Circulating human pregnancy-associated plasma protein-A is disulfide-bridged to the proform of eosinophil major basic protein.

A previously unrecognized association between pregnancy-associated plasma protein-A (PAPP-A) and the proform of eosinophil major basic protein (proMBP) is demonstrated. PAPP-A isolated from pooled pregnancy serum is shown to be a disulfide-bridged complex with proMBP (PAPP-A/proMBP) in which the subunits of the constituents are present in a 1:1 molar ratio. The results are the outcome of analysis of tryptic and CNBr/tryptic peptides from PAPP-A/proMBP, sequence analysis of intact and reduced and carboxymethylated PAPP-A/proMBP, and reaction with monoclonal antibodies directed against MBP and its proform. In addition, it is shown that commercial polyclonal anti-PAPP-A is polyspecific, also reacting with MBP.

nonpregnant individuals PUP-A has been reported to be present in low amounts in the Graafian follicle and corpus luteum (10, ll), and in the testes and the seminal fluid (12,131. The major basic protein (MBP) of the eosinophil granule is derived from the 222-residue preproMBP (14-16). In addition to mature MBP (residues 106-2221, preproMBP contains a presumed 15or 16-residue signal peptide and an acidic propiece (residues 16/17-105). MBP isolated from the eosinophil granule is cytotoxic to mammalian cells and has been implicated in tissue damage associated with eosinophil infiltrates (17)(18)(19). The plasma level of immunoreactive MBP is greatly elevated in pregnancy, and immunohistochemically, MBP is located to the X-cells and giant cells of placenta (20-22). MBP has been purified from placental tissue (231, and it seems to be tightly associated with unknown large proteins ( 2 3 , 24).
To initiate cDNA cloning of PAPP-A,2 tryptic peptides of protein isolated from pregnancy serum were characterized. Here we show that several of the peptides in fact are derived from proMBP. We conclude that circulating PUP-A is a disulfidebound complex between PUP-A and proMBP.
Materials-Sepharose CL-GB was from Pharmacia. ['4C]Iodoacetic acid was from Amersham. Standard chemicals were from Sigma, Serva, and Merck. PVDF membranes were from Applied Biosystems.
Miscellaneous Procedures-For SDS-PAGE the Tris-glycine system (28) was used (10-20% acrylamide, gel size 0.5 X 80 X 100 mm). Coomassie Brilliant Blue was used for staining. Electroblotting was done for 45 min at 100 V/500 mA using the standard Tris, glycine, 0.1% SDS buffer, pH 8.3. For immunological detection of proteins blotted or spotted onto PVDF, 2% Tween 20 and 1% fetal calf serum were used for blocking. Peroxidase activity was detected by incubation with H,02 and J6-8A4 and J163-15E10 were diluted 20and 2000-fold, respectively, 3,3'-diaminobenzidine. The cell culture supernatants containing mAbs and anti-PAPP-A was diluted 2000-fold. Incubations with either of two mAbs of irrelevant specificity (26, 271, with rabbit anti-complement C~C , or without primary antibody were all negative. Amino Acid and Sequence Analysis-Analysis of amino acids and amino sugars was done by cation exchange (29). Edman degradations of peptides were carried out using Beckman 890C (30) and Applied Biograded after being spotted on PVDF. systems 477A instruments. Proteins (less than 150 pmol) were de-Preparation of Peptides for Sequence Analysis-Reduced and carboxymethylated PAPP-NproMBP was digested with 1/50 (w/w) trypsin for 2 h at room temperature. The digest was fractionated by DEAE-Sephacel chromatography using gradient elution with NH4HC03 followed by reverse phase HPLC on Nucleosil C-18 (30L2 Fifteen pure peptides were subjected to sequence analysis (TP and TproMBP series, Table I

Complex between P M P -A and proMBP
PAPP-NproMBP was also degraded with CNBr and further digested with 1/200 (w/w) trypsin for 5 h a t 37 "C. Peptides less than 30-40 residues in length were subjected to cation exchange chromatography (31). Peptides CBTl and CBT2 were purified by reverse phase HPLC on Nucleosil C-18 (not shown). 1 and 2 ) shows an SDS-PAGE analysis of the preparation of PAPP-NproMBP. Including approximately 1 9 9 carbohydrate (3) the size of the 1547 residue PAPP-A subunit' is estimated at 215 kDa after reduction. Apart from a band of that size, a t the high load used, a smear corresponding to species of approximately 50-90 kDa was seen with reduced material, but not with nonreduced material.

