From the A C-terminally truncated form of yapsin 1 (yeast
aspartic protease 3), the first member of the novel sub-class of
aspartic proteases with specificity for basic residues (designated the Yapsins), was overexpressed and purified to apparent homogeneity, yielding ~1 µg of yapsin 1/g of wet yeast. N-terminal amino acid analysis of the purified protein confirmed that the propeptide was
absent and that the mature enzyme began at Ala68. The
mature enzyme was shown to be composed of approximately equimolar
amounts of two subunits, designated Peptide hormones are synthesized as larger precursors that require
endoproteolytic cleavage at basic residues in the secretory pathway. In
yeast, the Golgi resident, subtilisin-like serine protease, Kex2p (1,
2), is responsible for the processing of pro- Yapsin 1 has previously been expressed in Saccharomyces
cerevisiae as a carboxyl-terminally truncated soluble form and
purified from the cell extract (14). This yapsin 1 enzyme was shown to process the mammalian prohormone, pro-opiomelanocortin and its fragments (14). It was characterized as a ~70-kDa glycoprotein with a
pH optimum of 4.0-4.5 and observed to have a limited shelf life. In
the same expression system, yapsin 1 activity was also found in the
culture supernatant and was characterized as ~150-180- and ~90-kDa
hyperglycosylated forms that appeared to be highly stable
(8).2 Secreted yapsin 1 has
been shown to process a variety of mammalian prohormones in
vitro with catalytic efficiencies
(kcat/Km) of 1.6 × 104 M Our recent work has shown that yapsin 1 is immunologically related to
pro-opiomelanocortin converting enzyme, an enzyme with similar backbone
size and specificity. Moreover, immunocytochemical studies showed
colocalization of immunoreactive yapsin 1-related processing enzymes in
neuropeptide-rich regions of mammalian brain and pituitary (13)
supporting the existence of mammalian homologues of yapsin 1 involved
in pro-neuropeptide processing. We also showed that yapsin 1 can
function in vivo to process pro-opiomelanocortin in the
mammalian PC12 cell line (17). This indicates a close structural and
functional relationship of yapsin 1 with mammalian yapsins. Thus yapsin
1, which has been the most extensively studied member of the yapsin
family, represents an ideal model enzyme for learning how the yapsin
class of aspartic proteases are synthesized. Studies on the
biosynthesis of yapsin 1 will also further define the structure of the
enzyme and shed light on the best substrates that can interact with the
enzyme. This will ultimately provide an insight into the physiological
role that yapsin 1 plays in yeast.
Purification and Analysis of Yapsin 1 Secreted from
Overexpressing Yeast
Carboxyl-terminally truncated soluble yapsin 1 enzymatic
activity was found to be secreted into the culture supernatant of a
previously described yeast expression system (14) and was used as the
starting material for the purification of the enzyme.
Concentration--
The yeast culture supernatant containing the
secreted yapsin 1 enzyme was concentrated by tangential flow filtration
at room temperature using a 30-kDa molecular mass cutoff omega membrane in an ultrasette cassette (Filtron, Northborough, MA). A 4-fold diafiltration step to replace the media with 20 mM sodium
phosphate buffer, pH 7.0, was also performed. The sample was further
concentrated by centrifugation filtration using a Filtron 30-kDa
Macrosep omega membrane filter. All subsequent procedures were carried
out at 4 °C.
Anion Exchange Chromatography (MonoQ1)--
The concentrated
medium was applied to a MonoQ HR 5/5 column (Pharmacia Biotech Inc.)
connected to a fast protein liquid chromatography system (Pharmacia).
The column was allowed to re-equilibrate in buffer A (20 mM
sodium phosphate, pH 7.0) before a gradient of 0-25% buffer B (buffer
A with 1 M NaCl) was applied. The flow rate was 1 ml/min,
and 0.5-ml fractions were collected. The fractions were assayed by the
standard ACTH1-39 assay, previously described (15). Two
fractions on either side of the peak of activity were pooled and re-run
on the MonoQ. The peaks of activity were combined and concentrated by
centrifugation filtration through a Filtron 30-kDa Microsep omega
membrane filter.
