 5:
Relative level of mature papain prior to
and following in vitro processing. Protease activity was
measured immediately after extraction of the papain precursor from
yeast and compared to the maximum level of activity recovered after
in vitro processing. Mutant amino acids are indicated above
the position of the wild type residue.
Figure 6:
Rescue of the PheHis
propapain mutant in a vph1 yeast strain. Western blot
analysis. Total cellular extracts after treatment with endo H (/).
Strain BJ3501 is wild type with respect to the VPH 1 gene.
Propapain was processed in vitro using a trans ( T) or a cis ( C) reaction. Molecular
masses of standards (kDa) are indicated in the left
margin.
Figure 7:
Comparison of the pH profile of activation
of the PheHis mutant and wild type precursor. The
initial rate of activation was measured at different pH values for the
Phe
His mutant ( filled circle) and wild
type ( empty circle) precursor of
papain.
Cleavage of a Synthetic Peptide Mimetic of the Conserved
Motif
Our results suggest that pH-dependent intramolecular
cleavage of the pro region might take place within the conserved motif.
To test this possibility, we incubated with papain a 12-mer synthetic
peptide encompassing the sequence of the conserved motif. Two cleavage
sites were identified in the peptide: the
Gly/Leu
and
Ala
/Asp
bonds (papain precursor
numbering). The two possible pathways for hydrolysis are represented in
Fig. 8A, where ABC is used to represent the intact
peptide. Under the HPLC conditions used, fragments ABC, AB, BC, and B
could be easily detected, allowing complete monitoring of both reaction
pathways. After 10 min of incubation at pH 6.5
(Fig. 8 B), peaks were detected for fragments ABC, AB,
BC, and B, indicating that hydrolysis occurs at both sites and at
comparable rates (assessed from peak integration, the reaction via
pathway 1 can be estimated to be approximately twice as fast as
reaction via pathway 2 at pH 6.5). However, for 10 min of incubation at
pH 4.0 (Fig. 8 B), the peptide is completely hydrolyzed
(no ABC fragment detected) predominantly into fragments AB and B
indicating that cleavage between
Ala
/Asp
is much faster at this
pH (pathway 2). By using varying time intervals at both pH values, the
rate of hydrolysis of the Ala
/Asp
peptide bond can be estimated to increase approximately 10-fold
on going from pH 6.5 to 4.0. At both pH values, complete hydrolysis
leading to fragment B is observed upon longer incubation times or by
using higher enzyme concentrations.
Figure 8:
pH-dependent cleavage by papain of a
synthetic peptide containing the sequence of the conserved
heptapeptide. A, sequence of the synthetic peptide and
cleavage pathways by papain. Amino acid positions are those of the
corresponding sequence in the pro region of papain. Peptide fragments
produced following hydrolysis by papain are designated by capital
letters underneath the sequence. B, HPLC profiles of
peptides resulting from the hydrolysis of the conserved synthetic
peptide by papain incubated under various incubation conditions.
RT, retention time.
and Asp
being the most constrained positions. Thus, the motif
participates in the folding and/or stability of the papain precursor.
These results are consistent with our observation that papain expressed
in the absence of its pro region is not functional and is sensitive to
protease degradation.
(
)
Pro regions of a variety
of other protease precursors have been shown to act as intramolecular
chaperone (Baker et al., 1993). Our data suggest that point
mutations within pro regions can affect their molecular chaperone
activity.
where similar amino acid patterns are found in
the sequence alignment and in the mutagenesis experiment. This
indicates that the random mutagenesis was not grossly biased and that
our screening for functional mutants was efficient.
alters the pH dependence for
processing. We conclude that the negative charge of position -36
of the motif is important for maintaining the precursor of papain in a
latent form. The inability of the Asp
Glu mutation
to restore function of the precursor indicates that both electrostatic
and steric constraints prevail at position -36.
and Asp
is the primary
cleavage site during the pH-dependent intramolecular processing of the
papain precursor. Three lines of evidence support this hypothesis: (i)
during processing a transient immunoreactive species of about 30 kDa
has been observed (Vernet et al., 1991) suggesting that
conversion of the precursor into papain is a stepwise process. The
molecular mass of the intermediate molecule is compatible with a
cleavage within the heptapeptide motif. We have been unable to isolate
sufficient quantities of the 30 kDa processing intermediate to obtain
its N-terminal sequence. (ii) A synthetic peptide possessing the
sequence of the conserved motif is cleaved by papain more efficiently
between position Ala
and Asp
at
pH 4.0 than at pH 6.5 indicating an increase in substrate reactivity of
the free motif when the Asp
is not ionized. (iii)
Phe
would be located in the S2 subsite of the
proposed substrate region and is the most favorable side chain for the
papain substrates at this position (Baker and Drenth, 1987).
on the rate of cleavage as discussed
above. The maximal rate of papain precursor processing is obtained at a
pH value remote from the optimal value for mature papain (pH 6.5). This
could indicate that the optimal pH of papain activity within the
precursor is lower than for the free enzyme. More likely, the low pH
optimum could be the result of the combined effects of decrease in
activity of the enzyme and increase in hydrolysis rate of the substrate
at low pH.
residue is expected to be
negatively charged whereas processing of the pro enzyme under the same
conditions is much slower. Specificity studies have shown that papain
can accept a deprotonated aspartic acid residue in the P1` position of
a substrate (Koga et al., 1990; Desmazeaud, 1972; Johansen and
Ottesen, 1968). In addition, considering the nature of the preferred
residues in the S2 subsite of papain, it is not clear why mutant
Phe
His, bearing a positive charge on the His side
chain, would process so easily. Therefore, one must consider the
possibility that the motif could participate in the pH regulation of
processing without being directly involved as a cleavage site during
the event. The function of the motif could be to protect the pro
peptide against proteolysis at neutral pH through a yet unknown
mechanism. When the pH is lowered, protonation of residue
Asp
could trigger a conformational change
effectively ``switching-on'' the processing of propapain by
allowing proteolysis to occur at a cleavage site that is not
necessarily located at the Ala
/Asp
bond. Other ionizable groups, in addition to Asp
might be involved in the process as suggested from the shape of
the pH dependence curve for processing. A similar observation was made
in a study of cathepsin B inhibition by its pro peptide, where it was
concluded that the pH dependence of the inhibition must be due to
ionization of more than one group on either the enzyme or the pro
peptide (Fox et al., 1992). This second pathway is supported
by the observation that cleavage of mammalian cystatin C by papain at
low pH takes place at sites remote from the conserved motif (Berti and
Storer, 1994). Moreover, the three-dimensional complex between human
stefin B (also named cystatin B) and papain revealed that the active
site of the protease does not interact directly with the conserved
heptapeptide (Stubbs et al., 1990).
Table:
Oligonucleotides used for site-directed
mutagenesis
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.