Complex of Pregnancy-associated Plasma Protein-A and the Proform of Eosinophil Major Basic Protein

DISULFIDE STRUCTURE AND CARBOHYDRATE ATTACHMENT SITES*

Michael T. Overgaard, Esben S. Sørensen, Damian StachowiakDagger, Henning B. Boldt, Lene Kristensen, Lars Sottrup-Jensen, and Claus Oxvig§

From the Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark

Received for publication, August 27, 2002, and in revised form, October 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pregnancy-associated plasma protein-A (PAPP-A) is a metzincin superfamily metalloproteinase responsible for cleavage of insulin-like growth factor-binding protein-4, thus causing release of bound insulin-like growth factor. PAPP-A is secreted as a dimer of 400 kDa but circulates in pregnancy as a disulfide-bound 500-kDa 2:2 complex with the proform of eosinophil major basic protein (pro-MBP), recently shown to function as a proteinase inhibitor of PAPP-A. Except for PAPP-A2, PAPP-A does not share global similarity with other proteins. Three lin-notch (LNR or LIN-12) modules and five complement control protein modules (also known as SCR modules) have been identified in PAPP-A by sequence similarity with other proteins, but no data are available that allow unambiguous prediction of disulfide bonds of these modules. To establish the connectivities of cysteine residues of the PAPP-A·pro-MBP complex, biochemical analyses of peptides derived from purified protein were performed. The PAPP-A subunit contains a total of 82 cysteine residues, of which 81 have been accounted for. The pro-MBP subunit contains 12 cysteine residues, of which 10 have been accounted for. Within the 2:2 complex, PAPP-A is dimerized by a single disulfide bond; pro-MBP is dimerized by two disulfides, and each PAPP-A subunit is connected to a pro-MBP subunit by two disulfide bonds. All other disulfides are intrachain bridges. We also show that of 13 potential sites for N-linked carbohydrate substitution of the PAPP-A subunit, 11 are occupied. The large number of disulfide bonds of the PAPP-A·pro-MBP complex imposes many restraints on polypeptide folding, and knowledge of the disulfide pattern of PAPP-A will facilitate structural studies based on recombinant expression of individual, putative PAPP-A domains. Furthermore, it will allow rational experimental design of functional studies aimed at understanding the formation of the PAPP-A·pro-MBP complex, as well as the inhibitory mechanism of pro-MBP.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The proteolytic activity of pregnancy-associated plasma protein-A (PAPP-A)1 was demonstrated with its isolation from medium conditioned by human fibroblasts (1) and by recombinant expression (2). PAPP-A was shown to be the proteinase responsible for cleavage of insulin-like growth factor-binding protein (IGFBP)-4, one of six binding proteins (IGFBP-1 to -6) that function as regulators of the biological activities of insulin-like growth factors (IGF)-I and -II (3). Cleavage of IGFBP-4 causes release of bound IGF, and PAPP-A thus antagonizes the inhibitory effect of IGFBP-4. PAPP-A, IGFBP-4, and IGF appear to function together in several systems, in particular the reproductive system (4-7) and the cardiovascular system (8, 9). Cleavage of IGFBP-5 (10) and IGFBP-2 (46) by PAPP-A has been demonstrated recently, but the physiological role of these findings is currently less substantiated.

The 1547-residue PAPP-A polypeptide (11) is secreted as a disulfide-bound dimer of 400 kDa (2). PAPP-A contains the elongated zinc-binding motif (HEXXHXXGXXH) (12) and belongs to the metzincin superfamily of metalloproteinases, also including the astacins, the reprolysins, the serralysins, and the matrix metalloproteinases (13). As it cannot be grouped into any of these families, PAPP-A is the founding member of a fifth metzincin family, the pappalysins (14), which also include the recently discovered PAPP-A2 (15). In addition to a proteolytic domain (14), the PAPP-A subunit contains three lin-notch repeats (LNR-1-3, each of 26-27 residues) and five complement control protein modules (CCP-1-5, each of 57-77 residues) (11). The LNR module is known from the Notch-related receptors, which play important roles in the regulation of developmental programs (16). Of particular interest, the pappalysins are the only known proteins, besides the Notch receptors, in which LNR modules are present. The CCP modules, also known as short consensus repeats, are found in several other proteins, in particular proteins of the complement system (17). It has been found recently (18) that CCP-3 and -4 mediate cell surface adhesion of PAPP-A.

PAPP-A was first isolated from the serum of pregnant women (19), where its concentration rises throughout gestation. Interestingly, depressed circulating levels of PAPP-A antigen correlate with Down's syndrome (20) and low birth weight (21). In pregnancy serum, the vast majority of PAPP-A (>99% (2)) is covalently bound in a 2:2 complex to the 206-residue proform of eosinophil major basic protein, pro-MBP (22). The subunits of the 500-kDa PAPP-A·pro-MBP complex can be separated from each other only after denaturation and reduction (23). The mature 117-residue MBP polypeptide is well known from granules of the eosinophil leukocyte, where it functions as a cytotoxic effector molecule (24), but no evidence suggests that MBP is generated from pro-MBP of the PAPP-A·pro-MBP complex. Rather, pro-MBP functions as a proteinase inhibitor of PAPP-A (2, 25), whose mechanism of inhibition is currently unknown.

The PAPP-A subunit contains 82 cysteine residues, but their pairing cannot be predicted by comparison to other proteins with known disulfide structure, as PAPP-A shows global similarity only to the recently discovered PAPP-A2 (15); the disulfide structure of PAPP-A protein modules (LNRs and CCPs) cannot be inferred by similarity, because other similar modules have unknown cysteine pairing or differ from the modules of PAPP-A. The pro-MBP subunit contains a total of 12 cysteines. Proteins of known disulfide structure, relevant for comparison, include the mature MBP isolated from eosinophil leukocytes (26, 27).

To delineate the cysteine connectivity of the PAPP-A·pro-MBP complex, and to determine the occupancy of the 13 potential sites for N-linked carbohydrate in the PAPP-A subunit, biochemical analyses of peptides derived from purified protein were carried out. Knowledge of the cysteine pairing of the PAPP-A·pro-MBP complex likely will facilitate, for example, recombinant expression of individual pappalysin domains. Furthermore, it provides an experimental base for studying the process of complex formation by analysis of mutated proteins, and it will allow mechanistic studies of pro-MBP as an inhibitor of PAPP-A.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins and Chemicals-- Human PAPP-A·pro-MBP complex was purified from pooled third trimester pregnancy serum, as described previously in detail (23). Proteolytic enzymes used to generate peptides are as follows: bovine L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (Worthington), porcine pepsin (Worthington), and thermolysin (type X protease, Sigma). Cyanogen bromide, formic acid, guanidine hydrochloride, and urea were from Merck. Dithioerythritol and iodoacetamide were from Sigma.

