(Received for publication, December 13, 1994; and in revised form, March 20, 1995)
From the
The highly conserved polypeptide 7B2 and the subtilisin-related
prohormone convertases PC1/PC3 and PC2 are broadly distributed in
neurons and endocrine cells and are localized to secretory granules. We
recently showed that recombinant 7B2 is in vitro a potent
inhibitor of PC2 activity, but not of PC1/PC3, and that newly
synthesized 7B2 is transiently associated with proPC2 in vivo.
In the present study, in vitro mutagenesis was used to
identify the region within the 7B2 sequence responsible for the
inhibition of PC2. Mutant proteins were produced in a prokaryotic
expression system and their effects on PC1/PC3 and PC2 activities were
studied by two different in vitro enzyme assays. None of the
7B2 mutant proteins inhibited PC1/PC3 activity. Truncation studies
revealed that a short segment within the COOH-terminal portion of 7B2
is critical for its inhibitory effect on PC2. This segment contains a
pair of basic amino acid residues which may represent a recognition
motif for PC2. Single amino acid substitutions within this
Lys-Lys
site strongly diminished and a
double mutation abolished the inhibitory potency of 7B2. Our results
indicate that, although amino acid residues directly surrounding this
dibasic pair also contribute to PC2 inhibition, the
Lys
-Lys
site is particularly important for
the ability of 7B2 to inhibit PC2.
Many peptide hormones, neuropeptides, and other biologically active peptides and proteins are produced through intracellular proteolytic cleavage of larger precursor proteins at pairs of basic amino acid residues (Douglass et al., 1984). Recently, a mammalian family of processing enzymes related to bacterial subtilisins has been identified. This family specifically participates in the endoproteolytic cleavage of proproteins in the secretory pathway (Barr, 1991; Steiner et al., 1992; Seidah and Chrétien, 1992). The family includes the prohormone convertases PC1 (also known as PC3) and PC2, which are localized to the regulated secretory pathway of neuroendocrine cells, and the more broadly distributed transmembrane proteinase furin, involved in the proteolysis of proproteins in the constitutive pathway of secretion.
The neuroendocrine polypeptide
7B2 is exclusively present in prohormone-producing cells possessing a
regulated secretory pathway. In vivo, proteolytic processing
of the 27-kDa precursor form of 7B2 occurs at a site marked by
basic amino acids in the COOH-terminal domain by an unidentified
proteinase, resulting in the production of a
21-kDa 7B2 form
(Ayoubi et al., 1990; Lazure et al., 1991; Paquet et al., 1994). The processed
21-kDa 7B2 form was
initially purified from porcine and human pituitary glands (Hsi et
al., 1982; Seidah et al., 1983) and was found to be
released via the regulated secretory pathway (Ayoubi et al.,
1990; Iguchi et al., 1987; Vieau et al., 1991).
Immunohistochemical analyses have shown that 7B2, PC1/PC3, and PC2 are
present in a wide range of endocrine glands and in the central nervous
system (Waldbieser et al., 1991; Marcinkiewicz et
al., 1987; Steel et al., 1988; Smeekens et al.,
1991; Seidah et al., 1991; Cullinan et al., 1991).
The NH-terminal half of 7B2 is distantly related to a
subclass of molecular chaperones, the so-called chaperonins of the
60-kDa heat shock protein family (Braks and Martens, 1994). The
COOH-terminal half of the 7B2 protein sequence, on the other hand,
shares a low degree of homology with members of the potato inhibitor I
family, which includes subtilisin inhibitors (Martens et al.,
1994). Intact recombinant 27-kDa 7B2, but not processed 21-kDa 7B2, is in vitro a potent inhibitor of PC2 activity and prevents
pro-PC2 cleavage. The activity of PC1/PC3, however, was not affected by
either form of 7B2, indicating that 7B2 specifically inhibits PC2
(Martens et al., 1994). Furthermore, newly synthesized 7B2
transiently and selectively associates with pro-PC2 in vivo,
consistent with its putative role as a chaperone (Braks and Martens,
1994). In the present study, 7B2 mutant proteins were produced and
studied as possible inhibitors of PC2 and PC1/PC3 in order to define
the region within 7B2 responsible for the potent and selective
inhibition of PC2.
