From the School of Biosciences, Cardiff University,
P. O. Box 911, Cardiff CF1 3US, Wales, United Kingdom, the
¶ Department of Natural Sciences, Kalmar University, P. O. Box
905, S-39129 Kalmar, Sweden, and the
Department of
Immunology, Institute for Cell Biology, Eberhard Karls University,
D-72076 Tubingen, Germany
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ABSTRACT |
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The cDNA encoding the precursor of an
aspartic proteinase from the flowers of the cardoon, Cynara
cardunculus, was expressed in Pichia pastoris, and
the recombinant, mature cyprosin that accumulated in the culture medium
was purified and characterized. The resultant mixture of
microheterogeneous forms was shown to consist of glycosylated heavy
chains (34 or 32 kDa) plus associated light chains with molecular
weights in the region of 14,000-18,000, resulting from excision of
most, but not all, of the 104 residues contributed by the unique region
known as the plant specific insert. SDS-polyacrylamide gel
electrophoresis under non-reducing conditions indicated that disulfide
bonding held the heavy and light chains together in the heterodimeric
enzyme forms. In contrast, when a construct was expressed in which the
nucleotides encoding the 104 residues of the plant specific insert were
deleted, the inactive, unprocessed precursor form (procyprosin)
accumulated, indicating that the plant-specific insert has a role in
ensuring that the nascent polypeptide is folded properly and rendered
capable of being activated to generate mature, active proteinase.
Kinetic parameters were derived for the hydrolysis of a synthetic
peptide substrate by wild-type, recombinant cyprosin at a variety of pH and temperature values and the subsite requirements of the enzyme were
mapped using a systematic series of synthetic inhibitors. The
significance is discussed of the susceptibility of cyprosin to
inhibitors of human immunodeficiency virus proteinase and particularly of renin, some of which were found to have subnanomolar potencies against the plant enzyme.
Sequences have been elucidated recently for genes encoding
aspartic proteinases of plant origin, e.g. barley (1), rice (2), tomato (3), and oilseed rape and Arabidopsis (4). All
of these sequences predict that, by comparison with the well studied
aspartic proteinases from mammals, fungi, yeasts like Saccharomyces (5) and Candida (6), parasites like
Plasmodium falciparum (7), and viruses such as
HIV1 (8), approximately 100 extra amino acids are introduced into the C-terminal domain of the
newly synthesized plant polypeptides. The function of this region
or plant-specific insert (9) is currently unknown, but, as it does
share considerable sequence identity with a group of mammalian
sphingolipid activator proteins known as saposins (10, 11), it has been
postulated to bind selectively to certain lipids and thus direct the
precursor form of the aspartic proteinase into the appropriate
cytomorphological compartment in the plant cell (10).
Alignment of the sequences of the inserts predicted by the plant
aspartic proteinase genes with those of saposins correctly positions
all six cysteine residues and a glycosylation site and also maintains
the pattern of hydrophobic residues described previously for all known
saposins (12). However, each plant-specific insert does not consist of
a single saposin domain but appears to correspond to the C-terminal
portion of one saposin domain linked to the N-terminal portion of a
second saposin domain. On this basis, the plant-specific inserts have
been described as swaposins (12), indicating that they are likely to
have a structure similar to that of the saposins, but with N- and
C-terminal halves interchanged in sequence order. Relatively little
information has been reported to date on aspartic proteinases
originating from plant tissues, but, in the few enzymes that have been
isolated, this swaposin domain is not present and appears to have been
excised by post-translational processing. Nothing is known of the
enzyme(s) responsible in planta, but the excision of each insert is
imprecise, resulting in the generation of a complex mixture of
heterogeneous, mature aspartic proteinases within the tissues of each
of the plants that has been studied, e.g. from seeds of
barley (13), pumpkin (14), and Arabidopsis thaliana (15),
from rice (16) and from flowers of the cardoon, Cynara
cardunculus (9, 17, 18). Commonly, the enzymes thus generated are
heterodimers with molecular weights in the region of
40,000-45,000.
This complexity of natural isoforms is compounded even further by the
expression of several genes in the plant tissues, each encoding closely
related enzymes so that physicochemical and enzymatic characterization
of naturally occurring aspartic proteinases isolated directly from
plants has been made rather difficult. A recombinant approach was thus
employed in attempts to gain an initial insight into the significance
of the plant-specific insert and to examine the activity and
specificity of an aspartic proteinase from the plant kingdom. We have
expressed in the methylotrophic yeast, Pichia pastoris, the
cDNA encoding the precursor of one aspartic proteinase from the
flowers of the cardoon, C. cardunculus (19). Extracts of
these flowers have been documented previously to contain a number of
isoforms of aspartic proteinases called cyprosins and cardosins (9, 17,
18) and have been used for centuries as coagulants in traditional
cheese-making in regions of southern Europe, particularly the Iberian
peninsula (20).
Gene Cloning and Mutagenesis--
A cDNA clone encompassing
the full-length precursor of a cyprosin was isolated by courtesy of Dr.
