RESUMEN
Genome-wide studies have identified abundant small, noncoding RNAs, including small nuclear RNAs, small nucleolar RNAs (snoRNAs), cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs), that are transcribed by RNA polymerase II (pol II) and terminated by an Nrd1-dependent pathway. Here, we show that the prolyl isomerase Ess1 is required for Nrd1-dependent termination of noncoding RNAs. Ess1 binds the carboxy-terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD. In ess1 mutants, expression of approximately 10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, stable unannotated transcripts, and uRNAs. Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase. We also provide evidence for a competition between Nrd1 and Pcf11 for CTD binding that is regulated by Ess1. These data indicate that a prolyl isomerase is required for specifying the "CTD code."
Asunto(s)
Isomerasa de Peptidilprolil/metabolismo , ARN no Traducido/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transcripción Genética , Perfilación de la Expresión Génica , Genoma Fúngico/genética , Modelos Genéticos , Mutación/genética , Peptidilprolil Isomerasa de Interacción con NIMA , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Fosfoserina/metabolismo , Estructura Terciaria de Proteína , ARN Polimerasa II/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Nucleolar Pequeño/genética , Secuencias Reguladoras de Ácido Ribonucleico/genéticaRESUMEN
HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.
Asunto(s)
Aparato de Golgi/enzimología , Heparina/biosíntesis , Heparitina Sulfato/biosíntesis , Ingeniería Metabólica , Sulfotransferasas/biosíntesis , Amidohidrolasas/biosíntesis , Amidohidrolasas/genética , Animales , Células CHO , Cricetinae , Cricetulus , Aparato de Golgi/genética , Heparina/genética , Heparitina Sulfato/genética , Humanos , Ratones , Sulfotransferasas/genéticaRESUMEN
Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities.
Asunto(s)
Biotecnología/métodos , Heparina/biosíntesis , Sulfotransferasas/metabolismo , Animales , Anticoagulantes/química , Conformación de Carbohidratos , Línea Celular Tumoral , Heparina/química , Mastocitoma/metabolismo , Ratones , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfotransferasas/genéticaRESUMEN
Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity â¼100-fold and the heparin/heparan sulfate yield â¼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in â¼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of â¼90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.
Asunto(s)
Anticoagulantes , Células CHO , Heparina/biosíntesis , Ingeniería Metabólica , Animales , Reactores Biológicos , Vías Biosintéticas , Cricetinae , Cricetulus , Heparina/metabolismoRESUMEN
Ess1 is an essential peptidylprolyl-cis/trans-isomerase in the yeast Saccharomyces cerevisiae. Ess1 and its human homolog, Pin1, bind to phospho-Ser-Pro sites within proteins, including the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (pol II). Ess1 and Pin1 are thought to control mRNA synthesis by catalyzing conformational changes in Rpb1 that affect interaction of cofactors with the pol II transcription complex. Here we have characterized wild-type and mutant Ess1 proteins in vitro and in vivo. We found that Ess1 preferentially binds and isomerizes CTD heptad-repeat (YSPTSPS) peptides that are phosphorylated on Ser5. Binding by the mutant proteins in vitro was essentially normal, and the proteins were largely stable in vivo. However, their catalytic activities were reduced >1,000-fold. These data along with results of in vivo titration experiments indicate that Ess1 isomerase activity is required for growth, but only at vanishingly low levels. We found that although wild-type cells contain about approximately 200,000 molecules of Ess1, a level of fewer than 400 molecules per cell is sufficient for growth. In contrast, higher levels of Ess1 were required for growth in the presence of certain metabolic inhibitors, suggesting that Ess1 is important for tolerance to environmental challenge.
Asunto(s)
Isomerasa de Peptidilprolil/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Pared Celular/metabolismo , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/genética , Proteínas de Saccharomyces cerevisiaeRESUMEN
Ess1 is a peptidyl-prolyl cis/trans isomerase (PPIase) that binds to the carboxy-terminal domain (CTD) of RNA polymerase II. Ess1 is thought to function by inducing conformational changes in the CTD that control the assembly of cofactor complexes on the transcription unit. Ess1 (also called Pin1) is highly conserved throughout the eukaryotic kingdom and is required for growth in some species, including the human fungal pathogen Candida albicans. Here we report the crystal structure of the C. albicansEss1 protein, determined at 1.6 A resolution. The structure reveals two domains, the WW and the isomerase domain, that have conformations essentially identical to those of human Pin1. However, the linker region that joins the two domains is quite different. In human Pin1, this linker is short and flexible, and part of it is unstructured. In contrast, the fungal Ess1 linker is highly ordered and contains a long alpha-helix. This structure results in a rigid juxtaposition of the WW and isomerase domains, in an orientation that is distinct from that observed in Pin1, and that eliminates a hydrophobic pocket between the domains that was implicated as the main substrate recognition site. These differences suggest distinct modes of interaction with long substrate molecules, such as the CTD of RNA polymerase II. We also show that C. albicans ess1(-)() mutants are attenuated for in vivo survival in mice. Together, these results suggest that CaEss1 might constitute a useful antifungal drug target, and that structural differences between the fungal and human enzymes could be exploited for drug design.
