Structure and function of the long pentraxin PTX3 glycosidic moiety: fine-tuning of the interaction with C1q and complement activation

The prototypic long pentraxin PTX3 is a unique fluid-phase pattern recognition receptor that plays a nonredundant role in innate immunity and female fertility. The PTX3 C-terminal domain is required for C1q recognition and complement activation and contains a single N-glycosylation site on Asn 220. In the present study, we characterized the structure of the human PTX3 glycosidic moiety and investigated its relevance in C1q interaction and activation of the complement classical pathway. By specific endo and exoglycosidases digestion and direct mass spectrometric analysis, we found that both recombinant and naturally occurring PTX3 were N-linked to fucosylated and sialylated complex-type sugars. Interestingly, glycans showed heterogeneity mainly in the relative amount of bi, tri, and tetrantennary structures depending on the cell type and inflammatory stimulus. Enzymatic removal of sialic acid or the entire glycosidic moiety equally enhanced PTX3 binding to C1q compared to that in the native protein, thus indicating that glycosylation substantially contributes to modulate PTX3/C1q interaction and that sialic acid is the main determinant of this contribution. BIAcore kinetic measurements returned decreasing K(off) values as sugars were removed, pointing to a stabilization of the PTX3/C1q complex. No major rearrangement of PTX3 quaternary structure was observed after desialylation or deglycosylation as established by size exclusion chromatography. Consistent with C1q binding, PTX3 desialylation enhanced the activation of the classical complement pathway, as assessed by C4 and C3 deposition. In conclusion, our results provided evidence of an involvement of the PTX3 sugar moiety in C1q recognition and complement activation.     

Heterocomplexes of mannose-binding lectin and the pentraxins PTX3 or serum amyloid P component trigger cross-activation of the complement system

The long pentraxin 3 (PTX3), serum amyloid P component (SAP), and C-reactive protein belong to the pentraxin family of pattern recognition molecules involved in tissue homeostasis and innate immunity. They interact with C1q from the classical complement pathway. Whether this also occurs via the analogous mannose-binding lectin (MBL) from the lectin complement pathway is unknown. Thus, we investigated the possible interaction between MBL and the pentraxins. We report that MBL bound PTX3 and SAP partly via its collagen-like domain but not C-reactive protein. MBL-PTX3 complex formation resulted in recruitment of C1q, but this was not seen for the MBL-SAP complex. However, both MBL-PTX3 and MBL-SAP complexes enhanced C4 and C3 deposition and opsonophagocytosis of Candida albicans by polymorphonuclear leukocytes. Interaction between MBL and PTX3 led to communication between the lectin and classical complement pathways via recruitment of C1q, whereas SAP-enhanced complement activation occurs via a hitherto unknown mechanism. Taken together, MBL-pentraxin heterocomplexes trigger cross-activation of the complement system.     

Interaction of C1q with IgG1, C-reactive protein and pentraxin 3: mutational studies using recombinant globular head modules of human C1q A, B, and C chains

C1q is the first subcomponent of the classical complement pathway that can interact with a range of biochemically and structurally diverse self and nonself ligands. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has specific and differential binding properties toward C1q ligands. It is largely considered that C1q-ligand interactions are ionic in nature; however, the complementary ligand-binding sites on C1q and the mechanisms of interactions are still unclear. To identify the residues on the gC1q domain that are likely to be involved in ligand recognition, we have generated a number of substitution mutants of ghA, ghB, and ghC modules and examined their interactions with three selected ligands: IgG1, C-reactive protein (CRP), and pentraxin 3 (PTX3). Our results suggest that charged residues belonging to the apex of the gC1q heterotrimer (with participation of all three chains) as well as the side of the ghB are crucial for C1q binding to these ligands, and their contribution to each interaction is different. It is likely that a set of charged residues from the gC1q surface participate via different ionic and hydrogen bonds with corresponding residues from the ligand, instead of forming separate binding sites. Thus, a recently proposed model suggesting the rotation of the gC1q domain upon ligand recognition may be extended to C1q interaction with CRP and PTX3 in addition to IgG1.     

Human pentraxin 3 binds to the complement regulator c4b-binding protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.     

