(Received for publication, August 7, 1995; and in revised form, October 10, 1995)
From the
A human placental gt11 cDNA library was screened for
sequences encoding proteins related to human proteinase inhibitor 6
(PI6), and two plaques were identified that displayed weak
hybridization at high stringency. Isolation and characterization of the
DNA inserts revealed two novel sequences encoding proteins composed of
376 and 374 amino acids with predicted molecular masses of
42 kDa.
The novel proteins displayed all of the structural features unique to
the ovalbumin family of intracellular serpins including the apparent
absence of a cleavable N-terminal signal sequence. The degree of amino
acid sequence identity between the novel serpins and PI6 (63-68%)
significantly exceeds that of any other combination of known
intracellular serpins. The two novel serpins encoded by the two novel
cDNA sequences have been designated as proteinase inhibitor 8 (PI8) and
proteinase inhibitor 9 (PI9). The putative reactive center
P
-P
` residues for PI8 and PI9 were
identified as Arg
-Cys
and
Glu
-Cys
, respectively. PI9 appears to be
unique in that it is the first human serpin identified with an acidic
residue in the reactive center P
position. In addition, the
reactive center loop of PI9 exhibits 54% identity with residues found
in the reactive center loop of the cowpox virus CrmA serpin. Two PI8
transcripts of 1.4 kilobases (kb) and 3.8 kb were detected by Northern
analysis in equal and greatest abundance in liver and lung, while the
1.4-kb mRNA was in excess over the 3.8-kb mRNA in skeletal muscle and
heart. Two PI9 transcripts of 3.4 and 4.4 kb were detected in equal and
greatest abundance in lung and placenta and were weakly detected in all
other tissues.
PI8 and PI9 were expressed in baby hamster kidney and yeast cells, respectively. Immunoblot analyses using rabbit anti-PI6 IgG indicated the presence of PI8 in the cytosolic fraction of stably transfected cells that formed an SDS-stable 67-kDa complex with human thrombin. PI9 was purified to homogeneity from the yeast cell lysate by a combination of heparin-agarose chromatography and Mono Q fast protein liquid chromatography and migrated as a single band in SDS-polyacrylamide gel electrophoresis with an apparent molecular mass of 42 kDa. Purified recombinant PI9 failed to inhibit the amidolytic activities of trypsin, papain, thrombin, or Staphylococcus aureus endoproteinase Glu-C and did not form an SDS-stable complex when incubated with thrombin. The cognate intracellular proteinases that interact with PI8 and PI9 are unknown.
The mammalian serine proteinase inhibitors, or serpins, are a
superfamily of single chain proteins that typically range between 40
and 60 kDa in molecular mass, resemble -proteinase
inhibitor in overall structure, and include antithrombin III,
plasminogen activator inhibitors 1 and 2,
-antichymotrypsin, and
-antiplasmin(1) . The majority of serpins
participate in the regulation of several proteinase-activated
physiological processes including blood coagulation, fibrinolysis,
complement activation, extracellular matrix turnover, cell migration,
and prohormone activation, to name a few(2) . Serpins inhibit
proteolytic events by forming a 1:1 stoichiometric complex with the
active site of their cognate proteinases, which is resistant to
denaturants(3) .
In addition to the mammalian serpins,
several viral serpins have been identified and implicated as virulent
factors. These serpins include the SERP-1 gene product produced by
tumorigenic myxoma virus (4) and the CrmA serpin produced by
the cowpox virus(5) . Insight into the mode of action of these
viral serpins has been derived from the findings that the SERP-1 gene
product inhibits the serine proteinases of the fibrinolytic system and
also inhibits C1 esterase, the first enzyme in the complement
cascade(4) . Recently, CrmA was found to attenuate the host
inflammatory response by acting as a specific inhibitor of the
interleukin-1 converting enzyme (ICE), (
)a novel
cytosolic cysteine proteinase(5) . These findings are
significant since they suggest that the proteinase specificity of
mammalian serpins may extend beyond serine proteinases and include some
cysteine proteinases. In this regard, an
-antichymotrypsin-like serpin was recently purified
from bovine chromaffin granules of adrenal medulla and found to inhibit
a novel cysteine proteinase involved in enkephalin precursor
processing(6) .
Aside from the serpins that regulate
proteinase activity, several members of this superfamily lack a
proteinase inhibitory capability and have other physiological roles.
