RESUMEN
Purpose: Fibrosis or scarring is a pathological outcome of wound healing and is characterized by terminally differentiated myofibroblasts. Heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) is a unique matrix component purified from amniotic membrane that exerts an anti-inflammatory effect. Herein, we investigate whether HC-HA/PTX3 can also exert an antiscarring effect. Methods: Human corneal fibroblasts and myofibroblasts were seeded on plastic, immobilized HA or HC-HA/PTX3 or on plastic with or without soluble HA and HC-HA/PTX3 in DMEM+10% FBS, with or without AMD3100 or SB431542 in DMEM+ITS with or without transforming growth factor-ß1 (TGF-ß1). Transcript expression of keratocyte and signaling markers was determined by RT-qPCR. Immunostaining was performed to monitor cytolocalization of signaling markers and α-SMA. Western blotting was used to measure relative protein level. Results: Human corneal fibroblasts and myofibroblasts cultured in or on HC-HA/PTX3, but not HA, were refrained from cytoplasmic expression of αSMA and nuclear translocation of pSMAD2/3 when challenged with exogenous TGF-ß1. Such an antiscarring action by suppressing canonical TGF-ß1 signaling was surprisingly accompanied by phenotypic reversal to keratocan-expressing keratocytes through activation of BMP signaling. Further investigation disclosed that such phenotypic reversal was initiated by cell aggregation mediated by SDF1-CXCR4 signaling highlighted by nuclear translocation of CXCR4 and upregulation of CXCR4 transcript and protein followed by activation of canonical BMP signaling. Conclusions: These findings collectively provide mechanistic understanding explaining how amniotic membrane transplantation exerts an antiscarring action. In addition, HC-HA/PTX3 and derivatives may be developed into a new biologic to treat corneal blindness caused by stromal scar or opacity in the future.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Proteína C-Reactiva/aislamiento & purificación , Proteína C-Reactiva/fisiología , Diferenciación Celular , Córnea/citología , Queratocitos de la Córnea/citología , Fibroblastos/citología , Ácido Hialurónico/fisiología , Miofibroblastos/citología , Componente Amiloide P Sérico/aislamiento & purificación , Componente Amiloide P Sérico/fisiología , Amnios/química , Humanos , Transducción de SeñalRESUMEN
Chronic graft-versus-host disease (cGVHD) is a major complication of hematopoietic stem cell transplantation. Dry eye disease is the prominent ocular sequel of cGVHD and is caused by excessive inflammation and fibrosis in the lacrimal glands. Heavy chain-Hyaluronan/Pentraxin 3 (HC-HA/PTX3) is a complex purified from human amniotic membrane (AM) and known to exert anti-inflammatory and anti-scarring actions. In this study, we utilized a mouse model of cGVHD to examine whether HC-HA/PTX3 could attenuate dry eye disease elicited by cGVHD. Our results indicated that subconjunctival and subcutaneous injection of HC-HA/PTX3 preserved tear secretion and conjunctival goblet cell density and mitigated inflammation and scarring of the conjunctiva. Such therapeutic benefits were associated with suppression of scarring and infiltration of inflammatory/immune cells in the lacrimal glands. Furthermore, HC-HA/PTX3 significantly reduced the extent of infiltration of CD45+ CD4+ IL-17+ cells, CD45+ CD34+ collagen I+ CXCR4+ fibrocytes, and HSP47+ activated fibroblasts that were accompanied by upregulation of collagen type Iα1, collagen type IIIα1 and NF-kB in lacrimal glands. Collectively, these pre-clinical data help prove the concept that subcutaneous and subconjunctival injection of HC-HA/PTX3 is a novel approach to prevent dry eye disease caused by cGVHD and allow us to test its safety and efficacy in future human clinical trials.
