RESUMEN
Host cell proteins are inevitable contaminants of biopharmaceuticals. Here, we performed detailed analyses of the host cell proteome of moss ( Physcomitrella patens) bioreactor supernatants using mass spectrometry and subsequent bioinformatics analysis. Distinguishing between the apparent secretome and intracellular contaminants, a complex extracellular proteolytic network including subtilisin-like proteases, metallo-proteases, and aspartic proteases was identified. Knockout of a subtilisin-like protease affected the overall extracellular proteolytic activity. Besides proteases, also secreted protease-inhibiting proteins such as serpins were identified. Further, we confirmed predicted cleavage sites of 40 endogenous signal peptides employing an N-terminomics approach. The present data provide novel aspects to optimize both product stability of recombinant biopharmaceuticals as well as their maturation along the secretory pathway. Data are available via ProteomeXchange with identifier PXD009517.
Asunto(s)
Proteasas de Ácido Aspártico/aislamiento & purificación , Bryopsida/enzimología , Metaloproteasas/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Inhibidores de Proteasas/aislamiento & purificación , Serpinas/aislamiento & purificación , Subtilisinas/aislamiento & purificación , Proteasas de Ácido Aspártico/clasificación , Proteasas de Ácido Aspártico/genética , Proteasas de Ácido Aspártico/metabolismo , Reactores Biológicos , Bryopsida/química , Bryopsida/genética , Biología Computacional , Técnicas de Inactivación de Genes , Espectrometría de Masas/métodos , Metaloproteasas/clasificación , Metaloproteasas/genética , Metaloproteasas/metabolismo , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inhibidores de Proteasas/clasificación , Inhibidores de Proteasas/metabolismo , Análisis por Matrices de Proteínas , Proteolisis , Serpinas/clasificación , Serpinas/genética , Serpinas/metabolismo , Subtilisinas/clasificación , Subtilisinas/genética , Subtilisinas/metabolismoRESUMEN
Genetic defects in complement regulatory proteins can lead to severe renal diseases, including atypical hemolytic uremic syndrome and C3 glomerulopathies, and age-related macular degeneration. The majority of the mutations found in patients with these diseases affect the glycoprotein complement factor H, the main regulator of the alternative pathway of complement activation. Therapeutic options are limited, and novel treatments, specifically those targeting alternative pathway activation, are highly desirable. Substitution with biologically active factor H could potentially treat a variety of diseases that involve increased alternative pathway activation, but no therapeutic factor H is commercially available. We recently reported the expression of full-length recombinant factor H in moss (Physcomitrella patens). Here, we present the production of an improved moss-derived recombinant human factor H devoid of potentially immunogenic plant-specific sugar residues on protein N-glycans, yielding approximately 1 mg purified moss-derived human factor H per liter of initial P. patens culture after a multistep purification process. This glycosylation-optimized factor H showed full in vitro complement regulatory activity similar to that of plasma-derived factor H and efficiently blocked LPS-induced alternative pathway activation and hemolysis induced by sera from patients with atypical hemolytic uremic syndrome. Furthermore, injection of moss-derived factor H reduced C3 deposition and increased serum C3 levels in a murine model of C3 glomerulopathy. Thus, we consider moss-produced recombinant human factor H a promising pharmaceutical product for therapeutic intervention in patients suffering from complement dysregulation.
