(Received for publication, April 24, 1995; and in revised form, September 1, 1995)
From the
We have isolated by immunological screening of a ZAPII cDNA
library constructed from rat brain mRNAs a cDNA clone encoding
endopeptidase 3.4.24.16. The longest open reading frame encodes a
704-amino acid protein with a theoretical molecular mass of 80,202
daltons and bears the consensus sequence of the zinc metalloprotease
family. The sequence exhibits a 60.2% homology with those of another
zinc metallopeptidase, endopeptidase 3.4.24.15. Northern blot analysis
reveals two mRNA species of about 3 and 5 kilobases in rat brain,
ileum, kidney, and testis. We have transiently transfected COS-7 cells
with pcDNA
containing the cloned cDNA and established the
overexpression of a 70-75-kDa immunoreactive protein. This
protein hydrolyzes QFS, a quenched fluorimetric substrate of
endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide
bond, leading to the formation of
neurotensin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and
neurotensin (11, 12, 13) . QFS and
neurotensin hydrolysis are potently inhibited by the selective
endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile and by
dithiothreitol, while the enzymatic activity remains unaffected by
phosphoramidon and captopril, the specific inhibitors of endopeptidase
3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether,
these physicochemical, biochemical, and immunological properties
unambiguously identify endopeptidase 3.4.24.16 as the protein encoded
by the isolated cDNA clone.
Endopeptidase 3.4.24.16 is a metalloendopeptidase ubiquitously
distributed in the central nervous system and in peripheral organs of
mammals(1) . This enzyme was first detected (2) and
later purified (3) on the basis of its ability to cleave the
Pro-Tyr
bond of the tridecapeptide
neurotensin, leading to the formation of the biologically inactive
catabolites,
neurotensin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and
neurotensin (11, 12, 13) . Studies on
neurotensin catabolism in vitro by membrane fractions or cell
lines of central or peripheral origin indicated that endopeptidase
3.4.24.16 was the only peptidase that ubiquitously contributed to the
inactivation of this neuropeptide(4) . Several lines of
evidence later suggested that endopeptidase 3.4.24.16 indeed
participated to neurotensin inactivation in vivo in the
gastrointestinal tract(5) . Thus, by means of a vascularly
perfused model of dog ileum, we showed that the dipeptide Pro-Ile, a
fully selective blocker of endopeptidase 3.4.24.16(6) ,
inhibited the formation of one of the major catabolites, i.e. neurotensin (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 5) ,
leading to a drastic protection of neurotensin from degradation. In the
central nervous system, we recently showed that mixed inhibitors of
endopeptidases 3.4.24.16 and 3.4.24.15 potently enhanced the
neurotensin-induced analgesia in the hot plate-tested mice (7) . Altogether, this indicates that endopeptidase 3.4.24.16
contributes to the catabolism of neurotensin in vivo in the
periphery but also likely in the central nervous system.
The characterization of the biochemical and pharmacological properties of endopeptidase 3.4.24.16 indicated that the enzyme behaved as a 70-75-kDa monomer that was inhibited by metal chelators and dithiothreitol(3) . Several studies suggested that endopeptidase 3.4.24.16 resembles another metallopeptidase, endopeptidase 3.4.24.15. Particularly, studies on the specificity of endopeptidase 3.4.24.15 showed that the enzyme cleaved several neuropeptides at peptidyl bonds that were reminiscent of those targeted by endopeptidase 3.4.24.16(8, 9) . However, several aspects that included the nature of the cleavage site on neurotensin, the sensitivity to dipeptide inhibitors and dithiothreitol, as well as immunological data clearly distinguished the two peptidases(10) . The present paper reports on the molecular cloning and expression of rat brain endopeptidase 3.4.24.16 and establishes that the two peptidases are related but clearly distinct molecular entities.