Pentraxin-3 regulates the inflammatory activity of macrophages

Background and aimsPentraxin-3 (PTX3) reportedly has protective roles in atherosclerosis and myocardial infarction, and is a useful biomarker of vascular inflammation. However, the detailed functions of PTX3 in inflammation are yet to be elucidated. This study aimed to investigate the function of PTX3 in macrophages.MethodsPMA-treated THP-1 cell line (THP-1 macrophage) and monocyte-derived human primary macrophages were treated with recombinant PTX3. Cytokine and chemokine levels in the THP-1 culture medium were measured as well as monocyte chemoattractant protein (MCP-1) concentrations in the Raw 264.7 cell culture medium. PTX3-silenced apoptotic macrophages (THP-1 cell line) were generated to investigate the roles of PTX3 in phagocytosis.ResultsIn the presence of PTX3, macrophage interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and MCP-1 levels were reduced significantly (?39%, P=0.007; ?21%, P=0.008; and ?67%, P=0.0003, respectively), whilst activated transforming growth factor-β (TGF?β) was detected in the THP-1 macrophages (P=0.0004). Additionally, PTX3 induced Akt phosphorylation and reduced nuclear factor-kappa B (NF-κB) activation by 35% (P=0.002), which was induced by TNF-α in THP-1 macrophages. Furthermore, silencing of PTX3 in apoptotic cells resulted in increased macrophage binding, elevated expression rate of HLA-DR (+30%, P=0.015) and CD86 (+204%, P=0.004) positive cells, and induction of IL-1β (+36%, P=0.024) production. Conversely, adding recombinant PTX3 to macrophages reduced CD86 and HLA-DR expression in a dose-dependent manner.ConclusionsWe identified PTX3 as a novel regulator of macrophage activity, and this function suggests that PTX3 acts to resolve inflammation.     

PTX3, a key component of innate immunity, is induced by SAA via FPRL1-mediated signaling in HAECs

Serum amyloid A (SAA) is regarded as an important acute phase protein in coronary artery diseases. However, its involvement in the immune response of atherosclerosis is poorly understood. The present study was designed to investigate the influence of SAA on the secretion of long pentraxin 3 (PTX3), a key component of innate immunity, in human aortic endothelial cells (HAECs). Our study revealed that recombinant SAA up-regulated PTX3 production in a remarkable dose- and time-dependent manner and the activation of formyl peptide receptor-like 1 (FPRL1) was crucial for SAA-induced expression of PTX3 in HAECs. Meanwhile, SAA-induced PTX3 production could be significantly down-regulated by using the specific siRNA sequences for Jun N-terminal kinases (JNK). Furthermore, the activation of activator protein-1 (AP-1) was necessary for the up-regulation of PTX3 expression. We also found that the activation of nuclear factor-kappa B (NF-κB) played an important role in this process. Our findings demonstrate that SAA up-regulates PTX3 production via FPRL1 significantly, and thus, contributes to the inflammatory pathogenesis of atherosclerosis.     

Identification of an antiangiogenic FGF2-binding site in the N terminus of the soluble pattern recognition receptor PTX3

Long-pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 comprises a pentraxin-like C-terminal domain involved in complement activation via C1q interaction and an N-terminal extension with unknown functions. PTX3 binds fibroblast growth factor-2 (FGF2), inhibiting its pro-angiogenic and pro-restenotic activity. Here, retroviral transduced endothelial cells (ECs) overexpressing the N-terminal fragment PTX3-(1-178) showed reduced mitogenic activity in response to FGF2. Accordingly, purified recombinant PTX3-(1-178) binds FGF2, prevents PTX3/FGF2 interaction, and inhibits FGF2 mitogenic activity in ECs. Also, the monoclonal antibody mAb-MNB4, which recognizes the PTX3-(87-99) epitope, prevents FGF2/PTX3 interaction and abolishes the FGF2 antagonist activity of PTX3. Consistently, the synthetic peptides PTX3-(82-110) and PTX3-(97-110) bind FGF2 and inhibit the interaction of FGF2 with PTX3 immobilized to a BIAcore sensor chip, FGF2-dependent EC proliferation, and angiogenesis in vivo. Thus, the data identify a FGF2-binding domain in the N-terminal extension of PTX3 spanning the PTX3-(97-110) region, pointing to a novel function for the N-terminal extension of PTX3 and underlining the complexity of the PTX3 molecule for modular humoral pattern recognition.     

