RESUMO
N-Glycosylation plays a fundamental role in many biological processes. Human diamine oxidase (hDAO), required for histamine catabolism, has multiple N-glycosylation sites, but their roles, for example in DAO secretion, are unclear. We recently reported that the N-glycosylation sites Asn-168, Asn-538, and Asn-745 in recombinant hDAO (rhDAO) carry complex-type glycans, whereas Asn-110 carries only mammalian-atypical oligomannosidic glycans. Here, we show that Asn-110 in native hDAO from amniotic fluid and Caco-2 cells, DAO from porcine kidneys, and rhDAO produced in two different HEK293 cell lines is also consistently occupied by oligomannosidic glycans. Glycans at Asn-168 were predominantly sialylated with bi- to tetra-antennary branches, and Asn-538 and Asn-745 had similar complex-type glycans with some tissue- and cell line-specific variations. The related copper-containing amine oxidase human vascular adhesion protein-1 also exclusively displayed high-mannose glycosylation at Asn-137. X-ray structures revealed that the residues adjacent to Asn-110 and Asn-137 form a highly conserved hydrophobic cleft interacting with the core trisaccharide. Asn-110 replacement with Gln completely abrogated rhDAO secretion and caused retention in the endoplasmic reticulum. Mutations of Asn-168, Asn-538, and Asn-745 reduced rhDAO secretion by 13, 71, and 32%, respectively. Asn-538/745 double and Asn-168/538/745 triple substitutions reduced rhDAO secretion by 85 and 94%. Because of their locations in the DAO structure, Asn-538 and Asn-745 glycosylations might be important for efficient DAO dimer formation. These functional results are reflected in the high evolutionary conservation of all four glycosylation sites. Human DAO is abundant only in the gastrointestinal tract, kidney, and placenta, and glycosylation seems essential for reaching high enzyme expression levels in these tissues.
Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Células CACO-2 , Cristalografia por Raios X , Glicosilação , Células HEK293 , Humanos , Dobramento de ProteínaRESUMO
BACKGROUND: The yeast Pichia pastoris is a widely used host for the secretion of heterologous proteins. Despite being an efficient producer, we observed previously that certain recombinant proteins were mistargeted to the vacuole on their route to secretion. Simultaneous disruption of one vacuolar sorting pathway together with vacuolar proteases prevented this mis-sorting and resulted in higher levels of secreted heterologous protein. Inspired by the positive results, we now set out to investigate the influence of further parts of the vacuolar pathway, namely the Cvt-pathway and the homotypic fusion and protein sorting (HOPS) complex. RESULTS: Strains impaired in the Cvt pathway (∆atg11, ∆atg8) had no effect on secretion of the model protein carboxylesterase (CES), but resulted in lower secretion levels of the antibody fragment HyHEL-Fab. Disruption of genes involved in the HOPS complex led to vacuole-like compartments of the B category of vps mutants, which are characteristic for the deleted genes YPT7, VPS41 and VAM6. In particular ∆ypt7 and ∆vam6 strains showed an improvement in secreting the model proteins HyHEL-Fab and CES. Additional disruption of the vacuolar protease Pep4 and the potential protease Vps70 led to even further enhanced secretion in ∆ypt7 and ∆vam6 strains. Nevertheless, intracellular product accumulation was still observed. Therefore, the secretory route was strengthened by overexpression of early or late secretory genes in the vacuolar sorting mutants. Thereby, overexpression of Sbh1, a subunit of the ER translocation pore, significantly increased HyHEL-Fab secretion, leading up to fourfold higher extracellular Fab levels in the ∆ypt7 strain. The beneficial impact on protein secretion and the suitability of these strains for industrial applicability was confirmed in fed-batch cultivations. CONCLUSIONS: Disruption of genes involved in the HOPS complex, especially YPT7, has a great influence on the secretion of the two different model proteins HyHEL-Fab and CES. Therefore, disruption of HOPS genes shows a high potential to increase secretion of other recombinant proteins as well. Secretion of HyHEL-Fab was further enhanced when overexpressing secretion enhancing factors. As the positive effect was also present in fed-batch cultivations, these modifications likely have promising industrial relevance.