Gel Filtration Chromatography (Superdex G-200)--
The
concentrated sample of activity from MonoQ1 was applied to a High Load
16/60 Superdex G-200 superfine column (Pharmacia) that was connected to
the fast protein liquid chromatography system. The buffer used was 20 mM sodium phosphate, pH 7.0. The flow rate was 1 ml/min,
and 1-ml fractions were collected. The column was previously calibrated
with dextran blue 2000 (V0), bovine serum albumin (69 kDa), and ovalbumin (43 kDa), where
V0 = 44 ml and Vt = 124 ml.
Fractions containing yapsin 1 activity were pooled and used for the
next step in the purification.
Anion Exchange Chromatography (MonoQ2)--
The pool of activity
from the G-200 column was applied to the MonoQ2 using the same buffer
system as that in MonoQ1. After re-equilibration, however, the protein
was eluted in 0.5-ml fractions with a shallower gradient as follows:
0-10% buffer B in 5 min, 10-12% buffer B in 20 min, and 12-25%
buffer B in 5 min. A selected portion of the yapsin 1 activity peak was
pooled and designated as purified yapsin 1.
Analysis of Yapsin 1 from Yeast Culture Supernatant--
280 ng
of the purified yapsin 1 were run on a 12% Tris/glycine precast
SDS-polyacrylamide gel and analyzed by silver stain (Novex, San Diego,
CA). Also, an aliquot from the Superdex G-200 pool of activity (1.6 µg of protein) was deglycosylated as described before (8), run on an
8-16% Tris/glycine SDS-polyacrylamide gel in either the presence or
absence of 5% Expression and Analysis of an Active Site Mutant of Yapsin 1 in Yeast
An active site mutant of the carboxyl-terminally truncated
yapsin 1 was engineered by site-directed mutagenesis of
Asp101 to Glu101 using an "overlap
extension" mutagenesis procedure involving three PCR steps (18).
Primers 3 and 4 were used as "outside" primers (see Table
I), whereas primers 5 and 6, containing
the Asp to Glu point mutation, were used as "inside" primers. The resulting fragment from the PCR steps was digested with
BamHI and PstI and subcloned into the pEMBLyex4
vector previously described (14) and called
pYAP3LC(Asp101 Section on Cellular Neurobiology,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and
, that were associated
to each other by a disulfide bond. C-terminally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression
system and secreted as a zymogen that could be activated upon
incubation at an acidic pH with an optimum at ~4.0. When expressed
without its pro-region, it was localized intracellularly and lacked
activity, indicating that the pro-region was required for the correct
folding of the enzyme. The activation of proyapsin 1 in
vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-mating factor and
pro-killer toxin at specific basic residue cleavage sites (3-5).
However, in Kex2p-deficient mutants, two genes encoding aspartic
proteases, YAP3 and MKC7, have been cloned.
Yapsin 1 (previously named Yap3p) was able to suppress the processing
defect of pro-
-mating factor (6), and Mkc7p (now named yapsin 2) was
able to suppress the cold-sensitive phenotype (7) observed in these
mutant cells. Yapsin 1 and yapsin 2 are glycophosphatidylinositol
(GPI)1 membrane-anchored
proteins (7-9) and share 53% amino acid identity (7). Both enzymes
have specificity for basic amino acid residues and appear to have a
common substrate pool with that of Kex2p in vivo. Such
properties allow these enzymes to be characterized as members of a
sub-class of aspartic proteases involved in proprotein processing which
demonstrate a unique specificity for arginine and/or lysine residue
cleavage sites. This is in contrast to other aspartic proteases whose
specificity is for hydrophobic residues (10, 11). This sub-class of
aspartic proteases is now named the yapsin family due to its partial
homology with pepsin (12, 13).
1 s
1 for the
B-C junction of bovine proinsulin and 3.1 × 106
M
1 s
1 for human
adrenocorticotropin (ACTH1-39) (15), demonstrating an
enhancement of the efficiency by additional basic residues upstream and
especially downstream of the cleavage site (15, 16).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-mercaptoethanol, and stained with Coomassie Blue. A
similar gel was run in parallel but the proteins (6 µg) were analyzed
by amino-terminal amino acid sequencing after they had been transferred
to polyvinylidene difluoride membrane (Trans-Blot, Bio-Rad), in a
Tris/glycine buffer containing 20% methanol. The proteins were
visualized by staining with 0.2% Ponceau S in 1% acetic acid and
destaining in water. Direct amino-terminal amino acid sequencing of the
bands was carried out by Edman degradation using a Perkin-Elmer/Applied
Biosystems model 494A Procise 228 Protein Sequencer.
