(Received for publication, May 4, 1995; and in revised form, June 9, 1995)
From the
The substrate specificities of two human prohormone convertases,
furin and PC1, were examined with a series of
7-amino-4-methylcoumarinamide (MCA) containing peptidyl substrates.
Using acetyl-Arg-Ser-Lys-Arg-MCA as model, P4 Arg substitution by Lys
or Orn resulted for furin in a 538- and a 280-fold lower k/K
value, but
only in a 14- and 18-fold decrease for PC1. Substitution of P3 Ser by
either Pro, Glu, or Lys does not modify significantly the k
/K
value for PC1,
whereas furin activity is seriously impaired by the Glu substitution.
Elongating the peptidyl sequence up to the P8 position decreases the k
/K
value for
furin but not for PC1. In both the P3 or P5 Glu substitution, the
decrease of k
/K
was due primarily to lower k
rather
than higher K
, possibly because of the
presence of a negatively charged side chain. Finally, an octapeptidyl
chloromethane derivative proved to be a potent irreversible inhibitor
of either PC1 and furin. The 811-fold difference in the apparent K
/[I] (1.63
10
s
M
), and k
/K
determined
with the corresponding peptidyl MCA substrate (2.01
10
s
M
), supports the
proposal that cleavage of the acylenzyme represents the rate-limiting
step for PC1 and furin.
Recently, a number of convertases were discovered which share a
common role in carrying out processing of protein and prohormone
precursors at distinct single or pairs of basic residues. So far, six
mammalian processing enzymes are known which exhibit significant
functional and structural similarities to both yeast kexin and
bacterial subtilisins. These subtilisin/kexin-like convertases
(reviewed in (1, 2, 3, 4) ) are
called furin/PACE, ()PC1/PC3, PC2, PACE4, PC4 and PC5/PC6.
Apart from their structural relatedness to kexin, further evidence in
favor of their roles in precursor processing in either the constitutive
or the regulated pathway of secretion was deduced from cellular
co-expression and localization studies. Thus, for example,
overexpression of PC1 or PC2 together with proopiomelanocortin in
mammalian cells led to specific processing of this precursor at the
COOH-terminal side of pairs of basic residues(5) . Similarly,
furin when expressed in both endocrine and non-endocrine cell types can
also process various precursors at sites characterized by the presence
of an Arg-Xaa-Lys/Arg-Arg sequence(6, 7) . In fact,
the number of potential substrates recognized by furin is expanding
rapidly to include peptide growth factors such as
-nerve growth
factor (8) and viral envelope glycoproteins such as
hemagglutinin of influenza virus(9) . Analysis of the
poly(A)
mRNAs of the convertases in rat tissues and
various cell lines by Northern blot analysis demonstrated a unique
pattern for each enzyme(4) . Thus, although furin, PACE4, and
PC5 mRNAs exhibit a widespread tissue distribution, only furin is
ubiquitously expressed. In contrast, Northern and in situ hybridization analysis of PC1 and PC2 indicate that they are
primarily expressed in tissues and cells containing secretory granules.
Finally, PC4 mRNA is only expressed in germ cells of the testis and
ovary.
The cellular co-expression of enzyme and substrate was extremely revealing in terms of intracellular activities of the convertases and their cleavage specificities. However, the limiting level of some intracellular convertases, such as furin, suggests that overexpression systems might not be the physiologically relevant way to analyze the functioning of a given convertase(10) . Therefore, in order to better understand the physiological role as well as the molecular characteristics of these enzymes, an in vitro analysis is mandatory. For this purpose, the use of heterologous expression systems offered a way to produce these enzymes at levels above that of endogenous contaminating enzymes. Until now, only few reports are available regarding the substrate specificities of recombinant furin or recombinant PC1 using in vitro experiments. Those include substrate precursors such as hproinsulin(11) , anthrax-protective antigen(12) , and human immunodeficiency virus envelope glycoprotein gp160 (13) and/or smaller peptidyl substrates(14, 15, 16) . Interestingly, as we and others have demonstrated previously, both recombinant furin and PC1 prefer substrates with an Arg residue occupying the P4 position relative to the cleavage site characterized by the presence of a pair of basic residues such as in Arg-Xaa-Lys/Arg-Arg or of a single basic residue such as in Arg-Xaa-Xaa-Arg.
The present comparative study of
both human furin and PC1 using fluorogenic substrates aims at
describing more precisely the biochemical basis behind their apparent
overlapping substrate specificities. The preparation as well as the
comparison of the cleavage efficiency as determined by measuring k/K
of several
7-amino-4-methylcoumarinamide derivatives are reported. Finally, based
upon this study, a peptidyl chloromethane derivative was prepared and
shown to be a very potent irreversible inhibitor for both PC1 and
furin, useful for specific labeling and titration purposes.