Digestion and Degradation of Protein-- For cleavage on the C-terminal side of methionine residues, cyanogen bromide was added to lyophilized protein dissolved in 70% formic acid at 100-fold molar excess over methionine. Following incubation for 20 h at room temperature in the dark, the protein was freeze-dried and then redissolved as specified. Digests were carried out at 37 (trypsin and pepsin) or at 55 °C (thermolysin) in appropriate buffers as specified, using an enzyme/substrate ratio of 1:200-1:50 (w/w).

Amino Acid and Amino Sugar Analysis-- Amino acids were quantified by cation exchange after peptide hydrolysis at 110 °C for 18 h with 6 M HCl, 0.1% phenol, 5% thioglycolic acid (28). The amino sugar GlcNAc was determined in the same system after hydrolysis at 110 °C with 6 M HCl, 0.1% phenol for 3 h. For detection of chromatographic fractions containing the amino acid cysteine (present as cystine, Cys2), samples were treated with performic acid, to convert all Cys residues into cysteic acid (Cya); hydrogen peroxide (30%) was mixed with formic acid (1:9 (v/v)), left on ice for 10 min, and then added to lyophilized sample. Following incubation on ice for 2 h, the sample was lyophilized again and then hydrolyzed. For screening of fractions, only Cya was quantified, using a short gradient on the amino acid analyzer.

N-terminal Sequence Analysis-- Edman degradation was performed on an Applied Biosystems 477A sequencer equipped with an on-line HPLC (29). The number of cycles was sufficient to identify the origin of the observed N-terminal sequence(s) and in some cases to resolve cysteine pairing based on the presence or absence of bis-PTH-Cys2 (30). For sample loading, isolated peptides (10-200 pmol) were pipetted onto Polybrene-coated glass filters, and protein separated by SDS-PAGE was blotted onto a ProBlott membrane (Applied Biosystems), Coomassie-stained, and excised.

Mass Spectrometry-- Mass spectra were acquired with a Bruker BIFLEX matrix-assisted laser desorption ionization time-of-flight instrument (Bruker-Franzen, Bremen, Germany) equipped with a 1-m flight tube, a reflector, a 337-nm nitrogen laser, and a 500-MHz digitizer. Thin film matrix surfaces were prepared from alpha -cyano-4-hydroxycinnamic acid (Sigma) dissolved in acetone/water (99:1) to 30 µg/µl. A 0.5-µl volume of the analyte (0.1-10 pmol/µl) was deposited on the matrix surface and allowed to dry onto the crystals. Spectra were obtained by averaging 20-50 single shot spectra and were calibrated internally by co-crystallizing angiotensin II (Sigma) and adrenocorticotropic hormone, fragment 18-39 (Sigma), with the analyte and by using the calibration constants of well known matrix ions. Some mass spectra were acquired on a Voyager DE-PRO (Applied Biosystems), using a similar technique for sample preparation. All peptides were observed as MH+ species.

Miscellaneous Procedures-- SDS-PAGE was performed in Tris glycine gels (10-20%) or in precast 3-8% Tris acetate gels (NOVEX). Separated proteins were visualized by Coomassie staining or blotted onto a PVDF membrane for sequence analysis or immunovisualization (see below).

Column Chromatography-- For gel filtration on Sephadex G-50-SF (Amersham Biosciences) (2.5 × 40 cm), the column was equilibrated and eluted with 1 M formic acid, and the eluate was monitored at 280 nm. The flow rate was 0.5 ml/min, and fractions of 3 ml were collected.

Cation exchange chromatography was carried out on a 4 × 250 mm column packed with polySULFOETHYL-aspartamide (PolyLC) (31). The gradient was formed from 5 mM phosphoric acid, 25% acetonitrile (pH approx 3) (solvent A), and 5 mM phosphoric acid, 25% acetonitrile, 1000 mM sodium chloride (solvent B) (0 min/1% B, 20 min/8% B, 40 min/30% B, 60 min/100% B, 61 min/1% B) at a flow rate of 1 ml/min. The column was operated at 50 °C, and the eluate was monitored at 226 nm. Fractions of 0.5 ml were collected.

Reversed-phase liquid high pressure chromatography (RP-HPLC) was carried out on a 4 × 250 mm column packed with Nucleosil C18 100-5 (Macherey-Nagel). Gradients were formed from 0.1% (v/v) trifluoroacetic acid (solvent A) and 0.075% (v/v) trifluoroacetic acid in 90% (v/v) acetonitrile (solvent B) increasing the amount of solvent B to 90% over 30-90 min at a flow rate of 1 ml/min. For rechromatography, when required, 0.05% heptafluorobutyric acid (HFBA) was used rather than trifluoroacetic acid as the ion pairing agent. The column was operated at 50 °C, and the eluate was monitored at 220 nm. Fractions were collected manually.

For gel filtration on Superose 12 HR 10/30 (Amersham Biosciences), the column was equilibrated and eluted with 8 M urea, 20 mM ammonium acetate, pH 5.0. The flow rate was 0.5 ml/min; the fraction size was 0.5 ml, and the eluate was monitored at 280 nm.

For cation exchange chromatography on Mono S HR 5/5 (Amersham Biosciences), the column was eluted with an increasing gradient of sodium chloride (0-1000 mM) in 8 M urea, 50 mM formic acid (pH 3.7). The total elution volume was 40 ml; the flow rate was 0.5 ml/min; the fraction size was 0.5 ml, and the eluate was monitored at 280 nm.

For gel filtration on Superdex Peptide HR 10/30 (Amersham Biosciences), the column was equilibrated and eluted with 50% formic acid. The flow rate was 0.5 ml/min; the fraction size was 0.5 ml, and the eluate was monitored at 280 nm.