-Lactoglobulin
was used to determine the sequencing efficiency.
Glu101).
Nucleotide sequences of primers used in the construction of the various
YAP3 constructs
The strain JHRY20-2C-yap3::LEU2 (Mat a,
his3-
200, leu2-3-112,
yap3::LEU2) (19, 20) was
transformed with either the pYAP3LC (8) or
pYAP3LC(Asp101
Glu101)
using standard procedures (21). Growth of the yeast and expression of
the enzymes were performed as described before (8). Aliquots of the
media (0.07 µg of protein) from each clone were analyzed by Western
blot with two different antisera. Anti-yapsin 1 serum, MW283,
previously characterized (8), and antiserum, VO2377, raised against a
synthetic peptide corresponding to amino acids Tyr47-Glu61 within the yapsin 1 propeptide.
Endoglycosidase H (Sigma) treatment of the enzymes was performed prior
to the Western blots in order that a clearer size comparison could be
made between the two forms of the enzyme. The media (1.1 µg of
protein) was also assayed for yapsin 1 enzymatic activity by the
standard ACTH1-39 assay (15).
Expression and Analysis of Proyapsin 1 from Baculovirus
Proyapsin 1 and yapsin 1(proyapsin 1) were engineered for
expression in the baculovirus expression system. Recombinant
YAP3 gene constructs, encoding amino acids 22-532
(proyapsin 1) and 68-532 (
proyapsin 1), were engineered by PCR
using the primer pairs 1/3 and 2/3, respectively (see Table I for
primer sequences), and the previously characterized expression vector,
pYAP3LC (8) as template. Both PCR fragments containing a
BamHI and a PstI restriction site at their 5
and
3
ends, respectively, were subcloned into the pAcGP67 baculovirus
transfer vector downstream of the strong signal peptide of the acidic
glycoprotein, gp67. The pAcGP67 plasmids containing the YAP3
inserts were cotransfected with the BaculoGold viral DNA (Pharmingen,
San Diego, CA) into Spodoptera frugiperda (Sf9)
cells. Recombinant viral particles were identified by plaque assay and
harvested for high titer stock generation. For the expression of the
recombinant proteins, Sf9 cells were grown in an
80-cm2 canted neck tissue flask at 27 °C with 18 ml of
serum-free medium (Sf-900 II SFM, Life Technologies, Inc.) supplemented
with penicillin and streptomycin. After reaching 80% confluency, the
cells were infected with 18 ml of fresh media containing 2 ml of
purified recombinant baculovirus (1.1×108 pfu/ml). Two
days postinfection, the cells and media were collected for analysis by
Western blot and by the yapsin 1 activity assay. The media were
concentrated and partially purified by concanavalin A (ConA) affinity
chromatography (22) prior to analysis, and the soluble cell extracts
were analyzed without manipulation except for an aliquot of the
proyapsin 1 sample which was treated with and without
N-glycanase, according to the company's guidelines (Genzyme, Cambridge, MA) and analyzed by Western blot.
Other experiments described below utilized the culture supernatant,
from multiple flasks of cells expressing proyapsin 1, that was
concentrated by centrifugation through a Macrosep 10-kDa omega membrane
and frozen at 20 °C until analysis. A fresh preparation of a
protease inhibitor mixture (CompleteTM, Boeringer Mannheim,
Germany) containing EDTA but not pepstatin A was added prior to the
experiments to minimize nonspecific degradation during the
procedures.
pH-dependent Activation of Proyapsin 1-- The effect of pH on the activation of proyapsin 1 was investigated. Five µl of the proyapsin 1 sample were incubated in 20 µl of 0.1 M sodium citrate or sodium citrate/sodium phosphate buffers with pH values ranging from pH 2.6 to 7.2 for 20 h at 30 °C. Five µl from each incubate were adjusted to pH 4.3 and assayed for yapsin 1 activity by the ACTH1-39 assay which was carried out at 30 °C for 30 min.