The crude peptidyl derivatives were
purified by RP-HPLC as described previously(17) . TLC was
performed on silica gel precoated (0.2 mm thickness) aluminium sheets
(Kiesel gel, Merck Co.). The following solvent systems were used for
ascending TLC (v/v): A, CHCl/MeOH (24:1); B,
CHCl
/MeOH (2:1); C, CHCl
/MeOH (12:1); D, n-BuOH/HOAc/H
O/pyridine (15:3:12:10); and E,
CHCl
/MeOH (10:7). The spots were revealed by UV
illumination or by spraying with molybdate/sulfuric acid or dilute
ninhydrin.
The two human endoproteases, prohormone convertase 1
(hPC1) and hfurin, were obtained from the medium of somatomammotroph
GHC
cells following infection with the
respective recombinant vaccinia virus as described(15) . The
two proteases were partially purified through anion exchange
chromatography prior to use and their enzymatic activities determined
using a fluorometric assay as described previously(15) . One
unit of enzymatic activity is defined as the amount of enzyme necessary
to release 1 pmol of AMC from the fluorogenic substrate
acetyl-Arg-Ser-Lys-Arg-MCA per minute.
Figure 1: Synthetic scheme for the preparation of acetyl-Arg-Ser-Lys-Arg-MCA (II). A, synthesis of the fluorogenic amide (I). Part B: Synthesis of the fully protected tripeptide (IIa). Part C: Condensation of peptide segments and final deprotection of the peptidyl-substrate (IIb).
The
acetyl-Arg(Pmc)-Ser(tBu)-Lys(Boc)-Arg(Pmc)-MCA (IIb) was
obtained by condensing Ia and IIa using the activating
reagent TBTU(19) . Compound I (74.1 mg; 90.5 mmol) was
added to a solution of 20% piperidine, dimethylacetamide. The reaction
mixture was concentrated under reduced pressure and the residue washed
with dimethylacetamide several times. The free amine Ia was then
added to a solution of compound IIa (75 mg; 90.4 mmol),
triethylamine (91 mg; 87.8 mmol) and TBTU (29 mg; 90.4 mmol) in 3 ml of
dimethylacetamide, and the resulting mixture was stirred at ambient
temperature. After 30 min, TLC analysis showed a major compound; R(C): 0.42. The reaction mixture was
precipitated using an ice-cold acid solution (pH 3; HCl). The resulting
precipitate was filtered and dried in vacuo. Purification
through preparative TLC plates using solvent system C afforded compound IIb (60-65% yield based on weight). FAB-MS: m/z 1434 (M + H)
. Its
H NMR spectrum
was fully consistent with its structure.
The final deprotection of
the peptidyl-MCA derivative IIb was performed by reacting with
reagent K (20) for 4 h at room temperature. After appropriate
dilution with water and lyophilization, the residue was repetitively
washed with ether to remove the excess of scavengers. The material was
finally purified by RP-HPLC and eluted at 31.7 min (32%
CHCN). FAB-MS: m/z 745 (M +
H)
. Amino acid analysis: Ser
(1),
Lys
(1), Arg
(2). The
H NMR
spectrum was fully consistent with its structure.
For
acetyl-Ser-Lys-Arg-MCA (III): FAB-MS, m/z 589
(M+H); RP-HPLC, R
= 29.05 min (29% CH
CN). Amino acid
analysis: Ser
(1), Lys
(1),
Arg
(1).
For acetyl-Arg-Glu-Lys-Arg-MCA (IV):
FAB-MS, m/z 787 (M + H); RP-HPLC, R
= 29.05 min (29%
CH
CN). Amino acid analysis: Glu
(1),
Lys
(1), Arg
(2).
For
acetyl-Arg-Lys-Lys-Arg-MCA (V): FAB-MS: m/z 786 (M +
H). RP-HPLC: R
= 30.95 min (31%
CH
CN). Amino acid analysis: Lys
(2),
Arg
(2).
For acetyl-Arg-Pro-Lys-Arg-MCA (VI):
FAB-MS, m/z 755 (M + H); RP-HPLC, R
= 30.95 min (31%
CH
CN). Amino acid analysis: Pro
(1),
Lys
(1), Arg
(2).
For
acetyl-Arg-Phe-Ala-Arg-MCA (VII): FAB-MS, m/z 748 (M
+ H); RP-HPLC, R
= 38.95 min (39% CH
CN). Amino acid
analysis: Ala
(1), Phe
(1),
Arg
(2).
