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1.
Science ; 369(6506): 942-949, 2020 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-32820120

RESUMEN

Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αß and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αß T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αß T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αß and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.


Asunto(s)
Antígenos CD/inmunología , Butirofilinas/antagonistas & inhibidores , Butirofilinas/inmunología , Linfocitos Intraepiteliales/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/genética , Butirofilinas/genética , Femenino , Humanos , Inmunoterapia/métodos , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
2.
JCI Insight ; 5(5)2020 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-32161196

RESUMEN

CD137 (4-1BB) is a member of the TNFR superfamily that represents a promising target for cancer immunotherapy. Recent insights into the function of TNFR agonist antibodies implicate epitope, affinity, and IgG subclass as critical features, and these observations help explain the limited activity and toxicity seen with clinically tested CD137 agonists. Here, we describe the preclinical characterization of CTX-471, a fully human IgG4 agonist of CD137 that engages a unique epitope that is shared by human, cynomolgus monkey, and mouse and is associated with a differentiated pharmacology and toxicology profile. In vitro, CTX-471 increased IFN-γ production by human T cells in an Fcγ receptor-dependent (FcγR-dependent) manner, displaying an intermediate level of activity between 2 clinical-stage anti-CD137 antibodies. In mice, CTX-471 exhibited curative monotherapy activity in various syngeneic tumor models and showed a unique ability to cure mice of very large (~500 mm3) tumors compared with validated antibodies against checkpoints and TNFR superfamily members. Extremely high doses of CTX-471 were well tolerated, with no signs of hepatic toxicity. Collectively, these data demonstrate that CTX-471 is a unique CD137 agonist that displays an excellent safety profile and an unprecedented level of monotherapy efficacy against very large tumors.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Inmunoterapia/métodos , Neoplasias/terapia , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Mapeo Epitopo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Inmunoterapia/efectos adversos , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/metabolismo , Macaca fascicularis , Ratones , Ratones Desnudos , Neoplasias/inmunología , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/química , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Protein Eng Des Sel ; 26(10): 663-70, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24046438

RESUMEN

Low expression, poor solubility, and polyspecificity are significant obstacles that have impeded the development of antibodies discovered from in vitro display libraries. Current biophysical characterization tools that identify these 'developability' problems are typically only applied after the discovery process, and thus limited to perhaps a few hundred candidates. We report a flow cytometric assay using a polyspecificity reagent (PSR) that allows for the identification and counter selection of polyspecific antibodies both during and after the selection process. The reported assay correlates well with cross-interaction chromatography, a surrogate for antibody solubility, as well as a baculovirus particle enzyme-linked immunosorbent assay, a surrogate for in vivo clearance. However, unlike these assays, PSR labeling is compatible both with screening of individual antibodies as well as selections of large antibody libraries. To this end, we demonstrate the ability to counter-select against polyspecificity while enriching for antigen affinity from a diverse antibody library, which enables simultaneous evolution of both antigen binding and superior non-target-related properties during the discovery process.


Asunto(s)
Anticuerpos/genética , Técnicas de Visualización de Superficie Celular/métodos , Citometría de Flujo , Levaduras/citología , Anticuerpos/química , Anticuerpos/inmunología , Línea Celular , Membrana Celular/metabolismo , Reacciones Cruzadas , Evolución Molecular Dirigida , Humanos , Estabilidad Proteica , Solubilidad , Temperatura
4.
Glycobiology ; 23(10): 1192-203, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23893788

RESUMEN

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with ß- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose ß-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.


Asunto(s)
Manosa/biosíntesis , Ingeniería Metabólica/métodos , Pichia/metabolismo , alfa-Manosidasa/metabolismo , Pichia/crecimiento & desarrollo , Ingeniería de Proteínas , alfa-Manosidasa/genética
5.
PLoS One ; 8(7): e68325, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23840891

RESUMEN

The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of ß-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins.


Asunto(s)
Manosiltransferasas/genética , Pichia/enzimología , Pichia/genética , Candida albicans/enzimología , Candida albicans/genética , Técnicas de Inactivación de Genes , Genes Fúngicos , Glicosilación , Manosiltransferasas/metabolismo , Filogenia , Pichia/metabolismo , Polisacáridos/metabolismo
6.
Glycobiology ; 22(10): 1332-42, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22745283

RESUMEN

A family of nine genes encoding proteins involved in the synthesis of ß-1,2 mannose adhesins of Candida albicans has been identified. Four of these genes, BMT1-4, encode enzymes acting stepwise to add ß-mannoses on to cell-wall phosphopeptidomannan (PPM). None of these acts on phospholipomannan (PLM), a glycosphingolipid member of the mannose-inositol-phosphoceramide family, which contributes with PPM to ß-mannose surface expression. We show that deletion of BMT5 and BMT6 led to a dramatic reduction of PLM glycosylation and accumulation of PLM with a truncated ß-oligomannoside chain, respectively. Disruptions had no effect on sphingolipid biosynthesis and on PPM ß-mannosylation. ß-Mannose surface expression was not affected, confirming that ß-mannosylation is a process based on specificity of acceptor molecules, but liable to global regulation.