Isolation and Analysis of Cysteine-containing Peptides Derived from PAPP-A·Pro-MBP-- First, purified PAPP-A·pro-MBP complex (45 mg) in 50 mM Tris, 50 mM sodium chloride, pH 7.5, was digested (20 h) with trypsin. The digest was lyophilized, redissolved in 70% formic acid, and then degraded with cyanogen bromide. The reaction mixture was lyophilized again, digested further (10 h) with trypsin, and then separated by gel filtration on a G-50-SF column (Fig. 1). The pools made were analyzed for the contents of cysteine (determined as Cya following performic acid oxidation) and GlcNAc (Fig. 1). The majority of cysteine was found in pool CBT-1, but this pool also contained the majority of GlcNAc. Pools CBT-2 to -5 were each lyophilized, redissolved in 5 mM phosphoric acid, 25% acetonitrile, and separated by cation exchange chromatography collecting 120 fractions, as shown in Fig. 2. The amount of cysteine in each fraction was determined, and pools of two to four fractions were further separated by RP-HPLC (Fig. 3). Some fractions (manually collected) contained peptides sufficiently pure for unequivocal identification by two or three of the following techniques: compositional amino acid analysis, N-terminal sequence analysis, and mass spectrometry. However, other peptides required rechromatography (on the same column using HFBA rather than trifluoroacetic acid as the ion pairing agent). Second, the material of pool CBT-1 (contained in 1 M formic acid) was further digested with pepsin and then separated by gel filtration with a G-50-SF (not shown). Pools of fractions 31-75 were processed as described above by cation exchange chromatography and RP-HPLC, and isolated peptides were identified. Third, because the majority of cysteine still eluted early (in fractions 18-30, corresponding to the position of pool CBT-1 of Fig. 1) after digestion with pepsin, this material was lyophilized, redissolved in 50 mM NaCl, 50 mM Tris, pH 8.0, digested (20 h) with thermolysin, and then separated by gel filtration on a G-50-SF (Fig. 4). Peptides in pools CBTPTL-2, -3, and -4 (Fig. 4) were separated by cation exchange chromatography and RP-HPLC, and isolated, cysteine-containing peptides were identified.

Isolation and Analysis of Cyanogen Bromide Fragments Derived from the PAPP-A·Pro-MBP Complex-- Purified PAPP-A·pro-MBP complex (25 mg) was degraded with cyanogen bromide, separated by denaturing gel filtration on Superose 12 HR 10/30, and then analyzed by nonreducing SDS-PAGE (Fig. 5) and N-terminal sequence analysis of selected bands after blotting. The material in pool CB-1 (Fig. 5) was further separated by cation exchange chromatography on a Mono S column following analysis of fractions by nonreducing SDS-PAGE and sequence analysis of bands after blotting (not shown). Thirteen pools of the eluate from the Mono S column were separately dialyzed against 50 mM NaCl, 50 mM Tris, pH 8.0, and then 1) digested (20 h) with thermolysin and separated by RP-HPLC or 2) digested with trypsin (20 h) and separated by gel filtration on a Superdex peptide column. Fractions (selected by their contents of cysteine) were lyophilized, redissolved in 50 mM NaCl, 50 mM Tris, pH 8.0, digested with thermolysin, and further separated by RP-HPLC. Isolated, Cys-containing peptides were identified by sequence analysis and mass spectrometry.

Mutagenesis, Transfection, and Analysis of Recombinant Protein-- A plasmid construct (pA-C1130A) encoding a PAPP-A variant with Cys-11302 substituted with alanine was made with QuikChange (Stratagene) using the PAPP-A plasmid pB989-1547 as a template (14). The primers were 5-TGCGTGCACTTCGCAgcTGAGAAAACTGACTGTCC-3' and 5'-CAGTTTTCTCAgcTGCGAAGTGCACGCAGCTCTGC-3' (mutated nucleotides are shown in lowercase). The mutated PAPP-A cDNA fragment was then swapped into the wild-type construct, pcDNA3.1-PAPP-A (14), to generate pA-C1130A. Human embryonic kidney 293T cells (293tsA1609neo) (32) were maintained in high glucose DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, nonessential amino acids, and gentamicin (Invitrogen). Cells were plated onto 6-cm tissue culture dishes and were transfected 18 h later by calcium phosphate co-precipitation (33), using 10 µg of plasmid DNA. Plasmid constructs used for transfection are as follows: pcDNA3.1-PAPP-A and pA-C1130A. Recombinant proteins were analyzed by Western blotting after separation by reducing or nonreducing SDS-PAGE (3-8%). Protein was blotted onto a polyvinylidene difluoride membrane (Millipore). The blots were blocked with 2% Tween 20, equilibrated in 50 mM Tris-HCl, 500 mM NaCl, 0.1% Tween 20, pH 9.0 (TST), and then incubated with primary antibody (polyclonal anti(PAPP-A·pro-MBP) (23)) diluted in TST, 0.5% fetal bovine serum for 1 h at 37 °C. Blots were washed in TST and further incubated with secondary antibody (P217, DAKO) diluted in TST, 0.5% fetal bovine serum and then washed again with TST. The blots were developed using enhanced chemiluminescence (ECL, Amersham Biosciences).

Determination of Carbohydrate Attachment Sites of PAPP-A-- Purified PAPP-A·pro-MBP (20 mg) contained in 50 mM Tris, 50 mM sodium chloride, 6 M guanidine hydrochloride, pH 7.5, was reduced with 7 mM dithioerythritol and then alkylated using 15 mM iodoacetamide. Formic acid was added to 1 M, and the protein was digested with pepsin for 12 h, dialyzed against 50 mM Tris, 50 mM sodium chloride, pH 7.5, and further digested with trypsin for 12 h. The digest was then separated by gel filtration on a Sephadex G-50-SF column equilibrated and eluted with 1 M formic acid (not shown). Three pools (fractions 18-23, 24-29, and 30-35; compare with Fig. 1) were made, and each was further separated directly by RP-HPLC into 90 fractions (not shown). GlcNAc-containing fractions were subjected directly to N-terminal sequence analysis or further chromatographed on either the same column using HFBA rather than trifluoroacetic acid for ion pairing or on a polySULFOETHYL-aspartamide column, as detailed above. The absence of asparagine, when expected from the cDNA sequence, was taken as evidence for carbohydrate substitution. Mass spectrometry of peptides substituted with carbohydrate was not attempted.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of Disulfide Bound Peptides from the PAPP-A·Pro-MBP Complex-- To obtain isolated disulfide-bound peptides derived from the PAPP-A·pro-MBP complex, protein purified from pregnancy serum was digested with trypsin, degraded by cyanogen bromide, further digested with trypsin, and then fractionated by gel filtration (Fig. 1). The early eluting protein (pool CBT-1) contained the majority of cysteine residues, determined as Cya after oxidation (Fig. 1), but required further digestion for the generation of peptides suitable for analysis. However, peptides of pools CBT-2 to -5 were each separated by chromatography on a strong cation exchanger (Fig. 2). Fractions were pooled based on their contents of cysteine, and the peptides were further separated by at least one round of RP-HPLC (Fig. 3). Manually collected fractions were analyzed by amino acid analysis, and the peptides of cysteine-containing fractions were analyzed further. Unequivocal identification by sequence analysis was obtained for all peptides isolated, and the identities of most peptides were confirmed by the composition analyses and mass spectra. A total of 16 peptides, representing 36 PAPP-A cysteines and 5 pro-MBP cysteines, were isolated from pools CBT-2 to -5 (Table I, peptides marked 1).