Recombinant human complement subcomponent C1s lacking beta-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex.

In contrast to the human serum protein which is approximately one-half erythro-beta-hydroxyasparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant C1s expressed by insect cells after infection with recombinant baculovirus entirely lacks posttranslational modification at asparagine 134. It is also incompletely glycosylated, lacking, at least, sialic acid. Site-directed mutagenesis of one of the two sites of carbohydrate attachment (Asn 159 to Gln 159) yields a faster migrating recombinant C1s still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with C1q and either human serum or recombinant C1r, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between C1s and C1q, C1r, C2, and C4 required for reassembly of the C1 complex, activation, and initiation of the classical complement pathway. The 4.0S recombinant C1s dimerizes to yield 5.6S C1s2 in the presence of Ca2+ and forms the 9.1S C1s-C1r-C1r-C1s tetramer upon the addition of human serum C1r and the 15.6S C1 complex upon the addition of C1q to the tetramer. The recombinant C1s and human serum C1s have identical N-terminal amino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant C1s is secreted and isolated as the unactivated zymogen, and it may be activated by human serum C1r which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between C1-inhibitor and the light chain of recombinant C1s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complement dependent amplification of the innate response to a cognate microbial ligand by the long pentraxin PTX3

The long pentraxin PTX3 is a fluid-phase pattern recognition receptor, which plays a nonredundant role in resistance against selected pathogens. PTX3 has properties similar to Abs; its production is induced by pathogen recognition, it recognizes microbial moieties, activates complement, and facilitates cellular recognition by phagocytes. The mechanisms responsible for the effector function of PTX3 in vivo have not been elucidated. OmpA, a major outer membrane protein of Gram-negative Enterobacteriaceae, is a microbial moiety recognized by PTX3. In the air pouch model, KpOmpA induces an inflammatory response, which is amplified by coadministration of PTX3 in terms of leukocyte recruitment and proinflammatory cytokine production. PTX3 did not affect the inflammatory response to LPS, a microbial moiety not recognized by PTX3. As PTX3 binds to C1q and modulates the activation of the complement cascade, we assessed the involvement of complement in the amplification of the response elicited by KpOmpA and PTX3. Experiments performed using cobra venom factor, C1-esterase inhibitor, and soluble complement receptor 1 indicate that PTX3 amplifies the inflammatory response to KpOmpA through complement activation. The results reported here demonstrate that PTX3 activates a complement-dependent humoral amplification loop of the innate response to a microbial ligand.     

The therapeutic potential of the humoral pattern recognition molecule PTX3 in chronic lung infection caused by Pseudomonas aeruginosa

Chronic lung infections by Pseudomonas aeruginosa strains are a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Although there is no clear evidence for a primary defect in the immune system of CF patients, the host is generally unable to clear P. aeruginosa from the airways. PTX3 is a soluble pattern recognition receptor that plays nonredundant roles in the innate immune response to fungi, bacteria, and viruses. In particular, PTX3 deficiency is associated with increased susceptibility to P. aeruginosa lung infection. To address the potential therapeutic effect of PTX3 in P. aeruginosa lung infection, we established persistent and progressive infections in mice with the RP73 clinical strain RP73 isolated from a CF patient and treated them with recombinant human PTX3. The results indicated that PTX3 has a potential therapeutic effect in P. aeruginosa chronic lung infection by reducing lung colonization, proinflammatory cytokine levels (CXCL1, CXCL2, CCL2, and IL-1β), and leukocyte recruitment in the airways. In models of acute infections and in in vitro assays, the prophagocytic effect of PTX3 was maintained in C1q-deficient mice and was lost in C3- and Fc common γ-chain-deficient mice, suggesting that facilitated recognition and phagocytosis of pathogens through the interplay between complement and FcγRs are involved in the therapeutic effect mediated by PTX3. These data suggested that PTX3 is a potential therapeutic tool in chronic P. aeruginosa lung infections, such as those seen in CF patients.     

