DNA AND CELL BIOLOGY Volume 9, Number 4, 1990 Mary Ann I.¡chert. Inc., Publishers Pp. 243-250

Human Placental Protein 11, a Putative Serine Protease with Diagnostic Significance as a Tumor Marker

Cloning and Expression of a cDNA Encoding

ULRICH GRUNDMANN, JÜRGEN RÖMISCH, BERNHARD SIEBOLD, HANS BOHN, and EGON AMANN

ABSTRACT with various The placental protein 11 (PP11) can act as a tumor marker because of its specific association a with screened was polyclonal antiforms of cancer. A Xgtll cDNA library prepared from human placenta the antiserum. The anti-PPll with PP11 antiserum. Out of 106 independent clones, only one clone reacted to rescreen a used was subsequently isolated cDNA coded only for the carboxy-terminal part of PP11 and the identified encoding entire proXgtlO placental cDNA library. Two cDNA clones out of 10" screened were 18 amino acids. Expression of tein of 369 amino acids, including a typical hydrophobic signal sequence of in of a the synthesis protein with the expected the PP11 cDNA coding sequence in Escherichia coli resulted Fractionation experiments resize which can be specifically immunoprecipitated with anti-PPll antiserum. of apvealed that two forms of the protein are present in the bacterial cell: a higher-molecular-weight form in the kD of 42 form approximately proximately 45 kD in the cytoplasm and a smaller-molecular-weight of the is removal E. in coli and processed by periplasm. This result indicates that PP11 can be synthesized a PP11 exhibit prorecombinant protein hydrophobic signal sequence. Both the placental and the processed tease

activity.

INTRODUCTION was first isolated and from aqueous extracts of hufrac-

(PP11) Placental physically characterized placentae, employing ammonium sulfate protein

man term

be the major site of PP11 in the placenta. No enzymatic activity or biological role has yet been reported for PP11. PP11 could not be detected in extracts of other normal human tissues and therefore is considered a placenta-specific protein (Bohn et al., 1980, 1981). More importantly, however, PP11 was associated with various malignant neoplasms (Inaba et al., 1982). PP11 was detected by immunohistochemical techniques in 47% of all breast cancers studied (Inaba et al., 1981), in 67% of all ovarian carcinomas studied (Inaba et al., 1980), and in 38% of all testicular and gastric cancers studied (Inaba et al., 1980). On the basis of immunochemical and immunohistological studies, PP11 (together with PP5) was designated a trophoblast- and carcinoma-associated antigen (TCA) (Inaba et al., 1982). Employing the PAP method for subcellular localization of PP11 in these malignant cells after fixation of thin sections, positive staining was only observed in the cytoplasm (Inaba et al., 1982). From these results it is evident that PP11 has diagnostic significance as a marker. The present study reports the complete amino acid se-

to

tionation, gel filtration, ion-exchange chromatography, and immunosorbent techniques (Bohn and Winckler, 1980). The protein has a sedimentation coefficient of 3.5S, a molecular weight of 44,300 as determined by analytical ultracentrifugation, and an isoelectric point of pH 5.1-5.2. The authors reported a carbohydrate content of 3.9%. In the above study, the yield of extractable PP11 after purification per human term placenta averaged 11 mg. Immunohistochemical studies employing normal placentae of man and monkey detected PP11 in the trophoblast cells of chorion, in the amniotic epithelium, and in human term placental villi (Inaba et al., 1980; Bohn et al., 1981; Wahlström et al., 1982). Since staining using an enzyme-bridge immunoperoxidase technique (PAP method) was much stronger for the villi, the syncytiotrophoblast is considered Research Laboratories, Behringwerke AG, 3550 Marburg, FRG.

243

GRUNDMANN ET AL.

244 quence for human placental protein PP11 as deduced from cDNA analysis and the expression of PP11 in Escherichia

0 (5490)

coli. Moreover, an enzymatic function (putative serine protease) could be assigned for PP11 and is found for both the placental and for the recombinant protein as expressed in E. coli. The knowledge of the complete PP11 amino acid sequence and the expression of its cDNA in E. coli in functional form will help elucidating its biological role in more detail.