Asunto(s)
Proteína C-Reactiva/farmacología , Cicatriz/prevención & control , Conjuntiva/efectos de los fármacos , Síndromes de Ojo Seco/tratamiento farmacológico , Enfermedad Injerto contra Huésped/patología , Ácido Hialurónico/farmacología , Aparato Lagrimal/efectos de los fármacos , Componente Amiloide P Sérico/farmacología , Amnios/química , Animales , Trasplante de Médula Ósea/efectos adversos , Proteína C-Reactiva/aislamiento & purificación , Enfermedad Crónica , Cicatriz/etiología , Cicatriz/inmunología , Cicatriz/patología , Conjuntiva/inmunología , Conjuntiva/patología , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/etiología , Síndromes de Ojo Seco/inmunología , Síndromes de Ojo Seco/patología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/patología , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/inmunología , Humanos , Ácido Hialurónico/aislamiento & purificación , Inyecciones Intraoculares , Inyecciones Subcutáneas , Aparato Lagrimal/inmunología , Aparato Lagrimal/patología , Masculino , Ratones , Componente Amiloide P Sérico/aislamiento & purificación , Trasplante Homólogo , Irradiación Corporal TotalRESUMEN
The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Calcio/sangre , Inmunidad Innata , Componente Amiloide P Sérico/metabolismo , Adulto , Anticuerpos Inmovilizados/metabolismo , Anticuerpos Monoclonales/metabolismo , Antitrombinas/farmacología , Apolipoproteínas/sangre , Apolipoproteínas/química , Apolipoproteínas/aislamiento & purificación , Apolipoproteínas/metabolismo , Factores de Coagulación Sanguínea/análisis , Factores de Coagulación Sanguínea/química , Factores de Coagulación Sanguínea/aislamiento & purificación , Factores de Coagulación Sanguínea/metabolismo , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/química , Proteínas Sanguíneas/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Proteínas del Sistema Complemento/análisis , Proteínas del Sistema Complemento/química , Proteínas del Sistema Complemento/aislamiento & purificación , Proteínas del Sistema Complemento/metabolismo , Femenino , Hirudinas/farmacología , Humanos , Inmunoprecipitación , Ligandos , Masculino , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Proteolisis , Proteínas Recombinantes/farmacología , Componente Amiloide P Sérico/análisis , Componente Amiloide P Sérico/antagonistas & inhibidores , Componente Amiloide P Sérico/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Pentraxin 3 (PTX3), a soluble pattern recognition receptor, plays an important role in innate immunity and has been implicated to be a candidate resistance gene against Streptococcus suis 2 infection. To discover the antibacterial effect of porcine PTX3 against S. suis 2, the 42-kDa PTX3 protein was expressed by Chinese hamster ovary cells (CHO), and an additional eukaryotic expression vector pVAX-ptx3 was constructed. The expressed porcine PTX3 mediated a range of antibacterial activities including increasing phagocytic capacity of primary porcine alveolar macrophages (PAM) against S. suis 2 and inhibiting adhesion of S. suis 2 to human epidermoid cancer cells (Hep-2). In mouse model, pre-intramuscular injecting with pVAX-ptx3 reduced mortality and reduced bacteria loads in blood, spleen, lung and brain compared with that of control group during 2-12 h following intraperitoneal injection (i.p.) with S. suis 2. Meanwhile, the expressions of IL-6 and IL-8 in blood were increased in pVAX-ptx3 group, whereas no obvious changes about IL-10. In piglet model, bacteria load in blood of pVAX-ptx3 group was significantly lower than that of control group after i.p. with S. suis 2, correspondingly, expression of IL-6 and IL-8 were significantly increased in pVAX-ptx3 group. In contrast, white blood cell (WBC) and neutrophil cell (NEU) count of peripheral blood in pVAX-ptx3 group were lower than that of control group. These studies described a novel antibacterial role for porcine PTX3 against S. suis 2 both in vitro and in vivo and suggested that porcine PTX3 may be a potential biological agent against S. suis 2 in pig and be used for the clinical prevention and treatment of streptococcosis caused by S. suis 2.
Asunto(s)
Proteína C-Reactiva/farmacología , Factores Inmunológicos/farmacología , Componente Amiloide P Sérico/farmacología , Streptococcus suis/efectos de los fármacos , Estructuras Animales/microbiología , Animales , Adhesión Bacteriana/efectos de los fármacos , Carga Bacteriana , Proteína C-Reactiva/administración & dosificación , Proteína C-Reactiva/genética , Proteína C-Reactiva/aislamiento & purificación , Células Epiteliales/microbiología , Factores Inmunológicos/administración & dosificación , Inyecciones Intramusculares , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Ratones , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Componente Amiloide P Sérico/administración & dosificación , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/aislamiento & purificación , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/prevención & control , Streptococcus suis/inmunología , Streptococcus suis/fisiología , Análisis de Supervivencia , PorcinosRESUMEN
To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in three-dimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-α-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1(DCKO) mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency.
