Expression of the beta-chain of the complement regulator C4b-binding protein in human ovary.

Human C4b-binding protein (C4BP) is an important regulator of the complement system that also binds and inactivates the anticoagulant vitamin K-dependent protein S. These two activities are performed by two distinct polypeptides of 70 kDa and 45 kDa known as alpha and beta chains, respectively. C4BP is present in plasma in various isoforms with different alpha/beta composition. We report here that C4BPbeta, but not C4BPalpha, is expressed in adult human ovary. Expression of C4BPbeta was detected in all ovarian biopsies analyzed (n = 15), independently of age and phase of the menstrual cycle. In situ hybridization and immunostaining analyses on cryostat sections demonstrated expression of C4BPbeta in both regressing corpus luteum and corpus albicans, but not in the follicles, the corpus luteum, the ovary stroma or the vascular cells. In addition, we noted that the expression pattern of the C4BPbeta mRNA resembles that described for the connective tissue that invades the degenerating corpus luteum and causes a progressive fibrosis that gradually converts it into a scar, the corpus albicans. RT-PCR and immunostaining analyses of primary cultures derived from human ovaries demonstrated the presence of fibroblast-like cells that express C4BPbeta. As a whole, these data suggest a role for the C4BPbeta in human ovary during the healing and scar resorption processes that leads to the formation of the corpus albicans and its replacement by ovarian stroma.     

Interaction between protein S and complement C4b-binding protein (C4BP). Affinity studies using chimeras containing c4bp beta-chain short consensus repeats.

Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical alpha-chains and a single beta-chain (C4BPbeta), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPbeta. To further localize the protein S binding site, we constructed chimeras containing C4BPbeta SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, beta-chain SCR-2 contributes to the interaction of C4BP with protein S.     

The Gene Coding for the β-Chain of C4b-Binding Protein (C4BPB) Has Become a Pseudogene in the Mouse

C4BPβ is one of the two polypeptides that in humans compose the plasma glycoprotein C4b-binding protein (C4BP). C4BPβ binds the anticoagulant vitamin K-dependent protein S. Two, nonmutually exclusive, roles have been proposed for the C4BP-protein S interaction. It has been suggested to play a role in the control of the protein C anticoagulatory pathway. In addition, it may serve an important role in localizing C4BP to the surface of injured or activated cells. While the physiological significance of C4BP-protein S interaction is unclear, it has clinical relevance because elevated plasma levels of C4BP are associated with increased risk for thromboembolic disorders in humans, due to an inactivation of the protein C anticoagulatory pathway. Using a human C4BPβ cDNA probe, we have isolated and characterized a genomic DNA fragment that includes the murine C4BPB gene. Murine C4BPB is a single-copy gene that maps close to the C4BPA gene in chromosome 1. It contains two exons homologous to the exons coding for the SCR-1 and SCR-2 repeats of the human C4BPβ polypeptide chain. Sequence analysis of the C4BPB exons in the Mus musculus inbred strains CBA, Balb/c, and C57BL/6, in penbred Swiss mice, and in Mus spretus demonstrated the presence of two in-phase stop codons that are incompatible with the expression of a functional C4BPβ polypeptide. Thus, the characterization of the murine C4BPB gene documents the peculiar situation of a single-copy gene that is functional in humans but has become a pseudogene in the mouse. Interestingly, the loss of a functional C4BPB gene is a relatively recent event in the evolution of the mouse. In addition, our data indicate that this genetic change has been fixed in the mouse population, suggesting that those individuals lacking the C4BPβ polypeptide were conferred with some kind of selective advantage.     

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.     

C4b-binding protein protects coagulation factor Va from inactivation by activated protein C

We investigated the effect of C4BP on APC-mediated inactivation of factor Va (FVa) in the absence and presence of protein S. FVa inactivation was biphasic (k(506) = 4.4 x 10(8) M(-)(1) s(-)(1), k(306) = 2.7 x 10(7) M(-)(1) s(-)(1)), and protein S accelerated Arg(306) cleavage approximately 10-fold. Preincubation of protein S with C4BP resulted in a total abrogation of protein S cofactor activity. C4BP also protected FVa from inactivation by APC in the absence of protein S. Control experiments with CLB-PS13, a monoclonal anti-protein S antibody, indicated that inhibition of FVa inactivation by C4BP was not mediated through contaminating traces of protein S in our reaction systems. Protection of FVa was prevented by a monoclonal antibody directed against the C4BP alpha-chain. Recombinant rC4BPalpha comprised of only alpha-chains also protected FVa, but in the presence of protein S, the level of protection was decreased, since rC4BPalpha lacks the beta-chain responsible for C4BP binding to protein S. A truncated C4BP beta-chain (SCR-1+2) inhibited protein S cofactor activity, but had no effect on FVa inactivation by APC in the absence of protein S. In conclusion, C4BP protects FVa from APC-catalyzed cleavage in a protein S-independent way through direct interactions of the alpha-chaims of C4BP with FVa and/or APC.     