MATERIALS AND METHODS

Screening of human placenta cDNA libraries A commercial human placenta Xgtll cDNA library (Genofit, Heidelberg) was screened for cDNA inserts encoding PP11 with a rabbit antiserum raised against PP11 (Bohn and Winckler, 1980). The library was blotted onto nitrocellulose filters and the bound antibody was detected by the ProtoBlot Immunoscreening System (Promega, Madison, WI) using anti-rabbit IgG alkaline phosphatase conjugate. Only one plaque could be isolated and was rescreened by the same procedure. Since the cDNA insert did not encode the amino-terminal peptide sequence, it was isolated and labeled with [32P]dCTP (Amersham Buchler, Braunschweig) by the hexanucleotide method and was used to rescreen a full-length human placenta XgtlO cDNA library (Grundmann et al., 1986). For hybridization with DNA the library was blotted on duplicate filters and hy-

FIG. 1. Structure of expression vector pTrc99C-PPll. The PP11 coding region is indicated by the crosshatched area, trc, trp/lac hybrid promoter; 5S Tl T2, 5S rRNA and transcriptional terminators of the rrnB operon of E. coli (Brosius et al., 1981); lacIQ, the IQ alíele of the lac repressor acting in eis on its operator present downstream of the trc promoter.

bridized at 68°C. The filters were washed at the same temperature as described (Grundmann et al., 1986).

Characterization of cDNA inserts Recombinant phages were plaque-purified, grown in liquid culture, and the phage DNA was extracted as described (Grundmann et al., 1986). cDNA inserts were characterized by restriction enzyme analysis and the sequence was determined by the dideoxy sequencing method (Sänger et al., 1977) after subcloning into the Bluescript Ml3 "KS-" vector (Stratagene Cloning System, La Jolla, CA). Oligodeoxynucleotides were synthesized on a Biosearch 8750 DNA Synthesizer to provide sequencing primers from either end of the sequence. Sequence data were analyzed with the UWGCG sequence analysis software as implemented on the Micro Vax II (Devereux et al., 1984). Construction

of plasmids

For expression of PP11 cDNA in E. coli, we used expression vector pTrc99C (Amann et al., 1988). This vector employs the strong trc promoter (Brosius et al., 1985) and the lac operator as well as a plasmid-encoded lacIQ repressor gene for the regulated expression of cloned genes in E. coli. The 1,339-bp Eco Rl-Xba I fragment from XgtlO-169 was isolated and cloned between these sites in pTrc99C. The resulting plasmid, pTrc99C-PPll, carries the PP11 cDNA insert in the correct translational reading frame with the vector-encoded ATG start codon (see Results) and comprises 5,490 bp (Fig. 1). For controls, the same frag-

cloned into expression vectors pTrc99A and pTrc99B, respectively, which both display incorrect transment was

lational frames of the vector-encoded ATG and the PP11 cDNA insert. The resulting plasmids are termed pTrc99APPU and pTrc99B-PPll, respectively.

Bacterial expression

of PP11

cDNA

For expression experiments, pTrc99-PPll plasmids were transformed into E. coli strain RB791 (Brent and Ptashne, 1981). Single bacterial clones were inoculated into LBAmp medium and cultures were grown to an optical density of 0.8 (OD600), at which time IPTG (1 mM final concentration) was added to the cultures. The cultures were grown at 37°C for 2 hr. Cells were collected, lysed, and

subjected to NaDodS04/polyacrylamide gel electrophoresis (Laemmli, 1970). In some experiments, cells were labeled with [35S]methionine, lysed, and immunoprecipitated as described (Amann et al., 1984). Small-scale preparations of periplasmic fractions from 5-ml cultures were done following the protocol of Koshland and Botstein (1980); large-scale preparations from 1-liter cultures were done according to the method of Hsiung et al. (1986). For quantification of bacterially expressed PP11, the intensity of immunoprecipitation lines in Ouchterlony plates of serial dilutions of bacterial extracts were compared with standard precipitation lines produced with highly purified placental PP11 protein (Ouchterlony, 1962).

HUMAN PLACENTAL PROTEIN 11 cDNA

245

Amino-terminal sequence determination Automated amino acid sequence analysis was performed with an Applied Biosystems model 477A gas phase sequencer equipped with an on-line phenylthiohydantoin amino acid analyzer (model 120A).

Determination

of enzymatic activity of PP11

Table 1. Comparison

control rabbit antiserum. This was carried by preincubating 300 fil of placental PP11 (-20 fig) or of E. coli extract with 30 ¡A of the rabbit antiserum for 15 min at 37°C before measuring the amidolytic activity as described above. For calculating activities of the extracts, the basic amidolytic activities displayed by the rabbit antisera were subtracted from the overall activities. Inhibition tests were performed by preincubating the PP11 containing E. coli periplasmic fraction with DFP (2 ¡¡M) or jodacetamide (2 mM) for 15 min at 37°C. or a

out

Amino Acid Composition

Mole percent

(cDNA)