The cDNAs for NH- and COOH-terminally truncated
7B2 proteins were generated by PCR using specific primers: the 5`
primers Deg-5` (5`-GC AGG ATC CCC TGT/C CCT GTG/ATC GGG/ATC
AAA/G AC-3` for pCP93), CD125-5` (5`-TT CGA GCT CTA GAA GGA
TCC GAT CCG GAA CAT GAC TAT CC-3` for pCD125), and 7B2-5`
(5`-GCA GGA TCC ATG GGA CAT ATG TAC AGC CCC CGG ACC CCT GAC
CG-3`) and 3` primers NG162-3` (5`-T TCT AGA AGC TTA TCC
TTG TAG ATA TGG ATT GAC-3` for pNG162), NN167-3` (5`-T TCT AGA
AGC TTA ATT ATC CAG TCT CTG TCC TTG-3` for pNN167), NF177-3`
(5`-T TCT AGA AGC TTA AAA ATG GGG GAC AGA CTT CTT-3` for pNF177)
and 7B2-3` (5`-GTG TCT AGA AGC TTA CTC TGG GTC CTT ATC CTC
ATC-3`). The resulting PCR products were cloned into the BamHI
and HindIII sites of the appropriate prokaryotic expression
vectors pQE30 and pQE32. The plasmid pNG86 was constructed by digesting
the 27 kDa 7B2 construct with KpnI and HindIII
followed by low melting point-agarose purification and blunt-end
ligation. The plasmid pNK171 was constructed by digesting the PCR
product also used for the construct pSTTP with BamHI and SalI followed by cloning into pQE30 (SalI site
indicated in bold in primer STTP-5`).
Amplification of 7B2 cDNA
representing amino acids Tyr-Pro
of the
human 7B2 protein (numbering according to that in Martens(1988)) was
accomplished by PCR using the 5` primer 7B2-5` and 3` primer
Subt-3` (5`-CG GAT CCC GGG ATA GTC ATG TTC CGG ATC-3`). The
plasmid p7B2BS was constructed by cloning the resulting PCR product
into the BamHI and SmaI sites of pQE30. Mutations in
the potential PC2 recognition site Lys
-Lys
of human 7B2 were introduced by PCR using the following primers:
the 5` primers Fur-5` (5`-AG GAT CCC GGG TTG GGC AAG AGG AAC AAG
AGA CTC CTT TAC-3` for pFur), and Chymo-5` (5`-AG GAT CCC GGG TTG GGC AAG TGG AAC ATG GAA CTC CTT TAC GAG-3` for pChymo) and the
3` primer 7B2-3`. The resulting PCR products corresponding to
amino acids Pro
-Glu
of the human 7B2
protein were cloned into the SmaI and HindIII sites
of p7B2BS.
Mutations in the potential furin recognition site
Arg-Arg
-Lys
-Arg
-Arg
and in the potential PC2 recognition site
Lys
-Lys
of 7B2 were introduced by a PCR
strategy involving the production of a double-stranded DNA megaprimer
(Barik, 1993). The appropriate megaprimer was generated using the 5`
primers RLKLR-5` (5`-GGA GAG AGA CTA AAG CTG AGG AGT GTC-3` for
pRLKLR), KS-5` (5`-GTT GTT GCA AAG TCG TCT GTC CCC CAT-3` for pKS),
KDEG-5` (5`-GTT GTT GCA AAG (C/G) (A/T/C)G TCT GTC CCC CAT-3` for pKV),
KR-5` (5`GTT GTT GCA AAG AGG TCT GTC CCC CAT-3` for pKR), SS-5` (5`-AAT
GTT GTT GCA TCG TCG TCT GTC CCC CAT-3` for pSS), RSK-5` (5`-AAT GTT GTT
GCA AG(A/T) AAG TCT GTC CCC-3` for pRK and pSK), STTP-5` (5`-GTT GTT
GCA AAG TCG ACT ACC CCC CAT TTT TCA-3` for pSTTP), and RAKR-5`
(5`-CTG GAT AAT GTT CGT GCA AAG AGG TCT GTC CCC CAT-3` for pRAKR) and
the 3` primer 7B2-3` in the first round of PCR amplification. The
PCR product for the pRAKR mutant was generated by using pRLKLR cDNA as
template. After low melting point-agarose purification and
phenol-chloroform extraction, the resulting megaprimers served in the
second round of PCR amplification as the 3` primer introducing the
appropriate mutation in 7B2 cDNA in combination with primer
7B2-5`. Purification of the PCR products was performed with low
melting point-agarose, and reamplification was carried out with primers
7B2-5` and 7B2-3`. The PCR products were cloned into the BamHI and HindIII sites of pQE30.