Assuntos
Carboxilesterase/biossíntese , Fragmentos Fab das Imunoglobulinas/biossíntese , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Adaptadoras de Transporte Vesicular/genética , Deleção de Genes , Genes Fúngicos , Pichia/genética , Transporte Proteico , Vacúolos/enzimologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Mass spectrometry-based metabolomic profiling is a powerful strategy to quantify the concentrations of numerous primary metabolites in parallel. To avoid distortion of metabolite concentrations, quenching is applied to stop the cellular metabolism instantly. For yeasts, cold methanol quenching is accepted to be the most suitable method to stop metabolism, while keeping the cells intact for separation from the supernatant. During this treatment, metabolite loss may occur while the cells are suspended in the quenching solution. An experiment for measuring the time-dependent loss of selected primary metabolites in differently buffered quenching solutions was conducted to study pH and salt concentration-dependent effects. Molecular properties of the observed metabolites were correlated with the kinetics of loss to gain insight into the mechanisms of metabolite leakage. Size and charge-related properties play a major role in controlling metabolite loss. We found evidence that interaction with the cell wall is the main determinant to retain a molecule inside the cell. Besides suggesting an improved quenching protocol to keep loss at a minimum, we could establish a more general understanding of the process of metabolite loss during quenching, which will allow to predict optimal conditions for hitherto not analysed metabolites.
Assuntos
Parede Celular/efeitos dos fármacos , Metaboloma , Metabolômica/métodos , Metanol/farmacologia , Pichia/efeitos dos fármacos , Reatores Biológicos , Soluções Tampão , Parede Celular/química , Parede Celular/metabolismo , Cromatografia Líquida , Meios de Cultura/química , Fermentação , Concentração de Íons de Hidrogênio , Cinética , Pichia/química , Pichia/metabolismo , Cloreto de Sódio/farmacologia , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Bacterial inclusion bodies (IBs) are non-toxic protein aggregates commonly produced in recombinant bacteria. They are formed by a mixture of highly stable amyloid-like fibrils and releasable protein species with a significant extent of secondary structure, and are often functional. As nano structured materials, they are gaining biomedical interest because of the combination of submicron size, mechanical stability and biological activity, together with their ability to interact with mammalian cell membranes for subsequent cell penetration in absence of toxicity. Since essentially any protein species can be obtained as IBs, these entities, as well as related protein clusters (e.g., aggresomes), are being explored in biocatalysis and in biomedicine as mechanically stable sources of functional protein. One of the major bottlenecks for uses of IBs in biological interfaces is their potential contamination with endotoxins from producing bacteria. RESULTS: To overcome this hurdle, we have explored here the controlled production of functional IBs in the yeast Pichia pastoris (Komagataella spp.), an endotoxin-free host system for recombinant protein production, and determined the main physicochemical and biological traits of these materials. Quantitative and qualitative approaches clearly indicate the formation of IBs inside yeast, similar in morphology, size and biological activity to those produced in E. coli, that once purified, interact with mammalian cell membranes and penetrate cultured mammalian cells in absence of toxicity. CONCLUSIONS: Structurally and functionally similar from those produced in E. coli, the controlled production of IBs in P. pastoris demonstrates that yeasts can be used as convenient platforms for the biological fabrication of self-organizing protein materials in absence of potential endotoxin contamination and with additional advantages regarding, among others, post-translational modifications often required for protein functionality.