Residue A C D E F G H I K L M N P

=

=

=

Amidolytic activity of periplasmic or cytoplasmic fractions of extracts prepared from E. coli RB791 (pTrc99CPP11) and from RB791 (pTrc99C) and RB791(pATIII-5) control cultures as well as the activity of purified PP11 from placenta were measured by an optical test using the chromogenic substrate "Chromozym TH" from Boehringer Mannheim GmbH. A AOO-fA amount of triethanolamine buffer and 100 /A of substrate were mixed in a cuvette with 300 fil of E. coli extract (periplasmic or cytoplasmic fraction) diluted to a protein concentration of 16 mg/ml or with 300 fil (80 fig/ml) of PP11 isolated from placenta. The cuvette was incubated at 37°C and the absorbance changes per minute were measured at 405 nm. For antibody inhibi- Q tion studies, placental PP11, PP11-containing and E. coli R control extracts were preincubated with an anti-PPll rab- S bit antiserum

of

=

=

=

=

=

=

=

=

=

=

« =

=

T V W Y

=

=

=

=

Number

1-369

19-369

26 14 18 35 20 23 13 15 22 28 5 17 12 15 13 33 15 17 6 22

7.046 3.794 4.878 9.485 5.420 6.233 3.523 4.065 5.962 7.588 1.355 4.607 3.252 4.065 3.523 8.943 4.065 4.607 1.626 5.962

6.268 3.419 5.128 9.972 5.698 6.268 3.704 3.989 6.268 6.838 1.140 4.843 3.419 4.274 3.419 9.117 4.274 4.274 1.425 6.268

Ala

Cys Asp Glu Phe

Gly His He

Lys Leu Met Asn Pro Gin

Arg Ser Thr Val

Trp Tyr

Mole percent

(Bohn)

6.30 3.37 10.75a

13.81b 6.06 6.23 3.34 3.60 6.26 6.74 1.06 10.75a 4.10

13.81b 3.31 9.63 3.31 4.53 1.66 5.90

as determined by Bohn and Winckler (1980) and deduced from the cDNA from the present report. Amino acid composition for both the precursor form (amino acids 1-369) and the predicted processed form (amino acids 19-369) of PP11 as deduced from the cDNA are shown. ¡"Summarized values for Asp and Asn as calculated by Bohn and Winkler (1980). bSummarized values for Gin and Glu as calculated by Bohn and Winkler (1980).

Compositions

as

RESULTS

Isolation of human PP11 cDNA

Immunoscreening 106 recombinant plaques of a human placental Xgtll cDNA library with an anti-PPll antiserum yielded only a single recombinant plaque. This clone was designated Xgt 11-93 and carried a cDNA insert of 1.8 kb. By sequence analysis we found that this cDNA contained an open reading frame of 850 bases at the 5' end and a poly(A) tail at the 3' end. The deduced protein sequence was of similar amino acid composition as described for the placental protein PP11 (Bohn and Winckler, 1980; see also Table 1). We subsequently used the 1.8-kb cDNA insert of PP11-93 to probe a XgtlO placental cDNA library to detect

cDNA molecules with maximal 5' sequence information. Three positive X clones with cDNA inserts ranging from 1,600 to 2,400 bases were isolated. Two of those three clones (PP11-169 and PP11-318) code for the complete protein sequence of PP11. Compared to clone PP11-169, clone PP 11-318 also carries in addition to the complete coding sequence a longer 3' untranslated region and a poly(A) stretch of 79 bp. Figure 2 shows the restriction pattern of the PP11 cDNA and the extent and location of three cDNA inserts. The cDNA insert of PP11-93 was subse-

used for Northern blot analysis to determine the size of the mRNA. A single hybridizing RNA band corresponding to a size of 2.5 kb was found with RNA prepared from placental tissue, but not with RNA from liver (not shown). The observed size of 2.5 kb for a single hybridizing RNA band correlates with the size of the cDNA.

quently

Analysis of cDNA and deduced amino acid sequence The entire nucleotide sequence of PP11 cDNA as determined from the clones presented above and the deduced amino acid sequence of the PP11 coding region is shown in Fig. 3. The 2,399-bp cDNA insert from clone PP11-318 consists of a 154 bp 5' untranslated region, a coding region of 1,107 bp, and a 3' untranslated region of 1,138 bp, including the 79-bp-long poly(A) stretch. The polyadenylation signal ATTAAA starts 17 nucleotides upstream of the poly(A) tail. The translation initiation site is deduced as follows: (i) there is no ATG codon further upstream from the assigned start codon, (ii) termination codons are près-

GRUNDMANN ET AL.