M. Pietrzak (Basel, Switzerland) by rescreening of a cDNA library
prepared from flower buds of C. cardunculus, as described
previously (19, 21). This was amplified by PCR using Vent DNA
polymerase (New England Biolabs) with appropriate forward and reverse
primers (5'-GG AAT TCC GGA TCC TCA CCT ACT GCA TTT TCG GTC-3' and 5'-GA
ATT CCG GGA TCC TCA AGC TGC TTC TGC AAA-3', respectively; purchased
from Amersham Pharmacia Biotech, Cambridge, United Kingdom (UK)) and
subcloned into the pUC18 vector. In turn, this recombinant pUC18 was
used as template DNA for overlapping PCR mutagenesis reactions, as described previously (22). Mutations were introduced at appropriate locations by two initial and one subsequent PCR reaction. To modify the
sequence connecting the propart region to the mature cyprosin enzyme, a
241-bp fragment spanning from the pUC18 vector to the desired, mutated
cleavage junction was amplified using appropriate forward and reverse
primers (5'-GTT GGG TAA CGC CAG GG-3' = F1 and 5'-CCT CAG AAA AGC AGC
GAA GCC-3', respectively). The second PCR amplified a 478-bp fragment
using a forward primer (5'-GGC TTC GCT GCT TTT CTG AGG-3') and a
reverse primer (5'-GTT CTT GAA CAA GAC CTT G-3' = R1) complementary to
the sequence located downstream from the region into which the
mutations were to be introduced and downstream from a BglII
restriction site. The purified fragments were combined and used as
template DNA in a final PCR using the F1 and R1 flanking primers. The
resultant 698-bp amplicon was digested with the restriction
endonucleases HindIII and BglII and subcloned to
replace the corresponding wild-type segment in the original procyprosin construct.
The region encoding the 104 residues of the plant specific insert was
excised by an identical strategy. The primer pairs were 5'-GG AAT TCC
GGA TCC TCA CCT ACT GCA TTT TCG GTC-3' = F2 and 5'-CTG GAT GAG AGG TAC
CGC ACC AAT TGC ATG ATT GAT TTC-3'; and 5'-GTA CCT CTC ATC CAG GGA GAA
TCA GCA GTA GAC TGC AAC-3' and 5'-GA ATT CCG GGA TCC TCA AGC TGC TTC
TGC AAA-3' = R2. The resultant 911- and 296-bp fragments were combined
and used in the final reaction using the F2/R2 combination of primers.
The 1192-bp amplicon thus generated was digested with BamHI,
ligated into a similarly treated, dephosphorylated pUC18 vector, and
the reaction mixture was used to transform competent Escherichia
coli (DH5 Expression in E. coli--
Restriction digestion with
BamHI enabled subcloning of each desired cDNA into the
pET-3a expression vector (AMS Biotechnology, Witney, UK). Expression of
this recombinant plasmid was induced by the addition of
isopropyl-1-thio- Expression in P. pastoris--
The cDNA encoding each
procyprosin (wild-type or mutants) was subcloned into the expression
vector pPICZ
Yeast colonies that had undergone the appropriate recombination events
to incorporate the procyprosin gene into the host chromosome were used
to inoculate BMGY medium (100 ml) containing ampicillin (100 µg/ml).
The flasks (250 ml) were shaken at 30 °C until the cells attained an
A600 of 5.0. After harvesting by centrifugation at 3000 × g for 5 min at room temperature, the cell
pellets were resuspended in 20-ml aliquots of BMMY induction medium
containing ampicillin (100 µg/ml) in 100-ml flasks and shaken at
30 °C over a period of 7 days. Expression was induced over this time
period by the addition of methanol to a final concentration of 0.5%
every 24 h.
Recombinant Proteinase Purification and
Characterization--
Samples of medium from induced Pichia
cells were analyzed by SDS-PAGE followed by staining with Coomassie
Blue or by Western blotting using an anti-cyprosin antiserum that had
been raised in rabbits (17). Detection of immunoreactive bands used a
goat anti-rabbit IgG-alkaline phosphatase conjugate as described
previously (22). Proteinase activity was monitored during purification steps using the substrate
Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu2
at pH 4.0, with the cleavage being monitored by fast protein liquid
chromatography using a Pep-RPC reverse phase column (22). Aliquots of
conditioned medium were diluted by 10-fold prior to each assay in order
to circumvent the complications caused by the increasing amounts of a
yellow pigment that is released by the yeast cells into the growth
medium. This absorbs in the UV region of the spectrum and thus
interferes with detection of the products of peptide substrate digestion.