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Fig. 1.   Gel filtration of PAPP-A·pro-MBP complex degraded with cyanogen bromide and digested with trypsin. Degraded and digested PAPP-A·pro-MBP complex (45 mg) was loaded onto a column packed with Sephadex G-50-SF, equilibrated, and eluted with 1 M formic acid. The eluate was divided into six pools, CBT-1 to -6, each of which were analyzed for the contents of total cysteine, determined as Cya after performic acid oxidation, and for the contents of the amino sugar GlcNAc. Relative values of Cya and GlcNAc of each pool are listed below the chromatogram. Peptides of pools CBT-2, -3, -4, and -5 were isolated by cation exchange chromatography (shown for pool CBT-4 in Fig. 2) followed by RP-HPLC (Fig. 3). The material of pool CBT-1 (>10 kDa) was further digested prior to repeated gel filtration (Fig. 4) and further peptide separation.


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Fig. 2.   Example of cation exchange chromatography of pooled fractions from gel filtration. The material in pool CBT-4 (Fig. 1) was loaded onto a column packed with a strong cation exchanger (polySULFOETHYL-aspartamide) and eluted with a gradient of increasing salt concentration. Fractions of 0.5 ml were collected. Vertical bars show the relative amount of Cya in each fraction, as determined after performic acid oxidation. Pools of two to four fractions were further separated by RP-HPLC, as indicated for the pool of fractions 40-42 (see Fig. 3). Pools CBT-2, -3, and -5 (Fig. 1) were chromatographed and analyzed similarly.


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Fig. 3.   Example of RP-HPCL of pooled fractions from cation exchange chromatography. Pooled fractions of the eluate from ion exchange chromatography of pools CBT-2 to -5 were fractionated by RP-HPLC (Nucleosil C18) using trifluoroacetic acid as the ion pairing agent. This chromatogram shows the chromatogram of fractions 40-42, originating from pool CBT-4. The fractions were collected manually, and the amount of Cya in each peptide-containing fraction was determined. Amino acid analysis, N-terminal sequence analysis, and mass spectrometry identified peptides of selected fractions. The peak eluting at 15 min, corresponding to peptide P31 (Table I), is pointed out as an example of an identified disulfide-bound peptide. Some fractions, containing mixtures of peptides, required further separation (carried out on the same column using HFBA, rather than trifluoroacetic acid, as the ion pairing agent).

                              
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Table I
Summary of disulfide bond peptides derived from the PAPP-A/pro·MBP complex

The material contained in pool CBT-1 was further digested with pepsin and fractionated by gel filtration (not shown). Pools of smaller peptides were then separated by ion exchange and RP-HPLC chromatography and identified as described above. Digestion with pepsin resulted in the isolation of five peptides containing 12 PAPP-A cysteine residues not previously isolated (Table I, peptides marked 2). As digestion with this enzyme still left a resistant protein mass (containing 77% of eluting cysteine), the early eluting fractions were pooled, further digested with thermolysin, and the resulting peptides separated by a third run of gel filtration (Fig. 4). The majority of the cysteine now eluted as smaller peptides, and larger peptides were primarily peptides with carbohydrate substitutions, as determined by the contents of GlcNAc (Fig. 4). Further analysis of peptides in pools CBTPTL-2, -3, and -4, again using cation exchange chromatography and RP-HPLC, resulted in the identification of 15 peptides with new information, containing 16 more cysteine residues of PAPP-A and 5 of pro-MBP (Table I, peptides marked 3). In summary, peptides representing a total of 64 PAPP-A and 10 pro-MBP cysteine residues were isolated in these experiments.


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Fig. 4.   Gel filtration of material from pool CBT-1 further digested with pepsin and thermolysin. The material of pool CBT-1 (Fig. 1) was further digested with pepsin and then separated by gel filtration on Sephadex G-50-SF (not shown). The material of this eluate, which still eluted in fractions 18-30, was digested with thermolysin and reloaded onto the column, equilibrated, and eluted with 1 M formic acid, as shown here. The material in pool CBTPTL-1 and -2 contained the majority of the total contents of GlcNAc, whereas the majority of cysteine (Cya) eluted in fractions of pools CBTPTL-2, -3, and -4, as listed below the chromatogram. For isolation of peptides, the pools CBTPTL-2, -3, and -4 were further fractionated using cation exchange chromatography (as in Fig. 2) and RP-HPLC (as in Fig. 3) (not shown).

For some of the isolated peptides containing more than two cysteine residues, further analysis provided additional information about disulfide bonds. When peptide P3 was subjected to sequence analysis, bis-PTH-Cys2 was absent from cycle 6, showing that Cys-334 is not connected to Cys-344 but rather to Cys-348 or Cys-360. Importantly, peptide P4 was generated by digestion of peptide P3 (partially cleaved at Asp/Pro 345/346 by acid-induced cleavage (34) while dissolved in 70% formic acid) with thermolysin, demonstrating that Cys-334 is bound to Cys-348, and Cys-344 to Cys-360. Sequence analysis of peptide P5 showed the presence of bis-PTH-Cys2 in cycle 8, demonstrating that Cys-377 pairs with Cys-393 and Cys-394 with Cys-405. The isolation of peptide P9 (in which Cys-633 and Cys-801 are connected), derived from P8 by further digestion with thermolysin, showed that Cys-630 is connected to Cys-798. Similarly, peptide P26 was resolved because P27 accounts for pairing of two of its four cysteine residues. Furthermore, the isolation of peptide P36 (in which Cys-1478 and Cys-1496 are connected) resolved two of the cysteines of P35, but we were unable to further resolve the cysteines of P35 by enzymatic digestion.

Analysis of Larger Cyanogen Bromide Fragments of the PAPP-A·Pro-MBP Complex-- Peptides corresponding to 18 PAPP-A cysteine residues and 2 pro-MBP residues still had not been isolated. We anticipated that further analysis of the present fractionated digest would not result in identification of the missing peptides, and therefore we undertook a second round of degradation of intact PAPP-A·pro-MBP complex with cyanogen bromide. The resulting polypeptide fragments were separated by denaturing gel filtration without prior enzymatic digestion, and fractions were separated by nonreducing SDS-PAGE (Fig. 5).