Asunto(s)
Amnios/metabolismo , Proteínas Morfogenéticas Óseas/biosíntesis , Proteína C-Reactiva/biosíntesis , Células Epiteliales/metabolismo , Ácido Hialurónico/biosíntesis , Componente Amiloide P Sérico/biosíntesis , Nicho de Células Madre/fisiología , Células 3T3 , Animales , Proteína C-Reactiva/aislamiento & purificación , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Ácido Hialurónico/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Ratones , Ratones Transgénicos , Componente Amiloide P Sérico/aislamiento & purificación , Transducción de Señal/fisiología , Células Madre/metabolismoRESUMEN
In this study, we have investigated the isolation of serum amyloid P (SAP) and C-reactive protein (CRP) from rainbow trout. It has recently been found that SAP is deposited in atherosclerotic lesions or neurofibrillary tangles, which are related to aging process and Alzheimer's disease. Given the importance of CRP, the CRP level in blood is becoming recognized as a potential means of monitoring cardiovascular risk. These two proteins, members of the pentraxin family of oligomeric serum proteins, were isolated from rainbow trout using N-methacryloyl-phosphoserine (MA-pSer) immobilized poly (2-hydroxy ethylmethacrylate) (PHEMA) cryogels as a column material in a fast protein liquid chromatography system. The separation process was verified in two steps. First, SAP and CRP proteins were isolated together from serum sample of rainbow trout using MA-pSer/PHEMA cryogel columns. Second, SAP protein was separated chromatographically from CRP protein using the Ca(2+) ion immobilized PHEMA cryogel column. According to the data, a new and effective technique has been developed for the isolation of SAP and CRP proteins from a biological source, rainbow trout. Finally, purified SAP and CRP were loaded using sodium dodecyl sulfate-polyacrylamide gel and western blot analysis to investigate the purity of chromatographically isolated SAP and CRP compared with commertial SAP and CRP.
Asunto(s)
Proteína C-Reactiva/aislamiento & purificación , Cromatografía Líquida de Alta Presión/instrumentación , Componente Amiloide P Sérico/aislamiento & purificación , Adsorción , Animales , Proteína C-Reactiva/análisis , Proteína C-Reactiva/química , Cromatografía Líquida de Alta Presión/métodos , Humanos , Concentración de Iones de Hidrógeno , Oncorhynchus mykiss , Polihidroxietil Metacrilato/química , Componente Amiloide P Sérico/análisis , Componente Amiloide P Sérico/químicaRESUMEN
BACKGROUND: Serum amyloid P component (SAP) is a glycoprotein that is universally found associated with different types of amyloid deposits. It has been suggested that it stabilizes amyloid fibrils and therefore protects them from proteolytic degradation. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we show that SAP binds not only to mature amyloid fibrils but also to early aggregates of amyloidogenic mutants of the plasma protein transthyretin (TTR). It does not inhibit fibril formation of TTR mutants, which spontaneously form amyloid in vitro at physiological pH. We found that SAP prevents cell death induced by mutant TTR, while several other molecules that are also known to decorate amyloid fibrils do not have such effect. Using a Drosophila model for TTR-associated amyloidosis, we found a new role for SAP as a protective factor in inhibition of TTR-induced toxicity. Overexpression of mutated TTR leads to a neurological phenotype with changes in wing posture. SAP-transgenic flies were crossed with mutated TTR-expressing flies and the results clearly confirmed a protective effect of SAP on TTR-induced phenotype, with an almost complete reduction in abnormal wing posture. Furthermore, we found in vivo that binding of SAP to mutated TTR counteracts the otherwise detrimental effects of aggregation of amyloidogenic TTR on retinal structure. CONCLUSIONS/SIGNIFICANCE: Together, these two approaches firmly establish the protective effect of SAP on TTR-induced cell death and degenerative phenotypes, and suggest a novel role for SAP through which the toxicity of early amyloidogenic aggregates is attenuated.
Asunto(s)
Neuropatías Amiloides Familiares/metabolismo , Drosophila melanogaster/metabolismo , Prealbúmina/metabolismo , Componente Amiloide P Sérico/metabolismo , Alas de Animales/metabolismo , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/fisiopatología , Animales , Animales Modificados Genéticamente , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Drosophila melanogaster/genética , Floculación , Expresión Génica , Humanos , Fenotipo , Prealbúmina/genética , Prealbúmina/farmacología , Unión Proteica , Suero/química , Componente Amiloide P Sérico/aislamiento & purificación , Componente Amiloide P Sérico/farmacología , Alas de Animales/fisiopatologíaRESUMEN
The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non-specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL-6 or IL-8, nor does SAP cause release of IL-1ß or IL-10. Furthermore neither of our preparations was pro-inflammatory in mice in vivo.