These latter serpins were originally identified by data base searching
and include thyroxine-binding globulin(7) ,
angiotensinogen(8) , and ovalbumin(9) . Ovalbumin
represents the parent prototype of a unique family of serpins, within
the serpin superfamily, that lack a typical amino-terminal cleavable
signal sequence but have been found to reside intracellularly,
extracellularly, or both(10) . Therefore, members of the
ovalbumin serpin family may function as dualistic molecules with an
intracellular and/or extracellular function. The serpins previously
classified as members of the ovalbumin family are plasminogen activator
inhibitor-2 (PAI-2)(11) , an elastase inhibitor (EI) isolated
from monocyte-like cells(12, 13) , a squamous cell
carcinoma antigen (SCCA)(14) , maspin (15) , and a
novel cytoplasmic serpin isolated in this laboratory and designated as
cytoplasmic antiproteinase(16, 17) . A functionally
inactive form of cytoplasmic antiproteinase was purified earlier from
human placenta and designated as placental thrombin inhibitor or PTI (18) . Recently, the Genome Database organization has
recommended that this serpin be designated as proteinase inhibitor 6,
or PI6(19) . Purified PI6 inhibits the amidolytic activities of
a broad spectrum of trypsin-like serine proteinases including thrombin,
trypsin, urokinase, and factor Xa(16) . In addition, trypsin
inhibition by PI6 appears to involve a two-step mechanism that results
in the formation of a tight inhibitory complex that is pseudoreversible
and behaves similar to the proteinase inhibitory complex formed with
-antiplasmin(17) . In the present study, we
report the molecular cloning, expression, and partial characterization
of two novel human PI6 homologs that are divergent within their
reactive centers and are predicted to inhibit distinct proteinases. In
addition, the reactive center loop of one novel PI6 homolog exhibits a
high degree of structural similarity to the serpin encoded by the crmA gene carried by the cowpox virus. These two novel PI6
homologs have been designated as proteinase inhibitor 8 (PI8) and
proteinase inhibitor 9 (PI9) as recommended by the Genome Database
nomenclature committee.
Figure 1: DNA sequences and deduced amino acid sequences of the inserts in clones H3-1-11 and H2-2-11. The overlines designate Kozak consensus motifs containing nucleotide sequences for the initiation of translation. The asterisks denote termination of the open reading frames. The circled Asn residues represent potential sites for the covalent attachment of sugar moieties. A potential polyadenylation signal is underlined. A, the DNA insert of clone H3-1-11 encoding PI8; B, the DNA insert of clone H2-2-11 encoding PI9.
A computer
search of the NBRF protein data base revealed that the proteins encoded
by the H2-2-11 and H3-1-11 cDNAs were novel but showed
considerable amino acid sequence identity with members of the ovalbumin
branch of the serpin superfamily of proteinase inhibitors. The
H3-1-11 and H2-2-11-derived amino acid sequences showed 68
and 63% identity with PI-6, respectively. We have provisionally
designated the proteins encoded by the H3-1-11 and H2-2-11
cDNAs as PI8 and PI9, respectively, according to recommendations
provided by the Genome Database nomenclature committee. Similar to PI6,
PI8 and PI9 exhibit a high degree of amino acid sequence identity to
other human members of the ovalbumin family of cytoplasmic serpins
including EI (PI8, 51%; PI9, 49%), PAI-2 (PI8, 46%; PI9, 45%), and SCCA
(PI8, 46%; PI9, 45%). In addition, PI8 showed 63% amino acid sequence
identity to PI9. The two novel cytoplasmic antiproteinases exhibit all
the structural features previously demonstrated to be unique to the
ovalbumin family of serpins that can be summarized as
follows(10) : (a) PI8 and PI9 lack an N-terminal
extension, and the open reading frame begins at amino acid residue 23
of -proteinase inhibitor; (b) PI8 and PI9
lack a C-terminal extension terminating at Pro
, the
equivalent of Pro
in
-proteinase
inhibitor; (c) PI8 and PI9 both have a Ser at position 375 of
-proteinase inhibitor in place of a highly conserved
Asn found among serpins distantly related to the ovalbumin family; and (d) PI8 and PI9 appear to lack a typical N-terminal cleavable
signal sequence. The new cytoplasmic antiproteinases also have a
potential N-glycosylation consensus sequence
(NX(T/S)) at Asn
and Asn
of PI8 and
Asn
and Asn
of PI9 (Fig. 1, A and B).