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Fig. 5.   Denaturing gel filtration of cyanogen bromide-degraded PAPP-A·pro-MBP complex. Purified PAPP-A·pro-MBP complex (25 mg) was degraded with cyanogen bromide and then separated by gel filtration on Superose 12 HR 10/30, equilibrated, and eluted in 8 M urea at pH 5.0. This chromatogram shows an analytical run of the degraded complex. The inserted gel image shows nonreducing SDS-PAGE of selected fractions (21, 26, 40, 43, and 48). Bands were analyzed by N-terminal sequence analysis. Further chromatography and digestion of a pool of fractions 20-30 resulted in the isolation of a series of disulfide-bound peptides (marked 5 in Table I), not previously seen as different variants.

N-terminal sequence analysis of the protein in the ~18-kDa band of fraction 25, CNB1, shows one sequence (Table II). We conclude that this fragment contains PAPP-A residues Cys-1266 to Met-1394. We have accounted for pairing of all 12 cysteine residues of this fragment (cf. above), except Cys-1362 and Cys-1391, which therefore must link to each other. The calculated molecular mass of CNB1 is 14 kDa, suggesting that Asn-1385 is substituted with N-linked carbohydrate.

                              
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Table II
Characterization of cyanogen bromide fragments by SDS-PAGE and N-terminal sequence analysis

N-terminal sequence analysis of protein in the ~21-kDa band of fraction 40, CNB2, revealed four sequences (Table II) corresponding to four contiguous stretches covering all of PAPP-A residues from Gln-1395 to Gly-1547. Based on a calculated molecular mass of 17 kDa, we conclude that none of the cysteine residues of this fragment are engaged in interchain disulfide bonding between two PAPP-A subunits. The discrepancy of 4 kDa between observed and calculated molecular weights is in agreement with carbohydrate substitution of Asn-1449 (see below).

N-terminal sequence analysis of protein in the ~17-kDa band of fraction 48, CNB3, revealed two sequences (Table II) corresponding to two contiguous stretches covering PAPP-A residues from Glu-1 to Met-159, confirming that the only two cysteine residues of this fragment are connected (as found with peptide P1, Table I).

Next, the material in pool CB-1 (Fig. 5) was further separated by cation exchange chromatography on a Mono S column, and fractions were analyzed by nonreducing SDS-PAGE as above (not shown). N-terminal sequence analysis showed that a fragment of 34 kDa, CNB4, contained three N-terminal sequences of peptides held together by disulfide bonds (Table II). We conclude that in addition to cleavage after Met-831, -883, and -1014, acid-induced cleavage had occurred at Asp-1105/Pro-1106 (34), resulting in a disulfide-bound fragment set covering PAPP-A residues Asp-832 to Asp-1105 with a calculated molecular mass of 30 kDa. The discrepancy of 4 kDa between observed and calculated values is in agreement with carbohydrate substitution of Asn-946 (see below). All cysteine residues within this fragment set are paired to other cysteine residues within it (Table I), except Cys-880 and Cys-891. Based on the size of the fragment, these residues are not engaged in PAPP-A interchain bonding (i.e. dimerization), and we conclude that Cys-880 and Cys-891 are linked to each other.

A fragment of 45-50 kDa, CNB5, showed two N-terminal sequences (Table II). Importantly, the sequence starting at Pro-1106 confirmed the occurrence of the acid-induced split at Asp-1105/Pro-1106. The CNB5 fragment covers PAPP-A residues Pro-1106 to Met-1265 with a calculated molecular mass of 18 kDa. This suggested that one or more cysteine residues of the fragment is engaged in dimerization of the PAPP-A subunits, as carbohydrate substitution (two potential sites, see Table II) is not likely to be responsible for the difference between the observed and the calculated molecular weights. Cys-1130 is the only residue not previously isolated and is therefore responsible for dimerization of the PAPP-A subunit. The acid-induced split at Asp-1105/Pro-1106 was only partial, as a 70-75-kDa variant of the fragment was also identified with a Asp-1105/Pro-1106 peptide bond still intact.

A fragment set of 70 kDa, CNB6, showed several N-terminal sequences (Table II), which could be resolved because of the limited number of possibilities after degradation with cyanogen bromide only. Thus, again based on complete cyanogen bromide cleavage of peptide bonds on the C-terminal side of methionine, the fragment set covers 415 residues of PAPP-A (221-460, 481-556, 576-650, and 789-816) and 36 residues of pro-MBP (47-82), with a combined molecular mass of 51 kDa. The additional 20 kDa of observed molecular mass are likely caused by carbohydrate substitution of both PAPP-A (see below) and pro-MBP, known to be substituted with a glycosaminoglycan at Ser-62 (35). The fragment set thus defined contains two cysteine residues not previously isolated, Cys-247 and Cys-542, which therefore must form a disulfide bond. Four of five cysteines in the stretch from Ile-481 to Met-556 are bound to each other (P7, Table I). This stretch (with a calculated molecular mass of 8 kDa) migrates as part of the 70-kDa fragment set because the fifth cysteine (Cys-542) is linked to Cys-247. We cannot completely exclude, however, that CNB6 also partially includes PAPP-A residues 557-576 (19 residues, including Cys-563) due to the possibility of incomplete cleavage at Met-556/Ser-557.

Thus, analysis of the peptide fragments CNB1-6 demonstrated linkage of 6 additional PAPP-A cysteine residues and further suggested that Cys-1130 participates in dimerization of PAPP-A (Table I, peptides marked 4).

Further Enzymatic Cleavage of Cyanogen Bromide Treated PAPP-A·Pro-MBP-- To attempt isolation of peptides containing the remaining 12 (including Cys-1130) cysteines of PAPP-A and 2 of pro-MBP, fractions originating from the ion exchange chromatography of pool CB-1 (Fig. 5) were further digested with trypsin and/or thermolysin and separated using gel filtration followed by RP-HPLC, as in the first round of digestion (not shown). Five disulfide-bound peptides, of which variants had not been isolated previously, were identified (Table I, peptides marked 5). Thus, in summary, peptides containing Cys-563 of PAPP-A and Cys-128 and -201 of pro-MBP remained unidentified.

Recombinant PAPP-A with Cys-1130 Substituted into Ala Is Secreted as a Monomer-- To analyze further the involvement of Cys-1130 in dimerization of PAPP-A, we used a recently established mammalian expression system for PAPP-A (2). The PAPP-A expression plasmid was mutated to encode a mutant, C1130A, in which Cys-1130 was substituted with alanine. Mutated protein secreted by transfected cells was compared with wild-type PAPP-A by nonreducing SDS-PAGE and visualization by Western blotting (Fig. 6). Wild-type PAPP-A migrated as a dimer of 400 kDa, as observed previously (2), whereas the C1130A mutant migrated as a monomer of 200 kDa. Thus, this experiment supports our finding that Cys-1130 is responsible for dimerization of PAPP-A and that no other cysteine residues of PAPP-A participate in PAPP-A-PAPP-A dimerization. Of importance, Cys-563 is not involved in a hypothetical arrangement of antiparallel PAPP-A dimerization, as the C563A mutant does not migrate as a monomer (14).


