Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor

Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.     

Covalent interaction between proform of eosinophil major basic protein (proMBP) and pregnancy-associated plasma protein-A (PAPP-A) is a cell-mediated event and required for proMBP inhibition of the catalytic activity of PAPP-A

This study was undertaken to determine the mechanism by which proform of eosinophil major basic protein (proMBP) inhibits the IGFBP-4 proteolytic activity of pregnancy-associated plasma protein (PAPP)-A. Co-overexpression of PAPP-A with proMBP in 293T cells, or co-incubation of 293T cells, respectively, overexpressing proMBP and PAPP-A resulted in the formation of a covalent proMBP-PAPP-A complex and inhibition of IGFBP-4 proteolysis. Similar results were obtained when recombinant proMBP and PAPP-A were incubated in the presence of U2 osteosarcoma cells or when recombinant proMBP was added to the U2 cells overexpressing PAPP-A. In contrast, no formation of covalent proMBP-PAPP-A complex or inhibition of IGFBP-4 proteolysis was observed when recombinant proMBP and PAPP-A were incubated under cell-free conditions, although proMBP was able to interact with PAPP-A in a non-covalent manner. These new findings suggest that formation of covalent proMBP-PAPP-A complex is a cell-mediated event and is required for proMBP to inhibit the catalytic activity of PAPP-A.     

Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein

We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.     

Inhibition of proteolysis by the proform of eosinophil major basic protein (proMBP) requires covalent binding to its target proteinase

By proteolytic cleavage of insulin-like growth factor binding proteins, the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) is able to control the biological activity of insulin-like growth factors. PAPP-A circulates in pregnancy as a proteolytically inactive complex, disulfide bound to the proform of eosinophil major basic protein (proMBP). We here demonstrate that co-transfection of mammalian cells with PAPP-A and proMBP cDNA results in the formation of a covalent PAPP-A/proMBP complex in which PAPP-A is inhibited. Formation of the complex also occurs when PAPP-A and proMBP synthesized separately are incubated. Complex formation was monitored by Western blotting, and by using an immunoassay specific for the complex. Using mutagenesis, we further demonstrate that the complex forms in a specific manner and depends on the presence of two proMBP cysteine residues. Mutated proMBP, in which Cys-51 and -169 are replaced by serine, is unable to form the covalent complex with PAPP-A. Of particular interest, such mutated proMBP further lacks the ability to inhibit PAPP-A. For the first time, this conclusively demonstrates that proMBP is a proteinase inhibitor. We further conclude that proMBP inhibits PAPP-A in an unusual manner, not paralleled by other proteinase inhibitors of our knowledge, which requires proMBP to be covalently bound to PAPP-A by disulfide bonds. ProMBP binding to PAPP-A most likely either abrogates substrate access to the active site of PAPP-A or induces a conformational change in the structure of PAPP-A, as we, by further mutagenesis, were able to exclude that the inhibitory mechanism of proMBP is based on a cysteine switch-like mechanism.     

Pregnancy-associated plasma protein A and proform eosinophilic major basic protein in the detection of different types of coronary artery disease

Kryptor system was proven to be a rapid, standard method for pregnancy-associated plasma protein A and proform eosinophilic major basic protein (PAPP-A/proMBP) complex detection in coronary artery disease (CAD). No age and/or gender differences in 51 controls and 110 stable coronary artery disease (SCAD) patients were found. SCAD patients did not differ from controls and no difference in PAPP-A/proMBP levels with regards to the number of affected vessels was found. In 21 unstable angina pectoris (UAP), in 35 without and 66 with ST elevation acute myocardial infarctions (NSTEMI, STEMI respectively) patients PAPP-A/proMBP levels were increased (P=0.004 and P<0.0005, respectively). PAPP-A/proMBP levels did not correlate with cardiac troponin I (cTnI) in STEMI and NSTEMI patients. PAPP-A/ proMBP increase was more frequent than cTnI (P=0.036) within the early phase of STEMI. In NSTEMI patients PAPP-A/proMBP positivity was present in 50 % of cTnI negative cases. Receiver operating characteristic (ROC) analysis revealed the highest diagnostic accuracy of PAPP-A/proMBP (0.919) in STEMI cTnI positive cases. The highest specificity/sensitivity PAPP-A/proMBP levels for particular acute coronary syndrome (ACS) types were 10.65-14.75 mIU/l. Combination of PAPP-A/proMBP with cTnI increases their diagnostic efficacy within the early phase of ACS. Our results suggest that PAPP-A/proMBP complex is involved in processes preceding vulnerable plaque development in ACS.     