Alignment of the deduced primary structure of
PI8 and PI9 with the amino acid sequences of PI6 (Fig. 2) and
other human members of the ovalbumin serpin family (data not shown)
identified the putative reactive center
P-P
` residues of PI8 as
Arg
-Cys
, respectively, which are identical
to PI6. However, the regions flanking the
P
-P
` residues in PI6 and PI8 are highly
divergent. The P
-P
residues of PI6 and PI8 show
no identity, while Arg
in the P
` position was
conserved in both serpins. Since residues in the vicinity of P
have been previously shown to influence both proteinase target
specificity and the inhibitory potency of several serpins(31) ,
these findings suggest that PI6 and PI8 interact with the active sites
of distinct cognate proteinases that have trypsin-like substrate
specificity. In contrast, alignment of the PI9 amino acid sequence
identified the putative P
-P
` residues as
Glu
-Cys
, respectively. The identification
of an acidic P
residue in the PI9 reactive center is unique
to the human serpin superfamily. The only other serpins identified with
an acidic P
residue in their reactive centers are CrmA
(Asp) (32) and a recently described rabbit
-1-antiproteinase E (Glu) (33) . The latter serpin failed
to inhibit the amidolytic activities of trypsin, thrombin, and S.
aureus endoproteinase Glu-C (33) .
Figure 2: Comparison of the amino acid sequences for PI6, PI8, and PI9. Amino acid residues common to the three homologs are boxed. Hyphens are introduced for optimal alignment. The complete amino acid sequence for human PI6 has been reported previously(17, 48) .
These observations
prompted us to determine the overall structural relatedness of the
cytoplasmic antiproteinases and the crmA protein relative to
other intracellular serpins. Previous studies have reported 30%
amino acid sequence identity between CrmA and several extracellular
serpins including antithrombin III, human and murine
-antichymotrypsin, human and murine
-proteinase inhibitor, and human heparin cofactor
II(32) . However, to our knowledge, no study has compared the
primary structures of CrmA and recently discovered members of the
mammalian intracellular serpins. By employing the NBRF program ALIGN,
CrmA was found to share
39% amino acid sequence identity with PI6,
EI, and PAI-2,
37% identity with PI8 and PI9, and
35%
identity with SCCA (data not shown). As a result, these intracellular
serpins appear to represent the closest mammalian relatives of the
viral serpin reported to date. These findings are consistent with the
previous observation that CrmA lacks a cleavable N-terminal signal
sequence and an N-terminal extension common to the ovalbumin family of
intracellular serpins(10) . In addition, a comparison of the
CrmA reactive center loop with the reactive center loops of the
mammalian intracellular serpin family revealed a remarkable degree of
structural conservation with PI9 (Fig. 3). The amino acid
sequence of the PI9 reactive center loop shares
54% of the
structurally conserved residues found in the reactive center loop of
CrmA. An insignificant degree of structural conservation is found
between the amino acid residues in the reactive centers of CrmA, EI,
SCCA, and PAI-2, ranging from 0 to 7%. A noteworthy feature
characteristic of only the cytoplasmic antiproteinase homologs and CrmA
is the presence of a conserved Cys residue in the reactive center
P
` positions. Since the reactive center has been previously
demonstrated to be the most divergent domain of the serpin
superfamily(34) , the cowpox virus may have acquired a
mammalian intracellular serpin gene and the reactive center of the
viral serpin either converged or remained relatively conserved with the
reactive center of PI9.
Figure 3:
A comparison of the reactive center
sequences of the mammalian intracellular serpins and the viral serpin
encoded by the crmA gene of the cowpox virus. Residues
352-364 corresponding to the reactive center regions of the
mammalian intracellular serpins were aligned with the reactive center
of CrmA. The boxed residues designate amino acid residue
identities between the reactive center sequences of the mammalian
serpins and the CrmA reactive center sequence. The solid circles represent accepted mutations in the PI9 reactive center that are
structurally conserved to the correponding amino acid residues in CrmA.
The mammalian reactive center sequences were searched for structurally
conserved amino acid alignments with CrmA based on the grouping system
previously reported by Dayhoff(49) , including D, E; R, K; Y,
F; I, V; and S, T. The arrow designates the cleavable
P-P
` peptide bond in the reactive center
sequences.
Figure 4:
Immunoblot analysis of PI8 and PI9
following incubation with human thrombin. Samples of PI8 (cytosolic
fraction) and purified recombinant PI9 were incubated with either
buffer control (TBS) or 50 nM human thrombin dissolved in TBS
for 30 min at 37 °C. Each incubation mixture was subjected to 12%
SDS-PAGE following reduction and electrophoretically transferred to a
nitrocellulose membrane. PI8-, PI9-, and PI8-thrombin complexes were
visualized by incubating with rabbit anti-PI6 IgG followed by
incubation with I-labeled protein A and autoradiography. Lane 1, PI8 (+TBS); lane 2, PI8
(+thrombin); lane 3, PI9 (+TBS); lane 4,
PI9 (+thrombin).
