Milk transcriptome biomarker identification to enhance feed efficiency and reduce nutritional costs in dairy ewes.

In recent years, rising prices for high-quality protein-based feeds have significantly increased nutrition costs. Consequently, investigating strategies to reduce these expenses and improve feed efficiency (FE) have become increasingly important for the dairy sheep industry. This research investigates the impact of nutritional protein restriction (NPR) during prepuberty and FE on the milk transcriptome of dairy Assaf ewes (sampled during the first lactation). To this end, we first compared transcriptomic differences between NPR and control ewes. Subsequently, we evaluated gene expression differences between ewes with divergent FE, using feed conversion ratio (FCR), residual feed intake (RFI), and consensus classifications of high- and low-FE animals for both indices. Lastly, we assess milk gene expression as a predictor of FE phenotype using random forest. No effect was found for the prepubertal NPR on milk performance or FE. Moreover, at the milk transcriptome level, only one gene, HBB, was differentially expressed between the NPR (n = 14) and the control group (n = 14). Further, the transcriptomic analysis between divergent FE sheep revealed 114 differentially expressed genes (DEGs) for RFI index (high-FERFI = 10 vs low-FERFI = 10), 244 for FCR (high-FEFCR = 10 vs low-FEFCR = 10), and 1 016 DEGs between divergent consensus ewes for both indices (high-FEconsensus = 8 vs low-FEconsensus = 8). These results underscore the critical role of selected FE indices for RNA-Seq analyses, revealing that consensus divergent animals for both indices maximise differences in transcriptomic responses. Genes overexpressed in high-FEconsensus ewes were associated with milk production and mammary gland development, while low-FEconsensus genes were linked to higher metabolic expenditure for tissue organisation and repair. The best prediction accuracy for FE phenotype using random forest was obtained for a set of 44 genes consistently differentially expressed across lactations, with Spearman correlations of 0.37 and 0.22 for FCR and RFI, respectively. These findings provide insights into potential sustainability strategies for dairy sheep, highlighting the utility of transcriptomic markers as FE proxies.

Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.

The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6-/- mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6-/- mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis.

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.

Data on clinical characteristics of a heart failure patients' cohort with reduced ejection fraction and analysis of the circulating values of five different heart failure biomarkers; high sensitivity troponin T, galectin-3, C-terminal propeptide of type I procollagen, soluble AXL and BNP.

In this article, the full description of a heart failure with reduced ejection fraction (HF_REF) cohort of 192 patients is provided. Tables with the baseline demographic, prior history, ECG parameters, echocardiographic parameters, laboratory values and pharmacological treatment of these patients are included. Also, the quartile values of the analyzed circulating biomarkers: high sensitivity Troponin T (hs-TnT), galectin-3 (Gal-3), C-terminal propeptide of type I procollagen (CICP), soluble AXL (sAXL) and Brain Natriuretic Peptide (BNP) are given. The main demographic and clinical features of the patients' subgroups that have hs-TnT, Gal-3, CICP or BNP above the third quartile are described. Tables with Pearson correlation analysis of the HF_REF patients' biomarker levels are included. And Pearson correlation analysis of the HF_REF patients' hs-TnT, Gal-3, CICP levels with patients' biochemical parameters, blood count and inflammation parameters are also described. These data are related to the research articles (AXL receptor tyrosine kinase is increased in patients with heart failure (M. Batlle, P. Recarte-Pelz, E. Roig, M.A. Castel, M. Cardona, M. Farrero, et al., 2014) [1] and 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 (M. Batlle, B. Campos, M. Farrero, M. Cardona, B. González, M.A. Castel, et al., 2016) [2].

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.

cDNA structure of rabbit C4b-binding protein alpha-chain. Preserved sequence motive in complement regulatory protein modules which bind C4b.

A full length cDNA clone for the alpha-chain of the rabbit complement regulatory protein C4b-binding protein (C4BP) was isolated from a liver cDNA library. The clone encoded an open reading frame of 597 amino acids, which included a signal peptide, eight short consensus repeats (SCR) and a carboxy terminal non-repeat region. Gel filtration of rabbit plasma and testing of fractions for factor I cofactor activity (C4BP-like) revealed two peaks of activity, the one with highest molecular weight corresponding in size to that of human C4b-binding protein. Comparison of the rabbit C4BP alpha-chain sequence with other SCR containing C3b/C4b binding proteins revealed highest sequence similarities between the second SCRs in C4BP from rabbit, human and murine species and SCRs at corresponding position in complement receptor 1 (CR1) whereas in decay accelerating factor (DAF), the third SCR was most similar. A conserved sequence motive was identified in these C4b-binding SCRs.

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.

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.

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.

Topological studies of the amino terminal modules of vitamin K-dependent protein S using monoclonal antibody epitope mapping and molecular modeling.

Protein S is an important anticoagulant protein acting as cofactor to activated protein C (APC) in the degradation of membrane-bound factors Va and VIIIa. Binding of protein S to the membrane depends on the Gla-domain, whereas sites for APC-interaction are located in the thrombin-sensitive region (TSR) and the first EGF domain. The aims of the present investigation were to localize the sites on protein S which are involved in APC-cofactor function and to elucidate possible orientations of the TSR in relation to the membrane. For these purposes, we determined the epitope for a calcium-dependent monoclonal antibody (HPS67) against the TSR, which inhibits APC cofactor activity even though it does not impede protein S binding to the membrane. HPS67 did not recognize wild-type mouse protein S but gained reactivity against a recombinant mouse protein in which G49 and R52 were mutated to R and Q (found in human protein S), respectively, suggesting these two residues to be part of a surface exposed epitope for HPS67. This information helped in the validation and refinement of the structural model for the Gla-TSR-EGF1-modules of protein S. The X-ray structure of a Fab-fragment mimicking HPS67 was docked onto the protein S model. The observation that HPS67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface. In the proposed model for membrane-bound protein S, there is no contact between the TSR and the membrane. Rather, the TSR is free to interact with membrane-bound APC.