Samples of conditioned medium harvested after 6 days were adjusted to
pH 4.0 by the addition of 1 M sodium formate buffer, pH
3.0, and dialyzed at 4 °C against 10 mM sodium formate
buffer, pH 4.0, containing 50 mM sodium chloride with five
changes of buffer. Dialysates were applied to a Hi-Trap SP column
fitted into a fast protein liquid chromatography instrument (Amersham Pharmacia Biotech), and elution was continued with the same pH 4.0 buffer. Under these conditions, the yellow Pichia-derived pigment was not retained by the column. After extensive washing, recombinant cyprosin was eluted using a linear gradient of 50-500 mM NaCl in the sodium formate buffer at pH 4.0. Fractions
were monitored for activity and combined as appropriate.
Samples for N-terminal sequencing were subjected to SDS-PAGE under
reducing or non-reducing conditions and blotted onto polyvinylidene difluoride membrane, and appropriate bands were subjected to automated Edman degradation, as described previously (23). Attempts to derive
C-terminal sequence on relevant bands were carried out (24), using
a Hewlett-Packard G1009A C-terminal sequencing system. Deglycosylation
reactions were carried out with N-glycosidase F (Roche
Molecular Biochemicals, Mannheim, Germany) in 250 mM Tris-HCl buffer, pH 8.8, at 37 °C for 1 h.
A naturally occurring preparation of isoform 3 of cyprosin was purified
to homogeneity from the dried flowers of C. cardunculus as
described previously (17).
Kinetic parameters for hydrolysis of the chromogenic substrate
Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu were derived spectrophotometrically as
described previously (23). Values of kcat were
derived from the equation Vmax = kcat × [Et], where the
concentration of active enzyme Et was derived by
active site titration using preparations of isovaleryl-pepstatin of
precisely defined concentration (23). Inhibition constants were derived
at pH 5.0, and the estimated error on all measurements was always less
than ±15%. However, it was necessary to use final concentrations of
cyprosin in the assay cuvettes of approximately 5 nM, and
so Ki values for tight-binding inhibitors (where
Ki The nucleotide sequence of the procyprosin clone isolated by
rescreening of the cDNA library from flower buds of C. cardunculus (19, 21) has been deposited in the EMBL/GenBank data
bases under the accession number X81984. The amino acid sequence predicted by this clone is aligned with that of human procathepsin D in
Fig. 1. From this, it is apparent that
(i) the plant gene encodes an insert of 104 residues within the
C-terminal domain that is not present in the mammalian enzyme, and (ii)
both proteins are predicted to have one common glycosylation motif (at
Asn67-Gly68-Thr69); the other known
site of carbohydrate attachment in cathepsin D (at
Asn183-Val184-Thr185) is not
present in the cyprosin sequence, although an additional glycosylation
motif (at
Asn83I-Glu84I-Thr85I)3
is present in the plant-specific insert of cyprosin (Fig. 1 and see
Introduction). Excluding the plant-specific insert, the sequence of the
mature enzyme region of this cyprosin shares 52% identity with that of
cathepsin D; in contrast, the two prosegments have very little
similarity to one another.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
) cells. The authenticity of all manipulations was
verified by dideoxy sequencing of both DNA strands.
-D-galactopyranoside (0.4 mM final concentration) when the cells had reached an
A600 of 0.6. After 3 h, the cells were
harvested and lysed, and the resultant insoluble material was washed at
4 °C for 4 h with 100 mM Tris-HCl, pH 11.0, containing 50 mM
-mercaptoethanol. After centrifugation at 16,000 × g for 30 min, the resultant pellet was
solubilized by stirring at 25 °C for 16 h in 6 M
urea in 100 mM Tris-HCl buffer, pH 8.0, supplemented with 1 mM glycine, 1 mM EDTA, and 50 mM
-mercaptoethanol.
C (Invitrogen, Leek, Netherlands) and used to transform
E. coli (Top10F') cells. Selection of transformants
containing the pPICZ
C vector was made on low salt LB agar
containing zeocin (50 µg ml
1). Plasmid DNA was purified
from selected colonies, linearized by digestion with the restriction
endonuclease PmeI, and electroporated into P. pastoris (KM71) cells according to the manufacturer's instructions (Invitrogen).
0.1 × Et) are given as best estimates, indicated by the < symbol. The synthetic inhibitors of HIV proteinase, Saquinavir and Indinavir, were generously supplied by Dr. J. A. Martin (Roche Products Ltd., Welwyn Garden City, Herts, UK), Ritonavir by Dr. D. Kempf (Abbott), and HBY-793 by
Dr. J. Knolle (Hoechst AG, Frankfurt, Germany), respectively. All of
the other compounds used were the kind gifts of Dr. D. F. Veber,
formerly of Merck, Sharp and Dohme Research Laboratories, West Point,
PA. Protein inhibitors from potato, Ascaris lumbricoides, and Saccharomyces cerevisiae were the respective gifts of
Drs. J. Brzin and B. Strukelj (J. Stefan Institute, Ljubljana,
Slovenia), Dr. R. J. Peanasky (formerly of the University of South
Dakota, Vermillion, SD), and Dr. L. H. Phylip (School of
Biosciences, Cardiff University, Cardiff, Wales).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the deduced amino acid sequence
of procyprosin (CYP) with that of human procathepsin D
(HCD). The numbering system used is that of pig
pepsin. The absence of a residue is indicated by a dash. The
positions of the active site Asp residues and potential
N-glycosylation sites are indicated by and
,
respectively. Known disulfide bonds in cathepsin D and other eukaryotic
aspartic proteinases are between cysteine residues (indicated by
)
45-50, 206-210, and 250-283. The N-terminal sequences of heavy and
light chains determined by Edman degradation are denoted by
arrows.