Asunto(s)
Proteína C-Reactiva/análisis , Citocinas/metabolismo , Leucocitos Mononucleares/metabolismo , Componente Amiloide P Sérico/análisis , Proteínas de Fase Aguda/análisis , Proteínas de Fase Aguda/aislamiento & purificación , Proteínas de Fase Aguda/farmacología , Amiloidosis/sangre , Animales , Proteína C-Reactiva/aislamiento & purificación , Proteína C-Reactiva/farmacología , Células Cultivadas , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Componente Amiloide P Sérico/aislamiento & purificación , Componente Amiloide P Sérico/farmacología , Espectrometría de Masa por Ionización de Electrospray , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Monocytes are produced in the bone marrow and enter the blood. They generally leave the blood and enter a tissue, and then become macrophages. In healing wounds, circulating monocytes also enter the tissue and instead of becoming macrophages, can differentiate into fibroblast-like cells called fibrocytes. Fibrocytes are also present in the lesions associated with fibrosing diseases such as congestive heart failure, end stage kidney disease, and pulmonary fibrosis. We have found that culturing blood monocytes, or white blood cell preparations containing monocytes, in serum-free media permits some of the monocytes to differentiate into fibrocytes within 5 days, and that this differentiation is inhibited by the blood plasma protein serum amyloid P.
Asunto(s)
Diferenciación Celular , Fibroblastos/citología , Monocitos/citología , Animales , Diferenciación Celular/efectos de los fármacos , Separación Celular/métodos , Medio de Cultivo Libre de Suero , Humanos , Leucocitos Mononucleares/citología , Ratones , Ratas , Componente Amiloide P Sérico/aislamiento & purificación , Componente Amiloide P Sérico/farmacología , PorcinosRESUMEN
Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (~200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (K(D) = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/sangre , Proteína C-Reactiva/metabolismo , Proteínas Portadoras/sangre , Neutrófilos/metabolismo , Sepsis/sangre , Componente Amiloide P Sérico/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Péptidos Catiónicos Antimicrobianos/química , Biomarcadores/sangre , Proteínas Sanguíneas/química , Proteínas Sanguíneas/aislamiento & purificación , Proteínas Sanguíneas/metabolismo , Proteína C-Reactiva/química , Proteína C-Reactiva/aislamiento & purificación , Células COS , Calcio/química , Proteínas Portadoras/química , Chlorocebus aethiops , Femenino , Humanos , Inmunoprecipitación , Sustancias Macromoleculares/sangre , Masculino , Persona de Mediana Edad , Peroxidasa/sangre , Peroxidasa/aislamiento & purificación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteómica , Vesículas Secretoras/metabolismo , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/aislamiento & purificaciónRESUMEN
From the serum of Pangasianodon hypophthalmus, two proteins were isolated by affinity chromatography on Sepharose and phosphorylcholine-Sepharose. Their binding on the affinity matrices critically depends on the presence of Ca2+ ions. N-terminal sequencing and sequencing of internal tryptic peptides identified the proteins as pentraxins and from their binding properties they are identified as SAP (serum amyloid P component) and CRP (C-reactive protein). Per ml serum, 36 microg SAP and 56 microg CRP was purified. Upon gel filtration, both the SAP and CRP elute as trimers of respectively 24 kDa and 28 kDa subunits. Both proteins are devoid of inter-chain disulfide bonds. Both SAP and CRP are glycosylated and agglutinate rabbit erythrocytes and pathogenic bacteria Edwardsiella ictaluri and Aeromonas hydrophila, but not Micrococcus lysodeikticus or Escherichia coli. Haemagglutination of SAP and CRP is inhibited by galactose (MIC = 1 mM) and by phosphorylcholine (MIC = 1-2 mM), respectively. Circular dichroism studies revealed that antiparallel beta-pleated sheets are dominating the secondary structure. Upon removing the Ca(2+) ions by EDTA, slight structural changes are observed by CD spectroscopy in the near-UV region. Immunodiffusion shows that P. hypophthalmus SAP and CRP do not cross-react.