High affinity interaction between C4b-binding protein and vitamin K-dependent protein S in the presence of calcium. Suggestion of a third component in blood regulating the interaction

The anticoagulant vitamin K-dependent protein S interacts with the complement regulatory protein C4b-binding protein (C4BP), both in purified systems and in plasma. The concentrations of these proteins in plasma are approximately equimolar (0.3 microM) and 30-40% of protein S in plasma is found in the noncomplexed state. Only the uncomplexed form of protein S displays anticoagulant activity and studies have shown that patients with a selective deficiency of free protein S have a high incidence of thrombosis. In this study, we report that the protein S-C4BP interaction is at least 100-fold tighter in the presence of Ca2+ than in EDTA. The KD in the presence of Ca2+ was estimated with a gel filtration technique to be less than 5 x 10(-10) M, whereas in the presence of EDTA, it was approximately 100-fold higher. Ca2+ titration experiments suggested that the Ca2+ sites which function in the protein S-C4BP interaction are of high affinity which, in turn, suggests that they may be independent of the gamma-carboxyglutamic acid region and may be present in the epidermal growth factor-like domains of protein S. The high affinity of the protein S-C4BP interaction in the presence of Ca2+ suggested that virtually all of the protein S in whole blood should be complexed with C4BP. However, even though the protein S-C4BP interaction in Ca2(+)-containing serum was shown to have the same high affinity as in purified systems, approximately 30-40% of the protein S in serum was free. These results appear best explained by the presence of a third component in whole blood which regulates the protein S-C4BP interaction, keeping approximately 30-40% of circulating protein S in its free, functionally anticoagulant form. It is speculated that persons with little free protein S may be deficient in this hypothetical third component.     

Alphabet, a Ser/Thr phosphatase of the protein phosphatase 2C family, negatively regulates RAS/MAPK signaling in Drosophila

Signal transduction through the RAS/mitogen-activated protein kinase (MAPK) pathway depends on a diverse collection of proteins regulating positively and negatively signaling flow. We previously conducted a genetic screen in Drosophila to identify novel components of this signaling pathway. Here, we present the identification and characterization of a new gene, alphabet (alph), whose activity negatively regulates RAS/MAPK-dependent developmental processes in Drosophila and this, at a step downstream or in parallel to RAS. alph encodes a protein phosphatase 2C (PP2C) family member closely related to the mammalian PP2C alpha and beta isoforms. Interestingly, although alph gene product does not appear to be essential for viability, its elimination leads to weak but significant developmental defects reminiscent of an overactivated RAS/MAPK pathway. Consistent with this interpretation, strong genetic interactions are observed between alph alleles and mutations in bona fide components of the pathway. Together, this work identifies a PP2C of the alpha/beta subfamily as a novel negative regulator of the RAS/MAPK pathway and suggests that these evolutionarily conserved enzymes play a similar role in other metazoans. Finally, despite the relatively large size of the PP2C gene family in metazoans, this study represents only the second genetic characterization of a PP2C in these organisms.     

The C4b-binding protein-protein S complex inhibits the phagocytosis of apoptotic cells

The phagocytosis of apoptotic cells is a complex process involving numerous interactions between the target cell and the macrophage. We have examined a role of the major soluble inhibitor of the classic and lectin complement pathways, C4b-binding protein (C4BP), in the clearance of apoptotic cells. The major form of C4BP present in blood is composed of seven alpha-chains and one beta-chain, which binds protein S (PS). Approximately 70% of all PS in human plasma is trapped in such a complex and is able to localize C4BP to the surface of apoptotic cells due to the high affinity to phosphatidylserine. Free PS has recently been shown to enhance phagocytosis of apoptotic cells by macrophages. We observed a stimulatory effect of free PS on the engulfment of apoptotic cells (BL-41 and Jurkat) by primary human macrophages or THP-1 cells and a decrease of activity in serum depleted of PS in agreement with previous results. However, we also show that the process is strongly inhibited in the presence of the C4BP-PS complex. Addition of the C4BP-PS complex to serum deficient in both molecules abolished the enhancing effect of serum on phagocytosis. The effect of both free PS and the C4BP-PS complex could be inhibited with monoclonal antibody directed against the Gla domain of PS. Although the presence of the C4BP-PS complex on apoptotic cells may lead to decreased phagocytosis, it may still be beneficial to the host, since it could prevent secondary necrosis because it inhibits further complement attack.     

Dependence on Vitamin K-dependent Protein S for Eukaryotic Cell Secretion of the β-Chain of C4b-binding Protein

The anticoagulant vitamin K-dependent protein S (PS) circulates in plasma in two forms, 30% free and 70% being bound to the complement regulatory protein C4b-binding protein (C4BP). The major C4BP isoform consists of 7 α-chains and 1 β-chain (C4BPβ⁺), the chains being linked by disulfide bridges. PS binds to the β-chain with high affinity. In plasma, PS is in molar excess over C4BPβ⁺ and due to the high affinity, all C4BPβ⁺ molecules contain a bound PS. Taken together with the observation that PS-deficient patients have decreased levels of C4BPβ⁺, this raises the question of whether PS is important for secretion of the β-chain from the cell. To test this hypothesis, HEK293 cells were stably and transiently transfected with β-chain cDNA in combinations with cDNAs for PS and/or the α-chain. The concentration of β-chains in the medium increased after co-transfection with PS cDNA, but not by α-chain cDNA, suggesting secretion of the β-chains from the cells to be dependent on concomitant synthesis of PS, but not of the α-chains. Thus, β-chains that were not disulfide-linked to the α-chains were secreted in complex with PS, either as monomers or dimers. Pulse-chase demonstrated that the complexes between PS and β-chain were formed intracellularly, in the endoplasmic reticulum. In conclusion, our results demonstrate that successful secretion of β-chains depends on intracellular complex formation with PS, but not on the α-chains. This provides an explanation for the decreased β-chain levels observed in PS-deficient patients.     

Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells

C4b and C3b deposited on host cells undergo limited proteolytic cleavage by regulatory proteins. Membrane cofactor protein (MCP; CD46), factor H, and C4b binding protein mediate this reaction, known as cofactor activity, that also requires the plasma serine protease factor I. To explore the roles of the fluid phase regulators vs those expressed on host cells, a model system was used examining complement fragments deposited on cells transfected with human MCP as assessed by FACS and Western blotting. Following incubation with Ab and complement on MCP(+) cells, C4b was progressively cleaved over the first hour to C4d and C4c. There was no detectable cleavage of C4b on MCP(-) cells, indicating that MCP (and not C4BP in the serum) primarily mediates this cofactor activity. C3b deposition was not blocked on MCP(+) cells because classical pathway activation occurred before substantial C4b cleavage. Cleavage, though, of deposited C3b was rapid (<5 min) and iC3b was the dominant fragment on MCP(-) and MCP(+) cells. Studies using a function-blocking mAb further established factor H as the responsible cofactor. If the level of Ab sensitization was reduced 8-fold or if Mg(2+)-EGTA was used to block the classical pathway, MCP efficiently inhibited C3b deposition mediated by the alternative pathway. Thus, for the classical pathway, MCP is the cofactor for C4b cleavage and factor H for C3b cleavage. However, if the alternative pathway mediates C3b deposition, then MCP's cofactor activity is sufficient to restrict complement activation.     

Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase

A synthetic peptide of 18 amino acids corresponding to the inhibitory domain of the heat-stable protein kinase inhibitor was synthesized and shown to inhibit both the C alpha and C beta isoforms of the catalytic (C) subunit of cAMP-dependent protein kinase. Extracts from cells transfected with expression vectors coding for the C alpha or the C beta isoform of the C subunit required 200 nM protein kinase inhibitor peptide for half-maximal inhibition of kinase activity in extracts from these cells. An affinity column was constructed using this synthetic peptide, and the column was incubated with protein extracts from cells overexpressing C alpha or C beta. Elution of the affinity column with arginine allowed single step isolation of purified C alpha and C beta subunits. The C alpha and C beta proteins were enriched 200-400-fold from cellular extracts by this single step of affinity chromatography. No residual inhibitory peptide activity could be detected in the purified protein. The purified C subunit isoforms were used to demonstrate preferential antibody reactivity with the C alpha isoform by Western blot analysis. Furthermore, preliminary characterization showed both isoforms have similar apparent Km values for ATP (4 microM) and for Kemptide (5.6 microM). These results demonstrate that a combination of affinity chromatography employing peptides derived from the heat-stable protein kinase inhibitor protein and the use of cells overexpressing C subunit related proteins may be an effective means for purification and characterization of the C subunit isoforms. Furthermore, this method of purification may be applicable to other kinases which are known to be specifically inhibited by small peptides.     

Pig apolipoprotein R: a new member of the short consensus repeat family of proteins.

Apolipoprotein R (apoR) is a 23-kDa protein found on very low-density lipoprotein (VLDL), on chylomicrons, and in the d > 1.21 g/mL fraction of pig plasma. The plasma concentration of apoR is 5.1 micrograms/mL, with 11.5% of apoR found on VLDL. In vitro, apoR can transfer from the d > 1.21 g/mL infranatant onto artificial lipid emulsions or human chylomicrons but not onto human VLDL. An apoR cDNA was isolated from a pig liver lambda gt11 expression library. DNA sequence analysis of the apoR cDNA revealed 67% identity with the 3'-terminal region of human C4b-binding protein alpha-chain cDNA (C4BP alpha). C4BP alpha is a 70-kDa glycoprotein that regulates both the coagulation and the complement cascades. In plasma, C4BP alpha exists as disulfide-linked multimers consisting of seven C4BP alpha chains and a single C4BP beta chain. Like C4BP, apoR forms high molecular weight disulfide-linked complexes in plasma. However, unlike C4BP alpha, apoR complexes do not appear to contain C4BP beta. ApoR mRNA was detected in pig liver, spleen, lung, bone marrow, and lymph node, but was absent in intestine and white blood cells. This distribution is consistent with the production of apoR in terminally differentiated macrophages but not in blood monocytes. ApoR mRNA was not detected in RNA isolated from human liver or lung. ApoR may be a lipoprotein-borne regulator of either the coagulation or the complement cascades.     

Ischemia-reperfusion alters gene expression of Na+-K+ ATPase isoforms in rat heart

The present study investigated whether oxidative stress plays a role in ischemia-reperfusion-induced changes in cardiac gene expression of Na(+)-K(+) ATPase isoforms. The levels of mRNA for Na(+)-K(+) ATPase isoforms were assessed in the isolated rat heart subjected to global ischemia (30 min) followed by reperfusion (60 min) in the presence or absence of superoxide dismutase (5 x 10(4)U/L) plus catalase (7.5 x 10(4)U/L), an antioxidant mixture. The levels of mRNA for the alpha(2), alpha(3), and beta(1) isoforms of Na(+)-K(+) ATPase were significantly reduced in the ischemia-reperfusion hearts, unlike the alpha(1) isoform. Pretreatment with superoxide dismutase+catalase preserved the ischemia-reperfusion-induced changes in alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, whereas the alpha(1) mRNA levels were unaffected. In order to test if oxidative stress produced effects similar to those seen with ischemia-reperfusion, hearts were perfused with an oxidant, H(2)O(2) (300 microM), or a free radical generator, xanthine (2mM) plus xanthine oxidase (0.03 U/ml) for 20 min. Perfusion of hearts with H(2)O(2) or xanthine/xanthine oxidase depressed the alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, but had lesser effects on alpha(1) mRNA levels. These results indicate that Na(+)-K(+) ATPase isoform gene expression is altered differentially in the ischemia-reperfusion hearts and that antioxidant treatment appears to attenuate these changes. It is suggested that alterations in Na(+)-K(+) ATPase isoform gene expression by ischemia-reperfusion may be mediated by oxidative stress.     

Early and delayed glutamate effects in rat primary cortical neurons. Changes in the subcellular distribution of protein kinase C isoforms and in intracellular calcium concentration

Glutamate-induced changes in the subcellular distribution of protein kinase C isoforms and in the intracellular calcium concentration were investigated in rat primary cortical neurons. Western blot analysis of protein kinase C isoforms (alpha, beta1, beta2, gamma, delta, epsilon, zeta and theta), performed 30 min after a 10 min treatment with 30 microM glutamate, revealed a decrease in the total beta1 (-24%) and beta2 (-40%) isoform levels, without any significant change in any of the other isozymes. All conventional isoforms translocated to the membrane compartment, while delta, epsilon, zeta and theta; maintained their initial subcellular distribution. Twenty-four hours after glutamate treatment, the total protein kinase C labelling had increased, particularly the epsilon isoform, which accounted for 34% of the total densitometric signal. At this time, protein kinase C beta1, delta, epsilon and zeta isoforms were mainly detected in the membrane compartment, while gamma and theta; signals were displayed almost solely in the cytosol. Basal intracellular calcium concentration (FURA 2 assay) was concentration-dependently increased (maximum effect +77%) 30 min, but not 24h after a 10 min glutamate (10-100 microM) treatment, while the net increase induced by electrical stimulation (10 Hz, 10s) was consistently reduced (maximum effect -64%). The N-methyl-d-aspartate receptor antagonist, MK-801, 1 microM, prevented glutamate action both 30 min and 24 h after treatment, while non-selective protein kinase C inhibitors, ineffective at 30 min, potentiated it at 24 h. These findings show that protein kinase C isoforms are differently activated and involved in the early and delayed glutamate actions, and that the prevailing effect of their activation is neuroprotective.     

Sodium transport systems in human chondrocytes. I. Morphological and functional expression of the Na+,K(+)-ATPase alpha and beta subunit isoforms in healthy and arthritic chondrocytes.

The chondrocyte is the cell responsible for the maintenance of the articular cartilage matrix. The negative charges of proteoglycans of the matrix draw cations, principally Na+, into the matrix to balance the negative charge distribution. The Na+,K(+)-ATPase is the plasma membrane enzyme that maintains the intracellular Na+ and K+ concentrations. The enzyme is composed of an alpha and a beta subunit, so far, 4 alpha and 3 beta isoforms have been identified in mammals. Chondrocytes are sensitive to their ionic and osmotic environment and are capable of adaptive responses to ionic environmental perturbations particularly changes to extracellular [Na+]. In this article we show that human fetal and adult chondrocytes express three alpha (alpha 1, alpha 2 and the neural form of alpha 3) and the three beta isoforms (beta 1, beta 2 and beta 3) of the Na+,K(+)-ATPase. The presence of multiple Na+,K(+)-ATPase isoforms in the plasma membrane of chondrocytes suggests a variety of kinetic properties that reflects a cartilage specific and very fine specialization in order to maintain the Na+/K+ gradients. Changes in the ionic and osmotic environment of chondrocytes occur in osteoarthritis and rheumatoid arthritis as result of tissue hydration and proteoglycan loss leading to a fall in tissue Na+ and K+ content. Although the expression levels and cellular distribution of the proteins tested do not vary, we detect changes in p-nitrophenylphosphatase activity "in situ" between control and pathological samples. This change in the sodium pump enzymatic activity suggests that the chondrocyte responds to these cationic environmental changes with a variation of the active isozyme types present in the plasma membrane.     

Brefeldin A influences the cell surface abundance and intracellular pools of low and high ouabain affinity Na+, K(+)-ATPase alpha subunit isoforms in articular chondrocytes

The catalytic alpha isoforms of the Na+, K(+)-ATPase and stimuli controlling the plasma membrane abundance and intracellular distribution of the enzyme were studied in isolated bovine articular chondrocytes which have previously been shown to express low and high ouabain affinity alpha isoforms (alpha 1 and alpha 3 respectively; alpha 1 > alpha 3). The Na+, K(+)-ATPase density of isolated chondrocyte preparations was quantified by specific 3H-ouabain binding. Long-term elevation of extracellular medium [Na+] resulted in a significant (31%; p < 0.05) upregulation of Na+, K(+)-ATPase density and treatment with various pharmacological inhibitors (Brefeldin A, monensin and cycloheximide) significantly (p < 0.001) blocked the upregulation. The subcellular distribution of the Na+, K(+)-ATPase alpha isoforms was examined by immunofluorescence confocal laser scanning microscopy which revealed predominantly plasma membrane immunostaining of alpha subunits in control chondrocytes. In Brefeldin A treated chondrocytes exposed to high [Na+], Na+, K(+)-ATPase alpha isoforms accumulated in juxta-nuclear pools and plasma membrane Na+, K(+)-ATPase density monitored by 3H-ouabain binding was significantly down-regulated due to Brefeldin A mediated disruption of vesicular transport. There was a marked increase in intracellular alpha 1 and alpha 3 staining suggesting that these isoforms are preferentially upregulated following long-term exposure to high extracellular [Na+]. The results demonstrate that Na+, K(+)-ATPase density in chondrocytes is elevated in response to increased extracellular [Na+] through de novo protein synthesis of new pumps containing alpha 1 and alpha 3 isoforms, delivery via the endoplasmic reticulum-Golgi complex constitutive secretory pathway and insertion into the plasma membrane.