(Received for publication, July 7, 1995; and in revised form, September 22, 1995)
From the
The serum level of placental leucine aminopeptidase (P-LAP) increases during pregnancy. P-LAP degrades several peptide hormones such as oxytocin and vasopresin, suggesting a role in maintaining homeostasis during pregnancy. In the study reported here, we have isolated a cDNA clone with 4084 base pairs encoding P-LAP from a human placental cDNA library. The amino acid sequence deduced from the cDNA contained all of the sequences of the peptide fragments obtained by digestion of the purified protein with trypsin. The predicted P-LAP contains the HEXXH consensus sequence of zinc metallopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase N and aminopeptidase A. The deduced sequence also contains a hydrophobic region near the N terminus, suggesting that the enzyme is a type II integral membrane protein. Northern blot analysis revealed that P-LAP was expressed in several tissues, some of which expressed two forms of mRNAs. These results suggest that the enzyme is synthesized as an integral membrane protein and is released into blood under some physiological conditions.
Aminopeptidases catalyze the cleavage of amino acids from the N terminus of protein or peptide substrates. They are widely distributed in animal tissues and found in many subcellular organelles, in cytoplasm, and as membrane components. They are shown to be essential for protein maturation, degradation of nonhormonal and hormonal peptides, and determination of protein stability(1) . It is well recognized that placental aminopeptidases play a role in degradation of many active peptide hormones, such as kinins, angiotensin, and oxytocin, and thus maintain the physiological conditions during normal pregnancy(2) . There are also several aminopeptidases in maternal serum, and their contents are shown to increase during pregnancy (3, 4, 5) . Among them, cysteine aminopeptidase is shown to degrade oxytocin and is regarded as oxytocinase (EC 3.4.11.3)(2, 6, 7) . Oxytocinase is defined as an enzyme that inactivates oxytocin by hydrolysis of the peptide bond between half cysteine and tyrosine.
Placental leucine
aminopeptidase (P-LAP), ()whose activity is measured by its
action on the hydrolysis of L-leucine
-naphthylamide, is
known to increase in maternal serum during pregnancy(7) . In
our previous work, we have purified P-LAP from retroplacental serum and
found that P-LAP was identical with cysteine
aminopeptidase(8) . Purified P-LAP degraded several peptide
hormones such as oxytocin and vasopressin, whose increase may have a
significant effect on the uterine tonus and uteroplacental blood flow,
suggesting that the enzyme might regulate the serum level of the
hormones and thus maintain homeostasis during pregnancy. The enzyme is
a glycoprotein and exists as an oligomer, presumably a dimer.
In the current study, we have cloned P-LAP cDNA and found that the enzyme belongs to the family of zinc metallopeptidases. Our data suggest that the enzyme is first synthesized as a type II integral membrane protein and then secreted into blood. Moreover, we found by Northern blot analysis that transcripts of P-LAP were expressed in several tissues, some of which expressed two different-sized mRNAs, suggesting that P-LAP gene is regulated in a tissue-specific manner.
The digested materials were separated by reversed-phase HPLC on a
Vydac C column using a linear gradient of acetonitorile in
0.1% (v/v) trifluoroacetic acid. The amino acid sequences of the
purified tryptic fragments were determined using an Applied Biosystems
477A gas phase sequencer.
The coding area of the cDNA for P-LAP was amplified by polymerase chain reaction(9) . Two degenerate oligonucleotides were synthesized based on the peptide sequences determined above. The sequence of the 5`-oligomer was 5`-AACATGAAGCCTGA(G/A)AT(C/T/A)CA(G/A)CC-3` and that of 3`-oligomer was 5`-CTGGAT(C/T)TG(G/A)TT(C/T)TG-3`. The reaction was carried out on a Perkin-Elmer thermal cycler. The following amplification protocol was employed: 30 cycles of denaturing at 94 °C (1 min), primer annealing at 57 °C (1.5 min), and primer extension at 72 °C (3 min). Initial amplification of the cDNA revealed the presence of a 477-base pair amplified product. This polymerase chain reaction product was sequenced and used as a probe to screen the cDNA library.
Bacteriophages were plated at a density of approximately 2
10
plaque forming units/150-mm plate. They were transferred
to a nitrocellulose filter and then treated as described
previously(10) . Hybridization was carried out using ECL random
prime labeling and detection systems (Amersham Corp.) as recommended by
the manufacturer. DNA sequence was determined by the method of Sanger et al.(11) using a Taq dye primer cycle
sequencing kit and an Applied Biosystems model 373A DNA sequencer.
Figure 1: Partial restriction map and sequencing strategy of 7-3. Only the restriction endonuclease sites relevant to the present work are shown. Arrows indicate the direction and extent of each sequence determination. The protein coding region is indicated by a closed bar.