The sequences of all 7B2 constructs were confirmed by double-stranded DNA sequence analysis using T7 DNA polymerase.
Recombinant human 7B2 proteins
were produced in Escherichia coli as fusion proteins with an
NH-terminal hexahistidine tag by induction of the cells
with 1 mM isopropyl-
-D-thiogalactopyranoside.
Recombinant proteins were purified by nickel nitriloacetate-agarose
affinity chromatography according to the manufacturer's
instructions (Qiagen).
For the
second assay, the proinsulin endoproteinases PC1/PC3 and PC2 were
solubilized by detergent extraction of insulin secretory granules
prepared from rat insulinoma cells (Bailyes and Hutton, 1992). PC1/PC3
and PC2 proteinase activities were measured in the presence or absence
of recombinant 7B2 through analysis of human I-proinsulin
cleavage essentially as described previously for PC1/PC3 (Bailyes and
Hutton, 1992). The assay mixture contained 50 mM sodium
acetate, pH 5.5; 1 mM calcium chloride; 0.1% Triton X-100; 10
µM E-64; 10 µM pepstatin A; 100 µM TPCK and 1 mM phenylmethylsulfonyl fluoride. In control
incubations, calcium chloride was replaced by 2 mM EDTA.
Enzyme and recombinant 7B2 proteins were preincubated for 30 min before
the enzyme assay was started by the addition of substrate. Incubations
(total volume: 55 µl) were conducted at 30 °C for 2 h and
terminated by the addition of Tris, pH 8.0, to a final concentration of
0.1 M. Proinsulin and its cleavage products were separated
through immunoprecipitation using the cellulose-coupled monoclonal
antibodies A6 (directed against the B-chain/C-peptide junction of
proinsulin) or ANT-1 (directed against the C-peptide/A-chain junction
of proinsulin; Crowther et al., 1994) in order to determine
PC1/PC3 or PC2 enzyme activity, respectively. Radioactivity remaining
in the supernatant after immunoprecipitation was determined and the
activity was expressed as the percentage conversion of the initial
radioactivity.
The effect of recombinant 7B2 on PC2 activity was studied by
two different in vitro assays. One assay was based on
PC2-mediated cleavage of a fluorogenic substrate (Z-Arg-Ser-Lys-Arg-AMC) (Martens et al., 1994) using
PC2 obtained by immunopurification (Shen et al., 1993;
Lindberg et al., 1995) from the conditioned medium of the
mouse pancreatic cell line TC3. The second assay used
I-proinsulin as a substrate and was based on the
separation of cleavage products by immunoprecipitation (with a
monoclonal antibody directed against the PC2 cleavage site in the
C-peptide/A-chain junction of proinsulin), followed by determination of
the radioactivity that remained in the supernatant. As a source for the
enzyme in this assay, solubilized secretory granules of rat insulinoma
cells were used (Bailyes and Hutton, 1992). In both assays, recombinant
27-kDa 7B2 produced half-maximal inhibition of PC2 at nanomolar
concentrations (Fig. 1A and Table I). In contrast,
at submicromolar concentrations, the COOH-terminally truncated
recombinant 21-kDa form of 7B2 did not affect PC2 activity (Fig. 1A and Table I). Granular PC1/PC3 was not
inhibited by either form of 7B2 (Fig. 1B).
Figure 1:
Recombinant
27-kDa 7B2 inhibits granular PC2 enzyme activity. Solubilized insulin
secretory granules were incubated with recombinant 7B2 proteins. A, recombinant 27-kDa 7B2 () but not COOH-terminally
truncated 21-kDa 7B2 (
) inhibited PC2-mediated cleavage of
proinsulin at nanomolar concentrations. B, recombinant 27-kDa
7B2 (
) and recombinant 21-kDa 7B2 (
) did not inhibit
PC1/PC3-mediated cleavage of proinsulin. PC2 and PC1/PC3 activities are
expressed as a percentage of the activity obtained without added 7B2
protein.