The cDNAs of 14 isolated positive clones
were subcloned into pBluescript by in vivo excision according
to the manufacturer's procedures (Stratagene). A clone, 7a
with an insert of 1806 bp, (
)was sequenced and showed an
open reading frame of 1613 bp, lacking the 5`-region coding for the
N-terminal domain of the protein. Using two synthetic oligonucleotides,
a polymerase chain reaction fragment of 1390 bp was derived from the
7a clone, labeled with
P by random-priming
(Appligene), and used as a probe to screen 6
10
clones of the above
ZAPII cDNA library. Hybridization was
carried out overnight at 65 °C in 6
SSC, 0.1% SDS, 5
Denhardt's solution, and 0.2 mg/ml heat-denatured herring
sperm DNA. The filters were washed in 3
SSC, 0.1% SDS at room
temperature and autoradiographied. A clone,
B1 containing an
insert of 2158 bp, was isolated. This clone encompassed 1516 bp of
clone
7a (Fig. 1) but lacks the complete 3`-region as
illustrated by the absence of a stop codon. Therefore the full-length
cDNA was reconstituted with these two overlapping cDNAs by ligating a
380-bp NcoI-EcoRI fragment of
7a with a 2120-bp KpnI-NcoI fragment of
B1. The resulting insert,
7aB1, was subcloned in pBluescript previously digested with KpnI-EcoRI. This construction allowed us to confirm
the whole sequence of the cDNA and to verify that the ligation of the
two fragments occurred without introduction of errors in the coding
phase.
Figure 1:
Schematic
representation of endopeptidase 3.4.24.16 cDNA clones and sequencing
strategy. The 5`- and 3`-untranslated sequences of 7aB1 are
represented by a line, and the open reading frame is indicated
by an open bar, on which the position of the NcoI
restriction site is indicated. The whole cDNA was reconstituted from
two independent overlapping clones,
7a and
B1, as described
under ``Experimental Procedures.'' Horizontal arrows indicate the direction and the extent of the sequences determined
by the use of internal oligonucleotides.
Figure 2: Nucleotide and deduced amino acid sequences of rat brain endopeptidase 3.4.24.16 cDNA. Nucleotides and amino acid residues are numbered on the right column. Amino acids are numbered from the first methionine residue and identified with the single letter code. Three possible initiation sites of the translation are indicated by the three circled methionines presented in bold. The stretches of charged amino acids are underlined. The consensus sequence of zinc metallopeptidases is boxed. An asterisk indicates the stop codon (TAA) of the open reading frame.
Figure 3: Hydropathic profile of endopeptidase 3.4.24.16 protein sequence. Hydropathy analysis of the amino acid-deduced sequence was obtained by the method of Kyte and Doolittle (44) with a window size of 10 residues.
Northern blot analysis performed with rat brain, ileum,
kidney, and testis poly(A) mRNAs consistently revealed
two mRNA species of about 3 and 5 kilobases, the lower molecular weight
label always being prominent (Fig. 4). It is noticeable that the
label was lower in brain than in other tissues (Fig. 4). This is
in agreement with our data concerning the relative endopeptidase
3.4.24.16 activities detected in various tissues(14) . It is
not yet clear as to whether the discrepancy observed between the sizes
of these two mRNA species reflects a variable length of their
non-coding 3`-region, consequently, to two distinct polyadenylation
sites. An alternative hypothesis could be that the higher molecular
weight mRNA represents an intermediate immature form of the mRNA.
Finally, the possibility that the mRNAs encode two distinct proteins
could be evoked. However, such a hypothesis is not sustained by our
previous data indicating that in whole rat brain homogenate, a tissue
that would be expected to contain all the various putative molecular
forms of the peptidase, we consistently detected a single immunolabeled
protein migrating with the apparent molecular weight of the recombinant
peptidase(1) .
Figure 4:
Northern blot analysis of endopeptidase
3.4.24.16 mRNA. Poly(A) RNA (5 µg) from various
rat tissues was fractionated on a 1% formaldehyde/agarose gel, blotted
on a nylon membrane, and hybridized with the
P-labeled
polymerase chain reaction fragment derived from a
7a clone as
described under ``Experimental Procedures.'' RNA molecular
weight markers are indicated in kilobases (kb) on the left.