Time Course Studies on the Activation of Proyapsin 1--
The
time course of ACTH1-39 cleavage was investigated to
determine the profile of proyapsin 1 activation. An aliquot (5-45
µl) of the proyapsin 1 was incubated in 900 µl of 0.1 M
sodium citrate/sodium phosphate buffer, pH 4.3, at 30 °C, in the
presence of ACTH1-39 (22 µM). At regular
intervals, 100-µl aliquots were removed, and the presence of
ACTH1-15, as a specific indicator of yapsin 1 activity,
was quantitated by HPLC. Note that the lower amounts of proyapsin 1 were used so that sufficient substrate remained when the longer
incubations were performed. It was calculated that 70% of the
substrate remained at the end of the time courses. The amount of
ACTH1-15 that was generated was then normalized to 5 µl
of the proyapsin 1/100-µl reaction.
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RESULTS |
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Purification and Analysis of Yapsin 1 from Yeast
Yapsin 1 was purified from the culture supernatant of the yeast
expression system yielding ~1 µg of yapsin 1/g of wet yeast. The
purification procedure resulted in an overall recovery of 29.7% of
yapsin 1 from the starting media with an overall fold purification of
43.6 (Table II). Anion exchange
chromatography of the concentrated culture supernatant on the MonoQ1
column resulted in the step with the highest fold purification of 8.7 and a recovery of ~64%. Yapsin 1 eluted from the column in 100-150
mM NaCl (Fig. 1A).
Application of the MonoQ1 pool of activity on the Superdex G-200 column
resulted in a bimodal distribution of yapsin 1 activity (Fig.
1B) which was attributed to the two hyperglycosylated forms of secreted yapsin 1, a ~150-180-kDa and a ~90-kDa form that have previously been characterized (8). Fractions 54-63, corresponding to
the ~150-180-kDa molecular mass form of yapsin 1, were pooled for
the next purification step resulting in a ~57% recovery of activity
in the G-200 step. The inclusion of a second MonoQ column at this time
was necessary to remove a contaminating protein, identified by amino
acid sequencing as glucan-1,3--glucosidase (Fig. 2,
lanes 2 and 3), that appeared to be co-purifying
with yapsin 1. The shallow NaCl gradient used to elute the protein from
the column generated a broad peak of yapsin 1 activity (data not
shown); however, only the first half of the peak (fractions 52-57) was
pooled, to avoid the glucosidase, and referred to as purified yapsin
1.
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Silver stain analysis demonstrated the apparent homogeneity of the
yapsin 1 preparation by staining only one protein (Fig. 2, lane
1), and amino-terminal amino acid sequence analysis resulted in
two yapsin 1 sequences as follows: sequence 1, A68DGYEEIIITNQQSF; sequence 2, D145INPFGWL(T)GTGSAI. The internal sequence of yapsin
1, starting at Asp145, has been obtained on four different
occasions, each with a different enzyme preparation, rendering this
cleavage consistent and specific. When the picomole recovery of amino
acids from each cycle of the sequencer was plotted, a least squared
linear regression fit allowed an initial concentration of each sequence
to be calculated by extrapolation. The average relative ratio of the
two sequences was 1.22:1 for sequence 1 relative to sequence 2, demonstrating that ~90% of the yapsin 1 molecules has been processed
into two subunits, and
. To determine if the two yapsin 1 subunits were associated by a disulfide bond, a sample of a partially
purified preparation of yapsin 1 was analyzed by SDS-PAGE under
nonreducing and reducing conditions followed by Coomassie Blue staining
and amino-terminal amino acid sequencing. Coomassie Blue staining of
1.6 µg of unreduced protein from the Superdex G-200 pool of yapsin 1 activity that had been deglycosylated by endoglycosidase H showed a
major protein between the 66- and 97-kDa molecular mass standards
(Mark12, Novex, San Diego, CA) and a minor protein at ~31
kDa (Fig. 2, lane 2). Amino-terminal amino acid sequencing
of the upper band gave a similar relative picomole ratio of the
-
and
-subunit sequences of yapsin 1 as the purified preparation
described above, whereas the lower band contained only yeast
glucan-1,3-
-glucosidase. When a similar aliquot of the protein was
run in the presence of
-mercaptoethanol, a reduction in the
molecular mass of the yapsin 1 band to ~65 kDa was observed
concomitant with the appearance of a diffuse protein at ~34 kDa (Fig.
2, lane 3). Amino-terminal amino acid sequencing of the
upper band resulted in the
-subunit sequence of yapsin 1, whereas
the lower band resulted in the
-subunit sequence of yapsin 1 in
addition to the glucosidase sequence.