As an
initial step toward the identification of the region within the 7B2
sequence responsible for the inhibition of PC2, we have produced the
NH- and COOH-terminal halves of 7B2 in E. coli and
purified the recombinant proteins to near homogeneity by affinity
chromatography. The NH
-terminal domain of 7B2 did not
inhibit PC2 activity (mutant pNG86, representing amino acid residues
Tyr
to Gly
; Table Iand Fig. 2).
This portion of the protein is chaperonin-related and may function in vivo as a molecular chaperone to act in the proper folding
or prevent aggregation of proPC2 (Braks and Martens, 1994). The
COOH-terminal half of 7B2 (mutants pCP93 and pCD125 corresponding to
Pro
-Glu
and
Asp
-Glu
, respectively) which carries
regions structurally related to members of the potato inhibitor I
family (Martens et al., 1994), inhibited PC2 activity with
comparable potency to intact 7B2 ( Table Iand Fig. 2). Note
that with these 7B2 fragments, similar effects on PC2 activity were
observed in the two different assays ( Table Iand Fig. 2).
This was in fact found to hold for all truncated and mutated 7B2
proteins examined in the present study (see below). Next,
COOH-terminally truncated forms of 7B2 were produced to further
localize the inhibitory domain within the COOH-terminal half of 7B2.
The inhibitory potencies of the truncated proteins encompassing
Tyr
to Gly
, Tyr
to
Asn
, and Tyr
to Lys
(mutants
pNG162, pNN167, and pNK171, respectively) were dramatically diminished,
as compared with the potency of intact 7B2 ( Table Iand Fig. 2). In contrast, the truncated 7B2 protein representing
amino acid residues Tyr
to Phe
(mutant
pNF177) was a relatively potent inhibitor of PC2 ( Table Iand Fig. 2). These results indicate that a region of six amino acid
residues within 7B2 (Lys
to Phe
) is crucial
for PC2 inhibition. Interestingly, the COOH-terminal portion of 7B2
encompassing amino acid residues Val
to His
is identical among all mammalian, fish, and amphibian 7B2
sequences identified thus far, whereas the region surrounding this
portion is much less conserved (Waldbieser et al., 1991;
Brayton et al., 1988; Mbikay et al., 1989; Martens et al., 1989).
Figure 2: Inhibitory effect of 7B2 mutant proteins on granular PC2. Solubilized insulin secretory granules were incubated with 100 nM recombinant 7B2 proteins. PC2 convertase activities are expressed as a percentage of the activity obtained without added 7B2. The results shown are the mean ± S.E. of triplicates. Recombinant 7B2 proteins added are: -, no 7B2 protein added; 1, pFur; 2, 21-kDa 7B2; 3, 27-kDa 7B2; 4, pNG86; 5, pCP93; 6, pNG162; 7, pNN167; 8, pNK171; 9, pNF177; 10, pCD125; 11, pSTTP; 12, pKS; 13, pKV; 14, pRAKR; 15, pChymo; 16, pRLKLR; 17, pKR; 18, pSS; 19, pSK; and 20, pRK.
In prohormones, pairs of basic amino acid
residues are thought to be potential recognition sites for prohormone
convertases (Barr, 1991; Steiner et al., 1992; Seidah and
Chretien, 1992). The potential PC2 binding site
Lys-Lys
is present within the portion of
7B2 which is highly conserved and now suspected to be responsible for
the in vitro inhibition of PC2 activity. These observations
prompted us to mutate this site. Substitution of
Lys
-Lys
by Ser
-Ser
(mutant pSS) indeed abolished the inhibitory potency of 7B2 ( Table IIand Fig. 2). The potency of 7B2 was also impaired
through the substitution of Lys
with Val (pKV), but to a
lesser extent than with the Lys
to Ser mutant ( Table IIand Fig. 2). This indicates that the effects of the
Lys
mutations apparently depend on the nature of the
substituting amino acid residue. Remarkably, substitution of
Lys
by Arg (mutant pKR) attenuated the inhibitory effect
of 7B2, whereas the Lys
to Arg mutant protein (mutant
pRK) was as potent as nonmutated 7B2 ( Table IIand Fig. 2).