Fig. 2shows the nucleotide sequence and the deduced amino acid sequence of the cDNA. DNA sequence analysis revealed that this cDNA contained a 3` poly(A) tail preceded by a poly(A) signal 19 nucleotides upstream and an open reading frame of 2832 base pairs, ending with a TAG stop codon at nucleotides 2833-2835. The deduced amino acid sequence contained the sequences of all of the peptide fragments determined. The N-terminal amino acid of the P-LAP purified from retroplacental serum was putatively assigned to be Ala-74. Although there is no methionine codon in the N-terminal region that matches the criteria for a consensus initiator methinonine(12) , we assigned the first Met as shown in the figure by analogy with other type II membrane-spanning aminopeptidases, since it is the last ATG codon that precedes the putative transmembrane region of the enzyme. Indeed aminopeptidase N and aminopeptidase A, which show 33.8 and 32.1% identity at the amino acid level and 45.6 and 45.1% at the nucleotide level with P-LAP, respectively (using the algorithm of Needleman and Wunsch)(13, 14, 15, 16) , have short cytoplasmic regions at the N terminus, and the last methionine codons preceding the transmembrane regions were assigned as the initiation codons. It should be noted here, however, that unlike the other cases(14, 15) , the putative initiation codon for P-LAP is not preceded by any termination codon within the cloned sequence that could code for 47 amino acids including two methionines. The identification of the real translation initiation site is left for future studies.
Figure 2: Nucleotide sequence of the cDNA and the deduced primary structure of P-LAP/oxytocinase. Nucleotide residues are numbered from 5` to 3` with the first residue of the ATG codon encoding the putative initiating methionine. The deduced amino acid sequence is displayed below the nucleotide sequence as a single-letter code starting from the methionine. The putative transmembrane-spanning sequence is underlined, the potential glycosylation sites are encircled, and the HEXXH consensus sequence and conserved glutamic acid are boxed. The dotted line represents amino acid sequences determined. The putative N-terminal amino acid residue of P-LAP purified from retroplacental serum is indicated by an asterisk. In the 3`-nontranslated region, the putative polyadenylation signal is represented by a double line.
Figure 3: Schematic primary structure of P-LAP. The protein is represented with its putative N-terminal domain facing the cytoplasm. The putative membrane-spanning domain (black box) is followed by a large extracellular domain. The possible N-glycosylation sites are indicated by lollipops. C, cysteine residues. Position of the consensus sequence of the active site (HELAH) is also shown.
Hydropathy analysis of P-LAP using the Kyte and Doolittle algorithm (17) is shown in Fig. 4. As is clear, P-LAP carries a significantly hydrophobic region only near the N terminus. By analogy to aminopeptidase N and aminopeptidase A, we predict that the P-LAP is a type II integral membrane protein. After a close look at this region, we assigned the membrane-spanning domain to the sequence starting from amino acid 29 to 51, which is flanked by arginine residues at both ends. It is likely that this domain might act both as a signal and membrane anchor as in the case of neuraminidase and asialoglycoprotein receptor(18, 19) .
Figure 4: Hydropathy profile of the amino acid sequence of P-LAP. The method of Kyte and Doolittle (16) with averaging over a window of 6 residues was used. Negative values correspond to hydrophilic regions and positive values to hydrophobic regions. The arrow indicates the only potential membrane-spanning region of P-LAP primary sequence.
There are 8 cysteine residues in the P-LAP molecule, 4 of which are clustered in the N-terminal region of the enzyme. Although it is possible that these 4 cysteine residues, including Cys-22 and Cys-33 (which occur in the putative cytoplasmic and transmembrane domains, respectively) are involved in the dimerization of the enzyme, no other Cys residues contribute to the dimerization, because, in the presence of SDS, the soluble enzyme lacking these domains dissociated into its monomeric form(8) .
Within the large extracellular domain is contained the consensus sequence of the zinc metallopeptidase family. Namely, there is a putative zinc-binding site (HEXXH) of the protease at amino acid residues 383-387 with a second glutamic acid separated by 18 amino acids(20, 21) . Unlike human aminopeptidase A(15) , there are no potential sites for the addition of glycosaminoglycans (i.e. SGXG)(22) .
Figure 5:
Southern blot analysis of human P-LAP
gene. Human genomic DNA (10 µg/lane) was digested with various
restriction endonucleases and probed with P-labeled cDNA
fragment corresponding to the nucleotide sequence of 1425-1794. Arrows indicate the positions of the bands hybridized with the
probe. Positions of molecular size markers are indicated at right.
Figure 6:
Northern blot analysis of
poly(A) RNA from various human tissues. A human adult
tissue Northern blot (Clontech) was probed with
P-labeled
cDNA as described under ``Experimental Procedures.''
Positions of molecular size markers are indicated at right.