Asunto(s)
Candida albicans/enzimología , Pared Celular/química , Glucolípidos/metabolismo , Mananos/metabolismo , Fosfopéptidos/metabolismo , Acetiltransferasas , Proteínas Bacterianas , Activación Enzimática , Glicosilación , Especificidad de la Especie
7.
Yeast ; 28(3): 237-52, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21360735

RESUMEN

To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N-terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N-glycosylation.


Asunto(s)
Retículo Endoplásmico/enzimología , Glucosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Manosidasas/metabolismo , Pichia/enzimología , Ingeniería de Proteínas , Glucosiltransferasas/genética , Manosidasas/genética , Pichia/genética , Señales de Clasificación de Proteína/genética , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
8.
J Ind Microbiol Biotechnol ; 37(9): 961-71, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20711797

RESUMEN

The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Ingeniería Genética , Glicoproteínas/biosíntesis , Pichia/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Anticuerpos Monoclonales/genética , Reactores Biológicos , Técnicas de Cultivo de Célula , Línea Celular , ADN de Hongos/genética , Fermentación , Expresión Génica , Glicoproteínas/genética , Glicosilación , Humanos , Técnicas Microbiológicas , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Selección Genética , Transformación Genética
9.
J Immunol Methods ; 358(1-2): 66-74, 2010 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-20338179

RESUMEN

A simple cell labeling method for sorting yeast Pichia pastoris antibody expressing strains is described. A small portion of secreted recombinant antibody retained on the cell surface was labeled with fluorescence detection antibody. The signal intensity of the labeled cell was correlated with the cell's antibody productivity. Using this labeling technique to sort a mixture model induced in the same fermenter where the cells of high producing strain were spiked into a population of a low producing strain at the frequency of 1:100,000, one round of sorting achieved a approximately 5000-fold enrichment of the high producing strain. A variety of P.pastoris strains expressing antibody sorted based on the signal intensity on the cell surface yielded titer improvements by 30% to 300%. Our data demonstrate that Pichia cell surface labeling is a simple, effective and reliable method for sorting Pichia antibody expressing strains for productivity improvement.


Asunto(s)
Inmunoglobulina G/biosíntesis , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Pichia/aislamiento & purificación , Pichia/metabolismo , Proteínas Recombinantes/biosíntesis , Coloración y Etiquetado/métodos , Animales , Anticuerpos/inmunología , Reactores Biológicos , Citometría de Flujo , Cabras , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Pichia/clasificación , Pichia/citología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología
10.
J Biol Chem ; 283(15): 9724-36, 2008 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-18234669

RESUMEN

Structural studies of cell wall components of the pathogenic yeast Candida albicans have demonstrated the presence of beta-1,2-linked oligomannosides in phosphopeptidomannan and phospholipomannan. During C. albicans infection, beta-1,2-oligomannosides play an important role in host/pathogen interactions by acting as adhesins and by interfering with the host immune response. Despite the importance of beta-1,2-oligomannosides, the genes responsible for their synthesis have not been identified. The main reason is that the reference species Saccharomyces cerevisiae does not synthesize beta-linked mannoses. On the other hand, the presence of beta-1,2-oligomannosides has been reported in the cell wall of the more genetically tractable C. albicans relative, P. pastoris. Here we present the identification, cloning, and characterization of a novel family of fungal genes involved in beta-mannose transfer. Employing in silico analysis, we identified a family of four related new genes in P. pastoris and subsequently nine homologs in C. albicans. Biochemical, immunological, and structural analyses following deletion of four genes in P. pastoris and deletion of four genes acting specifically on C. albicans mannan demonstrated the involvement of these new genes in beta-1,2-oligomannoside synthesis. Phenotypic characterization of the strains deleted in beta-mannosyltransferase genes (BMTs) allowed us to describe the stepwise activity of Bmtps and acceptor specificity. For C. albicans, despite structural similarities between mannan and phospholipomannan, phospholipomannan beta-mannosylation was not affected by any of the CaBMT1-4 deletions. Surprisingly, depletion in mannan major beta-1,2-oligomannoside epitopes had little impact on cell wall surface beta-1,2-oligomannoside antigenic expression.