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Fig. 6.   Western blot of wild-type PAPP-A and PAPP-A mutant C1130A. Culture media from 293T cells transfected with empty vector (lane 1), cDNA encoding PAPP-A wild-type (lane 2), or cDNA encoding PAPP-A mutant C1130A (lane 3) were separated by 3-8% SDS-PAGE and analyzed using polyclonal antibodies against PAPP-A.

Isolation of PAPP-A Peptides Containing N-Linked Carbohydrate-- Finally, to determine the occupancy of the 13 potential sites of N-linked glycosylation (Asn-X-Ser/Thr, X not Pro), reduced and alkylated PAPP-A·pro-MBP complex was digested with pepsin and trypsin. The digest was separated into three pools by gel filtration, each of which was further separated by RP-HPLC (not shown), and all fractions were analyzed for the presence of GlcNAc (diagnostic for the presence of N-linked carbohydrate). Peptides of GlcNAc-containing fractions were identified by sequence analysis without further separation or after a second round of chromatography. One peptide (CBH11) had been isolated from the first PAPP-A·pro-MBP digest, in which cyanogen bromide and trypsin were used. A total of 11 isolated PAPP-A peptides were found to carry N-linked carbohydrate (Table III).

                              
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Table III
Summary of isolated PAPP-A peptides with N-linked carbohydrate derived from reduced and carboxymethylated PAPP-A/pro-MBP


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have determined the connectivity of cysteine residues of the 2:2 complex between PAPP-A and pro-MBP. The PAPP-A subunit contains 82 cysteine residues, of which 81 have been accounted for. We were unable, however, to locate Cys-563 in any peptide derived from the PAPP-A·pro-MBP complex, and therefore its status remains unknown. The pro-MBP subunit contains 12 cysteine residues, of which two, Cys-128 and Cys-201, were not identified. Our results, summarized in Fig. 7, were obtained by chromatographic analyses of peptides generated by chemical and enzymatic cleavage of the purified PAPP-A·pro-MBP complex. In addition, our finding that Cys-1130 is responsible for dimerization of the PAPP-A subunit was verified by showing that when Cys-1130 is mutated into alanine, PAPP-A is secreted as a monomer (Fig. 6). Thus, within the 2:2 complex, containing a total of 188 cysteine residues, PAPP-A is dimerized by a single disulfide bond; pro-MBP is dimerized by two disulfides, and each PAPP-A subunit is connected to a pro-MBP subunit by two disulfide bonds. All other identified disulfide bonds are intrachain bridges (Fig. 7).


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Fig. 7.   Disulfide structure of the 2:2 PAPP-A·pro-MBP complex. One PAPP-A subunit (1547 residues, including 82 cysteines) and one pro-MBP subunit (206 residues, including 12 cysteines) are illustrated as open bars. The locations in the PAPP-A subunit of LNR-1 to -3 and CCP-1 to -5, both defined by sequence similarity, are indicated by bars and shading, and the proteolytic domain (PD), defined by prediction of secondary structure (14), is indicated by a bar. In addition, the location of a putative N-terminal domain (N), two central domains (M1 and M2), and a C-terminal domain, as discussed in the text, are indicated by bars. Disulfide bonds are shown as lines connecting individual cysteine residues. The pro-MBP dimer of the complex contains two interchain disulfide bridges (broken lines marked Pro-MBP dimerization), and the PAPP-A dimer of the complex contains one interchain disulfide bridge (broken line marked PAPP-A dimerization). The PAPP-A dimer and the pro-MBP dimer are connected to each other by two interchain bridges, as shown. Two cysteines of pro-MBP are bound either to a Cys-Gly dipeptide (CG) or to a single cysteine residue (C), as indicated. The figure is based on isolated disulfide-bound peptides (Table I), on analysis of larger disulfide bound fragments, as detailed in the main text. The connectivity of three cysteine residues (*) remains unresolved, as they were not contained in any isolated peptides or fragments. The middle part of the figure shows the exon structure of PAPP-A, according to the PAPP-A gene structure (Table IV). The lower part of the figure lists sequentially the pairing of all cysteine residues of PAPP-A and pro-MBP. PAPP-A cysteines at positions 1487, 1503, 1504, 1520, 1526, and 1528 were isolated in a partially resolved cluster (peptide P35, Table I), as indicated by linkage to CP35.

The disulfide connectivity of the majority of the cysteines was delineated to the level of single residues. However, we were unable to resolve completely the pairing of cysteines in two peptides, P7 (containing 4 cysteines) and P35 (containing 6 partially resolved cysteines) (Table I and Fig. 7), but cysteines of these peptides were present within relatively short sequence stretches of 30 and 42 residues, respectively.

We have also isolated peptides derived from reduced and carboxymethylated PAPP-A showing that 11 of 13 potential sites for N-linked carbohydrate are substituted (Table III). PAPP-A is most likely not O-glycosylated, as N-acetylgalactosamine is absent from the isolated PAPP-A subunit (23). In contrast, besides a single N-linked carbohydrate group, pro-MBP isolated from the PAPP-A·pro-MBP complex contains four O-linked glycan moieties and one O-linked glycosaminoglycan (35).

The proteolytic domain of PAPP-A does not show global sequence similarity to other proteins. But based on secondary structure prediction and on the assumption that the proteolytic domain of PAPP-A has the topology common to metzincins (13), its boundaries have been tentatively located (14). The three alpha -helices and five beta -strands common to metzincins are all predicted within the 312-residue sequence stretch from Val-272 to Tyr-583. Our data show that two disulfide bonds, Cys-247/Cys-542 and Cys-252/Cys-577 (Table I and Fig. 7), bring the N- and C-terminal ends of the predicted proteolytic domain in proximity to each other, consistent with the tertiary structure of the proteolytic domain of metzincins known from crystal structures (13). Of particular interest, the disulfide bridge connecting Cys-252 and Cys-577 could be equivalent to a salt bridge, present in two other metzincin families, which connects residues preceding the first secondary structure element (beta -strand S1) with the last element (alpha -helix HC) (13, 14). The predicted proteolytic domain of PAPP-A contains 14 more cysteine residues (Fig. 7). Of these, 12 form intradomain disulfide bonds, one connects to a cysteine residue of pro-MBP, and one has unidentified status (Cys-563). In contrast, the proteolytic domains of metzincins in general contain very few, if any, disulfide bonds (13).