Analysis of an Active Site Mutant of Yapsin 1 Expressed in
Yapsin 1 Yeast
It was demonstrated by Western blot using antiserum MW283 that
yapsin 1 and yapsin 1(Asp101 Glu101) were
successfully expressed and secreted at similar levels in the
yapsin
1 yeast strain (Fig. 3A, lanes
3 and 4). Yapsin 1 was ~5 kDa smaller than the active
site mutant. No enzymatic activity was present from the culture
supernatant of the mutant yapsin 1, although abundant activity was
observed with the normal yapsin 1 (Fig. 3B). Western blot
analysis using antiserum VO2377, which is specific for the propeptide
of yapsin 1, showed strong immunostaining of yapsin
1(Asp101
Glu101) (Fig. 3A, lane
1), whereas only trace amounts were evident in the normal yapsin 1 sample (Fig. 3A, lane 2).
|
Analysis of Baculovirus-expressed Yapsin 1 Recombinants
Western blot analysis of ConA-purified media (1.6 µg of protein)
from Sf9 cells expressing proyapsin 1 and proyapsin 1 demonstrated that proyapsin 1 was secreted into the growth media as a
~60-kDa protein (Fig. 4A, lane
1), whereas the
proyapsin 1 was not secreted (Fig. 4A,
lane 2). Western blot analysis of the corresponding cell extracts
(3.8 and 4.3 µg of protein) showed that proyapsin 1 was present
as a ~75-kDa protein and
proyapsin 1 was present as a ~70-kDa
protein (Fig. 4A, lanes 3 and 4). Furthermore,
proyapsin 1 shifted to a lower molecular mass upon treatment by
N-glycanase indicating the presence of N-linked
sugars on
proyapsin 1 (data not shown).
|
The ConA-purified media and the soluble cell extracts (1.3 and ~12 µg of protein, respectively) were further analyzed by the ACTH1-39 activity assay with and without a pre-activation step at pH 4.3 and 37 °C. The ConA-purified media were pre-activated for 12 h, and the soluble cell extracts were pre-activated for 2 h. Yapsin 1 enzymatic activity was found only in the proyapsin 1 samples yielding a ~31-fold increase in yapsin 1 activity from the ConA-purified media and a 5.6-fold increase in yapsin 1 activity from the soluble cell extract as a result of the pre-activations (Table III).
|
pH-dependent Activation of Proyapsin 1-- After incubation of 5 µl of proyapsin 1 for 20 h at various pH values, an aliquot of each incubate was assayed for yapsin 1 activity. A plot of nanogram of ACTH1-15 generated versus preincubation pH demonstrated a pH-dependent generation of yapsin 1 activity (Fig. 5). Activity was generated at acidic pH values between 3.0 and 5.0 with an optimum at pH 4.0. The samples that were incubated below pH 3.0 were unable to be activated by a subsequent incubation at pH 4.3 indicating that the enzyme had been irreversibly denatured. However, the samples that were incubated above pH 5.0 were able to be activated by a subsequent incubation at pH 4.3 indicating that the enzyme had not been denatured (data not shown).
|
Time Course Studies on the Activation of Proyapsin 1-- When proyapsin 1 was incubated at 30 °C and pH 4.3 in the presence of ACTH1-39 for various time points, the activity profile obtained demonstrated an initial lag in the generation of ACTH1-15 after which the product was generated in an apparent linear fashion (Fig. 6A). In reality, however, the mechanism governing the generation of product is a function of two processes, i.e. the activation of the enzyme and the activity of the enzyme, and should be described by nonlinear second-order kinetics. The observed apparent linearity is due to the fact that the rate of activation of the proyapsin 1 is significantly lower than the rate of ACTH1-39 hydrolysis. In such a case, the second-order process would appear as a single-order process.