Figure 5:
Western blot analysis of the protein
expressed by pcDNA-
7aB1 transfected COS-7 cells. A, homogenate proteins (10 µg) of pcDNA
(lane 1) or pcDNA
-7aB1 (lane 2)
transfected COS-7 cells were electrophoresed on a 8% SDS-polyacrylamide
gel and then blotted onto a nylon membrane. The recombinant protein was
labeled with the IgG-purified fraction of an antiserum raised against
endopeptidase 3.4.24.16 as described under ``Experimental
Procedures.'' Molecular weight standards are indicated in kDa on
the right. B, proteins (10 µg) from transfected
cell homogenate (total) and subcellular fractions (mb,
membrane-associated; sol, soluble; 100,000
g supernatant) were incubated for 10 h at 37 °C in
absence(-) or in the presence (+) of 0.15 units of
endoglycosidase F as described under ``Experimental
Procedures.'' Samples were submitted to SDS-PAGE and Western blot
analysis in the conditions described above.
Figure 6:
Hydrolysis of QFS by
pcDNA-7aB1 transfected COS-7 cells and the effect of
Pro-Ile and dithiothreitol. QFS (50 µM) was incubated for
the indicated times at 37 °C with 10 µg of protein homogenates
from pcDNA
(
) and pcDNA
-7aB1 (
)
transfected COS-7 cells, and then hydrolysis was fluorimetrically
monitored as described under ``Experimental Procedures'' (A). Mcc-Pro-Leu release was quantified by comparing the
fluorescence with that obtained with known amounts of the synthetic
peptide. QFS hydrolysis by pcDNA
-7aB1 transfected COS-7
cells was performed as described under ``Experimental
Procedures'' and HPLC analyzed. Arrows indicate the
elution times of synthetic Mcc-Pro-Leu and QFS run in the same HPLC
conditions. Small arrows indicate background absorbance
obtained with hydrolysis of QFS with mock-transfected pcDNA
COS-7 cells (B). Hydrolysis of QFS by
pcDNA
-7aB1 transfected COS-7 cells in absence (control) or
in the presence of the indicated concentrations of Pro-Ile (C)
and dithiothreitol (D) was monitored by fluorimetry as
described under ``Experimental Procedures.'' Data are
expressed as the percent of control fluorescence recovered in absence
of competing agent.
Figure 7:
Hydrolysis of neurotensin (NT) by
pcDNA-7aB1 transfected COS-7 cells and the effect of
Pro-Ile and dithiothreitol. Neurotensin (2 nmol) was incubated for 2 h
at 37 °C with 10 µg of protein homogenate from pcDNA
(A) and pcDNA
-7aB1 transfected COS-7 cells (B-D) in absence (B) or in the presence of 10
mM Pro-Ile (C) or 5 mM dithiothreitol (D). HPLC analysis was performed as described under
``Experimental Procedures.'' Arrows indicate the
elution times of synthetic peptides run in the same HPLC
conditions.
The immunological approach used in the present study has led us to isolate a cDNA clone that unambiguously encodes endopeptidase 3.4.24.16. First, the protein overexpressed in transfected COS-7 cells is recognized by the IgG-purified fraction of a specific polyclonal antibody developed toward rat brain endopeptidase 3.4.24.16(1) . Second, transfectant cells hydrolyze two peptides (QFS and neurotensin) at peptide bonds identical with those targeted by purified endopeptidase 3.4.24.16(3, 16) . Third, the catalytic activity of the protein produced by transfectants is fully inhibited by Pro-Ile, a dipeptide that selectively blocks endopeptidase 3.4.24.16(6) , and by dithiothreitol (19) in agreement with the pharmacological spectrum previously established for rat endopeptidase 3.4.24.16(14) . Furthermore, the activity remains insensitive to the specific inhibitors of angiotensin-converting enzyme, endopeptidase 3.4.24.11, and endopeptidase 3.4.24.15.