PAPP-A is encoded by 22 exons (Table IV), whose relation to the amino acid sequence of PAPP-A is depicted in Fig. 7. The 3'-half of exon 2, the largest exon of the PAPP-A gene, encodes the N-terminal half of the proteolytic domain. With exon 1, the 5'-half of exon 2 encodes a region of PAPP-A, designated domain N (Fig. 7), in which a LamG-like jellyroll fold domain has been predicted recently (36). This domain is known from several other proteins, including human sex hormone-binding globulin (37). The LamGL domain does not make disulfide bond connection to the proteolytic domain or other regions of PAPP-A (Fig. 7), but association with a proteolytic domain, as evident from the gene structure, is not known from other proteins.

                              
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Table IV
Structure of the gene encoding PAPP-A
The PAPP-A gene structure is derived from available genomic data (accession number NT_017568.9).

In contrast to the proteolytic domain, the three LNR and the five CCP modules of PAPP-A have been identified because they show weak sequence similarity to such modules in other proteins (11). The LNR module of about 35 amino acids, including 6 cysteine residues and other highly conserved residues (Fig. 8), is known from proteins related to the Notch receptor, important in signaling of cell fates (16). It occurs as a triplication in the extracellular portion of these receptors, close to the site of ligand-induced proteolysis involved in Notch signaling, and is thought to negatively regulate proteolysis (38, 39). Biochemical characterization of the LNR module has been lacking, but analysis of an isolated, recombinant LNR-1 module from human Notch1 shows that in the presence of calcium ions (>1 mM), only one disulfide isomer is formed (C1-C5, C2-C4, and C3-C6, local numbering) (Fig. 8) (40). Interestingly, the calcium-dependent folding was abolished when three conserved residues with acidic side chains (Fig. 8) were individually substituted with alanine. In PAPP-A and PAPP-A2, the only other proteins containing LNR modules, LNR-1 and -2, are located together within the proteolytic domain, and LNR-3 is located near the C terminus, in the sequence stretch designated domain C (Fig. 7). Based on the alignment of LNR sequences (Fig. 8), LNR-1 and -2 lack the C1-C5 disulfide, and LNR-3 lacks the C2-C4 disulfide, but the disulfide pairing observed is consistent with that of recombinant Notch1 LNR-1. Furthermore, all modules share conserved residues with the modules of the Notch-related proteins (Fig. 8). It is unknown whether LNR-3 is located in proximity to LNR-1 and -2 in the three-dimensional structure of PAPP-A, but it is tempting to speculate that it forms a functional unit with LNR-1 and -2. Interestingly, the importance of PAPP-A LNR-3 is underscored by a 100% conservation of this module between man and zebrafish (Fig. 8).


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Fig. 8.   Alignment of LNR modules. LNR-1, -2, and -3 modules of human PAPP-A, human PAPP-A2, the human Notch1 receptor (GenBankTM accession number P46531), and the Lin-12 protein from Caenorhabditis elegans (GenBankTM accession number P14585) are aligned to emphasize the conservation of cysteine residues (in boldface), and other conserved residues (shaded). An EST clone originating from zebrafish (accession number BI846276) shows that PAPP-A is also present in this species (z.f.PA). The isolated clone encodes 26 residues of LNR-3 (corresponding to Lys-1476 to Gly-1501 of human PAPP-A), all of which are identical in human PAPP-A. The disulfide pairing of PAPP-A cysteines, as found here, and the disulfide pairing of a recombinant, refolded LNR-1 module of human Notch1 (40) are indicated with lines. The positions of conserved residues with acidic (or amide) side chains, proposed to be involved in binding of calcium ions (40), are indicated with bullets. Local numbering of cysteine residues 1-6 is indicated. (The LNR module is also denoted the NL, the Notch, or the LIN-12 module.)

Our data show that disulfide bonds are formed between the first and the fifth (C1-C5, local numbering), the second and the third (C2-C3), and the fourth and the sixth (C4-C6) cysteine in each of the five CCP modules of PAPP-A. The standard CCP module (of complement proteins) contains four cysteines in a C1-C3/C2-C4 arrangement (17), but in the PAPP-A CCPs, two additional cysteine residues are present between the first and the second of these four cysteines (11). Thus, the arrangement of disulfide bonds known from the standard CCP module is conserved in the CCP modules of PAPP-A, but in PAPP-A, one additional disulfide bond is present in each module. Although none of the 6 cysteine residues of each CCP module links to neighboring domains, the domain boundaries do not coincide with exon junctions (Fig. 7). Interestingly, CCP modules of selectins also have 6 cysteine residues (41). None of these have experimentally established disulfide structures, however, but are likely to share the C1-C5/C2-C3/C4-C6 pattern with PAPP-A. For comparison, we have aligned the CCP modules of PAPP-A and PAPP-A2 with selected CCP modules from other proteins with know or unknown disulfide structure (Fig. 9).


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Fig. 9.   Alignment of CCP modules. CCP-1 to -5 of human PAPP-A (PA-1 to -5) and PAPP-A2 (P2-1 to -5), CCP-1 to -4 of human decay-accelerating factor (DAF, GenBankTM accession number P08174) (DAF-1 to -4), and CCP-1 to -4 of human E-selectin (GenBankTM accession number P16581) (ESE-1 to -4) are aligned to emphasize the conservation of cysteine residues (in boldface) and other particularly conserved residues (shaded). Local numbering of cysteine residues 1-6 or 1-4 is indicated. The C1-C3/C2-C4 disulfide arrangement of the complement CCPs with four cysteine residues (including decay-accelerating factor) has been determined experimentally (17, 45). In contrast, the pairing of four of the six cysteines of selectin CCPs (including E-selectin) has been inferred by similarity to the complement CCPs. Based on comparison with PAPP-A, which has the same number of cysteine residues, the selectin CCPs most likely share the C1-C5/C2-C3/C4-C6 pattern with PAPP-A CCPs. The disulfide pattern of PAPP-A CCPs and complement CCPs, and the predicted pattern of the selectin CCPs, is indicated with lines.