Complement C1q-target proteins recognition is inhibited by electric moment effectors

Classical complement pathway is an important innate immune mechanism, which is usually triggered by binding of C1q to immunoglobulins, pentraxins and other target molecules. Although the activation of the classical pathway is crucial in the host defence, its undesirable and uncontrolled activation can lead to tissue damage. Thus, understanding the molecular basis of complement activation and its inhibition are of great biomedical importance. Recently, we proposed a mechanism for target recognition and classical pathway activation by C1q, which is likely governed by calcium-controlled reorientation of macromolecular electric moment vectors. Here we sought to define the mechanism of C1q inhibition by low molecular weight disulphate compounds that bind to the globular (gC1q) domain, using experimental, computational docking and theoretical modelling approaches. Our experimental results demonstrate that betulin disulphate (B2S) and 9,9-bis(4'-hydroxyphenyl)fluorene disulphate (F2S) inhibit the interaction of C1q and its recombinant globular modules with target molecules IgG1, C-reactive protein (CRP) and long pentraxin 3 (PTX3). In most C1q-inhibitor docked complexes, there is a reduction of electric moment scalar values and similarly altered direction of electric/dipole moment vectors. This could explain the inhibitory effect by impaired electrostatic steering, lacking optimal target recognition and formation of functional complex. In the presence of the inhibitor, the tilt of gC1q domains is likely to be blocked by the altered direction of the electric moment vector. Thus, the transition from the inactive (closed) towards the active (open) conformation of C1q (i.e. the complement activation signal transmission) will be impaired and the cascade initiation disrupted. These results could serve as a starting point for the exploration of a new form of 'electric moment inhibitors/effectors'.     

Regulation of CD93 cell surface expression by protein kinase C isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.     

Pentoxifylline inhibits Ca2+-dependent and ATP proteasome-dependent proteolysis in skeletal muscle from acutely diabetic rats

Previous studies from this laboratory have shown that catecholamines exert an inhibitory effect on muscle protein degradation through a pathway involving the cAMP cascade. The present work investigated the systemic effect of pentoxifylline (PTX; cAMP-phosphodiesterase inhibitor) treatment on the rate of overall proteolysis, the activity of proteolytic systems, and the process of protein synthesis in extensor digitorum longus muscles from normal and acutely diabetic rats. The direct in vitro effect of this drug on the rates of muscle protein degradation was also investigated. Muscles from diabetic rats treated with PTX showed an increase (22%) in the cAMP content and reduction in total rates of protein breakdown and in activity of Ca2+-dependent (47%) and ATP proteasome-dependent (23%) proteolytic pathways. The high content of m-calpain observed in muscles from diabetic rats was abolished by PTX treatment. The addition of PTX (10(-3) M) to the incubation medium increased the cAMP content in muscles from normal (22%) and diabetic (51%) rats and induced a reduction in the rates of overall proteolysis that was accompanied by decreased activity of the Ca2+-dependent and ATP proteasome-dependent proteolytic systems, in both groups. The in vitro addition of H-89, an inhibitor of protein kinase A (PKA), completely blocked the effect of PTX on the reduction of proteolysis in muscles from normal and diabetic rats. The present data suggest that PTX exerts a direct inhibitory effect on protein degradative systems in muscles from acutely diabetic rats, probably involving the participation of cAMP intracellular pathways and activation of PKA, independently of tumor necrosis factor-alpha inhibition.     

A synthetic complement C1q-like peptide selectively interacts with immune complexes

A water-soluble peptide possessing an immune complex selective affinity was synthesized and its primary structure established as: Leu-Glu-Gln-Gly-Glu-Asn-Val-Phe-Leu-Gln-Ala-Thr-Ser-Asp-Asp-Cys. This peptide, designated as C1q-like peptide (CLP), represents a possible immune complex binding epitope of complement C1q. CLP has a hydrophilicity value of 0.21. At 0.5 M, it inhibited by 50% natural human C1q from binding to horseradish peroxidase-rabbit anti-peroxidase immune complex. CLP failed to inhibit Staphylococcus aureus protein A from binding monomeric IgG. When coated to a microplate, CLP showed selective binding to the immune complex, and could be used for application in immunochemical detection of immune complex. © Rapid Science Ltd. 1998     

Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.     