For acetyl-Lys-Ser-Lys-Arg-MCA (VIII):
FAB-MS, m/z 717 (M + H). RP-HPLC, R
= 29.75 min (30%
CH
CN). Amino acid analysis: Ser
(1),
Lys
(2), Arg
(1).
For
acetyl-Orn-Ser-Lys-Arg-MCA (IX): FAB-MS, m/z 703 (M
+ H); RP-HPLC, R
= 31.75 min (32% CH
CN). Amino acid
analysis: Ser
(1), (Orn + Lys)
(1),
Arg
(1).
For
acetyl-Tyr-Glu-Lys-Glu-Arg-Ser-Lys-Arg-MCA (X): FAB-MS, m/z 1294 (M + H). RP-HPLC, R
= 31.70 min (32% CH
CN). Amino acid
analysis: Ser
(1), Glu
(2),
Tyr
(1), Lys
(2), Arg
(2).
Figure 2:
Time-dependence of inhibition of hPC1
enzymatic activity toward acetyl-Arg-Ser-Lys-Arg-MCA by the
octapeptidyl chloromethane inhibitor. The progressive development of
inhibition produced by the reaction of hPC1 with the octapeptidyl
chloromethane inhibitor is plotted as a semilogarithmic curve in
accordance with Kitz and Wilson's representation(24) .
The enclosed graph represents the graphical determination of the
pseudo-first-order inactivation rate constant (K) derived
from the plot of ln v/v
versus time.
Figure 3:
Active site-titration and affinity
labeling of hPC1 and hfurin with the octapeptidyl chloromethane
inhibitor. Panel A: active site titration of hPC1 using the
procedure as described in Materials and Methods. Panel B:
RP-HPLC chromatogram of the radioiodinated octapeptidyl chloromethane
inhibitor used as labeling agent for both hPC1 and hfurin. Elution
conditions are described under Materials and Methods. The arrow
indicates the elution position of the Tyr-containing unlabeled peptide
as identified by amino acid analysis. Panel C: affinity
labeling of hPC1 (10.2U) and hfurin (6.9U) obtained from the medium of
recombinant vaccinia virus infected GHC
cells.
Aliquots of each medium were incubated in the presence of 10 mM EDTA (lane A) or 1 mM CaCl
(lane B) at pH 7.0
for hfurin or in the presence of 10 mM EDTA (lane D) or 5
mM CaCl
(lane C) at pH 6.0 for hPC1 with 2.4
10
cpm of the radiolabeled octapeptidyl
chloromethylketone at 37 °C for 90 min. Each sample was then boiled
in Laemli sample buffer and 1/3 was loaded on a 6% SDS-polyacrylamide
gel and electrophoresed. The gel was then dried and the radioactive
bands revealed by radiography.
The use of the radioiodinated inhibitor (Fig. 3B) resulted in the labeling of three molecular weight forms of hPC1, as shown in Fig. 3C, corresponding to 85, 74, and 66 kDa. The labeling of these three forms is found to be calcium-dependent as it is blocked by addition of 10 mM EDTA and represents, so far, the first example of an active site labeling agent for hPC1. The same set of experiments, done in parallel with the secreted shed form of hfurin, led to a calcium-dependent labeling of a molecular form corresponding to the predicted molecular mass form of 80 kDa (Fig. 3C).
The data of Table 1and Table 2reveal
substantially reduced values of k, when measured
with pGlu-Arg-Thr-Lys-Arg-MCA, for both hPC1 and hfurin as compared
with those obtained with the yeast-related enzyme kexin (21-45
s
) with similar peptidyl-MCA
substrates(28) . One possible explanation could be related to
the bulkiness of the MCA moiety rendering it a poor leaving group in
the case of the mammalian prohormone convertases. In agreement, we
recently demonstrated that substitution at the P`1 position by a
sterically hindered group can lead to a competitive
inhibitor(16) . Alternatively, these mammalian enzymes may
require more extensive interactions with the substrate brought about by
(i) stabilization of the substrate by elongation in the COOH-terminal
direction or (ii) the presence of an yet undefined
specificity-determining residues in the COOH-terminal region of the
substrate. In support of hypothesis (i), the cleavage with hPC1 of an
intramolecularly quenched fluorogenic peptidyl substrate containing the
peptidyl sequence corresponding to the native hproPC1 sequence
(positions 77-90) results in a 6-fold increase in the V
when compared with X without any
substantial change in the K
(29) .
