RESUMEN
We previously developed a unique recombinant protein vaccine against plague composed of a fusion between the Fraction 1 capsular antigen (F1) and the V antigen. To determine if overall expression, solubility, and recovery of the F1-V fusion protein could be enhanced, we modified the original fusion. Standard recombinant DNA techniques were used to reverse the gene order such that the V antigen coding sequence was fused at its C-terminus to the N-terminus of F1. The F1 secretion signal sequence (F1S) was subsequently fused to the N-terminus of V. This new fusion protein, designated F1S-V-F1, was then co-expressed with the Y. pestis Caf1M periplasmic chaperone protein in BL21-Star Escherichia coli. Recombinant strains expressing F1-V, F1S-F1-V, or F1S-V-F1 were compared by cell fractionation, SDS-PAGE, Western blotting, and suspension immunolabelling. F1S-V-F1 exhibited enhanced solubility and secretion when co-expressed with Caf1M resulting in a recombinant protein that is processed in a similar manner to the native F1 protein. Purification of F1S-V-F1 was accomplished by anion-exchange and hydrophobic interaction chromatography. The purification method produced greater than 1mg of purified soluble protein per liter of induced culture. F1S-V-F1 polymerization characteristics were comparable to the native F1. The purified F1S-V-F1 protein appeared equivalent to F1-V in its ability to be recognized by neutralizing antibodies.
Asunto(s)
Antígenos Bacterianos/genética , Vacuna contra la Peste/biosíntesis , Peste/prevención & control , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Recombinantes de Fusión/biosíntesis , Yersinia pestis/inmunología , Anticuerpos Neutralizantes/inmunología , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Vacuna contra la Peste/genética , Vacuna contra la Peste/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas de Subunidad/biosíntesis , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunologíaRESUMEN
Cell surface heparan sulfate (HS) proteoglycans are carbohydrate-rich regulators of cell migratory, mitogenic, secretory, and inflammatory activity that bind and present soluble heparin-binding growth factors (e.g., fibroblast growth factor, Wnt, Hh, transforming growth factor beta, amphiregulin, and hepatocyte growth factor) to their respective signaling receptors. We demonstrate that the deglycanated core protein of syndecan-1 (SDC1) and not HS chains nor SDC2 or -4, appears to target the epithelial selective prosecretory mitogen lacritin. An important and novel step in this mechanism is that binding necessitates prior partial or complete removal of HS chains by endogenous heparanase. This limits lacritin activity to sites where heparanase appears to predominate, such as sites of exocrine cell migration, secretion, renewal, and inflammation. Binding is mutually specified by lacritin's C-terminal mitogenic domain and SDC1's N terminus. Heparanase modification of the latter transforms a widely expressed HS proteoglycan into a highly selective surface-binding protein. This novel example of cell specification through extracellular modification of an HS proteoglycan has broad implications in development, homeostasis, and disease.
Asunto(s)
Células Epiteliales/metabolismo , Glucuronidasa/farmacología , Glicoproteínas/metabolismo , Sustancias de Crecimiento/metabolismo , Glicoproteínas de Membrana/metabolismo , Polisacáridos/metabolismo , Proteoglicanos/metabolismo , Línea Celular , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Glicoproteínas de Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Unión Proteica , Estructura Terciaria de Proteína , Proteoglicanos/efectos de los fármacos , Sindecano-1 , SindecanosRESUMEN
Renewal of nongermative epithelia is poorly understood. The novel mitogen "lacritin" is apically secreted by several nongermative epithelia. We tested 17 different cell types and discovered that lacritin is preferentially mitogenic or prosecretory for those types that normally contact lacritin during its glandular outward flow. Mitogenesis is dependent on lacritin's C-terminal domain, which can form an alpha-helix with a hydrophobic face, as per VEGF's and PTHLP's respective dimerization or receptor-binding domain. Lacritin targets downstream NFATC1 and mTOR. The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Galpha(i) or Galpha(o)-PKCalpha-PLC-Ca2+-calcineurin-NFATC1 and Galpha(i) or Galpha(o)-PKCalpha-PLC-phospholipase D (PLD)-mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1. This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi-translocated PKCalpha upstream of both Ca2+ mobilization and PLD activation in a complex with PLCgamma2. Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.
Asunto(s)
Proliferación Celular , Células Epiteliales/efectos de los fármacos , Glicoproteínas/farmacología , Sustancias de Crecimiento/farmacología , Factores de Transcripción NFATC/metabolismo , Proteína Quinasa C-alfa/metabolismo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Aparato de Golgi/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Fosfolipasa D/metabolismo , Conformación Proteica , Transporte de Proteínas , Glándulas Salivales/efectos de los fármacos , Glándulas Salivales/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TORRESUMEN
The type III secretion system (YscC) protein of Yersinia pestis plays an essential role in the translocation of Yersinia outer proteins (Yops) into eukaryotic target cells through a type III secretion mechanism. To assess the immunogenicity and potential protective efficacy of YscC against lethal plague challenge, we cloned, overexpressed, and purified YscC using two different bacterial expression and purification systems. The resulting expression plasmids for YscC, pETBlue-2-YscC and pTYB11-YscC, were regulated by robust T7 promoters that were induced with isopropyl-beta-D-thiogalactopyranoside. The intein-fusion pTYB11-YscC system and the six-histidine-tagging pETBlue-2-YscC system were both successful for producing and purifying YscC. The intein-mediated purification system produced about 1mg of soluble YscC per liter of bacterial culture while the YscC-His(6)-tag method resulted in 16mg of insoluble YscC per liter of bacterial culture. Protein identity for purified YscC-His(6) was confirmed by ion trap mass spectrometry. Antisera were produced against both YscC and YscC-His(6). The specific immune response generated in YscC-vaccinated mice was relative to the particular purified protein, YscC or YscC-His(6), which was used for vaccination as determined by Western blot analysis and ELISA. Regardless of the purification method, either form of the YscC protein failed to elicit a protective immune response against lethal plague challenge with either F1 capsule forming Y. pestis CO92 or the isogenic F1(-)Y. pestis C12.