(Received for publication, September 6, 1995; and in revised form, November 22, 1995)
From the
The specificity and relative efficiency of cleavage of mono- and
paired-basic residue processing sites by YAP3p was determined in
vitro for a number of prohormone substrates: human
ACTH, bovine proinsulin, porcine cholecystokinin
33, cholecystokinin (CCK) 13-33, dynorphin A(1-11),
dynorphin B(1-13), and amidorphin. YAP3p generated
ACTH
from ACTH
. It cleaved
proinsulin at the paired-basic residue sites of the B-C junction as
well as the C-A junction. Leu-enkephalin-Arg and Leu-enkephalin-Arg-Arg
were generated from dynorphin A and dynorphin B, respectively. YAP3p
generated Met-enkephalin-Lys-Lys from amidorphin showing that cleavage
by this enzyme can occur at a lone pair of Lys residues. CCK33 was
cleaved at Lys
and Arg
, each containing an
upstream Arg residue at the P6 and P5 position, respectively. K
values were between 10
and 10
M for the various substrates,
with the highest affinity exhibited for the tetrabasic site of
ACTH
(1.8
10
M). The tetrabasic residue site of ACTH
was cleaved with the highest relative efficiency (k
/K
= 3.1
10
M
s
), while that of the monobasic site of
CCK13-33 and the paired-basic site of proinsulin B-C junction,
were cleaved less efficiently at 4.2
10
M
s
and 1.6
10
M
s
, respectively.
Yeast cells express an alternate enzyme, an aspartic protease,
YAP3p, encoded by the YAP3 gene, which can process
pro--mating factor at paired-basic residues, when this prohormone
is overexpressed in a mutant yeast strain lacking the KEX2p serine
protease(1) . While the substrate that YAP3p normally processes
in yeast is not known, it most likely functions as a processing enzyme
since it is localized in the secretory pathway and can process
transfected anglerfish prosomatostatin correctly(2) . We have
previously overexpressed the YAP3 gene in the BJ3501 strain of Saccharomyces cerevisiae, purified the recombinant YAP3p
enzymatic activity, and investigated its ability to process the
prohormones, mouse pro-opiomelanocortin (POMC) (
)and its
fragments, and anglerfish prosomatostatin I and II (aPSS I and aPSS II) in vitro(3, 4) . Those studies revealed that
YAP3p specifically cleaved the paired-basic residues of POMC to yield
adrenocorticotropin (ACTH),
-endorphin, and
-melanocyte stimulating hormone, and aPSS I to yield
somatostatin 14. YAP3p cleaved aPSS II at a monobasic residue which has
an upstream Arg in the P6 position, to yield somatostatin 28, but
cleavage at the analogous monobasic residue cleavage site in aPSS I,
which has the upstream Arg substituted by a histidine, was not
observed(4) . This latter finding indicated that in addition to
recognizing the paired-basic residue motif, YAP3p also appeared to show
specificity for a monobasic residue that had an Arg present upstream.
Furthermore, YAP3p cleaved the monobasic residue site of aPSS II more
rapidly than the paired-basic residue site of aPSS I, raising the
possibility that YAP3p may have a greater preference for monobasic
residue sites.
However, in vitro analysis of the specificity of YAP3p on POMC, aPSS I, and aPSS II alone is insufficient to completely define the specificity of YAP3p or to conclude that YAP3p would in general cleave the monobasic residue motif of prohormones more efficiently than the paired-basic residue motif. In this study, we have investigated extensively the specificity and kinetic parameters governing the processing of six different substrates of physiological importance; proinsulin, cholecystokinin 33, dynorphin A, dynorphin B, amidorphin, and adrenocorticotropic hormone, containing mono-, paired-, and tetrabasic residue sites, by YAP3p. This study also provides the first kinetic information on the efficiency of cleavage of paired versus monobasic residue sites by an aspartic protease processing enzyme. In addition, YAP3p has similar specificity to the pro-opiomelanocortin converting enzyme (EC 3.4.23.17), a mammalian prohormone processing aspartic protease functionally and immunologically related to YAP3p(3) . Thus, kinetic studies on YAP3p as a model enzyme, which is obtainable in large quantities, will also likely provide a better understanding of the substrate parameters governing the specificity and efficiency of cleavage of prohormones by mammalian processing aspartic proteases.