Asunto(s)
Candida albicans/enzimología , Isomerasa de Peptidilprolil/química , Secuencia de Aminoácidos , Animales , Candida albicans/genética , Candida albicans/patogenicidad , Cristalografía por Rayos X , Femenino , Genes Fúngicos , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/fisiología , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Virulencia/genética , Virulencia/fisiologíaRESUMEN
The N-linked galactomannans of Schizosaccharomyces pombe have pyruvylated Galbeta1,3-(PvGal) caps on a portion of the Galalpha1,2-residues in their outer chains (Gemmill, T. R., and Trimble, R. B. (1998) Glycobiology 8, 1087-1095). PvGal biosynthesis was investigated by ethyl methanesulfonate mutagenesis of S. pombe, followed by the isolation of cells devoid of negatively charged N-glycans by Q-Sepharose exclusion and failure to bind human serum amyloid P component, which acts as a lectin for terminal PvGal residues. Mutant glycans were characterized by lectin binding, saccharide composition, exoglycosidase sensitivity, and NMR spectroscopy. Restoration of the cell surface negative charge by complementation with an S. pombe genomic library led to the identification of five genes involved in PvGal biosynthesis, which we designated pvg1-pvg5. Pvg1p may be a pyruvyltransferase, since NMR of pvg1(-) mutant N-glycans revealed the absence of only the pyruvyl moiety. Pvg2p-Pvg5p are crucial for attachment of the Galbeta1,3-residue that becomes pyruvylated. Pvg3p is predicted to be a member of the beta1,3-galactosyltransferase family, and Pvg3p-green fluorescent protein labeling was consistent with Golgi localization. Predicted Pvg1p and Pvg3p functions imply that Galbeta1,3-is added to the galactomannans and is then pyruvylated in situ, rather than by an en bloc addition of PvGalbeta1,3-caps to the outer chain. Pvg4p-green fluorescent protein targeted to the nucleus, and its sequence contains a MADS-box DNA-binding and dimerization domain; however, it does not appear to solely control transcription of the other identified genes. Pvg2p and/or Pvg5p may contribute to an enzyme complex. Whereas a functional role for the PvGal epitope in S. pombe remains unclear, it is nonessential for either cell growth or mating under laboratory conditions.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Mananos/biosíntesis , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Epítopos/biosíntesis , Epítopos/genética , Proteínas Fúngicas/metabolismo , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Glicosilación , Humanos , Mananos/genética , Datos de Secuencia Molecular , Ácido Pirúvico , Alineación de SecuenciaRESUMEN
Recombinant human bile salt-stimulated lipase (hBSSL) was expressed in and secreted by Pichia pastoris, an organism exploited for the large-scale production of recombinant (glyco)proteins by bioprocessing technology. The 76.3-kDa glycoprotein was associated with 75-80 Man and a small amount of GlcNAc. hBSSL has one N-glycosylation site at Asn187, which was 38-40% occupied with a Man(10)GlcNAc(2) structure defined previously in Pichia as the oligosaccharide-lipid form of Man(9)GlcNAc(2) trimmed of the middle-arm terminal alpha 1,2-Man and elongated with Man alpha 1,2Man alpha 1,6-disaccharide attached to the lower-arm core alpha 1,3-Man (Trimble et al. [1991], J. Biol. Chem., 266, 22807-22817). The C-terminal 192 residues of hBSSL contain 16 Pro-rich 11-amino-acid repeats, which include 32 Ser/Thr residues as potential O-glycosylation sites. Using hBSSL as a platform to study Pichia's O-glycosylation capabilities, we found that nearly all of these sites were occupied by mannose-containing O-glycans, whose structures, after beta-elimination and purification, were assigned by (1)H NMR and, in some cases, by linkage-specific exoglycosidases and methylation analysis. The most abundant O-glycan was alpha 1,2-mannobiitol (55%), followed by alpha 1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was alpha 1,2-mannotetraitol. Unexpectedly, Man(5) and Man(6) O-glycans were present, which had the structure Man beta 1,2Man beta 1,2Man alpha 1,2(Man alpha 1,2)(1,2)mannitol. Also a small amount of a phosphorylated Man(6) O-glycan was characterized by MALDI-TOF MS postsource decay analysis as having the reducing-end mannitol disubstituted with a glycosidically linked phosphorylated Man and an unbranched Man(4) polymer elongated from a different mannitol carbon. This is the first report of the synthesis of beta-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by P. pastoris.