These data are in full agreement with those recently published (30) and suggest that k
values are
affected in a positive way by the sequence of the peptidyl substrate
COOH-terminal to the cleavage site. Clearly more work is needed to
clarify this aspect and define what are the determinants responsible
for this substantial modulation of the k
value.
As indicated in Table 1and Table 2, a discrepancy is
observed for PC1 and furin in the overall efficiency of cleavage (k/K
) of synthetic
peptidyl substrates incorporating a negatively charged residue in the
P3 and P5 positions. Comparing compound II with compound X, the effect observed for furin is mostly related to a decrease
in the k
parameter, since the K
is more or less identical.
Interestingly, among the many sequences sensitive to cleavage by furin
identified by substrate phage display(31) , only one
incorporated a negatively charged residue at P5 and none at P3. This
observation can be rationalized in light of the recently proposed
three-dimensional model of the active site and substrate-binding region
of the convertases(32) . Thus, contrary to furin, of which
subsites S3 and S5 are proposed to contain a negatively charged residue
(Glu), PC1 appears to contain a neutral
-branched residue (Ile).
Therefore, with furin, the substrates IV and X in which a
Glu is occupying the P3 and P5 positions, respectively, will experience
an electrostatic repulsion upon entering the subsite cavities of the
enzyme, resulting in a substantially decreased value of k
. In fact, the relatively higher efficiency of
cleavage of compound XII by furin with respect to X also
indicates that, to observe the decrease in k
value, the free carboxylate moiety of the glutamic acid side
chain is needed as the presence of a pyroGlu moiety in P5 resulted in
an efficient cleavage by the enzyme. This last aspect was described
earlier by substitution of the trypsin active site S1 Asp-189 residue
by a Ser residue which led to a loss of complementary with the P1 Arg
or Lys residue and to a decrease in binding energy of 6.6 Kcal
mol
(33) . Similarly, these observations
could also be extended to the prohormone convertases PACE4 and PC5,
since an aspartic acid residue is predicted to occupy the S3/S5
subsites in contrast to PC2 and PC1, which contain a Phe and Ile
residue, respectively. Furthermore, recent studies by Walker et
al. (10) , using hemagglutinin mutants of a virulent avian
influenza virus as a model, indicated that replacement, within the
cleavage site of the P5 Lys residue by a Glu, completely abolished
cleavage by purified furin in vitro. Upon co-expression with
the vaccinia virus expression system, the same mutant hemagglutinin
was, however, cleaved, hinting that either other cellular convertases
can perform such processing or that the achieved cellular
furin:substrate ratio is high enough to allow such a cleavage.
Similarly, cellular proteolytic maturation of the HIV-1 envelope
glycoprotein gp160 into gp120 and gp41 is accomplished at a highly
conserved tetrapeptide sequence (Arg-Glu-Lys-Arg) for which furin has
been proposed as a candidate processing
enzyme(9, 13) . In both studies, only a small portion
of the gp160 was actually cleaved. Although it was reported to be due
to the slow exit of gp160 from the endoplasmic reticulum, (
)we further propose that the Glu at P3 may also contribute
to this phenomenon. Other structural features, including the
three-dimensional structure of gp160(34) , may also be
responsible. Noteworthy is the fact that when one compares compounds IV and V, substitution of the P3 Glu for a P3 Lys leads
to the recovery of catalytic efficiency; a similar effect of a P3 Lys
had been reported previously, since it had an enhancing effect on the
inhibitory activity of certain synthetic inhibitors(13) .
The substitution of Ser at P3 by a conformationally restricted amino
acid such as Pro results in an 11-fold decrease in k/K
for furin as
compared with only 2-fold for PC1. This effect of Pro substitution may
suggest that the active site of furin is more sterically restricted
than PC1 with respect to the topology in the S1 to S4 subsites of the
substrate binding region. Interestingly, Wasley et al.(35) showed that the endogenous chinese hamster ovary cell
processing enzymes are inefficient at processing overexpressed
profactor IX at the Arg-Pro-Lys-Arg site. This inefficiency can be
circumvented by increasing the amount of enzyme through co-expression
of furin, but not of PACE4. In addition, the three-domain vasopressin
precursor in guinea pig neurohypophysis is incompletely processed in
contrast to complete processing as observed in rat and other
mammals(36) . Interestingly, the interdomain sequence
comprising the guinea pig COOH-terminal sequence of MSEL-neurophysin
and NH
-terminal sequence of the copeptin contains a P3 Pro
residue as compared with, for example, an Arg in the bovine and ovine
sequences.