Figure 2:
Lineweaver-Burk plots of the cleavage of
each substrate by YAP3p. Data were obtained as explained under
``Experimental Procedures.'' A, 11.5-92
µM proinsulin, 4 nM YAP3p in 20 min; B,
43-344 µM CCK13-33, 1 nM YAP3p in 15
min; C, 36-294 µM dynorphin A, 0.4 nM YAP3p in 5 min; D, 32-254 µM dynorphin
B, 0.1 nM YAP3p in 10 min; E, 17-135 µM amidorphin, 0.2 nM YAP3p in 10 min; F,
11-88 µM ACTH, 0.1 nM YAP3p in 15 min. Error bars indicate standard deviation, n = 3.
Figure 3:
Lineweaver-Burk plot of the cleavage of
proinsulin B-C junction peptide. Product formation was measured as a
function of time in two experiments with overlapping substrate
concentrations (27-658 µM). From the curve fits of
the data points for each substrate concentration, V values were calculated and expressed as nanomol of product
generated/min. A double reciprocal plot of 1/nmol of product
generated/min versus 1/[substrate] was used to
obtain K
and V
values.
Figure 1: Proteolytic processsing of proinsulin. A, primary amino acid sequence of proinsulin cleavage sites. Big and small arrows indicate major and minor sites cleaved by YAP3p. B, HPLC profile of products generated from bovine proinsulin incubated with YAP3p in the absence (upper panel) and presence (lower panel) of pepstatin A. Ten µg of proinsulin was incubated with 0.7 pmol of YAP3p for 24 h. Gradient was 10-35% B in 30 min followed by 35-45% B in 30 min. * = peaks that were present in the enzyme alone control.
Figure 4: Cleavage of native and denatured/reduced proinsulin. Lane 1, 20 µg of proinsulin incubated in the absence of YAP3p. Lane 2, 20 µg of native proinsulin cleaved by YAP3p. Lane 3, 20 µg of denatured/reduced proinsulin cleaved by YAP3p. *, since the incubation conditions used were similar to Fig. 1, this band represents predominantly a mixture of the B chain of insulin and the C-A peptide of proinsulin. These two products were not resolved in this type of gel.
Figure 5: Time course of generation of CCK8-immunoreactive products from CCK33 by YAP3p. Products from each time point were first purified with DEAE-Sephadex ion-exchange resin and an aliquot assayed by RIA. Open squares indicate reactions in the absence of pepstatin A. Filled squares indicate reactions in the presence of pepstatin A.
Figure 6: Proteolytic processing of cholecystokinin 33. A, primary amino acid sequence of CCK33. Arrowheads indicate the sites cleaved by YAP3p. R = arginine, K = lysine, Y* = sulfated tyrosine. B, Sephadex G-50 gel filtration profile of CCK8-immunoreactive products generated by YAP3p. C, radioimmunoassay of fractions from reverse-phase HPLC separation of the CCK8-sized product generated from incubation of CCK33 with YAP3p. Products were purified by Sephadex G-50 filtration, and the CCK8-sized product was further analyzed by HPLC. Arrows in B and C indicate positions of standards. Numbering of the CCK peptides start from the C terminus.
Figure 7: Proteolytic processing of cholecystokinin 13-33. A, primary sequence of CCK13-33. Arrowhead indicates the site cleaved by YAP3p. B, HPLC profile of products generated from CCK13-33 incubated with YAP3p in the absence (upper panel) and presence (lower panel) of pepstatin A. Ten µg of CCK13-33 was incubated with 0.14 pmol of YAP3p for 2 h. Gradient was 10% B for 10 min, 10-30% B in 20 min, and 30-35% B in 10 min.
Figure 8: Proteolytic processing of dynorphin A(1-11). A, primary amino acid sequence of dynorphin A(1-11). Big and small arrows indicate major and minor sites cleaved by YAP3p. B, HPLC profile of products generated from dynorphin A incubated with YAP3p in the absence (upper panel) and presence (lower panel) of pepstatin A. Ten µg of dynorphin A was incubated with 0.014 pmol of YAP3p for 2 h. Gradient was 10% B for 10 min and 10-40% B in 50 min.
