©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Processing of the Papain Precursor
THE IONIZATION STATE OF A CONSERVED AMINO ACID MOTIF WITHIN THE Pro REGION PARTICIPATES IN THE REGULATION OF INTRAMOLECULAR PROCESSING (*)

Thierry Vernet (§) , Paul J. Berti (¶) , Chantal de Montigny , Roy Musil (**) , Daniel C. Tessier , Robert Ménard , Marie-Claude Magny , Andrew C. Storer , David Y. Thomas

From the (1) Biotechnology Research Institute, National Research Council of Canada, 6100 Avenue Royalmount, Montréal, Québec H4P 2R2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, 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, PheHis. The precursor of the PheHis mutant could be recovered by expression in a vph1 mutant yeast strain which has a vacuolar pH of about 7. The PheHis 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.


INTRODUCTION

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), -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 Srensen, 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.

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.


EXPERIMENTAL PROCEDURES

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 AspAsn/SerAla was constructed by site-directed mutagenesis in a separate reaction.

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).

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, HN-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% CHCN 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% CHCN 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).

Alignment of 56 cysteine protease pro regions was performed as a part of an overall alignment/phylogeny of the papain superfamily.() 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.


RESULTS

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.

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 (GlyLeu, AsnStop, PheSer, and AspMet) was chosen for the second round of mutagenesis. Between 170 and 360 clones from each new set of libraries were screened for functional mutants.

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.


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/SerAla, 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 SerAla 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).

As the mutant AspAsn 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 AspAsn/SerAla 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 GlyThr, 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 AsnIle and PhePro mutations is even more severe with little accumulation of propapain in the cell, which indicates that the protein is very unstable. The AspAsn/SerAla 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 PhePro 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).

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 (AspGly and AspCys) and mutation PheHis (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 PheHis 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 PheHis 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 PheHis mutant, and complete conversion to mature papain can be obtained by cis or trans processing. However, the PheHis mutant, accumulates in the vph1 yeast mutant as the papain precursor. The PheHis 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 PheHis Mutant-The availability of the mutant PheHis papain precursor now made it possible to measure the rate of autoprocessing as a function of pH. The rate of activation of PheHis 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 PheHis 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 PheHis 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.




DISCUSSION

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 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.

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 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.

Our analysis of functional mutants revealed that direct or indirect perturbation of the charge at position Asp 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 AspGlu mutation to restore function of the precursor indicates that both electrostatic and steric constraints prevail at position -36.

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 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).

The pH dependence of processing would reflect the influence of the charge state of Asp 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.

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 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 PheHis, 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).

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.

  
Table: Oligonucleotides used for site-directed mutagenesis

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).



FOOTNOTES

*
This work was supported as part of the Protein Engineering Network of Centres of Excellence sponsored by the government of Canada. This paper is issued as National Research Council of Canada publication No. 38531. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institut de Biologie Structurale, Avenue des Martyrs, 38027 Grenoble Cedex 1 France. E mail: Vernet@IBS.FR.

Present address, Dept. of Biochemistry, Albert Einstein College of Medecine, 1300 Morris Park Ave., Bronx, NY 10461.

**
Present address, The Immune Response Corp., 5935 Darwin Ct., Carlsbad, CA, 92008.

The abbreviations used are: E-64, 1-[[(L- trans-epoxysuccinyl)-L-Leucyl]amino]-4-guanidino)butane; HPLC, high performance liquid chromatography.

T. Vernet, D. C. Tessier, J. Chatellier, C. Plouffe, T. S. Lee, D. Y. Thomas, A. C. Storer, and R. Ménard, submitted for publication.

P. J. Berti and A. C. Storer, personal communication.

T. Vernet, D. C. Tessier, and D. Y. Thomas, unpublished results.


ACKNOWLEDGEMENTS

We thank L. Laramée for DNA sequencing and D. Proteau and E. W. Jones for strain BJ7537.