It is interesting to note that transfected
cells cleave neurotensin at a single peptide bond, giving rise to
neurotensin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and
neurotensin(11, 12, 13) . It was recently
suggested that endopeptidase 3.4.24.16 of porcine origin targeted the
same peptide bond but also triggered a minor production of
neurotensin(1, 2, 3, 4, 5, 6, 7, 8) and
neurotensin (9, 10, 11, 12, 13, 20) .
The present study clearly indicates that the production of
neurotensin(1, 2, 3, 4, 5, 6, 7, 8) and
neurotensin (9, 10, 11, 12, 13) by the
purified enzyme from porcine sources was indeed due to a peptidase
distinct from endopeptidase 3.4.24.16. This agrees well with our recent
work showing that such neurotensin(1, 2, 3, 4, 5, 6, 7, 8) and
neurotensin (9, 10, 11, 12, 13) production
derived from the participation of contaminating endopeptidase
3.4.24.15. ()
The purification of endopeptidase 3.4.24.16 from various rat organs allowed us to establish that the apparent molecular mass of the enzyme corresponded to 70-75 kDa(3, 21) . This appeared to be slightly lower than the molecular mass deduced from the longest open reading frame. Apparently, none of the nucleotidic sequences that abut to the ATG initiation codons fulfilled the structural features that unambiguously identify the eukaryotic Kozak sequence usually required to modulate the initiation of the translation(22) . However, according to the fact that a purinergic base appears generally required at the position 3 upstream to the initiation codon(22) , the second ATG codon appears the best candidate to initiate the genuine open reading frame encoding the enzyme. This will raise a protein of 77,724 daltons in good agreement with the reported molecular mass of the purified enzyme(3) .
We previously established that endopeptidase 3.4.24.16 was predominantly recovered in majority in a soluble form in the brain (23) . This also appears to be the case in transfected COS-7 cells as illustrated in Fig. 5B, which indicates a major soluble form of the protein, in agreement with the recovered activity in the two subcellular fractions (not shown). Previous immunological data clearly indicate that a minor fraction of endopeptidase 3.4.24.16 could exist in a genuine membrane-associated form in the brain(24) . This hypothesis was reinforced by light and electron microscopic analysis of the localization of endopeptidase 3.4.24.16 in rat mesencephalon(25) . Thus, it was shown that endopeptidase 3.4.24.16-like immunoreactivity could be characteristically associated with restricted zones of the plasma membrane of a subpopulation of neurons in the rat substantia nigra and ventral tegmental area(25) . Biochemical analysis of the type of association of endopeptidase 3.4.24.16 with the membrane of kidney microvilli indicated that the enzyme was not attached to the membrane by a glycosyl-phosphatidylinositol anchor (21) but partitioned in the detergent phase after Triton X-114 phase separation(21) , a physicochemical behavior that appears to be common to various intrinsic membrane proteins(26) .
Sequence analysis of endopeptidase 3.4.24.16 does not reveal the clearcut structural requirements generally fulfilled by intrinsic membrane-bound proteins. First, it is not possible to clearly delineate a N-terminal signal peptide that could serve as a membrane anchor, as has been shown for endopeptidase 3.4.24.11(27) . In agreement with this observation, it is noticeable that membrane-associated and soluble forms of endopeptidase 3.4.24.16 comigrate after SDS-PAGE and Western blot analysis experiments (Fig. 5B). Second, although there exist three putative glycosylation sites, deglycosylation experiments performed with the whole homogenate of transfected cells as well as with the membrane-associated and soluble enzymes did not affect the apparent molecular weight of the peptidase (Fig. 5B), in agreement with our previous biochemical data showing that endopeptidase 3.4.24.16 did not bind to various sugar-linked resins(3) . However, it is interesting to note that several clusters of hydrophobic residues can be deduced from the hydropathic profile of the protein that could be responsible for some protein-protein interactions. Furthermore, one can underline the presence of a stretch of charged residues at amino acids 331-335 and 341-348. This could be of importance with respect to a previous work showing that carboxypeptidase E displayed a similar domain rich in charged amino acids (28) that was shown to be responsible for the attachment of the ``membrane-bound'' carboxypeptidase E counterpart to the plasma membrane(29) . Mutagenesis analysis experiments should allow us to examine whether the above possibilities could account for the anchoring of the ``membrane-bound'' form of endopeptidase 3.4.24.16.