Figure 9: Proteolytic processing of dynorphin B(1-13). A, primary amino acid sequence of dynorphin B(1-13). Arrow indicates site cleaved by YAP3p. B, HPLC profile of products generated from dynorphin B incubated with YAP3p in the absence (upper panel) and presence (lower panel) of pepstatin A. Ten µg of dynorphin B was incubated with 0.014 pmol of YAP3p for 2 h. Gradient was 10% B for 10 min and 10-40% B in 50 min.
Figure 10: Proteolytic processing of Amidorphin. A, partial primary amino acid sequence of amidorphin. Arrow indicates the major site cleaved by YAP3p. B, HPLC profile of products generated from amidorphin incubated with YAP3p in the absence (upper panel) and presence (lower panel) of pepstatin A. Ten µg of amidorphin was incubated with 0.014 pmol of YAP3p for 2 h. Gradient was 10-45%B in 60 min. *, this peak was not sequenced for identification; however, it is predicted to be the other half of the amidorphin molecule.
Processing of prohormones to yield active hormones occurs
most commonly at paired-basic residues and to a lesser extent at
specific monobasic residue
sites(8, 9, 10, 11) . A number of
prohormone processing enzymes capable of carrying out these specific
mono/paired-basic residue cleavages have been described. They include
the serine/subtilisin-like enzymes, furin and the proprotein
convertases, (12, 13, 14, 15, 16, 17, 18, 19) ,
and representatives from the thiol (20, 21) and
aspartic protease
classes(1, 22, 23, 24, 25) .
YAP3p is a member of the aspartic protease family of prohormone
processing enzymes. In this study, we have analyzed a set of substrates
to further define the specificity of YAP3p and the mono/paired-basic
residue cleavage site motifs recognized by this enzyme. The catalytic
efficiency (k/K
) of YAP3p
for the cleavage of these substrates was determined.
YAP3p cleaved
bovine proinsulin at the B-C junction which contains the sequence
PKARRE (Fig. 1A). Cleavage occurred preferentially on
the carboxyl side of the Arg-Arg pair, putting the Lys at P4, while
some cleavage was observed in between, putting the Lys at P3. This
preference is similar to that of furin where an Arg at P4 enhances the
cleavage rate of synthetic proalbumin peptides (26) while an
Arg at P3 is deleterious(27) . The synthetic substrate,
Boc-RVRR-methylcoumarin amide was cleaved by YAP3p (3) exclusively in between the Arg-Arg pair, rendering the
assignment of the upstream Arg in this substrate in the P3 position (Table 2). The absence of any cleavage on the carboxyl side of
the pair of Arg residues is presumably due to the steric hindrance of
the 7-amino-4-methyl-coumarin moiety that would be in the S1` pocket of
the active site. Anglerfish pro-somatostatin I (aPSS I), containing the
sequence PRQRKA (Table 2), was cleaved by YAP3p, similar to the
proinsulin B-C junction, primarily on the carboxyl side of the Arg-Lys
cleavage site, although some cleavage in between the pair was
observed(4) , indicating an overall degree of tolerance but not
preference by YAP3p for a basic residue in the P3 position. In an
effort to determine the role that structural conformation plays in the
efficiency of this reaction, we compared the ability of YAP3p to cleave
native versus denatured/reduced proinsulin. YAP3p cleaved the
native proinsulin better than the denatured/reduced proinsulin (Fig. 4), demonstrating that disruption of the conformation of
the prohormone negatively affected the efficiency of its cleavage. When
we analyzed the cleavage of the proinsulin B-C junction peptide, we
found that the specificity was identical with that of the full-length
prohormone itself, demonstrating that the primary sequence around the
cleavage site was sufficient to dictate the specificity but the K was greatly increased.