Recombinant PI9 was purified to homogeneity from transformed yeast
cell lysates in a two-step procedure using heparin-agarose column
chromatography and Mono Q fast protein liquid chromatography. Initial
experiments indicated that 20% of the total protein in the
transformed yeast cell lysate migrated in SDS-PAGE with a molecular
mass of
42 kDa (Fig. 5). In contrast, this protein was not
observed in the mock-transfected yeast cell lysate by this technique
(data not shown), suggesting that the 42-kDa protein was PI9. In the
absence of a functional assay for PI9, the presence of high expression
levels of the putative PI9 in the yeast cell lysates facilitated
identification of this protein in column effluents by SDS-PAGE. As seen
in Fig. 5, the PI9 pool from heparin-agarose was
90% pure,
while the PI9 derived from Mono Q fast protein liquid chromatography
migrated as a single band in SDS-PAGE with an apparent molecular mass
of 42 kDa (Fig. 5). Amino-terminal amino acid sequence analysis
of the purified, putative PI9 indicated that the protein, like
PI6(16) , was derivitized at the amino terminus. Treatment of
the protein with methanolic HCl failed to release a potential N-formyl group, suggesting that the protein was acetylated at
the N terminus. In order to demonstrate that the isolated protein was
indeed PI9, the purified protein was cleaved with trypsin and the
tryptic digest fractionated on a C
reverse phase HPLC
column. One peptide (T28) was isolated and yielded an amino-terminal
sequence of LAHVGEV, which is identical to that observed in the PI9
deduced sequence at residues 206-212, thus confirming the
identity of the isolated protein as PI9.
Figure 5:
Expression and purification of recombinant
PI9 from yeast. Aliquots of PI9 fractions from various stages of
purification were subjected to 12% SDS-PAGE under reducing conditions. Lane 1, 100 µg of reduced PI9/pDPOT cell lysate; lane
2, 20 µg of reduced heparin-agarose PI9 pool; lane 3,
15 µg of reduced PI9 Mono Q pool; lane 4, mixture of
reduced standard proteins including phosphorylase b (94 kDa), bovine
serum albumin (67 kDa), ovalbumin (45 kDa), carbonic anhydrase (30
kDa), soybean trypsin inhibitor (20 kDa), and -lactalbumin (14
kDa).
The purified recombinant
PI9 was then tested for its ability to inhibit the amidolytic
activities of trypsin and papain toward S-2251 and N-benzoyl-DL-arginine p-nitroanilide, respectively. At a 500:1 inhibitor:enzyme
molar ratio, PI9 failed to inhibit the amidolytic activities of these
proteases under conditions where trypsin and papain amidolytic
activities were completely inhibited by a 10-fold molar excess of
soybean trypsin inhibitor and egg white cystatin, respectively (data
not shown). In addition, purified PI9, at a 500:1 inhibitor:enzyme
molar ratio, failed to inhibit the amidolytic activities of human
thrombin and S. aureus endoproteinase Glu-C and did not form a
high M
complex with human thrombin as judged by
Western blotting using the rabbit anti-PI6 IgG to detect PI9 (Fig. 4).
Figure 6:
Northern blot of human tissues. Blots were
screened with P-labeled DNA probes corresponding to
internal regions of PI8 (A) and PI9 (B). Mobilities
for the DNA markers are indicated in kilobases. Equivalent amounts of
mRNA from the following tissues were electrophoresed and probed as
described under ``Experimental Procedures.'' Lane 1,
pancreas; lane 2, kidney; lane 3, skeletal muscle; lane 4, liver; lane 5, lung; lane 6,
placenta; lane 7, brain; lane 8,
heart.
In the present study, we have cloned and sequenced two human
cDNAs encoding novel proteins that exhibit all the structural features
characteristic of the ovalbumin branch of the serpin superfamily. In
particular, the new proteins showed greatest amino acid sequence
identity with a recently discovered member of the ovalbumin serpins
that has been designated by the Genome Database organization as
proteinase inhibitor 6, or PI6. The extent of amino acid sequence
identity between the new serpins and PI6 (63 or 68%) significantly
exceeds that reported for any other combination of the ovalbumin serpin
family members, which typically range between 45 and 50%(10) .
The new serpins have been designated at PI8 and PI9 according to
recommendations made by the Genome Database nomenclature committee.