As described under "Materials and Methods," the cDNA encoding
this wild-type form of procyprosin was introduced into pET-3a. Expression in E. coli strain BL21 (DE3) pLysS resulted in
the accumulation of recombinant protein at a high level (estimated to
be 10-20 mg/liter of culture) but this was insoluble and misfolded. Despite extensive efforts, suitable conditions to prepare significant amounts of properly folded protein could not be established.
Consequently a different expression system was required and the
methylotrophic yeast, P. pastoris, was selected since this
has been used previously to produce an aspartic proteinase (precursor)
in a soluble, properly folded form (25). The procyprosin construct was
subcloned into the pPICZC plasmid, and appropriate recombinants of
the P. pastoris cells (KM71 strain) were selected as
described under "Materials and Methods."
Expression of the procyprosin gene resulted in the appearance in the
culture medium of a cluster of immunoreactive bands in the 32-34-kDa
region, together with a second cluster in the region between 14 and 18 kDa (Fig. 2A). The relative
proportions of each band in each of the clusters varied from
batch-to-batch of induced culture medium so the clusters are referred
to as heavy and light chains, respectively. The heavy chain (32-34
kDa) cluster was apparent as early as the second day of induction,
while the light chains (14-18 kDa) became visible after about day 5. The recombinant protein was purified from medium harvested after 6 days, as described under "Materials and Methods." Acidification and
dialysis of the medium at pH 4.0, followed by chromatography on a
HiTrap SP column, successfully removed the yellow pigment that is a
persistent contaminant released into the culture medium of induced
Pichia cells. The recombinant protein that was eluted by the
salt gradient from the HiTrap SP column did not emerge in a sharp peak
but rather was a broad smear of material that absorbed at 280 nm and
that reacted with the cyprosin antiserum (data not shown). Early
fractions of material contained a mixture of 34- and 32-kDa heavy
chains in which the 34-kDa band was predominant (Fig.
3A, lane
1), while in the later fractions the 32-kDa band was most
abundant (Fig. 3A, lane 2). All of the
fractions also contained light chains migrating in the 14-18-kDa
range, which stained only weakly with Coomassie Blue. The 34- and
32-kDa heavy chains and the light chains were all immunoreactive with
anti-cyprosin antiserum (data not shown). Thus, it was apparent that
all of the contaminating proteins had been removed and that the only
remaining proteins were derived from recombinant (pro)cyprosin. The
yield of (total) purified protein was approximately 1 mg/liter of
culture medium. However, microheterogeneity was clearly evident.
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The 34- and 32-kDa heavy chains were resolved from one another by SDS-PAGE under reducing conditions and blotted onto polyvinylidene difluoride membrane. N-terminal sequencing by Edman degradation revealed that both were identical and contained overlapping sequences (in the ratio 40:60).
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No data were obtained from attempts at C-terminal sequencing of the 34-kDa heavy chain, but, for the 32-kDa heavy chain, leucine was identified as the C-terminal residue, with threonine occupying the penultimate position. This ~Thr-Leu combination occurs only once in the sequence Cys3I-Lys4I-Thr5I-Leu6I near the predicted N terminus of the plant-specific insert region (Fig. 1). Proteolytic processing after this leucine residue would thus result in Cys3I remaining within the heavy chain, which would be predicted to have an Mr of 29,000 on SDS-PAGE under reducing conditions. Treatment (of a different preparation of recombinant cyprosin which contained both 34- and 32-kDa heavy chains) with N-glycosidase F resulted in a decrease in the sizes observed for the heavy chains from 34 and 32 kDa to approximately 32 and 29 kDa, respectively (Fig. 3C, lanes 7 and 8). The Pichia cells had thus carried out glycosylation of the heavy chains, compatible with the presence of the Asn67-Gly68-Thr69 motif (see above) and with our previous observation that naturally occurring cyprosins are glycoproteins (17). A disulfide bond between Cys3I near the C terminus of the heavy chains and Cys97I near the N terminus of the light chain would account for the increased size (46 kDa) of the recombinant cyprosin observed under non-reducing conditions (Fig. 3, lane 5). Six cysteine residues are predicted to be present within the plant-specific insert (Fig. 1) of cyprosin, and these are all conserved in the insert sequences of aspartic proteinase precursors from other plants (1-4). A putative assignment of these into disulfide-bonded pairs (3I-97I, 28I-69I, and 34I-66I) has been proposed (12) on the basis of the swaposin similarity (see Introduction).