No other domains of PAPP-A have been identified by sequence similarity. Thus, the region between the proteolytic domain and CCP-1 (Fig. 7), a total of about 550 amino acids corresponding to the central third of the sequence, does not share similarity with other known proteins. At least two clusters of disulfide bonds are apparent (Fig. 7): PAPP-A residues approx 600 to approx 800 compose one cluster (encoded by exons 6 and 7), and residues approx 850 to approx 1100 compose at least one other cluster (encoded by exons 8-13). Such clusters, in which disulfide bonds both restrain and stabilize the three-dimensional structure, may correspond to protein domains. We therefore tentatively define two central domains, M1 and M2 (see Fig. 7). Cys-1130, responsible for PAPP-A dimerization, is located at the very C-terminal end of M2, and Cys-652, linking to pro-MBP, is located at the N-terminal end of domain M1. All other cysteines, a total of 22, form disulfide bonds within domains M1 or M2. Importantly, knowledge of the pairing of cysteines will allow rational dissection of the putative domains for recombinant expression and further structural and functional studies.

In differentiating eosinophils, mature MBP is generated by proteolytic processing of pro-MBP (24). It contains residues 106-222 of pro-MBP and therefore 9 of the 12 pro-MBP cysteine residues. MBP, isolated from mature eosinophils, has two disulfide bridges and five cysteine residues with free sulfhydryl groups (Cys-107, -128, -147, -169, and -201) (26, 27). In the MBP portion of pro-MBP, as part of the PAPP-A·pro-MBP complex, we here demonstrate the same two disulfides by the isolation of peptides PM3 and PM5 (Table I). However, Cys-169 forms one of two bridges to the PAPP-A subunit (P10); Cys-107 forms one of two bridges to the other pro-MBP subunit (PM2), and Cys-147 links to a single cysteine residue, not part of a polypeptide (PM4). The status of Cys-128 and Cys-201 remains unknown, but they likely exist as unpaired cysteines, possibly oxidized by oxygen. The first of the three cysteine residues of the proportion of pro-MBP (Cys-51) forms the other bridge to PAPP-A (P6); the second (Cys-89) binds to a Cys-Gly dipeptide (possibly derived from glutathione) (PM1), and the third (Cys-104) forms the other bridge dimerizing pro-MBP (PM2).

Although accounted for in the PAPP-A·pro-MBP complex as responsible for linking to Cys-51 and Cys-169, the pro-MBP subunit, our results do not provide information about the status of PAPP-A residues Cys-381 and Cys-652 in the uncomplexed dimer. In one scenario, these two residues form a disulfide bond, which is broken in the process of complex formation by disulfide exchange with a sulfhydryl group of either Cys-169 or -51 of pro-MBP; the second interchain disulfide bond is then formed by oxidation of the remaining two cysteine residues with molecular oxygen. Another scenario involves participation of Cys-563 or of thiol- or disulfide-containing reactants such as glutathione. Of importance, our knowledge that the PAPP-A and pro-MBP subunits are linked by two identified disulfide bonds provides an experimental base for studying the process of complex formation by analysis of mutated proteins, as well as the mechanistic basis for the PAPP-A inhibitory activity of pro-MBP (2, 25). Inhibition may be based on interaction of a pro-MBP sulfhydryl group with the active site zinc ion, in analogy with the cysteine switch mechanism known from latent fibroblast collagenase, for example (42). Alternatively, the PAPP-A activity may be controlled by basic residues of MBP, as several basic residues of IGFBP-4 and -5 have recently been found to be important for substrate cleavage (43).

With 46% of PAPP-A residues identical, PAPP-A2 is the only known protein with global similarity to PAPP-A (15). Interestingly, all 82 cysteines of mature PAPP-A are also present in PAPP-A2, but PAPP-A2 has four additional cysteines: Cys-343,2 Cys-533, Cys-618, and Cys-1268 (corresponding to PAPP-A residues Lys-95, Tyr-281, Ala-367, and Tyr-1009). Cys-343 and -533 of PAPP-A2 likely form a disulfide bond linking the N-terminal domain with the proteolytic domain. PAPP-A2 is secreted as a monomer, and Cys-1390 of PAPP-A2 (corresponding to the dimerization cysteine of PAPP-A, Cys-1130) is therefore not engaged in dimerization. Based on a higher probability of making connection to a sequentially close residue, Cys-1390 may link to Cys-1268. A hypothetical linkage between Cys-1268 and -1390 of PAPP-A2 is compatible with the domain structure of PAPP-A, as dictated by the overall pattern of disulfide bonds (Fig. 7). Furthermore, unlike the deviating LNR-2 of PAPP-A (Fig. 8), LNR-2 of PAPP-A2 contains four cysteines, including Cys-618 as the first. This residue could pair with Cys-632, the third cysteine of LNR-2, which in PAPP-A links to pro-MBP. It is unknown whether PAPP-A2 interacts with pro-MBP. If it does not, the conserved Cys-903 of PAPP-A2 (corresponding to Cys-652 of PAPP-A, which links to pro-MBP) may be paired differently compared with PAPP-A.

In conclusion, we have delineated the connectivity of cysteine residues of the heterotetrameric PAPP-A·pro-MBP complex. The PAPP-A and pro-MBP subunits contain a total of 94 (82 + 12) cysteine residues, of which one from PAPP-A and two from pro-MBP were not encountered in any peptide. Knowledge of the PAPP-A disulfide pattern may help us define domain boundaries, thereby facilitating structural studies of individual domains. Furthermore, it will allow rational experimental design of functional studies aimed at understanding the formation of the PAPP-A·pro-MBP complex, as well as the inhibitory mechanism of pro-MBP.

    ACKNOWLEDGEMENT

We thank the staff at the Department of Gynaecology and Obstetrics at Aarhus University Hospital for donating pregnancy serum.

    FOOTNOTES

* This work was supported by grants from the Novo Nordic Foundation, the Danish Natural Science Research Council, and Danish Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Institute of Biochemistry and Molecular Biology, University of Wroclaw, 50-137 Wroclaw, Poland.

§ To whom correspondence should be addressed. E-mail: co@mb.au.dk.

Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M208777200

2 PAPP-A is numbered with its N-terminal Glu as residue 1 (11). Glu-1 of PAPP-A corresponds to Glu-81 of prepro-PAPP-A (GenBankTM accession number Q13219). For PAPP-A2, the numbering of prepro-PAPP-A2 (GenBankTM accession number AF311940) (15) is used; for pro-MBP, the numbering of prepro-MBP (GenBankTM accession number P13727) (44) is used.

    ABBREVIATIONS

The abbreviations used are: PAPP-A, pregnancy-associated plasma protein-A; pro-PAPP-A, proform of pregnancy-associated plasma protein-A; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor-binding protein; pro-MBP, the proform of eosinophil major basic protein; PAPP-A2, pregnancy-associated plasma protein-A2; LNR, Lin-Notch repeat; CCP, complement control protein; RP-HPLC, reversed-phase high pressure liquid chromatography; PTH, phenylthiohydantoin; HFBA, heptafluorobutyric acid; Cya, cysteic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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