Asunto(s)
Candida albicans/genética , Pared Celular/genética , Genes Fúngicos/fisiología , Oligosacáridos/genética , Pichia/genética , Polisacáridos/genética , Candida albicans/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Pared Celular/metabolismo , Clonación Molecular , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Eliminación de Gen , Glucolípidos/biosíntesis , Glucolípidos/genética , Glicosilación , Manosa/genética , Manosa/metabolismo , Manosiltransferasas/genética , Manosiltransferasas/metabolismo , Oligosacáridos/metabolismo , Pichia/metabolismo , Polisacáridos/biosíntesis
11.
Science ; 313(5792): 1441-3, 2006 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-16960007

RESUMEN

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.


Asunto(s)
Eritropoyetina/metabolismo , Pichia/genética , Ingeniería de Proteínas , Sialoglicoproteínas/biosíntesis , Animales , Línea Celular , Clonación Molecular , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Eritropoyetina/química , Eritropoyetina/genética , Vectores Genéticos , Glicosilación , Humanos , Pichia/metabolismo , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Ácidos Siálicos/metabolismo , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Transformación Genética
12.
Nat Biotechnol ; 24(2): 210-5, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16429149

RESUMEN

As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Mejoramiento Genético/métodos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Pichia/genética , Pichia/metabolismo , Ingeniería de Proteínas/métodos , Anticuerpos Monoclonales/genética , Glicosilación , Humanos , Proteínas Recombinantes/biosíntesis
13.
Genetics ; 170(3): 1091-104, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15802524

RESUMEN

MAP kinases homologous to Saccharomyces cerevisiae Fus3p/Kss1p have been identified in plant pathogenic fungi and are required for pathogenicity and sexual reproduction. To better understand the role of MAP kinase signaling in Neurospora crassa, and to identify downstream target genes of the pathway, we isolated, cloned, and disrupted the FUS3 homolog mak-2. Ste12p is a transcription factor target of Fus3p that activates genes of the mating pathway in yeast, and we also characterized the N. crassa STE12 homolog pp-1. The mak-2 and pp-1 mutants have reduced growth rate, produce short aerial hyphae, and fail to develop protoperithecia. In addition, ascospores carrying null mutations of either gene are inviable. Subtractive cloning was used to isolate genes having reduced expression in the mak-2 mutant. Expression of some of these genes is protoperithecia specific and three of them are part of a gene cluster potentially involved in the production of a polyketide secondary metabolite. Microarray analysis was used to extend the analysis of gene expression in mak-2 and pp-1 mutants. The role of the MAP kinase pathway in both sexual and asexual development as well as secondary metabolism is consistent with the dual regulation of the mating process and pathogencity observed in fungal pathogens.


Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Neurospora crassa/enzimología , Neurospora crassa/crecimiento & desarrollo , Transducción de Señal/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Vectores Genéticos , Análisis por Micromatrices , Datos de Secuencia Molecular , Reproducción/genética , Análisis de Secuencia de ADN
14.
Glycobiology ; 14(9): 757-66, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15190003

RESUMEN

A significant percentage of eukaryotic proteins contain posttranslational modifications, including glycosylation, which are required for biological function. However, the understanding of the structure-function relationships of N-glycans has lagged significantly due to the microheterogeneity of glycosylation in mammalian produced proteins. Recently we reported on the cellular engineering of yeast to replicate human N-glycosylation for the production of glycoproteins. Here we report the engineering of an artificial glycosylation pathway in Pichia pastoris blocked in dolichol oligosaccharide assembly. The PpALG3 gene encoding Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol mannosyltransferase was deleted in a strain that was previously engineered to produce hybrid GlcNAcMan(5)GlcNAc(2) human N-glycans. Employing this approach, combined with the use of combinatorial genetic libraries, we engineered P. pastoris strains that synthesize complex GlcNAc(2)Man(3)GlcNAc(2) N-glycans with striking homogeneity. Furthermore, through expression of a Golgi-localized fusion protein comprising UDP-glucose 4-epimerase and beta-1,4-galactosyl transferase activities we demonstrate that this structure is a substrate for highly efficient in vivo galactose addition. Taken together, these data demonstrate that the artificial in vivo glycoengineering of yeast represents a major advance in the production of glycoproteins and will emerge as a practical tool to systematically elucidate the structure-function relationship of N-glycans.