The bovine proinsulin C-A junction contains the sequence, PPQKRG, i.e. a lone pair of basic residues. YAP3p cleaved the C-A junction of proinsulin preferentially in between the Lys-Arg pair indicating that an Arg residue in the P1` position is an acceptable position. Cleavage at this site was relatively slow compared to the B-C junction, perhaps due to the presence of the two prolines immediately upstream from the cleavage site (P3 and P4). Further support of this hypothesis is borne out by our studies with aPSS I and aPSS II(4) . While aPSS I was cleaved by YAP3p at the Arg-Lys pair preferentially after the Lys to yield somatostatin 14 (Table 2), the cleavage of aPSS II at the analogous Arg-Lys site was not detected, possibly due to the presence of the two prolines immediately upstream (at P5 and P6) from the expected cleavage site (Table 2). The proline side chain is known to have less conformational freedom resulting in a more rigid structure around the peptide bond. This may prevent the substrate in the cleavage site region from assuming a structure that binds efficiently to the active site pocket of the aspartic protease which generally spans up to 10 amino acids(28, 29) . It is noteworthy that the specificity of YAP3p for the cleavage sites of bovine proinsulin is subtly different, especially at the C-A junction, to that observed in vivo where processing occurs exclusively on the carboxyl side of both sites.
YAP3p has been shown to cleave aPSS II in
vivo and in vitro at a monobasic Arg residue within a
motif containing an upstream Arg at P6(2, 4) .
However, in these two studies, cleavage of aPSS I, where the upstream
Arg at P6 is substituted by a histidine, was observed by one group (2) and not by the other(4) . This discrepancy
exemplifies the differences that can be observed between in vivo and in vitro studies where the regulation of enzyme to
substrate ratio dictates the efficacy of the reaction. The finding that
no cleavage by YAP3p was observed when the -mating
factor-leader-proinsulin fusion protein was mutated to delete the Arg
at the Lys-Arg junction (1) (Table 2), suggests that
YAP3p does not cleave monobasic sites without an additional basic
residue upstream or downstream.
To determine if YAP3p will cleave a
monobasic Arg, as well as a Lys, within a motif having an upstream
basic residue, CCK33 was tested as a substrate. YAP3p cleaved sulfated
CCK33 at two monobasic residue sites, each containing an upstream Arg
at either the P6 or P5 position (Fig. 6A and Table 2). Cleavage at Arg generated CCK8, while
cleavage at Lys
generated CCK22 (Fig. 6, B and C). YAP3p was also shown to cleave CCK13-33 to
CCK13-22 and CCK23-33 at Lys
(Fig. 7B). These results indicate that YAP3p can
recognize both a monobasic Lys or Arg apparently within a motif
containing an upstream Arg. This cleavage specificity exhibited by
YAP3p is similar to the CCK8 generating enzyme previously described
from rat brain synaptosomes which is capable of both these
cleavages(30) . Based on the relative concentrations of the
products generated at the 5-h time point (Fig. 6B), it
appears that YAP3p cleaved preferentially at Lys
to
generate CCK22 rather than at Arg
to generate CCK8. This
result may be an indication that YAP3p prefers mono-Lys sites over
mono-Arg sites, or simply that the upstream Arg in the P6 position is
more favorable than in the P5 position. The presence of a negatively
charged sulfated tyrosine close to Arg
may also render this
site difficult to cleave. While the presence of an Arg in the P5 and P6
positions of the cleavage sites of CCK33, and that of Arg in the P6
position of the monobasic cleavage site of aPSS II appear to correlate
with cleavage at these sites, the presence of other amino acids around
the cleavage site may also be important. For instance, a bulky
hydrophobic residue at the P2` position appears to also correlate with
the cleavage at monobasic residues (Table 2). The extent of the
effect of these residues surrounding the cleavage site is currently
being determined on one systematically varied substrate.
The
cleavage of dynorphin A(1-11) (dynA) and dynorphin B(1-13)
(dynB) was studied because both these substrates contain a potential
mono- and paired-basic cleavage site. The results show that YAP3p had a
preference for the paired-basic sites over the potential monobasic
sites in both substrates, generating Leu-enkephalin-Arg and
Leu-enkephalin-Arg-Arg from dynA and dynB, respectively ( Fig. 8and 9). It is interesting to note the presence of a basic
residue in the P3` position (Arg in dynA and Lys in dynB) relative to
the primary cleavage sites in both substrates. The preference for the
paired basic residues exhibited by YAP3p for dynA and dynB demonstrates
that a basic amino acid present downstream of the cleavage site (e.g. P3`) may also play a major role in determining the exact
cleavage site of YAP3p. Cleavage studies of synthetic proalbumin
peptides by YAP3p have verified the importance of downstream basic
residues. ()An almost 10-fold increase of relative cleavage
activity of YAP3p over the wild type proalbumin sequence was shown for
a peptide that substituted the Ala for an Arg in the P2` position.