REFERENCES
  1. Al-Janabi, J., Hartsuck, J. A., and Tang, J. (1972) J. Biol. Chem. 247, 4628-4632 [Abstract/Free Full Text]
  2. Baker, D., Silen, J., and Agard, D. A. (1992) Proteins Struct. Funct. Genet. 12, 339-344
  3. Baker, D., Shiau, A. K., and Agard, D. A. (1993) Curr. Biol. 5, 966-970
  4. Baker, E. N., and Drenth, J. (1987) in Biological Macromolecules and Assemblies: Active Sites of Enzymes (Jurnak, F. A., and McPherson, A. eds) Vol. 3, pp. 314-367, John Wiley & Sons, New York
  5. Berti, P. J., and Storer, A. C. (1994) Biochem. J. 302, 411-416 [Medline] [Order article via Infotrieve]
  6. Brocklehurst, K. Willenbrock, F., and Salih, E. (1987) in Hydrolytic Enzymes (Neuberber, A., and Brocklehurst, K., eds) pp. 39-158, Elsevier, Amsterdam
  7. Brömme, D., Bonneau, P. R., Wiederanders, B., Kirschke, H., Peters, C., Thomas, D. Y., Storer, A. C., and Vernet, T. (1993) J. Biol. Chem. 268, 4832-4838 [Abstract/Free Full Text]
  8. Casadaban, M., and Cohen, S. N. (1980) J. Mol. Biol. 138, 179-207 [Medline] [Order article via Infotrieve]
  9. Chang, S-C., Chang, P-C., and Lee Y-H. W. (1994) J. Biol. Chem. 269, 3548-3554 [Abstract/Free Full Text]
  10. Coll, M., Guasch, A., Avilès, F. X., and Huber, R. (1991) EMBO J. 10, 1-9 [Abstract]
  11. Cooner, G. E., and Richo, G. (1992) Biochemistry 31, 1142-1147 [Medline] [Order article via Infotrieve]
  12. Davis, R. W., Thomas, M., Cameron, J., St. John, T. P., Scherer, S., and Padgett, R. A. (1980) Methods Enzymol. 65, 404-411 [Medline] [Order article via Infotrieve]
  13. Desmazeaud, M. J. (1972) Biochimie (Paris) 54, 1109-1114 [Medline] [Order article via Infotrieve]
  14. Denizot, F., Brunet, J.-F., Roustan, P., Harper, K., Suzan, M., Luciani, M.-F., Mattei, M.-G., and Golstein, P. (1989) Eur. J. Biochem. 19, 631-635
  15. Fabre, E., Nicaud, J-M., Lopez, M. C., and Gaillardin, C. (1991) J. Mol. Biol. 266, 3782-3790
  16. Fabre, E., Nicaud, J-M., Tharaud, C., and Gaillardin, C. (1992) J. Mol. Biol. 267, 15049-15055
  17. Fox, T., de Miguel, E., Mort, J. S., and Storer, A. C. (1992) Biochemistry 31, 12571-12576 [Medline] [Order article via Infotrieve]
  18. Fukuda, R., Horiuchi, H., Ohta, A., and Tagagi, M. (1994) J. Biol. Chem. 269, 9551-9561
  19. Fusek, M., Mares, M., Vagner, J., Voburka, Z., and Baudys, M. (1991) FEBS Lett. 287, 160-162 [CrossRef][Medline] [Order article via Infotrieve]
  20. Germain, D., Dumas, F., Vernet, T., Bourbonnais, Y., Thomas, D. Y., and Boileau, G. (1992) FEBS Lett. 3, 283-286 [CrossRef]
  21. Hanada, K., Tamai, M., Morimoto, S., Adachi, T., Ohmura, S., Sawada, J., and Tanaka, I. (1978) Agric. Biol. Chem. 42, 537-541
  22. Ikemura, H., and Inouye, M. (1988) J. Biol. Chem. 263, 12959-12963 [Abstract/Free Full Text]
  23. Ito, H., Futuda, Y., Masata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168 [Medline] [Order article via Infotrieve]
  24. Johansen, J. T., and Ottesen, M. (1968) C. R. Trav. Lab. Carlsberg 36, 265-283 [Medline] [Order article via Infotrieve]
  25. Jones, E. W. (1991) Methods Enzymol. 194, 428-453 [Medline] [Order article via Infotrieve]
  26. Kassell, B., and Kay, J. (1973) Science 180, 1022-1027 [Medline] [Order article via Infotrieve]
  27. Keppler, D., Fondanèche, M. C., Dalet-Fumeron, V., Pagano, M., and Burtin, P. (1988) Cancer Res. 48, 6855-6862 [Abstract]
  28. Klionsky, D. J. Herman, P. K., and Emr, S. D. (1990) Microbiol. Rev. 54, 266-292
  29. Koga, H., Yukio, H, Nishimura, Y., Kato, K., and Imoto, T. (1990) J. Biochem. ( Tokyo) 108, 976-982 [Abstract]