All of these data can be interpreted on the basis of the scheme
depicted in Fig. 4. Processing of the
initial protein product translated in P. pastoris removes
the prosegment and generates mature cyprosin with microheterogeneity at
the N terminus of the heavy chain(s), as described previously. Much,
but not all, of the plant specific insert region is processed away, but
cleavage occurs in (at least) two locations near the predicted N
terminus of the plant-specific insert sequence, thereby generating 34- and 32-kDa heavy chains that differ at their C termini. Additionally, processing at a number of locations toward the C terminus of the insert
generates a complex mixture of light chains that remain attached by
disulfide bonding to the heavy chains to generate a series of isoforms
of mature cyprosin. In contrast, the heavy and light chains of a
naturally occurring cyprosin (isoform 3) isolated from flowers of
C. cardunculus are not held together by disulfide
bonding, since this heterodimeric protein was still resolved into heavy
and light chains on SDS-PAGE under non-reducing conditions
(Fig. 3B, lanes 4 and 6).
No N-terminal sequence was determined for the light chain of this
isoform, but the sequence PMGESAVD~ was identified at the N terminus
of the closely related isoform
14 that had been separated as
described previously (17) from the mixture of naturally occurring
enzyme forms extracted from the flowers of the plant. This sequence
begins at residue 103I of the plant-specific insert (Fig. 1) and is
located downstream from Cys97I.
Processing in planta to remove the plant-specific insert must therefore
have taken place at a slightly different location from those observed
in P. pastoris, i.e. downstream from
Cys97I, which serves therefore to indicate the
C-terminal boundary of the plant-specific insert. No C-terminal
sequence has been obtained for the heavy chain of any naturally
occurring cyprosin, but the homologous cardosin A heavy chain has been
reported recently (9) to have microheterogeneity at its C terminus,
corresponding to cleavage at the N-terminal end of the plant-specific
insert, after the residues (equivalent to) Lys238,
Val240, and Met241 (Fig. 1). This also suggests
that processing in planta may take place at adjacent site(s) to those
observed in Pichia, i.e. upstream from the
Cys3I-Lys4I-Thr5I-Leu6I
sequence at the beginning of the plant-specific insert.
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Processing in Pichia was only marginally affected by inclusion (at 400 nM final concentration) of the aspartic proteinase inhibitor, pepstatin, in the culture medium of the Pichia cells. This caused only a small increase in size of the 34- and 32-kDa bands to 36 and 33 kDa, respectively (Fig. 2B), but did not result in the accumulation of a protein band consistent in size with that of the unprocessed procyprosin precursor. As this report was nearing completion, Glathe et al. (26) reported the expression of the cDNA encoding the precursor of the aspartic proteinase, phytepsin, from barley (Hordeum vulgare L.) using baculovirus. In this heterologous system, the intact precursor accumulated in the medium, enabling it to be purified and its ability to be processed under defined conditions in vitro to be determined. It was shown to be capable of undergoing autoactivation to generate initially bands of 36 and 17 kDa, as monitored by SDS-PAGE under reducing conditions, followed by further processing to produce finally subunits of approximately 28 and 11 kDa, respectively. Microheterogeneity was detected at the N terminus of the 11-kDa subunit but the residues identified were (equivalent to) Tyr92I and Val93I (of cyprosin; Fig. 1); thus, autoprocessing had taken place finally at an identical location to that observed with recombinant procyprosin in the present study. In the prophytepsin case, no indication was given of the migration pattern of the subunits on SDS-PAGE under non-reducing conditions. However, these autoprocessing reactions of prophytepsin only occurred when the pH was below 4.5 (26). In our case, the medium of the Pichia cells was buffered well above pH 4.5 (pH 6.0), yet it was mature cyprosin and not the intact precursor that accumulated in the medium. Since the two plant proteinases are 78% identical, unless the initial translation product of procyprosin encounters pH values below 4.5 during its translocation through the lumen of the secretory apparatus in the Pichia cells, it would seem unlikely that the (multiple forms) of mature cyprosin that accumulated in the medium had been produced as a consequence of autoactivation of the recombinant precursor. A more likely explanation is that processing took place through the action of host cell proteinases at exposed sites that were susceptible not only to heterocatalytic attack under the conditions likely to be encountered in living cells but also to autocatalytic cleavage under the somewhat more extreme conditions that can be employed in vitro. Just as in the present case with recombinant and naturally occurring forms of cyprosin, the subunits of recombinant phytepsin differed slightly from those of the enzyme extracted from barley seeds (26).