In vitro characterisation of two naturally occurring mutations in the thrombin-sensitive region of anticoagulant protein S.

The molecular consequences of two naturally occurring mutations in the thrombin-sensitive region of protein S were investigated using a combination of recombinant protein expression, functional analysis and molecular modelling. Both mutations (R49H and R70S) have been found in thrombosis patients diagnosed as having type I protein S deficiency. Molecular modelling analysis suggested the R49H substitution not to disrupt the structure of thrombin-sensitive region, whereas the R70S substitution could affect the 3D structure mildly. To elucidate the molecular consequences of these substitutions experimentally, site directed mutagenesis of protein S cDNA and expression in mammalian cells created the two mutants. The secretion profiles and functional anticoagulant activities of the protein S mutants were characterised. Secretion of the R49H mutant was similar to that of wild type protein S, whereas the R70S mutant showed moderately decreased expression. Neither of the mutants showed any major functional defects as cofactors to activated protein C (APC) in an APTT-based assay or in degradation of factor Va. However, both mutants demonstrated decreased activity in a factor VIIIa degradation assay, which in addition to APC and protein S also included factor V as synergistic APC cofactor. In conclusion, the R49H substitution did not produce a quantitative abnormality in vitro, raising doubts as to whether it caused the type I deficiency. In contrast, the experimental data obtained for the R70S mutant agrees well with the observed type I deficiency. Our study illustrates that in vitro experimental characterisation together with computer-based structural analysis are useful tools in the analysis of the relationship between naturally occurring mutations and clinical phenotypes.

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.

Interaction between serum amyloid P component and C4b-binding protein associated with inhibition of factor I-mediated C4b degradation.

Serum amyloid P component (SAP) is a member of the pentraxin protein family. Although present in all types of amyloid deposits, it is also a normal constituent of blood and extravascular tissues. In blood, SAP forms a calcium-dependent, noncovalent complex with C4b-binding protein (C4BP). C4BP regulates the classical complement pathway as it binds C4b and functions as cofactor in its degradation by factor I. Although SAP and C4b bind to distinct sites on C4BP, it is not known whether the SAP-C4BP interaction affects the function of C4BP. We report that in a fluid phase system, SAP inhibited degradation of C4(H2O) (which is C4b-like) in a dose-dependent manner. Phosphorylethanolamine was found to alleviate the inhibitory effect of SAP on C4(H2O) degradation. Because this compound is known to inhibit the SAP-C4BP interaction, this indicated direct binding of SAP to C4BP to be required for inhibition of C4(H2O) degradation. Even though C4BP, C4(H2O), and SAP form a multimolecular complex in fluid phase, SAP was found to inhibit binding of C4BP to immobilized C4(H2O). The inhibitory effect was calcium dependent and alleviated by phosphorylethanolamine. Heparin, which is known to inhibit the interaction between SAP and C4BP, was also found to counteract the inhibitory effect of SAP on C4BP binding to C4(H2O). However, the effect of heparin was biphasic because high concentrations of heparin directly inhibited binding of C4(H2O) to C4BP. The inhibition of C4BP function by SAP suggests that SAP may be involved in regulation of the classical complement pathway C3 convertase.

6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase in liver of the teleost Sparus aurata.

6-Phosphofructo 2-kinase/fructose 2,6-bisphosphatase was purified from the liver of the teleost fish Sparus aurata and the enzymatic activities were characterized kinetically. Both activities copurify, being dimers of relative molecular mass of 98 kDa with subunits of M(r) 54 kDa. Although both specific activities are in the range of mammalian liver isozymes, the Kmfru 6-P of teleost 6-phosphofructo 2-kinase is 3 times that in rat liver. The S. aurata 6-phosphofructo 2-kinase is inhibited by ADP, citrate and phosphoenolpyruvate, and fructose-2,6-bisphosphatase presents inhibition by fru 6-P. Unlike the rat liver enzyme, the kinase reaction is scarcely inhibited by glycerol 3-P. The teleost isozyme is substrate for the cyclic-AMP-dependent protein kinase, as can be followed by the incorporation of 32P from ATP into the enzyme. Phosphorylation of the enzyme changes its kinetic behavior, leading to a form with a lower kinase/bisphosphatase activity ratio. No change is detected in the fru 6-P dependence of 6-phosphofructo 2-kinase, but the phosphorylated form is more sensitive to inhibition by effectors, especially by glycerol 3-phosphate. Phosphorylation enhances the fructose-2,6-bisphosphatase Vmax activity twofold. The implications of all these kinetic characteristics in the control of hepatic fructose-2,6-bisphosphate levels are discussed in the context of the studies in S. aurata in vivo. The results support the hypothesis that differences in the regulation of 6-phosphofructo 2-kinase/fructose-2,6-bisphosphatase are a key point for the specific adaptations of carbohydrate metabolism in this teleost fish.

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.