  30. Kunitz, M., and Northrop, J. H. (1936) J. Gen. Physiol. 19, 991-1007
  31. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492 [Abstract]
  32. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  33. Mach, L., Mort, J. S., and Glössl, J. (1994) J. Biol. Chem. 269, 13030-13035 [Abstract/Free Full Text]
  34. Mason, R., W., Gal, S., and Gottesman, M. M. (1987) Biochem. J. 248, 449-459 [Medline] [Order article via Infotrieve]
  35. Matthews, G., Shennan, K. I. J., Seal, A. J., Taylor, N. A., Colman, A., and Docherty, K. (1994) J. Biol. Chem. 269, 588-592 [Abstract/Free Full Text]
  36. McIntyre, G. F. Godbold, G. D., and Erickson, A. H. (1994) J. Mol. Biol. 269, 567-572
  37. Melchiori, A., Albini, A., Ray, J. M., and Stetler-Stevenson, W. G. (1992) Cancer Res. 52, 2353-2356 [Abstract]
  38. Ménard, R., Carrière, J., Laflamme, P., Plouffe, C., Khouri, H. E., Vernet, T., Tessier, D. C, Thomas, D. Y., and Storer, A. C. (1991) Biochemistry 30, 8924-8928 [Medline] [Order article via Infotrieve]
  39. Neurath, H. (1984) Science 224, 350-357 [Medline] [Order article via Infotrieve]
  40. Ohta, Y., Hojo, H., Aimoto, S., Kobayaski, T., Zhu, X, Jordan, F., and Inouye, M. (1991) Mol. Microbiol. 5, 1507-1510 [Medline] [Order article via Infotrieve]
  41. Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7027-7031 [Abstract]
  42. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  43. SanSegundo, Martinez, M. C., Vilanova, M., Cuchillo, C. M., and Aviles, F. X. (1982) Biochim. Biophys. Acta 707, 74-80 [Medline] [Order article via Infotrieve]
  44. Steiner, D. F., Smeekens, S. P., Ohagi, S., and Chan, S. J. (1992) J. Biol. Chem. 267, 23435-23438 [Free Full Text]
  45. Stetler-Stevenson, W. G., Krutzsch, H. C., Wacher, M. P., Marguiles, I. M., and Liotta, L. A. (1989) J. Biol. Chem. 264, 1353-1356 [Abstract/Free Full Text]
  46. Stubbs, M. T., Laber, B., Bode, W., Huber, R., Jerala, R., Lenarcic, B., and Turk, V. (1990) EMBO J. 6, 1939-1947
  47. Turk, V., and Bode, W. (1991) FEBS Lett. 285, 213-219 [CrossRef][Medline] [Order article via Infotrieve]
  48. van den Hazel, H. B., Kielland-Brandt, M. C., and Winther, J. R. (1992) Eur. J. Biochem. 207, 277-283 [Abstract]
  49. Vernet, T., Dignard, D., and Thomas, D. Y. (1987) Gene ( Amst.) 52, 225-233 [CrossRef][Medline] [Order article via Infotrieve]
  50. Vernet, T. Tessier, D. C., Laliberté, F., Dignard, D., and Thomas, D. (1989) Gene ( Amst.) 77, 229-236 [Medline] [Order article via Infotrieve]
  51. Vernet T., Tessier, D. C., Richardson, C., Laliberté, F., Khouri, H. E., Bell, A. W., Storer, A. C., and Thomas, D. Y. (1990) J. Biol. Chem. 265, 16661-16666 [Abstract/Free Full Text]
  52. Vernet, T. Khouri, H. E., Laflamme, P., Tessier, D. C., Gour-Salin, B., Storer, A. C., and Thomas, D. Y. (1991) J. Biol. Chem. 266, 21451-21457 [Abstract/Free Full Text]
  53. Vernet, T., Chatellier, J., Tessier, D. C., and Thomas, D. Y. (1993) Protein Eng. 6, 213-219 [Abstract]
  54. Wetmore, D. R., Wong, S-L., and Roche, R. S. (1992) Mol. Microbiol. 6, 1593-1604 [Medline] [Order article via Infotrieve]
  55. Wilcox, C. A., and Fuller, R. S. (1991) J. Cell Biol. 267, 297-307
  56. Winther, J. R., and S, P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9330-9334 [Abstract]
  57. Woolford, C. A., Noble, J. A., Garman, J., D., Tam, M. F., Innis, M. A., and Jones, E. W. (1993) J. Biol. Chem. 268, 8990-8998 [Abstract/Free Full Text]
  58. Zoller, M. J., and Smith, M. (1982) Nucleic Acids Res. 10, 6487-6500 [Abstract]

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