Some precursors of mammalian/fungal aspartic proteinases require the action of extrinsic proteinases to generate the mature enzyme, e.g. prorenin, whereas others such as pepsinogen undergo autocatalytic activation (27). In the case of procathepsin D, which is the mammalian enzyme most closely related to cyprosin and phytepsin, cysteine proteinases are required for activation to occur within the lysosomes of the cell although, in vitro, limited autoprocessing occurs under acidic conditions to generate an activation intermediate (known as pseudo-cathepsin D) in which only 26 of the 44 residues of the prosegment have been removed (28). In order to achieve complete autocatalytic removal of all 44 prosegment residues in vitro, it was necessary to introduce an autoactivation sequence at the junction between the prosegment and mature enzyme region of human procathepsin D (28). A comparable alteration was introduced into the procyprosin sequence by changing the residues linking the prosegment to the N terminus of the (heavy chain of the) mature cyprosin (Fig. 1) from ~Phe40P-Gly-Gly-Ala-Leu-Arg45P-Asp1~ to ~Phe40P-Ala-Ala-Phe-Leu-Arg45P-Asp1~. Expression of this construct in Pichia resulted in the appearance of active, mature enzyme that was indistinguishable in its features and behavior from wild-type cyprosin (data not shown). In order to gain some insight into the significance of the plant specific insert, a further adaptation was introduced into this mutant construct by deleting the nucleotides encoding the 104-residue plant-specific insert of procyprosin and replacing the five residues (~Lys238-Gly-Val-Met-Ser242~; Fig. 1) immediately preceding the plant insert by the corresponding residues (~Val238-Pro-Leu-Ile-Gln242~) from human cathepsin D, which are known from the crystal structure to form a surface loop in this region of the human enzyme (29). Expression of this insert-deleted construct in Pichia resulted in the production of lower amounts of recombinant protein. The protein that was detected (Fig. 3C, lane 9) had a molecular weight (46,000) which corresponded to that of unactivated (insert-deleted) procyprosin and it was inactive against the synthetic peptide substrate. Thus, it seems that, at least in Pichia cells where the processes do approximate those likely to be operative in plant cells, the residues of the plant-specific insert would appear to be essential to ensure that the nascent polypeptide is folded properly and rendered capable of being activated to generate mature enzyme, albeit in slightly altered forms from the multiple isoforms that are generated within the flowers of the plant (9, 17, 26).
Kinetic parameters (Km, kcat)
were determined for the hydrolysis of a synthetic chromogenic peptide
substrate by a purified preparation of wild-type, recombinant cyprosin
in which the heavy chain consisted predominantly of the 32-kDa form
(Table 1, A). Consistent values for
Km (varying by less than 2-fold) were observed
across the pH range 3.0-6.0. Similarly, the values for
kcat varied by only 2-fold in absolute magnitude but did increase progressively between pH 3.0 and 4.5-5.0, decreasing again at yet higher pH values, as has been observed previously for
aspartic proteinases from other species (30). Values were also measured
for the hydrolysis of the same substrate at pH 5.0 by one of the
naturally occurring isoforms (isoenzyme 3) of cyprosin, purified from
the flowers of the cardoon plant as described previously (17). The
Km value obtained (25 ± 5 µM)
was little different from that measured for the recombinant form of
cyprosin produced in Pichia (Table I, A). The
kcat value determined for the natural
isoform(29 ± 3 s1) was somewhat higher (by
~3-fold) than the corresponding value derived at pH 5.0 for the
recombinant form of the enzyme. A homology-based model for plant
aspartic proteinases constructed on the basis of their similarity to
mammalian/fungal aspartic proteinases for which structures have been
solved by x-ray crystallography, indicated that the plant-specific
insert residues are likely to be located adjacent to the active site
cleft (10). Since the specificity constants
(kcat/Km) measured for
isoform 3 and recombinant cyprosin at pH 5.0 were 1.2 and 0.18 µM
1 s
1, respectively (Table
I, A), it may be that the residual residues remaining after excision of
(most of) the plant-specific insert by the Pichia cells,
have a minor influence on kcat (and hence on
kcat/Km) but have relatively
little effect on substrate interaction (as reflected in
Km). Comparable situations have been reported for
pseudocathepsin D relative to cathepsin D (29) and from a comparison of
recombinant and naturally occurring forms of plasmepsin I, an aspartic
proteinase from the malaria parasite Plasmodium
falciparum.5
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The temperature dependence of the kinetic parameters for chromogenic substrate hydrolysis by the recombinant cyprosin was also determined. The specificity constant (kcat/Km) increased progressively in magnitude, reaching a maximum value at 55 °C but activity was still readily detectable at 65 °C (Table I, B). The stability of the recombinant proteinase at 55 °C was measured at pH 5.0, 6.0, and 7.5, and half-lives of 40, 40, and <2 min were determined, respectively. Recombinant cyprosin thus displays a remarkable stability at temperatures up to 55 °C and at pH values as high as 6.0.
The recombinant plant cyprosin was not affected (IC50
2,000 nM) by a plant derived protein isolated from
potatoes, which is a potent inhibitor (Ki~4
nM) of cathepsin D (32). Similarly, the naturally occurring
protein inhibitors of pepsin/cathepsin E and yeast proteinase A from
the parasitic worm A. lumbricoides (33) and S. cerevisiae (34), respectively, did not inhibit the recombinant
cyprosin to any significant extent (IC50
2,000 nM).