Asunto(s)
Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Bagres/genética , Bagres/metabolismo , Hipotálamo/metabolismo , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/metabolismo , Aglutinación , Secuencia de Aminoácidos , Animales , Bacterias/metabolismo , Western Blotting , Proteína C-Reactiva/química , Proteína C-Reactiva/aislamiento & purificación , Cromatografía en Gel , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Eritrocitos/metabolismo , Datos de Secuencia Molecular , Conejos , Alineación de Secuencia , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/aislamiento & purificación , Análisis EspectralRESUMEN
Serum amyloid P component (SAP) belongs to the pentraxin family of proteins, members of which are characterized by radial pentameric structure and calcium-dependent ligand binding. SAP is present in all types of amyloidosis and has been shown to bind to several ligands, but the physiological function of this protein has not been fully elucidated. The present study identified and characterized SAP in human semen and immunolocalized it to the male reproductive tract. SAP was also detected in seminal plasma by immunoblotting and purification by affinity chromatography followed by mass spectrometry. According to electroimmunoassay, the concentration of SAP in semen is approximately 2 mg/L, and flow cytometry revealed SAP attached to the surface of spermatozoa. Moreover, immunohistochemistry showed positive staining of spermatozoa, subsets of epithelial cells, and the stroma of accessory male genital glands and testis. Presence of mRNA supports local production of SAP, as shown with reverse transcription polymerase chain reaction. We identified SAP in a new setting - the human male reproductive system. SAP was detected on ejaculated spermatozoa, in seminal plasma and in tissue sections from the male reproductive tract. Further functional studies are needed to explain the role of SAP in human reproduction.
Asunto(s)
Componente Amiloide P Sérico/metabolismo , Espermatozoides/metabolismo , Sistema Urogenital/metabolismo , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoensayo/métodos , Inmunohistoquímica , Masculino , Espectrometría de Masas , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/aislamiento & purificaciónRESUMEN
Development of a rapid, effective, and highly specific platform for target identification in complex biofluids is one of the most important tasks in proteomic research. Taking advantage of the natural hydrophobic interaction of PVDF with probe protein, a simple and effective method was developed for protein quantitation and profiling. Using antibody-antigen interactions as a proof-of-concept system, the targeted plasma proteins, serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP), could be selectively isolated and enriched from human plasma by antibody-immobilized PVDF membrane and directly identified by MALDI-TOF MS without additional elution step. The approach was successfully applied to human plasma for rapid quantitation and variant screening of SAP, SAA, and CRP in healthy individuals and patients with gastric cancer. The triplexed on-probe quantitative analysis revealed significant overexpression of CRP and SAA in gastric cancer group, consistent with parallel ELISA measurements and pathological progression and prognostic significance reported in previous literatures. Furthermore, the variant mass profiling of the post-translationally modified forms revealed a high occurrence of de-sialic acid SAP in patients with gastric cancer. Due to the versatile assay design, ease of probe preparation without chemical synthesis, and compatibility with MALDI-TOF MS analysis, the methodology may be useful for target protein characterization, functional proteomics, and screening in clinical proteomics.
Asunto(s)
Proteína C-Reactiva/análisis , Polivinilos/química , Proteína Amiloide A Sérica/análisis , Componente Amiloide P Sérico/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteína C-Reactiva/aislamiento & purificación , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Humanos , Membranas Artificiales , Modelos Biológicos , Pronóstico , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Proteína Amiloide A Sérica/aislamiento & purificación , Proteína Amiloide A Sérica/metabolismo , Componente Amiloide P Sérico/aislamiento & purificación , Componente Amiloide P Sérico/metabolismo , Neoplasias Gástricas/sangre , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.