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DISCUSSION |
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Our previous work demonstrated that recombinant yapsin 1, devoid of its GPI membrane-anchoring domain, was efficiently overexpressed and secreted from a yeast expression system. These forms of yapsin 1 were found to be hyperglycosylated and stable to lyophilization and long term storage under a variety of conditions. In contrast to the yapsin 1 obtained from the cellular extract of the same expression system (14), the stability of the secreted enzyme rendered it useful for kinetic studies since the specific activity remained constant for long periods. In addition, the abundance of yapsin 1 activity in the culture supernatant as compared with the cell extract (8) represented a source of enzyme in quantities potentially sufficient for structural analysis. Yapsin 1 was purified to apparent homogeneity from the culture supernatant yielding ~1 µg of yapsin 1/g of wet yeast. The procedure utilized anion exchange chromatography (Fig. 1A) based on the finding that the isoelectric point (pI) of yapsin 1 was ~4.5 (8), thus rendering this enzyme negatively charged at pH 7.0. Gel filtration chromatography was also used (Fig. 1B), since secreted yapsin 1 was previously determined to be hyperglycosylated, primarily giving a molecular mass of ~150-180 kDa (8, 9). A combination of these procedures was used with an overall recovery of 29.7% and a fold purification of 43.6 (Table II). The unexpected increase in total activity observed in the concentration step (168%, Table II) was initially thought to be from the removal of a low molecular mass inhibitor; however, upon addition of the filtrate back to the retentate, no inhibition of the activity was observed (data not shown). The presence of a small amount of proyapsin 1 in the media (Fig. 3A, lane 2) that became activated during the initial stages of the concentration step may in part account for the increase. The major increase, however, is most likely due to compounded small errors in the pipetting introduced during the many dilutions of up to 1 × 104 which were required to obtain quantifiable yapsin 1 activity in the highly sensitive ACTH1-39 assay for yapsin 1 (picogram range). This was confirmed when the procedure was repeated on a smaller scale where such large dilutions were not necessary. In that instance the recovery of total yapsin 1 activity in the retentate was ~95% of the starting material.
Purity of the final preparation of enzyme was confirmed by silver stain
(Fig. 2, lane 1) and amino-terminal amino acid analysis. As
expected, the mature enzyme was shown to start at Ala68
indicating that the proregion had been removed, but surprisingly, an
additional internal sequence was obtained. Processing in this region
resulted in the generation of two subunits designated here as - and
-subunits each with one of the active site triads (Fig. 8D). The amino-terminal amino
acid of the
-subunit, identified as Asp145, is situated
within a unique loop domain of yapsin 1 arising from a large insertion
of ~76 residues in comparison with pepsin, rendering this site
susceptible to cleavage by proteases. Whether the loop gets cleaved at
this residue only or amino-terminally to this residue followed by
aminopeptidase trimming is still unknown. This loop domain is also
found in yapsin 2 and may represent an important domain of the yapsins
in general. Similar processing of cathepsin D has also been observed in
a species- and tissue-specific manner where both active site triads are
separated into two subunits by cleavage at a site within a 9-residue
insertion (23). Processing into subunits appears to be a rare event for
mammalian aspartic proteases, with cathepsin D as one exception, but it
has been documented for Aspergillus niger proteinase A (24)
and a barley aspartic protease (25). However, until now, association of
these subunits was found to be noncovalent. We have shown here that the
yapsin 1 subunits are associated by a disulfide bond (Fig. 2,
lanes 2 and 3), and by comparison with the known
structure of pepsin (26), the disulfide bond is most likely between
Cys117 of the
-subunit and Cys186 of the
-subunit. The relevance of such a processing is unknown; however,
preliminary pulse-chase
experiments3 have indicated
that it occurs early in the yeast secretory pathway and may represent a
key event in the structure/function relationship of yapsin 1 in
vivo.
|
The amino-terminal amino acid of the -subunit, identified as
Ala68, indicated that the proregion was removed at the
paired basic residue site, Lys66-Arg67, a site
similar to that in the activation of pro-renin to renin (27, 28). Since
this propeptide processing site represents an efficient motif for
yapsin 1 cleavage, RXXKR (15), it suggested that processing
of the propeptide may occur by autoactivation in contrast to renin
which relies on a trypsin-like activity, although active renin has been
reported to contain some intrinsic activity capable of activating
prorenin (29). We investigated this hypothesis by expressing an active
site mutant of yapsin 1 and demonstrated that the inactive enzyme was
~5 kDa bigger than the normal active yapsin 1 (Fig. 3, lanes
3 and 4). This expected difference in size corresponded
to the theoretical molecular mass of the propeptide, the presence of
which was confirmed by Western blot (Fig. 3, lane 1) and is
consistent with the inability of the active site mutated yapsin 1 to
remove its own propeptide. Also, since both constructs were expressed
in yapsin 1-deficient yeast strains, we can also conclude that the two
other known endogenous processing enzymes with specificity for this
type of cleavage site, Kex2p and Mkc7p (yapsin 2), were not able to
process the mutant proyapsin 1 either. It appears, therefore, that
removal of the propeptide is dependent upon active yapsin 1 molecules.