The proform of the eosinophil major basic protein binds the cell surface through a site distinct from its C-type lectin ligand-binding region

The highly basic eosinophil major basic protein (MBP), present in the crystalloid core of eosinophil leukocyte granules, has both cytotoxic and cytostimulatory properties and is directly implicated in a number of diseases. The crystal structure of MBP resembles that of the C-type lectin (CTL) superfamily, and recent data showed that MBP binds heparan sulfate glycosaminoglycan (GAG), with the CTL ligand-binding region as the binding site. MBP is synthesized as a proform (pro-MBP) containing an acidic propiece believed to neutralize the basic MBP domain. Using flow cytometry and site-directed mutagenesis, we demonstrate here that the MBP domain of pro-MBP binds to heparan sulfate GAG on the cell surface and that this is independent of GAG covalently bound to pro-MBP. Eight basic residues located in the CTL ligand-binding region of MBP were hypothesized previously to mediate GAG binding, but we found that surface binding was not compromised by the substitution of these residues with alanine. However, the analysis of a series of mutants with surface-exposed residues substituted with alanine showed that Ser-166, Arg-168, and Arg-171 are involved in surface binding. A binding site formed by these residues is located in the MBP domain between loop 1 and beta-strand 5, outside the CTL ligand-binding region. The binding of a cell-surface heparan sulfate proteoglycan may be important in MBP action, and our findings suggest that two regions shown previously to contain the cytotoxic and cytostimulatory properties of MBP are accessible for ligand interaction in cell surface-bound MBP.     

Proteinase inhibition by proform of eosinophil major basic protein (pro-MBP) is a multistep process of intra- and intermolecular disulfide rearrangements

The metzincin metalloproteinase pregnancy-associated plasma protein A (PAPP-A, pappalysin-1) promotes cell growth by the cleavage of insulin-like growth factor-binding proteins-4 and -5, causing the release of bound insulin-like growth factors. The proteolytic activity of PAPP-A is inhibited by the proform of eosinophil major basic protein (pro-MBP), which forms a covalent 2:2 proteinase-inhibitor complex based on disulfide bonds. To understand the process of complex formation, we determined the status of cysteine residues in both of the uncomplexed molecules. A comparison of the disulfide structure of the reactants with the known disulfide structure of the PAPP-A.pro-MBP complex reveals that six cysteine residues of the pro-MBP subunit (Cys-51, Cys-89, Cys-104, Cys-107, Cys-128, and Cys-169) and two cysteine residues of the PAPP-A subunit (Cys-381 and Cys-652) change their status from the uncomplexed to the complexed states. Upon complex formation, three disulfide bonds of pro-MBP, which connect the acidic propiece with the basic, mature portion, are disrupted. In the PAPP-A.pro-MBP complex, two of these form the basis of both two interchain disulfide bonds between the PAPP-A and the pro-MBP subunits and two disulfide bonds responsible for pro-MBP dimerization, respectively. Based on the status of the reactants, we investigated the role of individual cysteine residues upon complex formation by mutagenesis of specific cysteine residues of both subunits. Our findings allow us to depict a hypothetical model of how the PAPPA.pro-MBP complex is formed. In addition, we have demonstrated that complex formation is greatly enhanced by the addition of micromolar concentrations of reductants. It is therefore possible that the activity in vivo of PAPP-A is controlled by the redox potential, and it is further tempting to speculate that such mechanism operates under pathological conditions of altered redox potential.     

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.     

Characterization of pregnancy-associated plasma protein-A: comparison with alpha 2-macroglobulin.