Asunto(s)
Proteína C-Reactiva/biosíntesis , Proteína C-Reactiva/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Componente Amiloide P Sérico/biosíntesis , Componente Amiloide P Sérico/aislamiento & purificación , Animales , Proteína C-Reactiva/metabolismo , Células CHO , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Dicroismo Circular , Complemento C1q/metabolismo , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Humanos , Estructura Cuaternaria de Proteína , Componente Amiloide P Sérico/metabolismo , Espectrometría de FluorescenciaRESUMEN
Serum amyloid P-component (SAP), a pentraxin, is known to play an important role in innate immunity to microbial infections; however, nothing is known about it during tuberculosis (TB). Mice intratracheally infected with Mycobacterium tuberculosis Erdman, showed peak SAP levels (442+/-58.2 microg/ml) on day 21, which declined to background levels by day 60. Their serum interleukin-6 levels paralleled SAP levels, whereas, their serum transforming growth factor-beta levels were paradoxical. During the acute phase of infection, the SAP levels positively correlated with the lung mycobacterial load. Purified mouse SAP (1-50 microg/ml) treatment of M. tuberculosis-infected alveolar macrophages (AMs), in vitro, inhibited their intracellular mycobacterial growth; maximum inhibition (1.1 log10 CFU reduction) occurred at 10 microg/ml, and a 4-day treatment appeared optimal. Treatment of AMs with both rabbit anti-mouse SAP polyclonal antibody and mannose-derived simple sugars, separately, blocked the SAP-induced inhibition of mycobacterial growth. The mycobacterial growth inhibition appeared to be nitric oxide (NO)-dependent as NO synthase inhibitors, both aminoguanidine and N(G)-monomethyl-L-arginine, annulled it. Further, SAP treatment of infected AMs induced significant (P<0.05) elaboration of nitrite (72.1+/-8.3 nM/ml), compared to the controls, and these AMs showed augmented expression of inducible NO synthase. This first study demonstrates that during murine TB the SAP levels were increased, and purified mouse SAP inhibited the intra-AM M. tuberculosis growth, in vitro, apparently via NO-dependent mechanism(s). SAP may thus contribute both to the pathogenesis and pulmonary innate immunity in TB.
Asunto(s)
Macrófagos Alveolares/microbiología , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/patogenicidad , Componente Amiloide P Sérico/metabolismo , Animales , Células Cultivadas , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/efectos de los fármacos , Óxido Nítrico/biosíntesis , Componente Amiloide P Sérico/aislamiento & purificación , Componente Amiloide P Sérico/farmacología , Tuberculosis Pulmonar/microbiología , Regulación hacia ArribaRESUMEN
In mammals the pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP) are important components of the immune response. Although pentraxins have been isolated from a number of fish species few studies detail their functional immunological role. In this paper we report the establishment of a flow cytometry based assay to measure the phagocytic activity of isolated snapper head kidney leukocytes (HKLs). This assay was then used to examine the ability of a pentraxin-like protein isolated from the serum of snapper (P. auratus) (Sn-PLP) to act as an opsonin. Incubation of snapper head kidney leukocytes (HKL) with FITC-labelled beads resulted in uptake of these particles by approximately 35% of HKLs. Incubation of beads with Sn-PLP enhanced phagocytosis by snapper HKLs in a dose-dependant manner. Enhanced phagocytosis could be inhibited by addition of a rabbit anti-Sn-PLP antibody suggesting that Sn-PLP may act as a ligand for a HKL cell surface receptor. This study provides further evidence toward a functional role for pentraxins in the host defence repertoire of fish.
Asunto(s)
Proteína C-Reactiva/inmunología , Proteínas de Peces/sangre , Proteínas de Peces/inmunología , Peces/sangre , Peces/inmunología , Proteínas Opsoninas/inmunología , Componente Amiloide P Sérico/inmunología , Animales , Azidas/farmacología , Proteína C-Reactiva/aislamiento & purificación , Proteína C-Reactiva/farmacología , Proteínas de Peces/aislamiento & purificación , Proteínas de Peces/farmacología , Citometría de Flujo , Fluoresceína-5-Isotiocianato/metabolismo , Cabeza , Riñón , Leucocitos/citología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Proteínas Opsoninas/farmacología , Fagocitosis/efectos de los fármacos , Componente Amiloide P Sérico/aislamiento & purificación , Componente Amiloide P Sérico/farmacologíaRESUMEN
Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.
Asunto(s)
Componente Amiloide P Sérico/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Datos de Secuencia Molecular , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/aislamiento & purificaciónRESUMEN
Amyloid deposits are proteinaceous extra-cellular aggregates associated with a diverse range of disease states. These deposits are composed predominantly of amyloid fibrils, the unbranched, beta-sheet rich structures that result from the misfolding and subsequent aggregation of many proteins. In addition, amyloid deposits contain a number of non-fibrillar components that interact with amyloid fibrils and are incorporated into the deposits in their native folded state. The influence of a number of the non-fibrillar components in amyloid-related diseases is well established; however, the mechanisms underlying these effects are poorly understood. Here we describe the effect of two of the most important non-fibrillar components, serum amyloid P component and apolipoprotein E, upon the solution behavior of amyloid fibrils in an in vitro model system. Using analytical ultracentrifugation, electron microscopy, and rheological measurements, we demonstrate that these non-fibrillar components cause soluble fibrils to condense into localized fibrillar aggregates with a greatly enhanced local density of fibril entanglements. These results suggest a possible mechanism for the observed role of non-fibrillar components as mediators of amyloid deposition and deposit stability.