To study further the activation of yapsin 1 in vitro, yapsin
1 was expressed in the baculovirus/Sf9 system, with and without its proregion (proyapsin 1 and proyapsin 1, respectively), and analyzed with respect to its properties. Both recombinant proteins were
expressed in this system; however, only proyapsin 1 was secreted (Fig.
4, lane 1). Absence of the proregion resulted in the
expression of an intracellularly localized protein (Fig. 4, lane
4) that contained no apparent activity even when pre-activated
(Table III). These observations are consistent with a misfolded protein that was transport-incompetent. However, that it had entered the secretory pathway is supported by the result that it was
N-glycanase-sensitive (data not shown). In contrast to
proyapsin 1, proyapsin 1 was secreted from the cells and possessed
significant levels of activable yapsin 1 activity (Table III), showing
that the proregion was required for the correct folding and secretion
of yapsin 1, and only as a correctly folded enzyme was it able to be
activated. This activation process was characterized further and shown
to occur optimally at pH 4.0 with <10% activated in the pH range of
5.5-6.0 and no apparent activation at the more neutral pH values (Fig.
5). In addition, the enzyme was irreversibly denatured below pH 3.0 (data not shown), a result that had been observed previously (30). The
observed progressions of product formation in both the activity profile
experiment (Fig. 6A) and the pre-activation experiment (Fig.
6B) indicated that the mechanism of activation was
intra-molecular, a result that was further supported by the result that
purified yapsin 1 from yeast was unable to activate the proyapsin 1 (Fig. 6B, inset). Previous reports by us (8) and others (9)
have deduced that yapsin 1 is located to the extracellular side of the
plasma membrane via its GPI membrane anchor and is presumed to be its
site of action. Since the pH of yeast growth media is acidic (pH
5.0-6.0) and becomes more acidic with growth, the periplasmic space
may represent the primary compartment where autoactivation occurs.
However, yapsin 1 was first cloned based on its ability to process the
yeast prohormone, pro-
-mating factor (6), in the secretory pathway
of KEX2-deficient yeast. Since the synthesis of biologically
active
-mating factor requires the subsequent action of two other
Golgi resident enzymes, Kex1p (31), a carboxypeptidase B-like enzyme
and a dipeptidyl-aminopeptidase (32), it is assumed that a sufficient
amount of proyapsin 1 must become activated in the late Golgi in order
for mature
-factor to be generated. This is supported by the fact
that proyapsin 1 was expressed and functionally active in mammalian
PC12 cells (17) where the pH ranges from pH 6.2 in the trans-Golgi
network (33) to pH 5.0-5.5 in mature secretory vesicles (34).
Investigation of the biosynthesis and maturation of aspartic proteases such as endothiapepsin (35), cathepsin D (36), pepsin (37), Rhizopus niveus aspartic proteinase-I (38), and yeast proteinase A (39, 40) have all demonstrated the importance of their proregion in the folding of the enzyme and the regulation of their activity. For pepsin, the model aspartic protease, its proregion, which contains 11 basic residues, is partially stabilized by ionic interactions with the negatively charged aspartic acid residues of the active site (41). Upon acidification, the aspartic acid residues become protonated and less charged (pKa = 4.3) causing destabilization and an intramolecular cleavage at Leu16-Ile17 within the propeptide resulting in the generation of an intermediate form of the enzyme or pseudo-pepsin. The intermediate form undergoes a further intermolecular or intramolecular cleavage of the remainder of the proregion to generate the mature active enzyme. It is interesting to note the similarity to yapsin 1 in this respect in that a potential cleavage site exists at Lys16-Phe17 within the propeptide. Because of this structural similarity and that cleavage of a mono-lysine residue by yapsin 1 has previously been documented (15), it is predicted that a yapsin 1 intermediate, formed from an intramolecular cleavage presumably at this site, exists. Our activation studies support this prediction, since during the time course experiment, an initial lag phase (within 3 h) in the generation of yapsin 1 activity was observed (Fig. 6A). This observation is consistent with a molecular event that involves the generation of an intermediate, a process that has been documented for other aspartic proteases (36, 37, 42). Further support came from the pre-activation experiment where the immunoreactive proyapsin 1 was converted to a slightly smaller protein upon activation (Fig. 7A). Since this band still contained the propeptide antigenic site for antiserum VO2377, i.e. Tyr47-Glu61 (Fig. 7A, lane 4), the size shift is consistent with removal of an amino-terminal portion of the proregion and the assignment of this band as an intermediate, i.e. pseudo-yapsin 1. Although trimming at the carboxyl terminus cannot be completely ruled out, the use of serum-free medium, a 2-day infection to limit cell lysis and the addition of protease inhibitors, renders the presence of potential proteases specific for the carboxyl terminus of proyapsin 1 highly unlikely. Furthermore, the inability of 6 µM pepstatin A to inhibit formation of pseudo-yapsin 1 (data not shown) indicates that another aspartic protease was not responsible for the processing of proyapsin 1 and provides further evidence that the process was intra-molecular since the active site, already tightly bound to the proregion, was inaccessible to the inhibitor.