Pregnancy-associated plasma protein-A (PAPP-A), like alpha 2-macroglobulin (alpha 2M), is a large protein with homotetrameric molecular conformation. Each monomer has Mr 205 kDa. Total carbohydrate content of PAPP-A (19.4%) exceeded that of alpha 2M (8.6%). In addition to glucose (9.4%), fucose (3.1%), mannose (2.3%) and galactose (0.8%), PAPP-A contained glucuronic acid (3.8%). The amino acid composition of PAPP-A differed most significantly from alpha 2M, in the content of glutamate, glycine and lysine. Although the peptide core of both proteins were of similar size, the difference in size, of native molecules was due to the carbohydrate moiety. Whereas alpha 2M monomer was autolytically cleaved into two smaller non-identical subunits (Mr 128 and 65 kDa), no such breakdown products were observed with PAPP-A. Unlike alpha 2M, PAPP-A is not a broad spectrum protease inhibitor. Both proteins inhibited human granulocyte elastase (HGE) in a dose dependent relationship, with PAPP-A (Ki 0.2 x 10(-6) M) being a more potent inhibitor than alpha 2M (Ki 1.02 x 10(-6) M). Since PAPP-A lacked internal thiolester groups, the mechanism of HGE inhibition was unlikely to be entrapment, as defined for alpha 2M. Inhibition kinetics of HGE for PAPP-A (noncompetitive inhibitor) and alpha 2M (uncompetitive inhibitor) were distinct. Thus, these findings do not support the tenet of a common ancestral protein for PAPP-A and alpha 2M.     

Discovery and characterization of human antibody inhibitors of pregnancy-associated plasma protein-A

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease that cleaves insulin-like growth factor-binding proteins (IGFBPs) to release bioactive levels of free insulin-like growth factor. Specific and potent inhibitors of PAPP-A may further elucidate the biological functions of this protease and could prove to be of therapeutic value. Phage display was used to discover fully human antibody inhibitors of PAPP-A activity towards IGFBP4 cleavage. Estimates of the inhibition constants for these antibodies were subsequently determined using a novel continuous assay of PAPP-A protease activity that uses an internally quenched synthetic peptide substrate (DX-1655). DX-1655 was hydrolyzed by PAPP-A with a K(m) of 33 muM and a k(cat) of 0.3 s(-1) (k(cat)/K(m)=9.1x10(3) M(-1) s(-1)). PAPP-A activity towards DX-1655 displays a bell-shaped pH profile, with pK(a) values of 8.2 and 10.8 and a maximum rate at approximately pH 9.5. Using this continuous assay, we measured apparent K(i) values of 1.7+/-0.2 and 7.4+/-1.5 nM for the F2 and D9 antibodies, respectively.     

Peptides of major basic protein and eosinophil cationic protein activate human mast cells

The eosinophil granule proteins, major basic protein (MBP) and eosinophil cationic protein (ECP), activate mast cells during inflammation; however the mechanism responsible for this activity is poorly understood. We found that some theoretical tryptase-digested fragments of MBP and ECP induced degranulation of human cord blood-derived mast cells (HCMCs). The spectrum of activities of these peptides in HCMCs coincided with intracellular Ca²⁺ mobilization activities in Mas-related G-protein coupled receptor family member X2 (MRGPRX2)-expressing HEK293 cells. Two peptides corresponding to MBP residues 99–110 (MBP (99–110)) and ECP residues 29–45 (ECP (29–45)), respectively, induced degranulation of HCMCs and intracellular Ca²⁺ mobilization in MRGPRX2-expressing HEK293 cells in a concentration-dependent manner. Stimulation with MBP (99–110) or ECP (29–45) induced the production of prostaglandin D2 by HCMCs. The activities of MBP (99–110) and ECP (29–45) in both HCMCs and MRGPRX2-expressing HEK293 cells were inhibited by MRGPRX2-specific antagonists. In conclusion, these results indicated that MBP and ECP fragments activate HCMCs, and it may occur via MRGPRX2. Our findings suggest that tryptase-digested fragments of eosinophil cationic proteins acting via the MRGPRX2 pathway may further our understanding of mast cell/eosinophil communication.     

Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin

The human eosinophil granule contains a number of cationic proteins that have been identified and purified to homogeneity, including the major basic protein (MBP), the eosinophil cationic protein (ECP), and the eosinophil-derived neurotoxin (EDN). Because of confusion in the literature regarding the distinctiveness of MBP and ECP, we investigated the immunochemical and physicochemical properties of these purified proteins by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), by specific double antibody radioimmunoassays (RIA) for MBP and ECP, and by fractionation of acid-solubilized eosinophil granules on Sephadex G-50 columns. Analysis of a mixture of the three purified proteins by SDS-PAGE showed that they migrated as three distinct bands with differing m.w. Comparison by specific RIA for MBP and ECP did not demonstrate any appreciable immunochemical cross-reactivities among the three proteins. Sephadex G-50 column fractions of acid-solubilized eosinophil granules were analyzed by RIA and by SDS-PAGE analysis of individual column fractions. MBP, ECP, and EDN eluted at different volumes from Sephadex G-50 columns as determined by RIA and SDS-PAGE. Soluble extracts of eosinophil granules from patients with the hypereosinophilic syndrome contained between six and 64 times more MBP than ECP on a weight basis. These observations demonstrate that MBP, ECP, and EDN are distinctive cationic proteins of the human eosinophil granule and that eosinophil granules from patients with eosinophilia contain considerably greater quantities of MBP than ECP.     