In contrast, the naturally occurring peptide isovaleryl-pepstatin, which contains a central statine moiety as the transition state analogue occupying the P1-P1' positions in the acylated pentapeptide (35), showed subnanomolar potency as an inhibitor of recombinant cyprosin (inhibitor 1 in Table II). Whereas this response might perhaps have been expected as typical of an aspartic proteinase (30), a completely distinct acylated pentapeptide (inhibitor 2; Table II) was almost as effective as an inhibitor of cyprosin, despite having only the central statine residue in common with the sequence of isovaleryl pepstatin. Replacement of statine (which has a leucine side chain in P1) by its cyclohexyl alanine analogue (ACHPA, inhibitor 3) resulted in a 5-fold reduction in potency (compare inhibitors 3 and 2 in Table II). In longer inhibitors spanning nine subsites (Table III), replacement of statine by ACHPA also resulted in a reduction of potency (by 2.5-fold; compare inhibitors 8 and 6 in Table III); replacement by the variant containing a phenylalanine substituent in P1 (AHPPA, inhibitor 7; Table III) produced a compound with comparable inhibitory potency to that of the statine-containing inhibitor 6. Consequently, statine was retained as the centerpiece, occupying the P1-P1' positions of inhibitors, and the effect of substitutions in other positions was examined systematically.
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In the P6 position, truncation of the (CH3)2CH·CH2·CO~ (isovaleryl) substituent by removal of a methylene carbon (generating (CH3)2CH·CO~ = isobutyl) resulted in an ~3-fold improvement in Ki value toward recombinant cyprosin (cf. inhibitors 9 and 6 in Table III). Replacement of the methylene carbon atom with an oxygen (in (CH3)3C·O·CO~ = t-butoxycarbonyl) caused an additional 6-fold improvement in potency, resulting in an inhibitor with a potency of 1 nM (inhibitors 10 and 6 in Table III). It may be that the oxygen atom in the -O-CO~ arrangement of inhibitor 10 can enter into a hydrogen bonding arrangement with an H-bond donor in the enzyme that is not possible with the -CH2-CO~ equivalent in inhibitor 6.
At the P3 position, the replacement of phenylalanine by homophenylalanine (which has an additional -CH2- in its side chain) caused a significant improvement (10-fold) in Ki (cf. inhibitors 12 and 6 in Table III). Replacement of Phe with 2-naphthylalanine (inhibitor 13) had only a minor (3-fold) effect on potency, whereas a 1-naphthylalanine substituent in the P3 position resulted in an inhibitor (number 14) that showed almost equivalent potency toward cyprosin as that measured for pepstatin (Table II). In contrast to this increase in size upon replacing the benzene ring of phenylalanine with the 1-naphthylalanine substituent, removal of the benzene ring altogether, resulting in an alanine substituent in P3 (compare 11 and 10 in Table III), caused a reduction in potency of more than 3 orders of magnitude.
In the P2 position, substitution of His by the more
hydrophobic side chain of Tyr resulted in increased inhibitor potency toward cyprosin by about an order of magnitude (cf.
inhibitors 15 and 9; Table III). Replacement of the imidazole side
chain of His with the CH3 substituent of Ala generated an
inhibitor (number 16) with subnanomolar potency comparable to that of
pepstatin (Table II) and substitution with the longer
CH3-CH2~ side chain of -amino butyric acid
(in inhibitor 17) resulted in the best yet inhibitor of recombinant
cyprosin (Table III). Indeed, the interaction of this compound with the
enzyme was so tight that problems of mutual depletion were encountered,
so that an accurate Ki value could not be measured
using the methodology employed (see "Materials and Methods").
In the P2' position, substitution of Leu by its
-branched isomer, Ile, resulted in a 5-fold improvement in inhibitor
potency (cf. 18 and 6 in Table III), whereas replacement by the longer, straight-chain analogue,
-N-acetylornithine (inhibitor
19) had only a minimal effect. At the P3' position,
replacement of the hydrophobic Phe residue by the (charged),
hydrophilic imidazole side chain of His resulted in a reduction in
potency of 4-fold (cf. compounds 20 and 18; Table III)
toward recombinant cyprosin.
Thus, on the basis of the data presented in Tables II and III, it may
be concluded that the compound
ethoxycarbonyl-1-naphthylalanine--amino-n-butyric acid-statine-Ile-Phe-NH2 would be likely to be an
extremely potent, if somewhat water-insoluble inhibitor of recombinant
cyprosin. It will be apparent then that the active site cleft of the
plant enzyme is very hydrophobic.