246

5'

Bgl II Neo I

_I

Xbal

BamHI

I_I_|_3J 'a

C

N

Xgt11 -93

Agt10-169 \gt10-318 1500 2 000 1000 500 bp I_I_I_I_I_

O

FIG. 2. Restriction map of human cDNA encoding PP11 and the location of the coding sequence (solid bar). N and C, Amino and carboxyl termini of the protein PP11, respectively; black bar, the PP11 coding region; hatched area, protein signal sequence. The overlapping cDNA inserts of clones Xgtll-93, XgtlO-169, and -318 are shown, whereby the last one covers the complete cDNA sequence.

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CTTCCTGAAAGGATCTGGAGACACCAGCTCCACAAGTCCTGGTGTCTTTAAAAGGATCAGCTTGAGGAATAAGGCTCGTCTGAGAGCTGTGACATTCATCTGACTCTAGTGAAAGTCCAA 230

CAGCCACTCCCTTTTTGGCCTCCAACTGGGCACCATGAGGGCCTGCATCTCCCTGGTATTGGCCGTGCTGTGTGGCCTGGCCTGGGCTGAGGACCAGAAAGAGTCAGAGCCATTGCCACA HRACISLVLAVLCGLAWAEDHKESEPLPQ

GCTGGAGGAAGAGACAGAAGAGGCCCTCGCCAGCAACTTGTACTCGGCACCCACCTCCTGCCAGGGCCGCTGCTACGAAGCCTTTGACAAGCACCACCAATGTCACTGCAATGCCCGCTG LEEETEEALASNLYSAPTSCQGRCYEAFDKHHQCHCNARC

CCAAGAGTTTGGGAACTGCTGCAAGGATTTTGAGAGCCTGTGTAGTGACCACGAGGTCTCCCACAGCAGTGATGCCATAACAAAAGAGGAGATTCAGAGCATCTCTGAGAAGATCTACAG QEFGNCCKDFESLCSDHEVSHSSDAITKEEIQSISEKIYR GGCAGACACCAACAAAGCCCAGAAGGAAGACATCGTTCTCAATAGCCAAAACTGCATCTCCCCGTCAGAGACCAGAAACCAAGTGGATCGCTGCCCAAAGCCACTCTTCACTTATGTCAA ADTNKAQKEDIVLNSQNCISPSETRNQVDRCPKPLFTYVN 710

TGAGAAGCTGTTCTCCAAGCCCACCTATGCAGCCTTCATCAACCTCCTCAACAACTACCAGCGGGCAACAGGCCATGGGGAGCACTTCAGTGCCCAGGAGCTGGCCGAGCAGGACGCCTT EKLFSKPTYAAFINLLNNYQRATGHGEHFSAQELAEQDAF

CCTCAGAGAGATCATGAAGACAGCAGTCATGAAGGAGCTCTACAGCTTCCTCCATCACCAGAATCGCTATGGCTCAGAGCAAGAGTTTGTCGATGACTTGAAGAACATGTGGTTTGGGCT LREIHKTAVKKELYSFLHHQNRYGSEQEFVDDLKNMWFGL CTATTCGAGAGGCAATGAAGAGGGGGACTCGAGTGGCTTTGAACATGTCTTCTCAGGTGAGGTAAAAAAAGGCAAGGTTACTGGCTTCCATAACTGGATCCGCTTCTACCTGGAGGAGAA YSRGNEEGDSSGFEHVFSGEVKKGKVTGFHNWIRFYLEEK 970

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GGAGGGTCTGGTTGACTATTACAGTCACATCTACGATGGGCCTTGGGATTCTTACCCCGATGTGCTGGCAATGCAGTTCAACTGGGACGGCTACTATAAGGAAGTGGGCTCTGCTTTCAT EGLVDYYSHIYDGPWDSYPDVLAMQFNWDGYYKEVGSAFI

CGGCAGCAGCCCTGAGTTTGAGTTTGCACTCTACTCCCTGTGCTTCATCGCCAGGCCAGGCAAAGTGTGCCAGTTAAGCCTGGGAGGATATCCCTTAGCTGTCCGGACATATACCTGGGA GSSPEFEFALYSLCFIARPGKVCQLSLGGYPLAVRTYTWD 1310

CAAGTCCACCTATGGGAATGGCAAGAAGTACATCGCCACAGCCTACATAGTGTCTTCCACCTAATAGAACTTCGAGCCAGAAAGGGGCATGAGGGCTCTTGCGAGACTGAAGTGCTATCT KSTYGNGKKYIATAYIVSST 1330

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TCTCTGGACTAGAGAGAAGAGGGAGAGGACTGGAAGGGATCACCAAATCTCAAAGCAATGAGAAGCATTCCTAAATCCCAAAGTGCCCACATGGGAAAGAGATAAAATGTACAAATTAGA

