©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Placental Leucine Aminopeptidase/Oxytocinase
A NEW MEMBER OF TYPE II MEMBRANE-SPANNING ZINC METALLOPEPTIDASE FAMILY (*)

(Received for publication, July 7, 1995; and in revised form, September 22, 1995)

Tomohiro Rogi Masafumi Tsujimoto (§) Hiroshi Nakazato Shigehiko Mizutani (1)(¶) Yutaka Tomoda (1)

From the Suntory Institute for Biomedical Research, Mishima-gun, Osaka, 618, Japan Department of Obsterics and Gynecology, Nagoya University School of Medicine, Tsurumai-cho, Showa-ku, Nagoya, 466, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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), (^1)whose activity is measured by its action on the hydrolysis of L-leucine beta-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.


EXPERIMENTAL PROCEDURES

Amino Acid Sequences of Peptides Derived from P-LAP

P-LAP was purified from retroplacental serum by serial chromatography on Matrex Blue A, DEAE-Sepharose, phenyl-Sepharose, chelating-Sepharose, and gel filtration columns as described previously(8) . To determine the partial amino acid sequences, purified protein was carboxymethylated with dithiothreitol and iodoacetate in the presence of 6 M guanidine hydrochloride at pH 8.0. The protein was then dialyzed against 0.1% (v/v) trifluoroacetic acid, purified by reversed-phase HPLC, and lyophilized. Carboxymethylated P-LAP was dissolved in 0.2 ml of 0.1 M ammonium bicarbonate and digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin at a protein/enzyme ratio of 50:1 (w/w) for 16 h at 37 °C.

The digested materials were separated by reversed-phase HPLC on a Vydac C(18) 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.

cDNA Cloning of P-LAP

Poly(A) RNA was purified from total RNA extracted from human placenta using oligo(dT)-latex (Takara Shuzo). The human placental cDNA library was then synthesized from poly(A) RNA using a cDNA synthesis kit obtained from Life Technologies, Inc. The cDNA was ligated into gt22A, NotI-SalI arm, and packaged into -phage employing Gigapack II packaging extracts (Stratagene).

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 times 10^4 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.

Southern Blot Analysis

Human genomic DNA obtained from Clontech was digested with an excess of restriction endonucleases and size-fractionated by electrophoresis on 0.8% agarose gels. The fragments were denatured, transferred to a nylon membrane, hybridized with different cDNA fragment and washed under high stringency conditions (final washing conditions: 0.1 times SSC, 0.1% SDS for 60 min at 65 °C).

Northern Blot Analysis

The human multiple tissue blot was obtained from Clontech. The filter-bound RNA was hybridized with the P-labeled P-LAP fragment corresponding to the nucleotide sequence of 133-2437 of P-LAP employing Rapid-hyb buffer (Amersham Corp.) following the instruction manual.


RESULTS

Sequencing of the Peptide Fragments of P-LAP

P-LAP was purified to homogeneity from retroplacental serum as described previously(8) . Peptide fragments of the enzyme were generated by enzymatic digestion with trypsin and purified by reversed-phase HPLC. Several fragments were isolated, and their sequences were determined. The same or homologous amino acid sequences have not been found in known proteins, indicating that P-LAP is a novel protein(8) . The N-terminal sequence was also analyzed and determined as follows: XXXXXLFXWAQIXL. Because of the limited availability of the enzyme, we could not identify first five amino acid residues in the analysis.

Cloning and Sequencing of cDNA for P-LAP

Based on the amino acid sequence determined (i.e. FFLNMKPEIQPSD and LLQNQIQQQTXTDEGTP), a polymerase chain reaction experiment with degenerate primers was performed, resulting in the amplification of a cDNA fragment. The obtained fragment coded for the additional amino acids that had not been used for the primer construction. Using this fragment as a probe, we screened a placental cDNA library, identified one positive clone (designated as 7-3), having an insert of 4.1 kilobases and sequenced (Fig. 1). Most of the final sequence (92%) was determined from both strands.


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.



Primary Structure of P-LAP

Fig. 3shows the principal features of the primary structure of P-LAP. The predicted translation product of the P-LAP cDNA encodes a protein of 944 amino acids (excluding the initiator methionine), with a calculated molecular weight (M(r)) of 107,886. The predicted primary structure of the protein contains 18 potential N-glycosylation sites distributed throughout the molecule, which might account for the difference between the M(r) calculated from the amino acid composition and that estimated by SDS-polyacrylamide gel electrophoresis of the native enzyme.


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) .

Southern Blot Analysis

Several fragments from different regions of the cDNA were used for Southern analysis with human genomic DNA. As shown in Fig. 5, BglII digestion gave a single DNA fragment of about 12 kb, suggesting the presence of a single gene. Detection of two bands in the PstI digest also suggested a single gene because the probe employed contained one PstI site. The existence of multiple DNA fragments in EcoRI and HindIII digests should reflect the presence of restriction sites in the introns that are present in the genomic DNA.


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.



Distribution of P-LAP in Human Tissues

Expression of P-LAP mRNA in various human tissues was examined by Northern blot analysis employing the truncated cDNA insert corresponding to the nucleotide sequence of 133-2437 as a probe (Fig. 6). In lung, liver, and kidney, we could not detect any bands that hybridized with the probe. On the other hand, two forms of transcripts corresponding to 3.6 and 10.5 kb were detected in placenta, heart, and skeletal muscle, and the larger species was expressed more than the smaller one. The size of 3.6-kb transcript corresponded to that of cDNA we cloned. In brain, although a 10.5 kb band was detectable, we could not observe a 3.6 kb band. To confirm the presence of two forms of mRNA, we have performed Northern analysis extensively employing newly extracted mRNA from human placenta and different cDNA fragments. Although the relative amount of two mRNAs was not constant, we could reproducibly detect two bands. In other tissues such as spleen, colon, and small intestine, the 10.5 kb band was also predominantly expressed (data not shown). These results suggest that there are at least two types of mRNA encoding P-LAP, and expression of these mRNA may be regulated in a tissue-specific manner.


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.




DISCUSSION

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 alpha 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 alpha and beta subunits of meprin are encoded by two different but closely related genes and that both subunits have transmembrane domains(32, 33) . While the meprin beta subunit is anchored in the membrane, the alpha 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D50810[GenBank].

§
To whom correspondence should be addressed: Suntory Inst. for Biomedical Research, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, 618, Japan. Tel.: 81-75-962-9285; Fax: 81-75-962-6448.

To whom reprint request should be addressed.

(^1)
The abbreviations used are: P-LAP, placental leucine aminopeptidase; HPLC, high performance liquid chromatography; kb, kilobase pair(s).


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