Having demonstrated the critical role of
Lys
-Lys
, we then decided to investigate
whether other amino acid residues directly surrounding this dibasic
pair are important for PC2 inhibition. Indeed, additional substitution
of Val
or Ser
-Val
(mutants
pRAKR and pSTTP, respectively) further diminished the inhibitory
potency of 7B2 ( Table IIand Fig. 2). The
Lys
-Lys
site is probably a site of
proteolytic cleavage in vivo, since the COOH-terminal 7B2
peptide corresponding to Ser
to Glu
has
been recently identified in bovine adrenal medulla chromaffin vesicles
(Sigafoos et al., 1993). The enzyme responsible for this
cleavage remains to be identified. In contrast to the substitutions
within Lys
-Lys
, mutations within the two
other potential PC2 recognition sites in 7B2, namely
Lys
-Lys
(mutants pChymo and pFur) and
Arg
-Arg-Lys-Arg-Arg
(mutant pRLKLR), did
not impair the inhibitory capacity of 7B2 ( Table IIand Fig. 2). This indicates that paired basic amino acids other than
Lys
-Lys
do not contribute to the ability of
7B2 to inhibit PC2. Again, in the two different enzyme assays for both
PC2 and PC1/PC3, similar results were obtained with these 7B2 mutants.
None of the 7B2 mutant proteins described here was able to inhibit
PC1/PC3 (data not shown).
We recently reported that intact
recombinant 7B2 was cleaved in vitro by PC1/PC3 but not by PC2
(Martens et al., 1994). In order to examine whether our 7B2
mutants were cleaved, we incubated the recombinant proteins in the
presence of PC2 or PC1/PC3 and analyzed the cleavage products by
Western blotting. No PC2-mediated cleavage of recombinant 27 kDa 7B2
nor of the pFur, pChymo, and pRLKLR mutant proteins was observed under
the present conditions (data not shown). These results are in line with
the finding that these recombinant proteins potently inhibit PC2
activity. In vitro, PC1/PC3 has been shown to cleave intact
recombinant 7B2 at Arg-Arg
but not at
Lys
-Lys
(Martens et al., 1994).
Western blot analysis revealed that the pFur and pRLKLR mutant proteins
were effectively cleaved by PC1/PC3 (Fig. 3). The size of the
fragment (18 kDa) generated from the pFur mutant protein indicated that
the introduction of a potential furin recognition site through
replacement of Trp
-Asn-Lys-Lys
by
Arg
-Asn-Lys-Arg
resulted in cleavage at
this site by PC1/PC3. Interestingly, alteration of the natural furin
site in 7B2, Arg
-Arg-Lys-Arg-Arg
to
Arg
-Leu-Lys-Leu-Arg
(mutant pRLKLR), still
resulted in the production of a fragment of 21 kDa by PC1/PC3 (Fig. 3), consistent with other studies which indicate that
PC1/PC3 is able to cleave at single basic amino acid residues (Nakayama et al., 1992; Dupuy et al., 1994). The findings of
these cleavage studies are in accordance with the observation that none
of the 7B2 mutant proteins examined in the present study affected
PC1/PC3 activity (data not shown).
Figure 3: 7B2 proteins are cleaved in vitro by PC1/PC3. After incubation of recombinant 7B2 proteins with PC1/PC3, the samples were subjected to Western blot analysis using anti-7B2 monoclonal antibodies. From left to right: lanes 1 and 2, pRLKLR cleavage; lanes 3 and 4, pFur cleavage; and lanes 5 and 6, recombinant 27-kDa 7B2 cleavage. Either calcium or EDTA was included in the reaction mixture, as indicated.
Taken together, our data indicate
that a short segment located near the COOH terminus of 7B2 is
responsible for the potent and selective inhibition of PC2 activity.
The potential PC2 binding site Lys-Lys
present within this segment appears to be essential, but not
fully responsible, for the inhibitory potency of 7B2 and probably acts
in concert with other amino acid residues in the direct vicinity of
this site.
Table II: Inhibitory potency of mutated 7B2 proteins