Fig. lA (lunes
The complete or partial sequences of a random set of 15 tryptic peptides from PAPP-NproMBP are shown in Table I. The sequences of 12 of these peptides were located in the sequence of PAPP-A determined from cDNA cloning.2 However, the sequences of the remaining peptides exactly matched the sequences of three of the predicted tryptic peptides from proMBP (14-16).  From a digest aimed a t determining the disulfide bridges of PAPP-NproMBP one peptide (CBT1) connected the PAPP-A and proMBP subunits (Table 11). Another peptide (CBT2) spanned the processing site in proMBP (residues 105-106; Refs. 15 and 16). Curiously, that peptide was a dimer containing two disulfide bridges ( Table 11). The combined results suggested that proMBP is a constituent of circulating PAPP-A. As judged from the average yields of the 12 PAPP-Apeptides (22%) and the three proMBP peptides (26%), circulating PAPP-Ais a disulfide-bound complex in which the subunits of PAPP-A and proMBP are present in approximately equimolar amounts. PAPP-NproMBP showed, in addition to the sequence corresponding to the PAPP-A subunit,2 two NH,-terminal sequences in an approximately 2:l molar ratio (Table 111) starting at residues 17 and 18 of preproMBP.

17
14C-Carboxymethylated PAPP-NproMBP was subjected to denaturing gel chromatography to separate its subunits (Fig.  2). Approximately 84% of the radioactivity associated with protein eluted in fractions 20-29, the remainder in fractions 30-37. Upon SDS-PAGE the material in pool 1 had the expected size of the PAPP-A subunit (not shown), whereas the material in pool 2 ( Fig. L A , lane 4 ) migrated a s a smear corresponding to species of approximately 50-90 kDa rather than 23.4 kDa expected for proMBP (15, 16). The pool 2 material evidently corresponded to the 50-90-kDa material obtained from PAPP-N proMBP by reduction (Fig. LA, lane 2 ) . Sequence analysis confirmed that the PAPP-A subunit constituted the material in pool 1 and that proMBP, apart from a minor contamination with an approximately 500-residue fragment from PUP-A, constituted the material in pool 2 (Table 111).
By dot blot analysis carboxymethylated PAPP-NproMBP reacted strongly with mAbs recognizing epitopes in the propiece of MBP and in mature MBP (not shown). Using the same mAbs it was found in Western blot analysis (Fig. 1, B and C), that nonreduced PAPP-NproMBP reacted poorly with both mAbs (lane 1 ), that the 215-kDa subunit of PAPP-A did not react a t all, and that the 50-90-kDa material reacted strongly with both mAbs (lanes 2 and 4 ) . MBP (Fig. lB, lune 3 ) served as a control for mAb J6-8A4. Hence, consistent with the results described above, the 50-90-kDa material dissociated from PAPP-NproMBP by reduction and carboxymethylation contains epitopes reacting with mAbs specific for MBP and its propiece.
From the results presented above, it was likely that the Sequences of tryptic peptides from reduced and carboxymethylated PAPP-AlproMBP Peptides originating from PAPP-A are labeled T P peptides originating from proMBP are labeled TproMBP. The numbering in parentheses shows the position in mature PAPP-A or preproMBP. " Details of peptide purification will be presented elsewhere (see footnote 2).
The sequences of the residues in parentheses are taken from the cDNA sequence of PAPP-A (see footnote 2). Approximately 83 nmol of reduced and carboxymethylated PAPP-NproMBP was digested.

Complex between PMP-A and proMBP 12245
commercial rabbit anti-PAPP-AA23O would react with MBP. AS shown in Fig. 1D, this was indeed the case when examined by a dot blot analysis. PAPP-A served as a positive control.