In this study, we have cloned the cDNA encoding human P-LAP. The deduced amino acid sequence contained the sequences of all the peptide fragments determined. The predicted P-LAP contains three domains, a 28-amino acid N-terminal domain, a 23-amino acid hydrophobic domain, and a 893-amino acid C-terminal domain. The role of short N-terminal cytoplasmic domain is not clear at present and should be elucidated in the future studies. As with the other type II integral membrane proteins(18, 19) , the hydrophobic domain near the N terminus would function as both an internal signal peptide and a membrane-spanning domain. In the extracellular C-terminal domain, the cDNA predicts a protein that contains the HEXXH consensus sequence of zinc metallopeptidases with an additional glutamic acid residue 18 amino acids away, which constitutes the active site of metallopeptidases(20, 21, 23) . We are now conducting further studies by site-directed mutagenesis to identify the active site residues of the enzyme.
A computer search revealed that the amino acid sequence of P-LAP has homology to other members of the family of zinc metallopeptidases, such as aminopeptidase N and aminopeptidase A, both of which are also known to be type II integral membrane glycoproteins (Fig. 7) (13, 14, 15) . While aminopeptidase N shows 33.8% identity with P-LAP at the amino acid level, 32.1% identity is observed between P-LAP and aminopeptidase A. The homology is especially striking in the region containing the consensus zinc-binding motif. These homologies also suggest a possible evolutionary relationship between these enzymes and underline the importance of the zinc-binding region in the enzymatic activity.
Figure 7: Alignment of the predicted P-LAP protein with human aminopeptidase A and aminopeptidase N amino acid sequences. Identical residues are boxed. For optimal alignment, several gaps were inserted into the sequences. The potential zinc-binding motif is indicated by asterisks.
While cloned cDNA contains a
putative transmembrane domain, we have purified a soluble enzyme from
retroplacental serum. It is possible that the soluble enzyme derives
from the membrane-bound enzyme by post-translational proteolytic
processing near the anchoring domain, i.e. between Phe-73 and
Ala-74. Several membrane-bound zinc metallopeptidases, such as meprin
subunit (24) and angiotensin-converting
enzyme(25, 26) , were shown to be cleaved
proteolytically and secreted. However, proteases responsible for the
cleavage were not identified yet. Alternatively, alternative splicing
of mRNA can account for the presence of both membrane-bound and soluble
forms of enzymes and cytokines. Schauder et. al(27) recently suggested that the mechanism was operating in
case of thyrotropin-releasing hormone degrading enzyme, another type II
membrane bound zinc metallopeptidase. It was also reported that
macrophage colony stimulating factor was encoded by a single gene and
two forms of mRNAs, one coding for a rapidly secreted molecule and the
other primarily a membrane-bound form, were generated by alternative
splicing(28) .
We have detected at least two forms of mRNA
that might encode P-LAP by Northern blot analysis. At present we cannot
elucidate the biological significance of the presence of these two
mRNAs. However, multiple molecular species encoded by different mRNAs
might exhibit significantly different biological behavior. The two
forms of mRNA are most likely derived from a gene by alternative
splicing, since Southern blot analysis revealed the presence of a
single gene. However the wide difference in the size is rather
unexpected. In the case of human angiotensin-converting enzyme, which
has a gene consisting of 26 exons, the endothelial enzyme has two
highly similar tandem catalytic domains and is encoded by a 4.3-kb-long
transcript, while testicular enzyme has only one catalytic domain and
its transcript is 3.0 kb long. The former mRNA is transcribed from exon
1 to exon 26, excepting exon 13, while the latter is transcribed from
exon 13 to exon 26. The gene derived apparently from gene
duplication(29) . The occurrence of such duplicated structures
are also known in other brush-border anchored enzymes, including
sucrase isomaltase and lactase-phlorizin
hydrolase(30, 31) . By analogy with the above
mentioned enzymes, it is possible that the protein could have multiply
duplicated structures, since the 10.5-kb transcript is 3 times longer
than that of 3.6 kb. It is also possible that P-LAP is encoded by
closely related genes and that the soluble enzyme might derive from one
of the mRNA (presumably 3.6-kb species). It was shown that and
subunits of meprin are encoded by two different but closely
related genes and that both subunits have transmembrane
domains(32, 33) . While the meprin
subunit is
anchored in the membrane, the
subunit is secreted rapidly via
proteolytic process(34, 35) . Alternatively, the
difference could be due to different sizes of nontranslated regions of
the mRNAs. Cloning and expression of cDNAs of both forms of mRNA will
make it possible to elucidate the relationship of these mRNAs and the
secretion mechanism of P-LAP.
In the present study, we have cloned the cDNA for P-LAP and elucidate the structural features of the enzyme. It is believed that P-LAP plays a role in maintenance of the placental homeostasis through degradation of peptide hormones and is an indicator of placental function(2, 8) . It is also noteworthy that several membrane-bound zinc metallopeptidases are shown to function as hematopoietic differentiation antigens and control cell proliferation(36, 37, 38) . The availability of cDNA and recombinant protein will make it possible to characterize this new enzyme with respect to its physiological and pathological function in detail.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D50810[GenBank].