Asunto(s)
Amiloide/fisiología , Apolipoproteínas C/química , Apolipoproteínas C/ultraestructura , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/ultraestructura , Amiloide/química , Apolipoproteína C-II , Apolipoproteínas C/aislamiento & purificación , Humanos , Cinética , Microscopía Electrónica , Ovillos Neurofibrilares/ultraestructura , Reología , Componente Amiloide P Sérico/aislamiento & purificación , Cloruro de Sodio/farmacología , ViscosidadRESUMEN
Wound healing and the dysregulated events leading to fibrosis both involve the proliferation and differentiation of fibroblasts and the deposition of extracellular matrix. Whether these fibroblasts are locally derived or from a circulating precursor population is unclear. Fibrocytes are a distinct population of fibroblast-like cells derived from peripheral blood monocytes that enter sites of tissue injury to promote angiogenesis and wound healing. We have found that CD14(+) peripheral blood monocytes cultured in the absence of serum or plasma differentiate into fibrocytes within 72 h. We purified the factor in serum and plasma that prevents the rapid appearance of fibrocytes, and identified it as serum amyloid P (SAP). Purified SAP inhibits fibrocyte differentiation at levels similar to those found in plasma, while depleting SAP reduces the ability of plasma to inhibit fibrocyte differentiation. Compared with sera from healthy individuals and patients with rheumatoid arthritis, sera from patients with scleroderma and mixed connective tissue disease, two systemic fibrotic diseases, were less able to inhibit fibrocyte differentiation in vitro and had correspondingly lower serum levels of SAP. These results suggest that low levels of SAP may thus augment pathological processes leading to fibrosis. These data also suggest mechanisms to inhibit fibrosis in chronic inflammatory conditions, or conversely to promote wound healing.
Asunto(s)
Fibroblastos/citología , Inhibidores de Crecimiento/fisiología , Componente Amiloide P Sérico/fisiología , Células Madre/citología , Adulto , Anciano , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Inhibidores de Crecimiento/sangre , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/aislamiento & purificación , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Monocitos/citología , Monocitos/fisiología , Plasma/metabolismo , Plasma/fisiología , Suero/fisiología , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/aislamiento & purificación , Componente Amiloide P Sérico/metabolismo , Células Madre/inmunología , Células Madre/metabolismo , Células Madre/fisiologíaRESUMEN
The serum amyloid P component (SAP)-like pentraxin Limulus polyphemus SAP is a recently discovered, distinct pentraxin species, of known structure, which does not bind phosphocholine and whose N-terminal sequence has been shown to differ markedly from the highly conserved N terminus of all other known horseshoe crab pentraxins. The complete cDNA sequence of Limulus SAP, and the derived amino acid sequence, the first invertebrate SAP-like pentraxin sequence, have been determined. Two sequences were identified that differed only in the length of the 3' untranslated region. Limulus SAP is synthesised as a precursor protein of 234 amino acid residues, the first 17 residues encoding a signal peptide that is absent from the mature protein. Phylogenetic analysis clusters Limulus SAP pentraxin with the horseshoe crab C-reactive proteins (CRPs) rather than the mammalian SAPs, which are clustered with mammalian CRPs. The deduced amino acid sequence shares 22% identity with both human SAP and CRP, which are 51% identical, and 31-35% with horseshoe crab CRPs. These analyses indicate that gene duplication of CRP (or SAP), followed by sequence divergence and the evolution of CRP and/or SAP function, occurred independently along the chordate and arthropod evolutionary lines rather than in a common ancestor. They further indicate that the CRP/SAP gene duplication event in Limulus occurred before both the emergence of the Limulus CRP variants and the mammalian CRP/SAP gene duplication. Limulus SAP, which does not exhibit the CRP characteristic of calcium-dependent binding to phosphocholine, is established as a pentraxin species distinct from all other known horseshoe crab pentraxins that exist in many variant forms sharing a high level of sequence homology.