Ozone-induced hyperresponsiveness and blockade of M2 muscarinic receptors by eosinophil major basic protein.

Control of airway smooth muscle is provided by parasympathetic nerves that release acetylcholine onto M(3) muscarinic receptors. Acetylcholine release is limited by inhibitory M(2) muscarinic receptors. In antigen-challenged guinea pigs, hyperresponsiveness is due to blockade of neuronal M(2) receptors by eosinophil major basic protein (MBP). Because exposure of guinea pigs to ozone also causes M(2) dysfunction and airway hyperresponsiveness, the role of eosinophils in ozone-induced hyperresponsiveness was tested. Animals were exposed to filtered air or to 2 parts/million ozone for 4 h. Twenty-four hours later, the muscarinic agonist pilocarpine no longer inhibited vagally induced bronchoconstriction in ozone-exposed animals, indicating M(2) dysfunction. M(2) receptor function in ozone-exposed animals was protected by depletion of eosinophils with antibody to interleukin-5 and by pretreatment with antibody to guinea pig MBP. M(2) function was acutely restored by removal of MBP with heparin. Ozone-induced hyperreactivity was also prevented by antibody to MBP and was reversed by heparin. These data show that loss of neuronal M(2) receptor function after ozone is due to release of eosinophil MBP.     

Crystal structure of the eosinophil major basic protein at 1.8 A. An atypical lectin with a paradigm shift in specificity

The eosinophil major basic protein (EMBP) is the predominant constituent of the crystalline core of the eosinophil primary granule. EMBP is directly implicated in epithelial cell damage, exfoliation, and bronchospasm in allergic diseases such as asthma. Here we report the crystal structure of EMBP at 1.8 A resolution, and show that it is similar to that of members of the C-type lectin superfamily with which it shares minimal amino acid sequence identity (approximately 15--28%). However, this protein lacks a Ca(2+)/carbohydrate-binding site. Our analysis suggests that EMBP specifically binds heparin. Based on our results, we propose a possible new function for this protein, which is likely to have implications for EMBP function.     

Localization of eosinophil cationic protein, major basic protein, and eosinophil peroxidase in human eosinophils by immunoelectron microscopic technique

An immunoelectron microscopic technique using protein A-gold as a specific marker was used for precise intracellular localization of eosinophil granule proteins. Eosinophils from healthy individuals were isolated in metrizamide gradients. Eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were clearly located in the matrix of the large crystalloid-containing granules. In addition, ECP was probably present in the small granules of eosinophils. Major basic protein (MBP) was present in the crystalloid structure of specific granules. This method can be applied in studies of eosinophil degranulation to trace the release of biological effector molecules.     

Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein

Eosinophil granule major basic protein (MBP) is a relatively low molecular weight cationic (pI greater than 10) protein present in the crystalloid core of the eosinophil granule. Amino acid sequence analysis of this protein was undertaken as part of an analysis of the structural basis of the potent cytotoxic activities of MBP on parasites and mammalian cells. Many conventional sequencing strategies were unworkable because of the unusual amino acid composition of MBP and its insolubility in solutions buffered at neutral pH. Less conventional chemical reactions, including cyanogen bromide-induced cleavage at tryptophan and acid-induced cleavage at aspartic acid, were used successfully to obtain peptides which allowed definition of the amino acid sequence of MBP. Characterization of MBP by reverse-phase high pressure liquid chromatography and two-dimensional gel analysis showed no microheterogeneity that might be attributed to post-translational modifications. Comparison of the MBP sequence with a protein sequence data base showed that MBP has no significant sequence homology with other characterized proteins. The basicity (pI 10.9) and hydrophobicity predicted from the MBP sequence are likely responsible for the observed affinity of this cytotoxic molecule for cell surfaces and some serum proteins.