DISCUSSION
The finding presented here that proMBP is a constituent of circulating PAPP-A is the first demonstration of this molecule in serum. The conclusion that circulating PAPP-A is complexed with proMBP through disulfide bridge formation is based on the following concordant evidence. 1) Tryptic peptides originating from proMBP were found in a digest of reduced and carboxymethylated PAPP-NproMBP; 2) a disulfide-bridged peptide connecting proMBP and PAPP-A and a peptide spanning the proMBP processing site have been isolated from nonreduced PAPP-Npro-MBP in high yield; 3) sequence analysis shows that PAPP-Npro-MBP contains three NH2-terminal sequences, one originating from the PAPP-A subunit, and two originating from proMBP; 4) the PAPP-A subunit can be separated from the proMBP subunit by denaturing gel chromatography of reduced and carboxymethylated PAPP-NproMBP; 5 ) mAbs directed against the propiece of MBP and against mature MBP react strongly with reduced PAPP-NproMBP and with the material separated from the PAPP-A subunit by reducing SDS-PAGE or denaturing gel chromatography.
In the complex, one PAPP-A subunit is likely to be bound to one molecule of proMBP as judged from the yield of tryptic peptides, the yields of the NH2-terminal residues upon sequence analysis of PAPP-NproMBP, and the recovery of radioactivity (82 half-cystines in PAPP-A and 12 half-cystines in  Two sequences were present in equimolar yield. Only PTH-Glu was seen in cycle 2, and bis-PTH-Cys2 was seen in cycle 5, hence establishing the disulfide bridge.
The amino acid composition of both peptide sets agreed with the sequences determined.
Only one sequence was present. Bis-PTH-Cys, was seen in cycles 7 and 10, hence establishing that the peptide is a dimer, in which Cys-104 in one mate is connected to Cys-104 in the other, and Cys-107 in one mate is connected to Cys-107 in the other. proMBP) in the two fractions obtained after denaturing gel chromatography.
Intriguingly, proMBP, when separated from the PAPP-A subunit after reduction, migrates in SDS-PAGE and elutes in denaturing gel chromatography as species of 50-90 kDa, rather than as the expected 23.4-kDa polypeptide. The propiece being acidic (PI = 4.0) and MBP being basic (PI = 11) may enable proMBP to form very stable oligomers even after denaturation. In addition, since approximately 30 residues of glucosamine and 2-3 residues of galactosamine were found in the pool 2 material (not shown), the propiece is extensively glycosylated.
It has been suggested that the signal sequence of preproMBP comprises residues 1-15 or 1-16 (15, 16). However, the present results show that proMBP associated with PAPP-A contains NH, termini reflecting cleavage at Leu-17-His-18 and at Ala-16-Leu-17 (approximately 2:l molar ratio). Both cleavage sites conform with the "-1 to -3 rule" (32), although Leu residues are rarely found in position -1 (33). Alternatively, the NH, terminus starting with His-18 may represent a secondary trimmed product.
Prior to or during storage in the granules of eosinophils proMBP is cleaved at Gln-105-Thr-106 (15, 16). As discussed above, proMBP associated with PAPP-A is probably unprocessed. However, since MBP can be recovered from the placental septal fluid (23), processing can take place in certain compartments of the placenta.
The intracellular or extracellular compartments where the assembly of the PAPP-NproMBP complex takes place are un- Absorbance at 280 nm (-) and radioactivity ( ---1 are shown. An aliquot of the material in the peak fractions (shown by burs) was desalted on a PDlO column (eluent 0.1% trifluoroacetic acid) and used for analysis. Carboxymethylated soybean trypsin inhibitor (20 kDa) peaked in fractions 39-40.

Sequence analysis of PMP-AlproMBe and of reduced and carboxyrnethylated Sepharosr CL-GB pools
The sequences were obtained from samples spotted on PVDF. Unequal amounts of the material in pools 1 and 2 were analyzed. The yields, corrected for the steady increase of background, are shown in parentheses (pmol). Pro ( The sequence Glu-Ala-Arg-Gly-Ala-Thr-Glu-Glu-Pro-was also observed (less than 5 pmol level), reflecting the presence uf a tnlllur -500residue NH,-terminal proteolytic fragment of PAPP-A (see Footnote 2). It has been indicated previously that placenta' MBP is 12. Bischof, P., Martin-Du-Pan, R., Lauber, K., Girard, J. P., H e m a n n , W. L., and The physiological significance of the interaction between PA-(Farmer, S. G., and Hay, D. W. P., eds) pp. 255-300, Marcel Dekker Inc., New