Since the aspartic proteases exhibit a high degree of symmetry in the
active site, it is possible that a given substrate can bind in a manner
such that the bond to be cleaved is regulated by basic residues in
downstream as well as upstream positions.
The cleavage specificity
of YAP3p for the substrates tested in this and previous studies (Table 2) indicates that YAP3p recognizes the following motifs: a
pair of basic residues or monobasic residues with an additional
upstream basic residue within the P2-P6 position. However, cleavage of
some monobasic sites without an upstream basic residue may be regulated
by additional amino acids in the cleavage site. Cleavage at paired
basic residues can occur either between or on the carboxyl side of the
pair of basic residues, the preference being substrate-dependent and
likely governed predominantly by the upstream and downstream basic
residues surrounding the cleavage site. However, most of the substrates
studied also contain nonpolar residues in the P3 position. The
importance of such residues remains unclear, but the finding that a
charged residue, Arg or Lys, as mentioned before, is tolerable in the
P3 position suggests that S3 does not have a strict requirement for a
given type of amino acid. Cleavage at a monobasic site occurs on the
carboxyl side of the basic residue. The specificity of YAP3p shows some
overlap with that of the mammalian pro-opiomelanocortin converting
enzyme(3, 31) . Pro-opiomelanocortin converting enzyme
has been shown to cleave at paired-basic residue sites of POMC,
proinsulin(22, 32) , and the monobasic residue motif
at Lys of CCK13-33. (
)However,
pro-opiomelanocortin converting enzyme did not cleave
ACTH
(22) or
-endorphin
(33) .
Analysis of
kinetic parameters (Fig. 2) for the cleavage of paired and
monobasic residues shows that YAP3p cleaves prohormone substrates at
these sites with k/K
values
comparable to monkey cathepsin E for a variety of synthetic peptides
mimicking the cleavage sites of some prohormone precursors (34) . Cathepsin E may represent another member of the aspartic
protease family of enzymes involved in intracellular precursor
processing(35) . A comparison of the catalytic efficiency (k
/K
) of cleavage of the
motifs in this study suggests that YAP3p cleaves the motifs containing
a paired-basic residue site, with or without a basic residue upstream
or downstream, as in ACTH
, dynA, dynB, and
amidorphin, 10-100 times more efficiently than the monobasic
residue motif as in CCK13-33. Moreover, having additional basic
residues flanking the cleavage site between P2-P6 and P2`-P6` enhances
the affinity of binding and catalytic efficiency. This is exemplified
by the decrease in K
and increase in k
/K
for ACTH
versus amidorphin, both of which are cleaved at a
Lys-Lys pair, but ACTH
has 4 additional basic
residues flanking the cleavage site. Tetrabasic residues may well be a
highly efficient cleavage site for YAP3p. CCK13-33 and amidorphin
were both cleaved on the carboxyl side of a Lys residue, but amidorphin
was cleaved with a >10-fold higher efficiency than CCK13-33.
This may be an indication of the preference by YAP3p for a basic
residue in the P2 position, Lys in amidorphin, rather than P6, Arg in
CCK13-33.
From the present studies it would appear that YAP3p can cleave all the motifs recognized by the subtilisin-like serine proteases, PC1/3, PC2, and furin. PC1/3 has been shown to cleave paired-basic residue sites (36, 37, 38) and at a monobasic residue site with an upstream Arg in the P4 position(39) , while PC2 cleaves only at paired-basic residues(15, 19) . Furin, on the other hand, prefers a paired-basic residue motif with an additional upstream Arg residue at the P4 position, although the basic residue at P2 appears not to be essential(12, 39, 40, 41, 42) . Future kinetic studies on the cleavage of these substrates by PC1 and PC2 will be important in assessing whether these enzymes cleave the same substrates with different efficiencies and the role the structural conformation plays in dictating efficiency of cleavages, independent of the enzyme. Such kinetic information will also be important in determining the relative role the aspartic proteases and the subtilisin-like serine processing enzymes play in cleaving various prohormones, since both families of proteases can be found in the same endocrine cells, e.g. the presence of pro-opiomelanocortin converting enzyme, PC1, and PC2 in the pituitary intermediate lobe cells(22, 43) .