AAAATGTGGATAAACAGTCAAACCTTTATCCTCTAGAATTTTGGCAATGTTGACTAAGAAACAGAGTCCAAGCAGAGAAGGTAGGAACCCTCCATAGCTCTCTGCCCTGATGTGTGGGGG AACTAGGAAGAAGTCCTTTGACCTCACCAGGCCTCATGCTTCCCTTTAATGTAAAGGGAAGGGGTTTGCCCACTTTCCTCTTTTTGGGGTTGGTGAGAGGGCAAACCCTGATATTTTTAC

TGTGAAGGTGTTTTCAGTTGTTCTTAGGAAGAACAGCTGATAGAAATTCAAGATTACTATAATGGCTGTTATTATACACAGCTCTGTAAACTACCACTCAGCCCTGTGTTGGGGTCCTCA 1810 AAGAAGTAAGGCCACAGTAATCAAGCAAGGGCCTTTGGTTTTTTCCAGAGTTAGATCCTCTCAGAACAGAGTCTGGGAGAACTCCAATGCTGAATGGAGAAGGGTAATAGGTTGGTTGCA

GTGAATGGGCTGGGGGTGGGGTGGCCTTCTCCAGGCCTGAGTGTTTTTGTGTCCAGCTCAGTATCTGCAACAAGAAGTTTCCCACTTGTGGATGTTTAGTGCAGCCACAGACTTGTATTT TGATCCCCAATTTTTTTTGAAAGAGTTCTCCTCATAGGAGGATGATTCAGCATCAGAAGAAGAAGGAACCCATAGCTTGGTGTCATTAACATAATTATTTTAAGCCTTATCCAGCAGCCA IAATTTGAATAACTCTACGAGACCAGAGAGACTGTAGTTCCCTATTTTAACCTCAATTATGCATTTGTCCCCAACCCCACTGAGAACTAAATGCTGTACCACAGAGCCGGGTGTGAACTA TGGTTTAGAAGGTTCAAGTTTCCAATTAAAGTCATTGAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Nucleotide sequence of human placental cDNA encoding PP11. The nucleotide sequence was determined by analysis of the overlapping inserts shown in Fig. 2. The deduced amino acid sequence (single-letter code) is displayed below the DNA sequence. The poly(A) addition site upstream from the poly(A) sequence and the consensus sequence CATTG are underlined by *** marks and-, respectively. FIG. 3.

247

HUMAN PLACENT AL PROTEIN 11 cDNA

in all three possible reading frames within the 154-bp 5' untranslated region preceding the ATG, and (iii) the consensus sequence 5'-GGCACC-3' typical for vertebrate mRNAs (Kozak, 1987) directly preceeds the initiator codon. The PP11 open reading frame encodes a protein of 369 amino acids, comprising a molecular weight of 42,120. A hydrophobic profile was calculated on the Micro Vax using the algorithm of Kyte and Doolittle (1982). The profile reveals an amino-terminal hydrophobic region of 18 amino acids typical for secretory proteins. PP11 does not contain AMinked glycosylation sites. There are 14 cysteine residues, of which 10 are clustered at the amino terminal quarter of the molecule.

The observed molecular weight (Fig. 4) of approximately 42 kD corresponds well with the expected molecular weight deduced from the cDNA sequence (Table 1). Furthermore, immunological identity of the fusion protein was demonstrated. The bacterially expressed protein can be specifically immunoprecipitated with a polyclonal antiserum raised against the purified placental PP11 protein (Fig. 4A), and it can also be seen in Western blot experiments

ent

Expression of PP11 cDNA

(not shown).

in E. coli

The 1,339-bp Eco Rl-Xba I fragment from PP11 clone 169 was cloned into expression vector pTrc99C. The resulting plasmid pTrc99C-PPll (Fig. 1) should, upon IPTG induction, express a fusion protein comprising 378 amino acids, of which 369 are encoded by the PP11 open reading frame, seven are specified by the 5' untranslated region of the PP11 cDNA, and two by the pTrc99 vector. The relevant region of translational initiation has the following sequence (vector-encoded RBS, vector-encoded ATG start codon, and PP11 cDNA encoded ATG start codon are

To prove the specificity of this immunoreactivity and to rule out the possibility that an internal ATG start codon of the PP11 cDNA is being utilized by the E. coli translational apparatus, control plasmids were constructed using the same Eco Rl-Xba I PP11 cDNA fragment as above, but in which the translational frames of the vector-encoded ATG and the PP11 cDNA were shifted by -1 (pTrc99B-PPll) and -2 (pTrc99A-PPll), respectively. Upon IPTG induction of bacterial cultures carrying the latter two plasmids, no immunoreactive protein could be detected in immunoprecipitation or in Western blot experiments (not shown). This result indicates that no other ATG start codon is used other than the one encoded by the expression vector (5' ATG in above sketch), in particular not the PP11 ATG start codon (3' ATG in the sketch

above).