Although the generation of pseudo-yapsin 1 appears to be fairly rapid,
within 3 h as evidenced by the lag in Fig. 6A and a time course study (data not shown), the generation of yapsin 1 from
pseudo-yapsin 1 appeared to be extremely slow. The concentration of
proyapsin 1 in the starting material was estimated by Western blot from
a standard curve made with purified yapsin 1 from yeast (data not
shown) and based on 100% conversion of this amount, the amount of
enzymatic activity obtained was found to be <1% of that expected.
Such inefficient activation explains why no mature processed yapsin 1 was detectable in the Western blot in the activated sample (Fig. 7).
Attempts at increasing the efficiency with either calcium or
dithiothreitol had no effect on the rate (data not shown) making it
apparent that either some other co-factor is necessary or that some
structural requirement has not been met by the baculovirus-expressed
proyapsin 1 for activation to occur efficiently. The most obvious
structural difference of proyapsin 1 expressed in Sf9 cells is
its lack of processing into - and
-subunits. We speculate that
the lack of this processing may result in conformational restraints on
pseudo-yapsin 1 to allow efficient removal of the remainder of the
proregion at Lys66-Arg67 and may be the reason
why pseudo-yapsin 1 appears to get stuck in that form. Other
pseudo-aspartic proteases, such as pseudo-chymosin and pseudo-cathepsin
D (42, 43) under certain conditions, have also been found to be poorly
converted.
In conclusion, our studies with yeast have demonstrated that mature yapsin 1 is a heterodimer stabilized by an intramolecular disulfide bond, and the final proregion cleavage site, which was yapsin 1-dependent, was confirmed to occur at the Lys66-Arg67 (Fig. 8D). From our baculovirus expression studies, the proregion of yapsin 1 was required for the formation of intact, single-chain proyapsin 1 which was then capable of being self-activated by incubation at acidic pH. The in vitro activation process resulted in the generation of pseudo-yapsin 1 (Fig. 8B) which appeared to be converted to mature yapsin 1 at an extremely slow rate by an apparent intramolecular mechanism (Fig. 8C). It is hypothesized that the slow rate of in vitro activation may be a result of the difference between single-chain proyapsin 1 and two-chain proyapsin 1 found in yeast. The enzyme(s) responsible for the processing of yapsin 1 into the subunits may ultimately play an important role in the regulation of yapsin 1 activity in vivo and is currently under investigation.
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ACKNOWLEDGEMENT |
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We thank Dr. Ying Zhang for expert help in the baculovirus expression of the yapsin 1 constructs.
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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.
§ To whom correspondence should be addressed: Bldg. 49, Rm. 5A38, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-8222; Fax: 301-496-9938; E-mail: cawley{at}codon.nih.gov.
1 The abbreviations used are: GPI, glycophosphatidylinositol; YAP3, yeast aspartic protease 3; ACTH, adrenocorticotropin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; ConA, concanavalin A; HPLC, high pressure liquid chromatography.
2 N. X. Cawley, V. Olsen, and Y. P. Loh, unpublished observations.
3 V. Olsen, N. X. Cawley, and Y. P. Loh, manuscript in preparation.
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REFERENCES |
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