Asunto(s)
Síndrome Hemolítico Urémico Atípico/tratamiento farmacológico , Bryopsida , Proteínas del Sistema Complemento , Enfermedades del Sistema Inmune/tratamiento farmacológico , Animales , Bryopsida/metabolismo , Factor H de Complemento/biosíntesis , Factor H de Complemento/metabolismo , Factor H de Complemento/uso terapéutico , Glicosilación , Humanos , RatonesRESUMEN
Enzyme replacement therapy (ERT) is an effective treatment for several lysosomal storage disorders (LSDs). Intravenously infused enzymes are taken up by tissues through either the mannose 6-phosphate receptor (M6PR) or the mannose receptor (MR). It is generally believed that M6PR-mediated endocytosis is a key mechanism for ERT in treating LSDs that affect the non-macrophage cells of visceral organs. However, the therapeutic efficacy of MR-mediated delivery of mannose-terminated enzymes in these diseases has not been fully evaluated. We tested the effectiveness of a non-phosphorylated α-galactosidase A produced from moss (referred to as moss-aGal) in vitro and in a mouse model of Fabry disease. Endocytosis of moss-aGal was MR-dependent. Compared to agalsidase alfa, a phosphorylated form of α-galactosidase A, moss-aGal was more preferentially targeted to the kidney. Cellular localization of moss-aGal and agalsidase alfa in the heart and kidney was essentially identical. A single injection of moss-aGal led to clearance of accumulated substrate in the heart and kidney to an extent comparable to that achieved by agalsidase alfa. This study suggested that mannose-terminated enzymes may be sufficiently effective for some LSDs in which non-macrophage cells are affected, and that M6P residues may not always be a prerequisite for ERT as previously considered.
Asunto(s)
Enfermedad de Fabry/enzimología , Enfermedad de Fabry/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Manosafosfatos/metabolismo , Receptores de Superficie Celular/metabolismo , alfa-Galactosidasa/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Terapia de Reemplazo Enzimático/métodos , Femenino , Humanos , Isoenzimas/metabolismo , Riñón/metabolismo , Enfermedades por Almacenamiento Lisosomal/enzimología , Enfermedades por Almacenamiento Lisosomal/metabolismo , Masculino , Receptor de Manosa , Ratones , Ratones Endogámicos C57BL , Receptor IGF Tipo 2/metabolismo , Proteínas RecombinantesRESUMEN
Trans-acyltransferase polyketide synthases (trans-AT PKSs) are an important group of bacterial enzymes producing bioactive polyketides. One difference from textbook PKSs is the presence of one or more free-standing AT-like enzymes. While one homolog loads the PKS with malonyl units, the function of the second copy (AT2) was unknown. We studied the two ATs PedC and PedD involved in pederin biosynthesis in an uncultivated symbiont. PedD displayed malonyl- but not acetyltransferase activity toward various acyl carrier proteins (ACPs). In contrast, the AT2 PedC efficiently hydrolyzed acyl units bound to N-acetylcysteamine or ACP. It accepted substrates with various chain lengths and functionalizations but did not cleave malonyl-ACP. These data are consistent with the role of PedC in PKS proofreading, suggesting a similar function for other AT2 homologs and providing strategies for polyketide titer improvement and biosynthetic investigations.
Asunto(s)
Aciltransferasas/metabolismo , Policétidos/metabolismo , Acetilcoenzima A/metabolismo , Bacillus/enzimología , Malonil Coenzima A/metabolismo , Filogenia , Polienos/metabolismo , Piranos/metabolismoRESUMEN
Many marine sponges contain massive numbers of largely uncultivated, phylogenetically diverse bacteria that seem to be important contributors to the chemistry of these animals. Insights into the diversity, origin, distribution, and function of their metabolic gene communities are crucial to dissect the chemical ecology and biotechnological potential of sponge symbionts. This study reveals a sharp dichotomy between high and low microbial abundance sponges with respect to polyketide synthase (PKS) gene content, the presence of methyl-branched fatty acids, and the presence of members of the symbiotic candidate phylum "Poribacteria". For the symbiont-rich sponge Cacospongia mycofijiensis, a source of the tubulin-inhibiting fijianolides (=laulimalides), near-exhaustive large-scale sequencing of PKS gene-derived PCR amplicons was conducted. Although these amplicons exhibit high diversity at the sequence level, almost all of them belong to a single, architecturally unique group of PKSs present in "Poribacteria" and are proposed to synthesize methyl-branched fatty acids. Three components of this PKS were studied in vitro, providing initial insight into its biochemistry.