The molecular size of the fusion

underlined):

protein obtained by the

MGNSLQLGTMRA

RBS

5 AGGAAAC AGACC ATG GGG AAT TCT CTC CAA CTG GGC ACC ATG AGG GCC-3' -

B 12

200 92



3

4

t

H

Pe Cv PI

69

M

46

ff]|

JJ^



PP11

45-»•»

30 4mm

14

^

an

42-

FIG. 4. Expression of PP11 from pTrc99C-PP11 in E. coli strain RB791. A. Analysis of [35S]methionine-labeled uninduced (lanes 1 and 2) or induced (lanes 3 and 4) cultures. Bacteria were lysed and protein extracts were loaded either directly (lanes 1 and 3) or after immunoprecipitation (lanes 2 and 4) using an antiPP11 antiserum on a 12% polyacrylamide gel. The position of the PP11 protein is indicated by an arrow. 14C-Labeled molecular weight standard proteins were from Amersham-Buchler, Inc. B. Western blot analysis of the PP11 protein present in bacterial fractions after osmotic shock treatment of induced cells. Bacterial cultures were grown, induced, and osmotically shocked. Aliquots of the periplasmic fraction (Pe), the cytoplasmic fraction (Cy), and a PP11 preparation purified from placental origin (PI) were boiled in Laemmli sample buffer and were run on a long 12% polyacrylamide gel. The gel was blotted onto a nitrocellulose filter. The filter was incubated with an anti-PPll antiserum and bound antibody was seen after incubation with 10 ¿tCi 35S-labeled Protein A by autoradiography. The molecular weight positions of the observed proteins are indicated. Molecular weight standards were as in A.

GRUNDMANN ET AL.

248

immunoprecipitation experiment shown in Fig. 4A indicates that the signal sequence of PP11 might be processed in E. coli. If the signal sequence indeed is recognized as such by the bacterial cell, then the protein should be transported through the inner membrane into the periplasm. To show the processing of PP11, we prepared periplasmic and cytoplasmic fractions after osmotic shock treatment of IPTG-induced bacterial cells (Fig. 4B). About 20% of the total PP11 protein was found as the 45-kD precursor protein in the cytoplasmic fraction and about 80% was present in the periplasmic fraction as the processed 42-kD protein. Approximately 2 mg of PP11 could be extracted by preparative osmotic shock treatment from the periplasmic fraction from a 1-liter culture grown in shaker flask. The analysis of bacterially expressed PP11 from the periplasmic fraction by the Ouchterlony plate diffusion technique showed identical precipitation lines when compared with purified placental PP11 (Fig. 5), indicating the immunological identity of the bacterially expressed PP11 protein with the placental protein.

Amino-terminal sequence

of placental PP11

The purified placental PP11 was subjected to amino-terminal sequence determination and 11 cycles of Edman degradation (Edman, 1956) were performed, revealing the sequence: EDHKESEPLPQ. This result shows that the placental protein is processed.

Amidolytic activity of PP11 In

tein,

assigning a

a

biological function

protease activity

was

to the placental prodemonstrated using a colori-

metric assay (H. Bohn et al., unpublished). To determine if the bacterially expressed PP11 also displays this activity, we prepared control and PP11-containing periplasmic fractions. At identical overall protein concentrations, the periplasmic fractions from E. coli containing PP11 caused a mean absorbance change compared to control periplasmic fractions, as shown in Fig. 6A. The absorbance change for the periplasmic fraction containing PP11 was A A 0.04/min and for the control periplasmic fraction AA 0.024/min, corresponding to 32 mU/ml and to 19.2 mU/ ml, respectively. The difference of 12.8 mU/ml was almost completely inhibited by preincubation of the extract containing PP11 with an anti-PPll antiserum, whereas the endogenous periplasmic E. coli amidolytic activity was not influenced by this serum (Fig. 6B). Moreover, a control serum that does not contain PP11 antibodies did not reduce the PP 11-specific amidolytic activity upon preincubation of the periplasmic fraction containing PP11 (not =

=

shown).

