Formation of high-molecular-weight angiotensinogen during pregnancy is a result of competing redox reactions with the proform of eosinophil major basic protein.

The plasma concentration of the placentally derived proMBP (proform of eosinophil major basic protein) increases in pregnancy, and three different complexes containing proMBP have been isolated from pregnancy plasma and serum: a 2:2 complex with the metalloproteinase, PAPP-A (pregnancy-associated plasma protein-A), a 2:2 complex with AGT (angiotensinogen) and a 2:2:2 complex with AGT and complement C3dg. In the present study we show that during human pregnancy, all of the circulating proMBP exists in covalent complexes, bound to either PAPP-A or AGT. We also show that the proMBP-AGT complex constitutes the major fraction of circulating HMW (high-molecular weight) AGT in late pregnancy, and that this complex is able to further associate with complement C3 derivatives post-sampling. Clearance experiments in mice suggest that complement C3-based complexes are removed faster from the circulation compared to monomeric AGT and the proMBP-AGT complex. Furthermore, we have used recombinant proteins to analyse the formation of the proMBP-PAPP-A and the proMBP-AGT complexes, and we demonstrate that they are competing reactions, depending on the same cysteine residue of proMBP, but differentially on the redox potential, potentially important for the relative amounts of the complexes in vivo. These findings may be important physiologically, since the biochemical properties of the proteins change as a consequence of complex formation.

Pregnancy-associated plasma protein-A (PAPP-A) and cardiovascular risk.

The search for markers to improve risk prediction for individuals at risk of developing serious cardiovascular events is ongoing. New markers of coronary artery disease progression have been identified in recent years, among which, circulating levels of pregnancy-associated plasma protein-A (PAPP-A) offer an interesting profile. PAPP-A may play a role in the development of atherosclerotic lesions and represent also a marker of atheromatous plaque instability and extent of cardiovascular disease. PAPP-A has been shown to be a marker of adverse outcome in both acute coronary syndrome and stable coronary disease patients. The present article reviews currently available evidence supporting a role for PAPP-A as a marker of cardiovascular risk and discusses some of the pitfalls that may limit its use in clinical practice.

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.

Eosinophil-granule major basic protein, a C-type lectin, binds heparin.

The eosinophil major basic protein (EMBP), a constituent of the eosinophil secondary granule, is implicated in cytotoxicity and mediation of allergic disorders such as asthma. It is a member of the C-type lectin family, but lacks a Ca(2+)- and carbohydrate-binding site as seen in other members of this family. Here, we report the crystal structure of EMBP in complex with a heparin disaccharide and in the absence of Ca(2+), the first such report of any C-lectin with this sugar. We also provide direct evidence of binding of EMBP to heparin and heparin disaccharide by surface plasmon resonance. We propose that the sugars recognized by EMBP are likely to be proteoglycans such as heparin, leading to new interpretations for EMBP function.

Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils.

The effects of platelet-activating factor (PAF) and IL-5 on intracellular pH were investigated in human eosinophils. Purified peripheral blood eosinophils were loaded with the ratiometric fluorescent pH indicator BCECF-AM ester. Stimulation of eosinophils with PAF produced time-dependent alkalinization of the cytoplasm from an initial pH of 7.1+/-0.04 to 7.5+/-0.05. A similar alkalinization response was produced by the calcium ionophore, ionomycin and by the calcium ATPase inhibitor, thapsigargin. These compounds as well as PAF produce significant increases in cytoplasmic calcium ([Ca2+]i). In contrast, IL-5 and the protein kinase C (PKC) activator phorbol myristate acetate (PMA) did not produce cytoplasmic alkalinization and had no effect on [Ca2+]i in eosinophils. PAF-stimulated alkalinization was not inhibited under conditions that blocked plasma membrane Na+-H+ exchange, proton channel or plasma membrane H+-ATPase activities. Measurements of intragranule pH with a cell permeant pH indicator (LysoSensor Yellow/Blue DND-160), which partitions into intracellular acidic compartments, revealed that PAF-stimulated cytosolic alkalinization correlated with intragranule acidification. These results suggest that the increase in [Ca2+]i after PAF stimulation activates a H+-ATPase present in the granule membranes, leading to enhanced granule acidification and cytoplasmic alkalinization. We propose that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins.