An electrochemical immunosensor for brain natriuretic peptide prepared with screen-printed carbon electrodes nanostructured with gold nanoparticles grafted through aryl diazonium salt chemistry.

A sensitive amperometric immunosensor has been prepared by immobilization of capture antibodies onto gold nanoparticles (AuNPs) grafted on a screen-printed carbon electrode (SPCE) through aryl diazonium salt chemistry using 4-aminothiophenol (AuNPs-S-Phe-SPCE). The immunosensor was designed for the accurate determination of clinically relevant levels of B-type natriuretic peptide (BNP) in human serum samples. The nanostructured electrochemical platform resulted in an ordered layer of AuNPs onto SPCEs which combined the advantages of high conductivity and improved stability of immobilized biomolecules. The resulting disposable immunosensor used a sandwich type immunoassay involving a peroxidase-labeled detector antibody. The amperometric transduction was carried out at -0.20V (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. The nanostructured immunosensors show a storage stability of at least 25 days, a linear range between 0.014 and 15ngmL-1, and a LOD of 4pgmL-1, which is 100 times lower than the established cut-off value for heart failure (HF) diagnosis. The performance of the immunosensor is advantageously compared with that provided with immunosensors prepared by grafting SPCE with p-phenylendiamine (H2N-Phe-SPCE) and attaching AuNPs by immersion into an AuNPs suspension or by electrochemical deposition, as well as with immunosensors constructed using commercial AuNPs-modified SPCEs. The developed immunosensor was applied to the successful analysis of human serum from heart failure (HF) patients upon just a 10-times dilution as sample treatment.

Use of serum levels of high sensitivity troponin T, galectin-3 and C-terminal propeptide of type I procollagen at long term follow-up in heart failure patients with reduced ejection fraction: Comparison with soluble AXL and BNP.

BACKGROUND: Prognostic biomarkers are needed to improve the management of the heart failure (HF) epidemic, being the brain natriuretic peptides the most valuable. Here we evaluate 3 biomarkers, high sensitivity troponin T (hs-TnT), galectin-3 (Gal-3) and C-terminal propeptide of type I procollagen (CICP), compare them with a recently described new candidate (sAXL), and analyze their relationship with BNP. METHODS: HF patients with reduced ejection fraction (n=192) were included in this prospective observational study, with measurements of candidate biomarkers, functional, clinical and echocardiographic variables. A Cox regression model was used to determine predictors for clinical events, i.e. all-cause mortality and heart transplantation. RESULTS: Hs-TnT circulating values were correlated to clinical characteristics indicative of more advanced HF. When analyzing the event-free survival at a mean follow-up of 3.6years, patients in the higher quartile of either BNP, hs-TnT, CICP and sAXL had increased risk of suffering a clinical event, but not Gal-3. Combination of high sAXL and BNP values had greater predictive value (HR 6.8) than high BNP alone (HR 4.9). In a multivariate Cox regression analysis, BNP, sAXL and NYHA class were independent risk factors for clinical events. CONCLUSIONS: In this HF cohort, hs-TnT is a good HF marker and has a very significant prognostic value. The prognostic value of CICP and sAXL was of less significance. However, hs-TnT did not add predictive value to BNP, while sAXL did. This suggests that elevated troponin has a common origin with BNP, while sAXL could represent an independent pathological mechanism.

AXL receptor tyrosine kinase is increased in patients with heart failure.

BACKGROUND: AXL is a membrane receptor tyrosine kinase highly expressed in the heart and has a conspicuous role in cardiovascular physiology. The role of AXL in heart failure (HF) has not been previously addressed. METHODS AND RESULTS: AXL protein was enhanced 6-fold in myocardial biopsies of end-stage HF patients undergoing heart transplantation compared to controls from heart donors (P<0.0001). Next, we performed a transversal study of patients with chronic HF (n=192) and a group of controls with no HF (n=67). sAXL and BNP circulating levels were quantified and clinical and demographic data were collected. sAXL levels in serum were higher in HF (86.3 ± 2.0 ng/mL) than in controls (67.8 ± 2.0 ng/mL; P<0.0001). Also, sAXL correlated with several parameters associated with worse prognosis in HF. Linear regression analysis indicated that serum creatinine, systolic blood pressure and atrial fibrillation, but not BNP levels, were predictive of sAXL levels. Cox regression analysis indicated that high sAXL values at enrollment time were related to the major HF events (all-cause mortality, heart transplantation and HF hospitalizations) at one year follow-up (P<0.001), adding predictive value to high BNP levels. CONCLUSIONS: Myocardial expression and serum concentration of AXL is elevated in HF patients compared to controls. Furthermore, peripheral sAXL correlates with parameters associated with the progression of HF and with HF events at short term follow-up. All together these results suggest that sAXL could belong to a new molecular pathway involved in myocardial damage in HF, independent from BNP.

Expression of the vitamin K-dependent proteins GAS6 and protein S and the TAM receptor tyrosine kinases in human atherosclerotic carotid plaques.

The GAS6/ProS-TAM system is composed of two vitamin K-dependent ligands (GAS6 and protein S) and their three protein tyrosine kinase receptors TYRO3, AXL and MERTK, known as the TAM receptors. The system plays a prominent role in conditions of injury, inflammation and repair. In murine models of atherosclerotic plaque formation, mutations in its components affect atherosclerosis severity. Here we used Taqman low-density arrays and immunoblotting to study mRNA and protein expression of GAS6, ProS and the TAM receptors in human carotid arteries with different degrees of atherosclerosis. The results show a clear down-regulation of the expression of AXL in atheroma plaques with respect to normal carotids that is matched by decreased abundance of AXL in protein extracts detected by immunoblotting. A similar decrease was observed in PROS1 mRNA expression in atherosclerotic carotids compared to the normal ones, but in this case protein S (ProS) was clearly increased in protein extracts of carotid arteries with increasing grade of atherosclerosis, suggesting that ProS is carried into the plaque. MERTK was also increased in atherosclerotic carotid arteries with respect to the normal ones, suggesting that the ProS-MERTK axis is functional in advanced human atherosclerotic plaques. MERTK was expressed in macrophages, frequently in association with ProS, while ProS was abundant also in the necrotic core. Our data suggest that the ProS-MERTK ligand-receptor pair was active in advanced stages of atherosclerosis, while AXL signalling is probably down-regulated.

Genetic loss of Gas6 induces plaque stability in experimental atherosclerosis.

The growth arrest-specific gene 6 (Gas6) plays a role in pro-atherogenic processes such as endothelial and leukocyte activation, smooth muscle cell migration and thrombosis, but its role in atherosclerosis remains uninvestigated. Here, we report that Gas6 is expressed in all stages of human and mouse atherosclerosis, in plaque endothelial cells, smooth muscle cells and macrophages. Gas6 expression is most abundant in lesions containing high amounts of macrophages, ie thin fibrous cap atheroma and ruptured plaque. Genetic loss of Gas6 does not affect the number and size of initial and advanced plaques in ApoE(-/-) mice, but alters its plaque composition. Compared to Gas6(+/+): ApoE(-/-) mice, initial and advanced plaques of Gas6(-/-): ApoE(-/-) mice contained more smooth muscle cells and more collagen and developed smaller lipid cores, while the expression of TGFbeta was increased. In addition, fewer macrophages were found in advanced plaques of Gas6(-/-): ApoE(-/-) mice. Hence, loss of Gas6 promotes the formation of more stable atherosclerotic lesions by increasing plaque fibrosis and by attenuating plaque inflammation. These findings identify a role for Gas6 in plaque composition and stability.

Protein S Thr103Asn mutation associated with type II deficiency reproduced in vitro and functionally characterised.

Protein S functions as a cofactor to activated protein C (APC) in the degradation of FVa and FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofactor activity. A naturally occurring Thr103Asn (T103N) mutation in the first EGF-like domain of protein S has been associated with functional (type II) protein S deficiency. To elucidate the functional consequences of the T103N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N from transiently transfected COS 1 cells was equal to that of wild type protein S. The mutant protein S and wild type protein S were also expressed in 293 cells after stable transfection, and the recombinant proteins purified. In APTT- and PT-based coagulation assays, the mutant protein demonstrated approximately 50% lower anticoagulant activity as compared to wild type protein S. The functional defect was further investigated in FVa- and FVIIIa-degradation assays. The functional defect of mutant protein S was attenuated at increasing concentrations of APC. The results demonstrate the region around residue 103 of protein S to be of functional importance, possibly through a direct interaction with APC.

The second laminin G-type domain of protein S is indispensable for expression of full cofactor activity in activated protein C-catalysed inactivation of factor Va and factor VIIIa.

Vitamin K-dependent protein S is a cofactor to the anticoagulant serine protease activated protein C (APC) in the proteolytic inactivation of the procoagulant, activated factor V (FVa) and factor VIII (FVIIIa). In the FVa degradation, protein S selectively accelerates the cleavage at Arg306, having no effect on the Arg506 cleavage. In the FVIIIa inactivation, the APC-cofactor activity of protein S is synergistically potentiated by FV, which thus has the capacity to function both as a pro- and an anticoagulant protein. The SHBG-like region of protein S, containing two laminin G-type domains, is required for the combined action of protein S and FV. To elucidate whether both G domains in protein S are needed for expression of APC-cofactor activities, chimeras of human protein S were created in which the individual G domains were replaced by the corresponding domain of the homologous Gas6, which in itself has no anticoagulant activity. In a plasma-based assay, chimera I (G1 from Gas6) was as efficient as wild-type recombinant protein S, whereas chimera II (G2 from Gas6) was less effective. The synergistic cofactor activity with FV in the inactivation of FVIIIa was lost by the replacement of the G2 domain in protein S (chimera II). However, chimera I did not exert full APC-cofactor activity in the FVIIa degradation, indicating involvement of both G domains or the entire SHBG-like region in this reaction. Chimera I was fully active in the degradation of FVa in contrast to chimera II, which exhibited reduced cofactor activity compared to protein S. In conclusion, by using protein S-Gas6 chimeric proteins, we have identified the G2 domain of protein S to be indispensable for an efficient inactivation of both FVIIa and FVa, whereas the G1 domain was found not to be of direct importance in the FVa-inactivation experiments.

The first laminin G-type domain in the SHBG-like region of protein S contains residues essential for activation of the receptor tyrosine kinase sky.

Vitamin K-dependent protein S and the product of growth-arrest-specific gene 6 (Gas6) both possess the ability to phosphorylate members of the Axl/Sky subfamily of receptor tyrosine kinases. However, Gas6 appears to be the bona fide ligand for these receptors in man, as human protein S has been demonstrated to activate murine Sky but not the human orthologue. In contrast, bovine protein S is able to stimulate human Sky despite its high degree of sequence identity with human protein S. The domain organisations of protein S and Gas6 are virtually identical and the C-terminal SHBG-like region, containing two globular (G) domains, has been shown to play a crucial role in the receptor stimulation. In order to further localise the area responsible for the interaction, a number of protein chimeras were used to stimulate human Sky. Each chimera had one part of the human protein S SHBG-like region replaced by the corresponding part of bovine protein S or human Gas6. We found that human protein S may indeed activate human Sky but only above physiological plasma concentrations. The human-bovine protein S chimeras provided new information implying that the first G domain contains critical residues for the interaction with the Sky receptor. Moreover, these residues do not seem to be clustered but rather to be distributed at various positions in the first G domain.

Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein.

Anticoagulant protein S interacts with the complement regulatory protein C4b-binding protein (C4BP) via its sex-hormone-binding globulin (SHB6)-like region, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4BP or to have any anticoagulant activity. To determine the relative importance of the two G domains in protein S for C4BP protein binding, three recombinant protein S chimeras were produced having either of the two globular domains, or the whole SHB6-like globulin region, replaced by corresponding parts from Gas6. The chimeras were tested for binding to immobilized C4BP using surface-plasmon-resonance technology and microtiter plate-based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild-type protein S. The affinities for C4BP of both chimeras containing individual G domains from protein S, were lower than that of wild-type protein S. Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, which had the protein S-derived G2 domain. The chimera containing the whole SHB6-like globulin region from Gas6 interacted considerably more weakly with C4BP. Our results demonstrate that both G domains of protein S are involved in the interaction between protein S and C4BP and that full affinity binding is dependent on contributions from both domains.

Thrombophilia as a multigenic disease.

BACKGROUND AND OBJECTIVE: Venous thrombosis is a common disease annually affecting 1 in 1000 individuals. The multifactorial nature of the disease is illustrated by the frequent identification of one or more predisposing genetic and/or environmental risk factors in thrombosis patients. Most of the genetic defects known today affect the function of the natural anticoagulant pathways and in particular the protein C system. This presentation focuses on the importance of the genetic factors in the pathogenesis of inherited thrombophilia with particular emphasis on those defects which affect the protein C system. INFORMATION SOURCES: Published results in articles covered by the Medline database have been integrated with our original studies in the field of thrombophilia. STATE OF THE ART AND PERSPECTIVES: The risk of venous thrombosis is increased when the hemostatic balance between pro- and anti-coagulant forces is shifted in favor of coagulation. When this is caused by an inherited defect, the resulting hypercoagulable state is a lifelong risk factor for thrombosis. Resistance to activated protein C (APC resistance) is the most common inherited hypercoagulable state found to be associated with venous thrombosis. It is caused by a single point mutation in the factor V (FV) gene, which predicts the substitution of Arg506 with a Gln. Arg506 is one of three APC-cleavage sites and the mutation results in the loss of this APC-cleavage site. The mutation is only found in Caucasians but the prevalence of the mutant FV allele (FV:Q506) varies between countries. It is found to be highly prevalent (up to 15%) in Scandinavian populations, in areas with high incidence of thrombosis. FV:Q506 is associated with a 5-10-fold increased risk of thrombosis and is found in 20-60% of Caucasian patients with thrombosis. The second most common inherited risk factor for thrombosis is a point mutation (G20210A) in the 3' untranslated region of the prothrombin gene. This mutation is present in approximately 2% of healthy individuals and in 6-7% of thrombosis patients, suggesting it to be a mild risk factor of thrombosis. Other less common genetic risk factors for thrombosis are the deficiencies of natural anticoagulant proteins such as antithrombin, protein C or protein S. Such defects are present in less than 1% of healthy individuals and together they account for 5-10% of genetic defects found in patients with venous thrombosis. Owing to the high prevalence of inherited APC resistance (FV:Q506) and of the G20210A mutation in the prothrombin gene, combinations of genetic defects are relatively common in the general population. As each genetic defect is an independent risk factor for thrombosis, individuals with multiple defects have a highly increased risk of thrombosis. As a consequence, multiple defects are often found in patients with thrombosis.

The SHBG-like region of protein S is crucial for factor V-dependent APC-cofactor function.

Activated protein C (APC) regulates blood coagulation by degrading factor Va (FVa) and factor VIIIa (FVIIIa). Protein S is a cofactor to APC in the FVa degradation, whereas FVIIIa degradation is potentiated by the synergistic APC-cofactor activity of protein S and factor V (FV). To elucidate the importance of the sex-hormone-binding globulin (SHBG)-like region in protein S for expression of anticoagulant activity, a recombinant protein S/Gas6 chimera was constructed. It comprised the amino-terminal half of protein S and the SHBG-like region of Gas6, a structurally similar protein having no known anticoagulant properties. The protein S/Gas6 chimera expressed 40-50%, APC-cofactor activity in plasma as compared to wild-type protein S. In the degradation of FVa by APC, the protein S/Gas6 chimera was only slightly less efficient than wild-type protein S. In contrast, the protein S/Gas6 chimera expressed no FV-dependent APC-cofactor activity in a FVIIIa-degradation system. This demonstrates the SHBG-like region to be important for expression of APC-cofactor activity of protein S and suggests that the SHBG-like region of protein S interacts with FV during the APC-mediated inactivation of FVIIIa.

Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites.

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP alpha-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the alpha-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions.

A common 4G allele in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene as a risk factor for pulmonary embolism and arterial thrombosis in hereditary protein S deficiency.

Reduced fibrinolytic capacity due to increased plasminogen activator inhibitor-1 (PAI-1) activity in plasma is a common finding in patients with coronary heart disease or venous thromboembolism, although its clinical significance is debated. Recently, a dimorphism in the PAI-1 promoter (4G-5G) has been reported and homozygosity for the 4G allele is associated with increased transcription and higher PAI-1 levels. Homozygous 4G genotype has been suggested to be a risk factor for myocardial infarction. In the present study, the 4G-5G dimorphism was determined in 349 individuals from 21 thrombophilic families with hereditary protein S deficiency and in 140 unrelated healthy controls. Among the 143 protein S deficient individuals, there was no relationship between deep or superficial venous thrombosis and the PAI-1 dimorphism. However, 26% (12/46) of individuals having protein S deficiency combined with homozygosity for the 4G allele had suffered pulmonary embolism as compared to 7% (7/97) of protein S deficient individuals carrying at least one 5G allele (p = 0.0019). In protein S deficient individuals, arterial thrombosis was found to be associated with smoking and 4G homozygosity. No association was found between the PAI-1 dimorphism and arterial or venous thromboembolism in family members without protein S deficiency. In conclusion, the PAI-1 genotype affects the phenotypic expression of thrombophilia in protein S deficient individuals.

Clarification of the risk for venous thrombosis associated with hereditary protein S deficiency by investigation of a large kindred with a characterized gene defect.

BACKGROUND: Protein S is an important regulatory protein of the coagulation cascade. The risk for venous thrombosis associated with protein S deficiency has been uncertain because all previous risk estimates used phenotypic evaluation alone, which can be ambiguous. OBJECTIVE: To quantitate the risk for thrombosis associated with a characterized protein S gene mutation that causes a Gly295-->Val substitution and protein S deficiency. DESIGN: Retrospective study of a single extended family. SETTING: University hospital referral center. PARTICIPANTS: A 122-member protein S-deficient family, in which 44 members had a recently characterized gene defect. MEASUREMENTS: Comprehensive history of thrombosis, history of exposure to acquired risk factors for thrombosis, levels of total and free protein S antigen, and genotype for the mutation causing the Gly295-->Val substitution. RESULTS: Kaplan-Meier analysis of thrombosis-free survival showed that the probability of remaining free of thrombosis at 30 years of age is 0.5 (95% CI, 0.33 to 0.66) for carriers of the Gly295-->Val mutation compared with 0.97 (CI, 0.93 to 1.0) for normal family members (P < 0.001). In a multivariate Cox regression model that included smoking and obesity, the mutation was a strong independent risk factor for thrombosis (hazard ratio, 11.5 [CI, 4.33 to 30.6]; P < 0.001). For free (but not total) protein S antigen levels, the distributions of persons with and persons without the mutation did not overlap. CONCLUSIONS: Protein S deficiency, as defined by the presence of a causative gene mutation or a reduced level of free protein S antigen, is a strong independent risk factor for venous thrombosis in a clinical affected family.

SHBG region of the anticoagulant cofactor protein S: secondary structure prediction, circular dichroism spectroscopy, and analysis of naturally occurring mutations.

Protein S (PS) and growth arrest specific factor 6 (GAS6) are vitamin K-dependent proteins with similar structures. They are mosaic proteins possessing a carboxyl-terminal region presenting sequence similarity with plasma sex hormone binding globulin (plasma SHBG), although apparently not involved in steroid binding. The SHBG-like modules have sequence similarity with the G repeats of the chain A of laminin. Laminin G repeats have been reported to contain mainly beta-strands (about 40-50%) but no or little alpha structure by circular dichroism (CD) spectroscopy. Secondary structure predictions carried out in the present work unexpectedly showed a 20 to 27% helices content in the SHBG region of PS/GAS6 (about 100 residues), while plasma SHBG and laminin G repeats had around 10% helices. CD measurements for human PS indicated also that its SHBG region had about 100 residues in alpha-helical structure. These data suggest that the SHBG region of PS/GAS6 on the one hand, and the laminin G repeats and possibly plasma SHBG on the other hand, could present important structural differences. Previously reported polymorphisms and point mutations leading to PS deficiency and thrombophilia have been analyzed with our structural predictions. We found a good agreement between these structural predictions, CD measurements, experimental and clinical data. This information allows us to gain insights into the three-dimensional structure of PS that will be helpful for the design of new experiments and future clinical investigations.

Stimulation of Sky tyrosine phosphorylation by bovine protein S--domains involved in the receptor-ligand interaction.

Protein S is an anticoagulant vitamin-K-dependent plasma glycoprotein, which acts as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. It has been proposed that protein S has an additional function as a growth factor. Protein S and a structurally similar protein, Gas6, have been found to stimulate members of the Axl/Sky family of receptor tyrosine kinases. Human Gas6 is able to activate Axl and Sky. In contrast, while bovine protein S activates human Sky and its murine homologue, human protein S activates murine Sky but not the human receptor. In the present investigation, we studied the structural background of this species difference. Using protein S chimeras with domains from human and bovine origin, we found that only those chimeras with the steroid-hormone-binding globulin-like (SHBG) region from bovine protein S activate human Sky, indicating that the SHBG region is essential for the interaction. This observation was confirmed by inhibition of Sky phosphorylation by C4b-binding protein, a plasma protein that interacts tightly with the SHBG region of protein S. Another chimeric molecule, composed of the N-terminal 4-carboxyglutamic-acid-containing domain (Gla domain) and the two epidermal-growth-factor-like domains of human factor IX, and the SHBG region of bovine protein S, stimulated the receptor less efficiently. Antibodies directed against the Gla domain of protein S, inhibited the activation of human Sky by bovine protein S. These results indicate that the N-terminal domains of protein S are not essential for activation of the receptor, but contribute to the affinity of the interaction. Our data suggest that protein S might be a ligand of Sky in some species despite the lack of activity of human protein S on human Sky. The bovine/human protein S species difference will be a useful model to establish the structural requirements for the interaction between Sky and its ligands.

Inhibition of Alzheimer beta-peptide fibril formation by serum amyloid P component.

A 39-43-amino acid residue-long fragment (beta-peptide) from the amyloid precursor protein is the predominant component of amyloid deposits in the brain of individuals with Alzheimer's disease. Serum amyloid P component (SAP) is present in all types of amyloid, including that of Alzheimer's disease. We have used an in vitro model to study the effects of purified SAP on the fibril formation of synthetic Alzheimer beta-peptide 1-42. SAP was found to inhibit fibril formation and to increase the solubility of the peptide in a dose-dependent manner. At a 5:1 molar ratio of A beta 1-42 peptide to SAP, fibril formation was completely inhibited, and approximately 80% of the peptide remained in solution even after 4 days of incubation. At lower SAP concentrations, e.g. at peptide to SAP ratio of 1000:1, short fibrillar like structures, lacking amyloid characteristics, were formed. These structures frequently contained associated SAP molecules, suggesting that SAP binds to the polymerizing peptide in a reaction which prevented further fibril formation.

Serum amyloid P component binding to C4b-binding protein.

Human C4b-binding protein (C4BP), which is a regulator of the classical complement pathway C3 convertase, forms high affinity complexes with anticoagulant protein S and with the pentraxin serum amyloid P component (SAP). SAP is a plasma protein present in all amyloid deposits. Recently, SAP was shown to inhibit the complement regulatory functions of C4BP. In this investigation, we have studied the structural requirements for the C4BP-SAP interaction. C4BP was subjected to chymotrypsin digestion, which yielded two major fragments corresponding to the central core (160 kDa) and to the cleaved-off tentacles (48 kDa). SAP-Sepharose specifically bound the 160-kDa fragment, suggesting that the central core of C4BP contains the binding site for SAP. In a quantitative affinity chromatography assay, the dissociation constants for binding of intact C4BP and of the 160-kDa central core fragment to SAP were found to be 30 and 70 nM, respectively. Recombinant C4BP composed of only alpha-chains bound SAP with similar affinity (Kd = 22 nM), whereas nonglycosylated recombinant alpha-chain C4BP (synthesized in the presence of tunicamycin) bound SAP with lower affinity (Kd = 126 nM). This suggests that the carbohydrate moiety of the central core of C4BP is important for binding of C4BP to SAP in contrast to the C4BP beta-chain, which is not required. EDTA, heparin, and phosphorylethanolamine as well as a peptide comprising amino acids 27-39 of SAP were found to completely displace C4BP from the SAP matrix. Moreover, the immobilized SAP peptide bound C4BP in a reaction that, in contrast to the C4BP-SAP interaction, was not dependent on calcium.

The muscle isoform of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase of the teleost Sparus aurata: relationship with the liver isoform.

The liver isoform of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase of the teleost fish Sparaus aurata has several characteristics similar to the skeletal muscle isoform of mammals. In order to ascertain the relation between muscle and liver isoforms in teleost, 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was purified from skeletal muscle of S. aurata. The muscle isozyme is composed of subunits with a molecular weight of 54 kDa, is bifunctional, and has an activity ratio kinase to bisphosphatase of 2.5. Muscle 6-phosphofructo 2-kinase is not sensitive to glycerol 3-phosphate inhibition and has noncooperative KmATP, higher than the liver isozyme. Thus, the kinetic characteristics of the muscle were distinguishable from the liver isozyme. Furthermore, the muscle isozyme is not a substrate of cAMP-dependent protein kinase. Despite those differences, two polyclonal antibodies raised against purified liver and muscle isozymes from S. aurata are not able to distinguish between them. Both antisera recognize with lower affinity recombinant rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase. A third antibody raised against the rat liver isozyme was also able to immunoprecipitate the teleost enzymes. The close immunological properties found suggest that S. aurata isozymes share epitopes in common. Considering the kinetic and immunological data reported, it is likely that the skeletal muscle/liver isozymes in teleost are products of a differentially spliced transcript of the same gene, as it is in rat. As those species are distant in vertebrate evolution, the similitude suggest that a common ancestral gene is involved in the muscle/liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase system in vertebrates.