To exclude further any nonspecific amidolytic activity present in the periplasmic fraction containing PP11, we

prepared

periplasmic extract from a strain expressing a protein at approximately the same level as PP11. RB791(pATIII-5) expresses and secretes the processed antithrombin III protein with the aid of the E. coli ompA signal sequence (E. Amann, unpublished). The periplasmic fraction prepared from this strain did not show any increase in amidolytic activity when compared to the standard control periplasmic fractions (Fig. 6C). These results demonstrate that the PP11 present in the periplasmic fraction displays an amidolytic enzymatic activity. To test a

different secreted

whether the 45-kD precursor form of PP11 present in the bacterial cytoplasm also displays the amidolytic activity, we prepared control and PP 11-containing cytoplasmic fractions. This time, no increase in amidolytic activity was observed (not shown; actual PP11 concentration in cytoplasmic fraction was identical to PP11 concentration in periplasmic fraction displaying the amidolytic activity shown in Fig. 6A). From these results we conclude that only the processed, periplasmic 42-kD form of PP11 exhibits the enzymatic activity observed, but not the 45-kD precursor form present in the cytoplasm. In the same test, activity was also observed with PP11 isolated from placenta (not shown). Since the amidolytic activity of both the placental and the recombinant protein is completely abolished by DFP, PP11 is most likely a serine protease.

DISCUSSION

FIG. 5. Ouchterlony immune diffusion analysis of placental and recombinant PP11 proteins. Well 1, Anti-PPll antiserum; well 2, RB791 (pTrc99C-PPll) extract, supernatant fraction; well 3, RB791 (pTrc99C-PPll) extract, pellet fraction; well 4, anti-PPll antiserum; well 5, RB791 (pTrc99C) control extract, supernatant fraction; well 6, RB791 (pTrc99C) control extract, pellet fraction; well 7, purified PP11 protein from placenta.

A full-length human cDNA encoding the placental protein PP11 was isolated by screening a placental cDNA library with a polyclonal antibody raised against PP11. The authenticity of the full-length cDNA was established by amino acid composition, by amino-terminal sequence determination of the placental protein and by immunoreactivity of the bacterially expressed protein, which was subsequently identified as a serine protease. Northern blot hybridization analysis of poly(A)*RNA

HUMAN PLACENTAL PROTEIN 11 cDNA

249

time(min)

Analysis of amidolytic activities of periplasmic fractions prepared from plasmid-bearing bacterial strains. A. Amidolytic activity in periplasmic fractions prepared from RB791(pTrc99C-PPll) (open circles) and from RB791(pTrc99C) control cells (stars). B. Amidolytic activity in periplasmic fractions prepared from RB791(pTrc99C-PPll) (crosses) and from RB791(pTrc99C) control cells (stars). Prior to testing, both extracts were preincubated with anti-PPll antiserum. C. Amidolytic activity in periplasmic control fractions prepared from RB791(pATIII-5) (filled circles) and from RB791(pTrc99C) (stars). FIG. 6.

from human placental tissue showed a mRNA for PP11 of about 2,500 bases, in good agreement with the length of the cloned cDNA of 2,399 bp. Poly(A)+RNA from human liver did not hybridize under the same conditions, confirming the placental-specific expression of PP11. The polyadenylation signal ATTAAA 17 bp upstream from the poly(A) addition site has been described for 10% of all polyadenylated mRNA molecules and represents the most commonly observed alternative next to the hexanucleotide AATAAA (Manley, 1988). In addition, a second consensus sequence (5'-CATTG-3') described for the 3' end of polyadenylated mRNAs (Benoist et al, 1980) is located between the polyadenylation signal and the poly(A) tail. The PP11 cDNA shows an open reading frame of 1,107 bp and encodes 369 amino acids. The molecular weight calculated from the amino acid composition is 42,120. Computer search in nucleic acid and protein data bases did not reveal any sequence homologies to known proteins. PP11 is most likely a serine protease. At present, however, it cannot be ruled out that the protease is an esterase, since esterases also exhibit an amidolytic, DFP-inhibitable ac-

vealed that the predicted signal sequence is indeed absent from the placental protein. The amino acid composition of PP11 as deduced from the cDNA corresponds to the one determined for the placental protein by Bohn and Winckler (1980; Table 1). There are no potential AMinked glycosylation sites in the protein sequence as deduced from the PP11 cDNA. Although Bohn and Winckler (1980) reported a carbohydrate content of 3.9%, a recent repetition of PP11 carbohydrate determination employing hydrolysis and conversion of the compounds to alditol acetates indicated complete absence