In this regard, however, it has long been held that, among the aspartic proteinases, cathepsin D has one of the most hydrophobic active site clefts in its specificity requirements (36). For the sake of comparison, then, the Ki values that we have reported previously (37) for the interaction of this human enzyme with the same set of inhibitors, are included in Tables II and III. From these data, it is apparent that each inhibitor binds substantially tighter (varying from ~5- to 800-fold) to recombinant cyprosin than to human cathepsin D, with the exception of pepstatin and inhibitor 11, which contains an Ala in the P3 position. The dislike of cyprosin for a small hydrophobic substituent in P3 reflects the situation reported previously (37) for human renin, gastricsin, and cathepsins D and E. In contrast, human pepsin exhibits a striking preference for Ala rather than Phe as a P3 substituent (37). The inhibitors described in Tables II and III were synthesized originally as potential inhibitors of human renin, which is one of, if not the, most specific proteinase so far described. It acts solely on one protein substrate angiotensinogen, to generate angiotensin I, and so there have been substantial efforts made to design specific renin inhibitors, such as those in Tables II and III, as potential anti-hypertensive agents. It seems all the more remarkable then that inhibitors designed on the basis of the sequence of residues in plasma angiotensinogen recognized by this highly specific enzyme involved in the mammalian cardiovascular system, should prove to be so effective against a proteinase from the flowers of the cardoon plant.
Following this rationale, with the advent of AIDS, several synthetic compounds have also been developed recently as inhibitors of the aspartic proteinase from human immunodeficiency virus (8). These have been shown to have differing potencies toward the human aspartic proteinases (38-41). Consequently, these were also examined for their effectiveness toward recombinant cyprosin. HBY-793 (compound 21), which is a symmetrical compound containing a central dihydroxyethylene moiety (38), was found to act as an inhibitor of cyprosin (Table IV). It is, however, much less effective toward the plant enzyme than HIV proteinase or some of the human enzymes for which subnanomolar Ki values were obtained. Ritonavir (compound 22) contains a hydroxyethylene transition state analogue and has been shown to have Ki values of 20 and 8 nM, respectively, for human cathepsin D and cathepsin E (39), so is not totally specific for HIV proteinase either. This trend was reflected with recombinant cyprosin against which Ritonavir was a weak (Ki = 110 nM) inhibitor. In total contrast, Saquinavir (inhibitor 23 in Table IV) has been determined previously (40) to be completely specific for the aspartic proteinases from HIV-1, HIV-2, and SIV and to have no measurable effect on any other aspartic proteinase, including those from other retroviruses (41). This hydroxyethylamine-containing inhibitor was found to inhibit recombinant cyprosin, albeit weakly (Table IV) at 140 nM. In contrast, Indinavir (compound 24 in Table IV), which also contains a hydroxyethylamine transition state analogue, did not inhibit cyprosin to any significant extent.
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From these data, it is evident that the active site cleft of cyprosin
can readily accommodate chemically synthesized inhibitors of mammalian
and retroviral aspartic proteinases although the plant enzyme is not
susceptible to protein inhibitors including one, itself of plant origin
(potato). Thus, it would seem that the most distinctive feature of the
plant aspartic proteinase is the plant-specific insert. This conserved
feature has been hypothesized to direct the newly translated plant
polypeptides into appropriate cytomorphological compartments within the
cell. However, in the present study, recombinant cyprosin was secreted by the yeast cells into the medium so that it did not appear to contain
appropriate intracellular targeting signals that were functional in the
Pichia cells. Thus, in order to gain further insight into
the significance of the plant-specific insert, it will be necessary to
express mutants encoding diminishing lengths of the insert, within
plant cells. Nevertheless, from the present study, the plant-specific
insert appears to be necessary for the production of the recombinant
precursor in such a form that it can be processed to produce the mature enzyme.
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ACKNOWLEDGEMENTS |
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We gratefully appreciate the respective contributions made by M. Pietrzak (Basel, Switzerland), Edy Segura (Protein Core Facility, University of Florida), and Wieland Keilholz (AnalyTeck, Tubingen, Germany) in clone isolation, N-terminal sequencing, and C-terminal sequencing. We also acknowledge the generosity of J. Brzin, B. Strukelj, R. J. Peanasky, L. H. Phylip, J. A. Martin, D. Kempf, J. Knolle, and D. F. Veber, for their kind provision of the inhibitors used.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a studentship award from the Biotechnology and Biological Sciences Research Council.
** To whom correspondence should be addressed. Tel.: 44-1222-874124; Fax: 44-1222-874116; E-mail: smbjk{at}cardiff.ac.uk.
2 The nomenclature system ~P6-P5-P4-P3-P2-P1*P1'-P2'-P3'~ is used to depict amino acids adjacent to the residues in the P1 and P1' positions that contribute the scissile peptide bond (indicated by an asterisk).
3 In keeping with IUB convention, putative plant-specific insert residues have been designated with the suffix I, and prosegment residues are assigned the suffix P.
4 M. C. Cordeiro and P. E. Brodelius, unpublished observation.
5 L. Tyas, R. P. Moon, K. Rupp, J. Westling, R. G. Ridley, J. Kay, D. E. Goldberg, and C. Berry, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: HIV, human immunodeficiency virus; ACHPA, 4-amino-5-cyclohexyl-3-hydroxypentanoic acid; AHPPA, 4-amino-3-hydroxy-5-phenylpentanoic acid; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).
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REFERENCES |
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