Analysis of binding sites in human C-reactive protein for Fc{gamma}RI, Fc{gamma}RIIA, and C1q by site-directed mutagenesis

Human C-reactive protein (CRP) is a classical, acute phase serum protein synthesized by the liver in response to infection, inflammation, or trauma. CRP binds to microbial antigens and damaged cells, opsonizes particles for phagocytosis and regulates the inflammatory response by the induction of cytokine synthesis. These activities of CRP depend on its ability to activate complement and to bind to Fcgamma receptors (FcgammaR). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human FcgammaRI (CD64) and FcgammaRIIa (CD32). Several mutations of the CRP structure were studied based on the published crystal structure of CRP. Mutant and wild-type recombinant CRP molecules were expressed in the baculovirus system and their interactions with FcgammaR and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu(176), critical for CRP binding to FcgammaRI and work by others (Agrawal, A., Shrive, A. K., Greenhough, T. J., and Volanakis, J. E. (2001) J. Immunol. 166, 3998-4004) determined several residues important for C1q binding. The amino acid residues important to CRP binding to FcgammaRIIa were previously unknown. This study newly identifies residues Thr(173) and Asn(186) as important for the binding of CRP to FcgammaRIIa and FcgammaRI. Lys(114), like Leu(176), was implicated in binding to FcgammaRI, but not FcgammaRIIa. Single mutations at amino acid positions Lys(114), Asp(169), Thr(173), Tyr(175), and Leu(176) affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for FcgammaRI, FcgammaRIIa, and C1q and indicate that these sites are overlapping.     

Paclitaxel Induces Primary and Postmitotic G1 Arrest in Human Arterial Smooth Muscle Cells

Paclitaxel (PTX), a microtubule-active drug, causes mitotic arrest leading to apoptosis in certain tumor cell lines. Here we investigated the effects of PTX on human arterial smooth muscle cell (SMC) cells. In SMC, PTX caused both (a) primary arrest in G1 and (b) post-mitotic arrest in G1. Post-mitotic cells were multinucleated (MN) with either 2C (near-diploid) or 4C (tetraploid) DNA content. At PTX concentrations above12 ng/ml, MN cells had 4C DNA content consistent with the lack of cytokinesis during abortive mitosis. Treatment with 6-12 ng/ml PTX yielded MN cells with 2C DNA content. Finally, 1-6 ng/ml of PTX, the lowest concentrations that affected cell proliferation, caused G1 arrest without multinucleation. It is important that PTX did not cause apoptosis in SMC. The absence of apoptosis could be explained by mitotic exit and G1 arrest as well as by low constitutive levels of caspase expression and by p53 and p21 induction. Thus, following transient mitotic arrest, SMC exit mitosis to form MN cells. These post-mitotic cells were subsequently arrested in G1 but maintained normal elongated morphology and were viable for at least 21 days. We conclude that in SMC PTX causes post-mitotic cell cycle arrest rather than cell death.     

Single-Step Purification of Two Functional Human Apolipoprotein E Variants Hyperexpressed inEscherichia coli

We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH₂-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into theEscherichia colistrain BL21(DE3). Recombinant strains were grown at 37°C in a Luria and Bertani medium and the addition of isopropyl β-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.     

Role of the soluble pattern recognition receptor PTX3 in vascular biology

Pentraxins act as soluble pattern recognition receptors with a wide range of functions in various pathophysiological conditions. The long-pentraxin PTX3 shares the C-terminal pentraxin-domain with short-pentraxins C-reactive protein and serum amyloid P component and possesses an unique N-terminal domain. These structural features suggest that PTX3 may have both overlapping and distinct biological/ligand recognition properties when compared to short-pentraxins. PTX3 serves as a mechanism of amplification of inflammation and innate immunity. Indeed, vessel wall elements produce high amounts of PTX3 during inflammation and the levels of circulating PTX3 increase in several pathological conditions affecting the cardiovascular system. PTX3 exists as a free or extracellular matrix-associated molecule and it binds the complement fraction C1q. PTX3 binds also apoptotic cells and selected pathogens, playing a role in innate immunity processes. In endothelial cells and macrophages, PTX3 upregulates tissue factor expression, suggesting its action as a regulator of endothelium during thrombogenesis and ischaemic vascular disease. Finally, PTX3 binds the angiogenic fibroblast growth factor-2, thus inhibiting its biological activity. Taken together, these properties point to a role for PTX3 during vascular damage, angiogenesis, atherosclerosis, and restenosis.