carbohydrate (H. Zilg, personal communication). Therefore, it is very likely that PP11 is an unglycosylated protein. There are other examples of unglycosylated, secreted proteins, for example human interferon-a (IFN-a) (Allen and Fantes, 1980) and FXIIIa (Takahashi et al, 1986). After expression of the full-length cDNA of PP11 in E. coli, an immunoreactive protein with the expected molecular weight was observed. Fractionation experiments revealed the presence of an apparently processed protein of 42 kD in the periplasm, whereas the 45-kD precursor form tivity. Although it has been reported that PP11 is present pre- is observed in the cytoplasm. This size difference of apdominantly in the cytoplasm of trophoblasts and specific proximately 3 kD corresponds well with the theoretical tumor cells and that concentrations of PP11 in serum of molecular weight of 2,792 for the 27-amino-acid "prepepsuch patients are very low, the protein has an amino-termi- tide," which includes the 18 amino acids of the natural nal signal sequence typical of secreted proteins. Proteolytic PP11 signal sequence. Quantification of the two protein cleavage of the 18-amino-acid signal peptide would result forms revealed that the majority (80%) of PP11 is present in a mature protein of 351 amino acids with a molecular in its processed form, indicating efficient recognition of a weight of 40,248. The structural prerequisites for this pro- signal sequence of the human protein by the E. coli signal teolytic cleavage of PP11 are in good agreement with the peptidase. Moreover, only the processed form exhibited rules postulated by von Heijne (1986) for predicting the the biological activity observed, which can be taken as furcleavage site at the junction of the signal sequence and the ther evidence for correct processing and possibly correct mature exported protein. The amino-terminal amino acid folding of the protein in the E. coli periplasm. Correct of from PP11 re,sequence purified placenta subsequently processing of eukaryotic proteins in E. coli has been deof

250

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scribed before (Talmadge et al., 1980; Linard et al., 1988). The knowledge of the complete PP11 amino acid sequence, the assignment of an enzymatic activity (putative serine protease), and the availability of the recombinant protein from E. coli will help in elucidating its biological role.

BOHN, H. (1980). Ectopic synthesis of pregnancy specific (31glycoprotein (SP1) and placental specific tissue proteins (PP5, PP10, PP11, PP12) in nontrophoblastic malignant tumors; possible markers in oncology. Klin. Wochenschr. 58, 789-791. INABA, N., RENK, T., DAUME, E., and BOHN, H. (1981). Ectopic production of placenta-"specific" tissue proteins (PP5 and Ppll) by malignant breast tumors. Arch. Gynecol. 231, 87-90.

ACKNOWLEDGMENTS We thank C. Nerlich, T. Rein, T. Schwarz, and B. Ochs for excellent technical assistance and K.J. Abel and M. Riihl for oligodeoxynucleotide synthesis. We acknowledge H. Zilg for carbohydrate analysis of PP11.

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LINARD, CG., MBIKAY, M., BENJANNET, S., LAZURE, C, SEIDAH, N.G., and CHRÉTIEN, M. (1988). Correct processing and secretion of a human prostatic secretory protein (PSP94) in Escherichia coli. Gene 73, 479-487. MANLEY, J.L. (1988). Polyadenylation of mRNA precursors. Biochim. Biophys. Acta 950, 1-12. OUCHTERLONY, Ö. (1962). Quantitative immunoelectrophoresis. Acta Pathol. Microbiol. Scandinavica (Suppl.) 54, 252-262. SANGER, F., NICKLEN, S., and COULSON, A.R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Nati. Acad. Sei. USA 74, 5463-5467. TAKAHASHI, N., TAKAHASHI, Y., and PUTNAM, F.W. (1986). Primary structure of blood coagulation factor XHIa (fibrinoligase, transglutaminase) from human placenta. Proc. Nati. Acad. Sei. USA 83, 8019-8023. TALMADGE, K., STAHL, S., and GILBERT, W. (1980). Eukaryotic signal sequence transports insulin antigen in Escherichia coli. Proc. Nati. Acad. Sei. USA 77, 3369-3373. VON HEIJNE, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683-4690. WAHLSTRÖM, T., BOHN, H., and SEPPÄLÄ, M. (1982). Immunohistochemical studies on pregnancy proteins. In Pregnancy Proteins, J.G. Grudzinskas, B. Teisner, and M. Seppälä, Eds. (Academic Press, New York) pp. 415-422. Address reprint requests to: Dr. Ulrich Grundmann Research Laboratories Behringwerke AG 3550 Received for publication January 30, 1990, February 21, 1990.

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and in revised form

Cloning and expression of a cDNA encoding human placental protein 11, a putative serine protease with diagnostic significance as a tumor marker.

The placental protein 11 (PP11) can act as a tumor marker because of its specific association with various forms of cancer. A lambda gt11 cDNA library...
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