Purified endopeptidase 3.4.24.16 is sensitive to metal chelators such as EDTA and o-phenanthroline(3) . We showed that the activity of the apoenzyme could be restored upon incubation with various divalent cations, the most efficient recovery being obtained with zinc(19) . The sequence of endopeptidase 3.4.24.16 reveals the presence of an HEFGH sequence that confirms that the enzyme belongs to the zinc metalloprotease family(15, 30) .
The current knowledge of the biochemical and physicochemical features of endopeptidases 3.4.24.16 and 3.4.24.15 and their specificity toward various neuropeptides underlined that the two enzymatic activities share some similar properties(8, 9) . On the other hand, the two peptidases can be distinguished by their distinct cleavage sites for neurotensin(3, 8) , their sensitivity to dipeptide inhibitors(16, 23) , and by the lack of recognition of endopeptidase 3.4.24.15 by the IgG-purified fraction of the antiserum raised against rat brain endopeptidase 3.4.24.16(1, 10) . Furthermore, Orlowski et al.(8) reported on the activation of endopeptidase 3.4.24.15 by low concentrations of dithiothreitol while this peptidase appeared inhibited by higher concentrations of such agents. This appeared not to be the case for endopeptidase 3.4.24.16, which is never activated by dithiothreitol whatever the concentration that were examined(19) . According to the above considerations, it is therefore not unexpected to find that the sequence of endopeptidase 3.4.24.16 displays a 60.2% homology with that of endopeptidase 3.4.24.15(31) .
It is interesting to note that endopeptidase 3.4.24.15 exhibits a 35.5% homology with proteinase YscD(32) . This protein is encoded by the PRD1 gene borne by the chromosome III of yeast and was therefore claimed to be the yeast analog of endopeptidase 3.4.24.15(32) . However, the purified yeast enzyme generated neurotensin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) from neurotensin and was not activated by dithiothreitol(33) , two properties reminiscent of endopeptidase 3.4.24.16. The fact that endopeptidase 3.4.24.16 displayed a 35.7% identity with the yeast enzyme strongly suggests the possibility that YscD could indeed be the yeast counterpart of endopeptidase 3.4.24.16.
Recently, the complete sequence of a microsomal metalloendopeptidase from rabbit liver was established (34) and shows 90.3% identity with that of endopeptidase 3.4.24.16. Sequence homology strongly suggests that microsomal metalloendopeptidase corresponds to the rabbit counterpart of rat endopeptidase 3.4.24.16. However, very limited information exists on the specificity of this enzyme toward natural neuropeptides since Kawabata and Davie (35) only reported on the ability of microsomal metalloendopeptidase to cleave a synthetic peptide that mimics the amino acid sequence encompassing the processing site of vitamin K-dependent proteins. Further studies are clearly needed to document the specificity of microsomal metalloendopeptidase with respect to the known properties of endopeptidase 3.4.24.16. The sequence of endopeptidase 3.4.24.16 exhibits 24.2 and 25.6% homology with those of a rat liver mitochondrial intermediate peptidase (36, 37) and a dipeptidyl carboxypeptidase from Escherichia coli(38) , respectively. Finally, the enzyme did not align with the sequences of endopeptidase 3.4.24.11 (39, 40) and angiotensin-converting enzyme (41, 42, 43) .
The isolation of the cDNA clone of endopeptidase 3.4.24.16 should allow us to express a high amount of the recombinant protein. The recent design of highly potent inhibitors of endopeptidase 3.4.24.16 and their use to affinity purify the enzyme should allow us to obtain high quantities of pure enzyme. This tool should be of importance to examine the detailed structural features of the enzyme and to envision crystallographic experiments. The cDNA should also prove useful to delineate the putative biological signals that could modulate the level of expression of the peptidase as it seems to occur during the differentiation processes of primary cultured neurons(24) .