Asunto(s)
Galactosa/metabolismo , Glicoproteínas/metabolismo , Oligosacáridos/química , Pichia/metabolismo , Secuencia de Bases , Cartilla de ADN , Glicoproteínas/química , Glicoproteínas/genética , Glicosilación , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
15.
Science ; 301(5637): 1244-6, 2003 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-12947202

RESUMEN

We report the humanization of the glycosylation pathway in the yeast Pichia pastoris to secrete a human glycoprotein with uniform complex N-glycosylation. The process involved eliminating endogenous yeast glycosylation pathways, while properly localizing five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyl transferases I and II, and uridine 5'-diphosphate (UDP)-N-acetylglucosamine transporter. Targeted localization of the enzymes enabled the generation of a synthetic in vivo glycosylation pathway, which produced the complex human N-glycan N-acetylglucosamine2-mannose3-N-acetylglucosamine2 (GlcNAc2Man3GlcNAc2). The ability to generate human glycoproteins with homogeneous N-glycan structures in a fungal host is a step toward producing therapeutic glycoproteins and could become a tool for elucidating the structure-function relation of glycoproteins.


Asunto(s)
Ingeniería Genética , Glicoproteínas/biosíntesis , Manosidasas/genética , Pichia/genética , Polisacáridos/metabolismo , Proteínas Recombinantes/biosíntesis , Animales , Dominio Catalítico , Retículo Endoplásmico/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Glicosilación , Aparato de Golgi/metabolismo , Humanos , Manosidasas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Biblioteca de Péptidos , Pichia/enzimología , Pichia/metabolismo , Polisacáridos/química , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Transformación Genética
16.
Proc Natl Acad Sci U S A ; 100(9): 5022-7, 2003 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-12702754

RESUMEN

The secretory pathway of Pichia pastoris was genetically re-engineered to perform sequential glycosylation reactions that mimic early processing of N-glycans in humans and other higher mammals. After eliminating nonhuman glycosylation by deleting the initiating alpha-1,6-mannosyltransferase gene from P. pastoris, several combinatorial genetic libraries were constructed to localize active alpha-1,2-mannosidase and human beta-1,2-N-acetylglucosaminyltransferase I (GnTI) in the secretory pathway. First, >32 N-terminal leader sequences of fungal type II membrane proteins were cloned to generate a leader library. Two additional libraries encoding catalytic domains of alpha-1,2-mannosidases and GnTI from mammals, insects, amphibians, worms, and fungi were cloned to generate catalytic domain libraries. In-frame fusions of the respective leader and catalytic domain libraries resulted in several hundred chimeric fusions of fungal targeting domains and catalytic domains. Although the majority of strains transformed with the mannosidase/leader library displayed only modest in vivo [i.e., low levels of mannose (Man)(5)-(GlcNAc)(2)] activity, we were able to isolate several yeast strains that produce almost homogeneous N-glycans of the (Man)(5)-(GlcNAc)(2) type. Transformation of these strains with a UDP-GlcNAc transporter and screening of a GnTI leader fusion library allowed for the isolation of strains that produce GlcNAc-(Man)(5)-(GlcNAc)(2) in high yield. Recombinant expression of a human reporter protein in these engineered strains led to the formation of a glycoprotein with GlcNAc-(Man)(5)-(GlcNAc)(2) as the primary N-glycan. Here we report a yeast able to synthesize hybrid glycans in high yield and open the door for engineering yeast to perform complex human-like glycosylation.


Asunto(s)
Pichia/genética , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Secuencia de Bases , Cartilla de ADN , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Glicosilación , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Humanos , Manosiltransferasas/genética , Pichia/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
17.
Mol Microbiol ; 45(3): 795-804, 2002 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12139624

RESUMEN

Pheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. To begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species Neurospora crassa. One gene, expressed exclusively in mat A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by Kex2 processing sites. Strains of the opposite mating type, mat a, express a pheromone precursor gene whose polypeptide contains a C-terminal CAAX motif predicted to produce a mature pheromone with a C-terminal carboxy-methyl isoprenylated cysteine. The predicted sequences of the pheromones are remarkably similar to those encoded by other filamentous ascomycetes. The expression of the pheromone precursor genes is mating type specific and is under the control of the mating type locus. Furthermore, the genes are highly expressed in conidia and under conditions that favour sexual development. Both pheromone precursor genes are also regulated by the endogenous circadian clock in a time-of-day-specific fashion, supporting a role for the clock in mating.


Asunto(s)
Ritmo Circadiano/fisiología , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Neurospora crassa/genética , Péptidos/fisiología , Feromonas/genética , Secuencia de Aminoácidos , Relojes Biológicos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Factor de Apareamiento , Datos de Secuencia Molecular , Morfogénesis/genética , Neurospora crassa/crecimiento & desarrollo , Neurospora crassa/fisiología , Feromonas/metabolismo , Feromonas/fisiología , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido
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