Based on primary structure identity and the presence of a unique Cys
residue conserved in the reactive center P`position of the
cytoplasmic antiproteinases, these serpins appear to represent a
distinct subfamily within the ovalbumin branch of the serpin
superfamily. Since serpins are generally classified by the reactive
center P
specificity residue(1, 2) , PI8
appears to be an Arg-serpin and, like PI6, inhibits trypsin amidolytic
activity and forms an SDS-stable complex with human thrombin. On the
other hand, PI9 was found to be unique in that it is the first human
serpin identified with an acidic residue in the reactive center P
position and has been classified as a Glu-serpin. PI6 has been
previously shown to function as a proteinase inhibitor of several
prototypical serine proteinases (16, 17) , and PI8 and
PI9 both show complete conservation of the PI6 reactive center hinge
region that conforms to the structural motif
P
EEGTEAAAATP
recently identified in the
majority of inhibitory serpins(35) . Serpins that carry
unconserved mutations in the reactive center hinge region typically
lack serine proteinase inhibitory activity since steric hindrance
impedes partial insertion of the hinge peptide into the antiparallel
A-
sheet(36, 37) , which normally provides a
source of favorable interactions that contribute to the overall
stability of the proteinase-serpin inhibitory complex. Therefore, since
the reactive center hinge sequence of PI9 conforms precisely with the
hinge sequences of other inhibitory serpins, this novel cytoplasmic
antiproteinase is likely to function as active site-directed proteinase
inhibitor.
The novel cytoplasmic antiproteinases displayed all of the structural characteristics common to the mammalian serpins of the ovalbumin family, including the apparent absence of a typical N-terminal cleavable signal sequence(10) . These findings suggest that, like PI6 (16, 17, 38) , EI(12, 13) , PAI-2(39, 40, 41) , and SCCA(14) , the new serpins also reside in the cytoplasm of cells. Nonetheless, similar to other members of the ovalbumin serpin family, PI8 and PI9 have consensus sites for the potential attachment of N-linked carbohydrate. Therefore, the possibility that PI8 and PI9 are secreted and function in the extracellular milieu under certain conditions cannot be ruled out at present. Previous studies have demonstrated that PAI-2 (40, 41) and SCCA (14) can exist intracellularly and/or extracellularly. For example, PAI-2 has been detected predominantly in the cytosolic fraction of resting monocytes as an unglycosylated functionally active inhibitor of urokinase(40, 41) . Upon activation of the monocytes with phorbol esters, the majority of intracellular PAI-2 is efficiently glycosylated and secreted(40, 41) . Consequently, the degree of glycosylation has no effect on the inhibition kinetics of urokinase by PAI-2(40) . Likewise, SCCA has been reported to be localized in the cytoplasm of normal squamous epithelial cells while the corresponding carcinoma cells secrete a glycosylated form of SCCA (14) , apparently in a manner similar to that described for PAI-2. To date, PI6 has been detected only in the cytosolic fraction of all cultured cells examined, and treatment of cells with phorbol esters had no effect on the intracellular localization of functionally active PI6 (16, 18, 38) . Given the high degree of amino acid sequence identity in regions outside the reactive centers of the cytoplasmic antiproteinases, PI8 and PI9 may also behave like PI6 and be confined to the cytoplasm of cells.
To date, the only
intracellular serpin with a defined intracellular proteinase target is
the viral serpin encoded by the crmA gene of the cowpox
virus(5) . CrmA functions as a specific inhibitor of the
ICE(5) , which has recently been shown to represent a prototype
of a larger family of ICE-like
homologs(42, 43, 44, 45, 46) .
The ICE family of cysteine proteinases have been intimately linked to
both the negative and positive regulation of apoptosis(44) .
Mammalian intracellular inhibitors of these cysteine proteinases have
not been identified. In the present study, we have made the previously
unreported observation that CrmA shows the greatest degree of amino
acid sequence identity to the mammalian intracellular serpins of the
ovalbumin family, suggesting a possible origin of the crmA gene. In addition, the reactive center of CrmA shows considerable
structural similarity to the reactive center of PI9, including a
conserved Asp to Glu switch in the P specificity site.
Moreover, all of the cytoplasmic antiproteinases have a unique Cys
residue conserved in the P
` position and found only in the
corrresponding position of CrmA. CrmA has been shown recently to form a
noncovalent tight inhibitory complex with ICE(47) , suggesting
that the geometric compatibility between serpins and some proteinases
is important in determining inhibitory potency. Therefore, since the
cytoplasmic antiproteinases are the only mammalian serpins with a Cys
residue in the P
` position, they may regulate proteinases
by a mechanism analogous to CrmA.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L40377 [GenBank]and L40378[GenBank].