Assuntos
Corpos de Inclusão/fisiologia , Pichia/genética , Pichia/metabolismo , Biocatálise , Endotoxinas/análise , Escherichia coli/genética , Escherichia coli/metabolismo , Corpos de Inclusão/química , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismoRESUMO
Yeasts are valuable hosts for recombinant protein production. Among them, Pichia pastoris is frequently used for production of secreted proteins, and much effort was made to improve the secretion efficiency of this expression platform. However, the knowledge on the secretion machinery is mainly based on studies in Saccharomyces cerevisiae. Therefore, it is of great interest for targeted improvement of the system to learn more about the secretion process in P. pastoris. Using human serum albumin, a protein which is produced in high quantities in P. pastoris, we show here the secretion pathway of this protein. During passage of the secretory route, the recombinant protein is mainly localized in the endoplasmic reticulum (ER) and in COPII vesicles, and is inherited to the daughter cell via the perinuclear ER. The final release to the cell exterior occurs at the bud, initiating at the bud tip and later spreading over the entire bud surface. The same polarized secretion pattern was observed for a recombinant antibody light chain and the native secretory protein Epx1 of P. pastoris. Clarifying the point of release of secretory proteins will have major impact on engineering the secretory pathway of P. pastoris and other budding yeasts.
Assuntos
Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo , Albuminas/metabolismo , Proteínas Fúngicas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismoRESUMO
Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.
Assuntos
Proteínas Fúngicas/metabolismo , Pichia/metabolismo , Sinais Direcionadores de Proteínas , Fusão Gênica Artificial , Análise Mutacional de DNA , Proteínas Fúngicas/genética , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Pichia/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genéticaRESUMO
The development of Pichia pastoris as a production platform for recombinant proteins has been a remarkable success story over the last three decades. Stable cheap production processes and the good protein secretion abilities were pacemakers of this development. However, limitations of protein folding, glycosylation or secretion have been identified quite early on. With the availability of genome sequences and the development of systems biology characterization in the last 5 years, remarkable success in strain improvement was achieved. Here, we focus on recent developments of characterization and improvement of P. pastoris production strains regarding protein folding, intracellular trafficking, glycosylation and proteolytic degradation.
Assuntos
Pichia/metabolismo , Engenharia de Proteínas/métodos , Dobramento de Proteína , Proteínas Recombinantes/metabolismo , Degradação Associada com o Retículo Endoplasmático , Exocitose , Glicosilação , Complexo de Golgi/metabolismo , Pichia/genética , Polissacarídeos/química , Polissacarídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Resposta a Proteínas não Dobradas/fisiologia , Vacúolos/metabolismoRESUMO
The M6P (mannose 6-phosphate)/IGF2R (insulin-like growth factor II receptor) interacts with a variety of factors that impinge on tumour invasion and metastasis. It has been shown that expression of wild-type M6P/IGF2R reduces the tumorigenic and invasive properties of receptor-deficient SCC-VII squamous cell carcinoma cells. We have now used mutant forms of M6P/IGF2R to assess the relevance of the different ligand-binding sites of the receptor for its biological activities in this cellular system. The results of the present study demonstrate that M6P/IGF2R does not require a functional binding site for insulin-like growth factor II for inhibition of anchorage-independent growth and matrix invasion by SCC-VII cells. In contrast, the simultaneous mutation of both M6P-binding sites is sufficient to impair all cellular functions of the receptor tested. These findings highlight that the interaction between M6P/IGF2R and M6P-modified ligands is not only important for intracellular accumulation of lysosomal enzymes and formation of dense lysosomes, but is also crucial for the ability of the receptor to suppress SCC-VII growth and invasion. The present study also shows that some of the biological activities of M6P/IGF2R in SCC-VII cells strongly depend on a functional M6P-binding site within domain 3, thus providing further evidence for the non-redundant cellular functions of the individual carbohydrate-binding domains of the receptor.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor IGF Tipo 2/metabolismo , Sítios de Ligação , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Matriz Extracelular/genética , Matriz Extracelular/patologia , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Mutação , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Estrutura Terciária de Proteína , Receptor IGF Tipo 2/genéticaRESUMO
BACKGROUND & AIMS: The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), a multifunctional protein, plays a central role in intracellular targeting of lysosomal enzymes and control of insulin-like growth factor II (IGF-II) bioactivity. Importantly, the gene encoding this receptor is frequently inactivated in a wide range of malignant tumors including hepatocellular carcinomas. Thus, M6P/IGF2R is considered a putative liver tumor suppressor. The aim of this study was to establish the impact of the receptor on the invasive properties of liver cells. METHODS: Reconstitution experiments were performed by expression of wild type and mutant M6P/IGF2R in receptor-deficient FRL14 fetal rat liver cells. RNA interference was used to induce M6P/IGF2R downregulation in receptor-positive MIM-1-4 mouse hepatocytes. RESULTS: We show that the M6P/IGF2R status exerts a strong impact on the invasiveness of tumorigenic rodent liver cells. M6P/IGF2R-deficient fetal rat liver cells hypersecrete lysosomal cathepsins and penetrate extracellular matrix barriers in a cathepsin-dependent manner. Forced expression of M6P/IGF2R restores intracellular transport of cathepsins to lysosomes and concomitantly reduces the tumorigenicity and invasive potential of these cells. Conversely, M6P/IGF2R knock-down in receptor-positive mouse hepatocytes causes increased cathepsin secretion as well as enhanced cell motility and invasiveness. We also demonstrate that functional M6P-binding sites are important for the anti-invasive properties of M6P/IGF2R, whereas the capacity to bind IGF-II is dispensable for the anti-invasive activity of the receptor in liver cells. CONCLUSIONS: M6P/IGF2R restricts liver cell invasion by preventing the pericellular action of M6P-modified proteins.
Assuntos
Hepatócitos/patologia , Neoplasias Hepáticas/patologia , Manosefosfatos/metabolismo , Receptor IGF Tipo 2/fisiologia , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Lisossomos/enzimologia , Camundongos , Invasividade Neoplásica , Ligação Proteica , RatosRESUMO
The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) mediates biosynthetic sorting and endocytosis of various factors that impinge on the proliferation, migration and invasiveness of tumour cells. The gene encoding M6P/IGF2R is frequently lost or mutated in a wide range of malignant tumours including squamous cell carcinomas. We have previously shown that M6P/IGF2R-deficient SCC-VII murine squamous cell carcinoma cells secrete large amounts of pro-invasive lysosomal proteinases. Furthermore, the formation of mature lysosomes is impaired in SCC-VII cells. To assess the link between M6P/IGF2R status and tumour invasion, we have now generated SCC-VII lines stably transfected with human M6P/IGF2R cDNA. Reconstitution of functional M6P/IGF2R expression in SCC-VII cells strongly improves the intracellular retention of lysosomal proteinases and restores the formation of mature lysosomes. In addition, the presence of heterologous M6P/IGF2R compromises the growth of SCC-VII cells both in vitro and in vivo. Remarkably, M6P/IGF2R expression also reduces the invasive capacity of SCC-VII cells in response to various chemoattractants. These results indicate that the M6P/IGF2R status influences the metastatic propensity of squamous cell carcinomas.
Assuntos
Carcinoma de Células Escamosas/patologia , Receptor IGF Tipo 2/fisiologia , Animais , Proliferação de Células , Matriz Extracelular/fisiologia , Feminino , Humanos , Lisossomos/enzimologia , Camundongos , Camundongos SCID , Invasividade Neoplásica , Receptor IGF Tipo 2/análise , Proteínas Supressoras de Tumor/fisiologia , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
As a possible viable and non-invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non-invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones.
Assuntos
Células CHO/citologia , Células CHO/ultraestrutura , Microscopia Confocal/métodos , Engenharia de Proteínas/métodos , Adalimumab/genética , Adalimumab/metabolismo , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Análise por Conglomerados , Cricetulus , Retículo Endoplasmático/ultraestrutura , Humanos , Lipídeos/química , Metais/química , Imagem Molecular/métodos , Análise de Componente Principal , Proteínas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise Espectral Raman/métodosRESUMO
The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography-mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.
Assuntos
Carboxilesterase/genética , Pichia/genética , Proteínas Recombinantes/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Biotecnologia/métodos , Carboxilesterase/metabolismo , Vesículas Citoplasmáticas/genética , Vesículas Citoplasmáticas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Pichia pastoris is the most frequently used yeast system for heterologous protein production today. The last few years have seen several products based on this platform reach approval as biopharmaceutical drugs. Successful glycoengineering to humanize N-glycans is further fuelling this development. However, detailed understanding of the yeast's physiology, genetics and regulation has only developed rapidly in the last few years since published genome sequences have become available. An expanding toolbox of genetic elements and strains for the improvement of protein production is being generated, including promoters, gene copy-number enhancement, gene knockout and high-throughput methods. Protein folding and secretion have been identified as significant bottlenecks in yeast expression systems, pinpointing a major target for strain optimization. At the same time, it has become obvious that P. pastoris, as an evolutionarily more 'ancient' yeast, may in some cases be a better model for human cell biology and disease than Saccharomyces cerevisiae.
Assuntos
Pesquisa Biomédica/métodos , Biotecnologia/métodos , Pichia/metabolismo , Tecnologia Farmacêutica/métodos , Engenharia Genética/métodos , Genética Microbiana/métodos , Humanos , Pichia/genética , Pichia/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Oxidative protein folding can exceed the cellular secretion machinery, inducing the unfolded protein response (UPR). Sustained endoplasmic reticulum (ER) stress leads to cell stress and disease, as described for Alzheimer, Parkinson, and diabetes mellitus, among others. It is currently assumed that the redox state of the ER is optimally balanced for formation of disulfide bonds using glutathione as the main redox buffer and that UPR causes a reduction of this organelle. The direct effect of oxidative protein folding in the ER, however, has not yet been dissected from UPR regulation. To measure in vivo redox conditions in the ER and cytosol of the yeast model organism Pichia pastoris we targeted redox-sensitive roGFP variants to the respective organelles. Thereby, we clearly demonstrate that induction of the UPR causes reduction of the cytosol in addition to ER reduction. Similarly, a more reduced redox state of the cytosol, but not of the ER, is observed during oxidative protein folding in the ER without UPR induction, as demonstrated by overexpressing genes of disulfide bond-rich secretory proteins such as porcine trypsinogen or protein disulfide isomerase (PDI1) and ER oxidase (ERO1). Cytosolic reduction seems not to be caused by the action of glutathione reductase (GLR1) and could not be compensated for by overexpression of cytosolic glutathione peroxidase (GPX1). Overexpression of GPX1 and PDI1 oxidizes the ER and increases the secretion of correctly folded proteins, demonstrating that oxidative protein folding per se is enhanced by a more oxidized ER and is counterbalanced by a more reduced cytosol. As the total glutathione concentration of these strains does not change significantly, but the ratio of GSH to GSSG is altered, either transport or redox signaling between the glutathione pools of ER and cytosol is assumed. These data clearly demonstrate that protein folding and ER stress have a severe impact on the cytosolic redox balance, which may be a major factor during development of folding-related diseases.
Assuntos
Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Pichia/metabolismo , Dobramento de Proteína , Saccharomyces cerevisiae/metabolismo , Resposta a Proteínas não Dobradas , Western Blotting , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Glutationa/metabolismo , Microscopia de Fluorescência , Oxirredução , Espectrometria de Massas em TandemRESUMO
Proteinases are known to be involved in many cancer-related processes, particularly in the breakdown of extracellular matrix barriers in the course of tumor invasion and metastasis. In this review we summarize the current knowledge about the role of the most important matrix-degrading proteinases (cathepsins, matrix metalloproteinases, plasmin/plasminogen activators) and their respective inhibitors in liver cancer progression and metastasis.