Another important aspect was the demonstration of the
highly selective nature of the S4 subsite of furin, when compared with
PC1. The significant decrease observed in the k/K
rate constant
for compounds VIII and IX relative to II indicates
that both the guanidinyl moiety and the spatial geometry of the side
chain are important components for an efficient cleavage. In fact, the
results obtained with a genetically engineered proinsulin (PI)
molecule, whereupon insertion of furin-like recognition sites at both
the B/C and/or C/A junctions led to correct and efficient cleavage by
the endogenous enzymes in the constitutive pathway(37) , are
confirmed by the data presented herein. On the other hand, in the case
of PC1, it can be seen that Lys (VIII) and even Orn (IX)
can substitute for the Arg in P4 without major effect on the k
. Nevertheless, it can be said that the
presence of a positively charged side chain containing residue at P4
appears advantageous as illustrated by studies centered on the
processing of proinsulin(38, 39) . These demonstrated
that mouse PC1 activity is dependent upon the presence of a Lys at P4
position as it cleaves very well human PI-I, but processes more slowly
the rat or murine PI-II (P4, Met) at the B/C junction and not at all at
the hPI-I C/A junction (P4, Leu).
Concerning the peptidyl sequence
Arg-Xaa-Xaa-Arg as a recognition motif for furin, we demonstrate with
the compound VII that PC1 not only cleaves this sequence but
does it in a more efficient way than furin (6 times more efficient (k/K
). Actually,
it was shown recently that PC1 can cleave the prodynorphin precursor at
a single Arg residue preceded by a P8 Arg, releasing the
C-peptide(40) . Therefore, one should be cautious in reporting (41, 42) the neuroendocrine PC1 as a processing enzyme
cleaving exclusively at pairs of basic sites. Furthermore, the peptidyl
substrate VII is identical to the last four residues of the
chicken proalbumin precursor sequence which was shown to be efficiently
and specifically cleaved in vitro by recombinant mouse
PC1(43) . The extremely low k
/K
observed for
furin (55 times lower than observed with II) can be best
explained by the high K
value (345
µM), suggesting that chicken proalbumin precursor will at
best be only moderately cleaved by furin in vivo.
Finally,
the usefulness of the chloromethane derivative as a tool for labeling
and titration purpose for both PC1 and furin was demonstrated in the
present paper. In the case of PC1, three molecular forms were labeled
by the I-chloromethane octapeptidyl inhibitor and
corresponded to molecular forms observed
previously(15, 44) , namely, the 80-85 kDa form
and the 74- as well as the 66-kDa forms (two carboxyl-terminally
truncated PC1). Moreover, the high value observed for the kinetic
parameter (K
/[I]: 1.63
10
s
M
)
underlines its potential usefulness as a complementary tool to Western
blotting techniques in order to investigate the different forms of
convertases found in various neuroendocrine cells and tissues. In the
case of furin, one molecular form of 80 kDa was labeled using the
iodinated chloromethane derivative. This shed form was observed
previously using Western blot analysis and corresponds to the
carboxyl-terminally truncated form secreted in the medium using the
full-length recombinant vaccinia cDNA of furin.
Clearly more work is
needed to define all of the structural subsites refinement of PC1 and
furin in order to get a better understanding of the mechanism of
recognition and cleavage of endogenous substrate by those two
convertases. The principal advantage of using synthetic peptidyl
substrate as a probe for mapping the substrate specificity of enzyme is
clearly highlighted by allowing access to kinetic constants such K, k
, and k
/K
as well as to
relative transition state binding energies,
(
G),
which measure the selectivity of catalysis(45) . As shown in Fig. 4, in the particular case of hPC1 and hfurin, the high
degree of selectivity of furin as compared with PC1 is well illustrated
by, for example,
(
G) values of up to 3.7 Kcal/mol for single
amino acid substitutions at P4 and of 3 Kcal/mol for discrimination
between Glu and Ser residue in P3. Furthermore, the good agreement
between the results described herein with those using (i) co-expression
studies of proprotein and convertases, (ii) in vitro studies
with native proproteins, as well as (iii) the three-dimensional model
proposed for furin, demonstrated that fluorescent peptidyl substrates
can help in elucidating some important elements of substrate
recognition in proprotein processing and can lead to the design of
potent irreversible inhibitors.
Figure 4:
The importance of the substrate side
chains at P position for the transition-state
stabilization. The difference in transition-state stabilization between
the preferred (acetyl-Arg-Ser-Lys-Arg-MCA denoted as A) and the other
substrates denoted as B is given by
(
G) = RT ln
[(k
/K
)
/(k
/K
)
]
as described previously in(45) . The solid triangles represent
values obtained with hfurin whereas the solid circles represent data
obtained with hPC1.