Regulation of the cellulase complex production by Saccoboloides

Se evaluó la capacidad de Saccobolus saccoboloides (Pezizales, Ascomycetes) de producir el sistema celulolítico completo en medio de cultivo líquido sintético. La caracterización de las enzimas producidas permite afirmar que este hongo coprófilo es un verdadero agente celulolítico, ya que su complejo enzimático presenta las tres actividades necesarias para la digestión del polímero (β-1,4 endoglucanasa, β-1,4 exoglucanasa y β-glucosidasa), y que estas enzimas son extracelulares, termoestables y (a diferencia de las de otros organismos) poco inhibidas por producto final. El crecimiento fue alto en glucosa y celobiosa, sin producción de enzimas, en tanto que en celulosa hubo muy buen crecimiento y actividad enzimática, evidenciándose diferencias de respuesta según el medio elegido. La nutrición nitrogenada no afecta la producción enzimática, sino el crecimiento: se investiga, por lo tanto, el control por fuente carbonada, en cultivos de reemplazo. En la inducción, cuando no pudo medirse endoglucanasa, se utilizó un método alternativo de degradación de CMC (Carboximetilcelulosa) en placa (CMCasa). La celulosa fue el mejor inductor, seguida por la lactosa. El sistema no se induce en presencia de glucosa, sorbosa, maltosa ni almidón, y se detecta una mínima actividad en xilano. La CMC es un pobre inductor, en tanto que la celobiosa tiene un efecto diferencial. Cuando se ensayó el efecto represor de los no inductores se ve que el sistema no se reprime fácilmente y que la glucosa y celobiosa tienen efecto diferencial. Finalmente la mezcla de dos inductores tiene un esperado efecto negativo. Las enzimas se producen en todos casos por síntesis de novo, las dos formas diferentes de β-glucosidasa se regulan independientemente, y existe inducción cruzada con el complejo xilanasa. Estos datos, junto al análisis de isoenzimas producidas en cada condición permite postular un novedoso modelo regulatorio que explicaría como los hongos filamentosos son estimulados a producir las hidrolasas específicas para degradar celulosa.The cellulase complex production by Saccobolus saccoboloides (Pezizales, Ascomycetes) was evaluated. This fungus produces a complete cellulase system. The enzyme characterisation shows that S. saccoboloides is a true cellulolytic organism, the three enzymes (β-glucosidase, β-1,4 endoglucanase and β-1,4 exoglucanase) required for the complete cellulose hydrolisis were produced. All the enzymes were thermostable and did not show end-product inhibition. Glucose and cellobiose were good sources for growth but not for enzyme production, cellulose was a suitable carbon source for growth and cellulase production: there was evidence for carbon regulation. The nitrogen source affected biomass production, and therefore enzyme production. For these reasons carbon nutrition was investigated as the primary control of cellulase production. When endoglucanase and exoglucanase could not be measured, an alternative Carboxymethylcellulose-clearing method was used. Crystalline cellulose was the best inducer, followed by lactose. The other mono- di- or polysaccharides were non inducers or poor inducers. Cellobiose or glucose repressed cellulase production in cellulose containing media. This repression was selective for some components of the cellulase complex, and was not observed using repressor concentrations below 3% w/v. Moreover, the degree of repression varied with the age of the cultures. The mixture of both inducers had an unexpected negative effect. Cellulase production by this fungus is inducible and subject to a complex repression by easily metabolized sugars. The enzyme production is due to the novo synthesis, two different β-glucosidase forms were observed and cellulase-xylanase cross-induction was observed as well. The data analysis shows that it is possible to present a new model of cellulase regulation, that would explain cellulose recognition and cellulase induction in filamentous fungi.Fil: Magnelli, Paula E.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submit- Avenue, Silver Spring, Maryland 20993; 22Glycoscience Research Laboratory, Genos, Borongajska cesta 83h, 10 000 Zagreb, Croatia; 23Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇ ic´ a 1, 10 000 Zagreb, Croatia; 24Department of Chemistry, Georgia State University, 100 Piedmont Avenue, Atlanta, Georgia 30303; 25glyXera GmbH, Brenneckestrasse 20 * ZENIT / 39120 Magdeburg, Germany; 26Health Products and Foods Branch, Health Canada, AL 2201E, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, K1A 0K9 Canada; 27Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama Higashi-Hiroshima 739–8530 Japan; 28ImmunoGen, 830 Winter Street, Waltham, Massachusetts 02451; 29Department of Medical Physiology, Jagiellonian University Medical College, ul. Michalowskiego 12, 31–126 Krakow, Poland; 30Department of Pathology, Johns Hopkins University, 400 N. Broadway Street Baltimore, Maryland 21287; 31Mass Spec Core Facility, KBI Biopharma, 1101 Hamlin Road Durham, North Carolina 27704; 32Division of Mass Spectrometry, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongwon-gu, Cheongju Chungbuk, 363–883 Korea (South); 33Advanced Therapy Products Research Division, Korea National Institute of Food and Drug Safety, 187 Osongsaengmyeong 2-ro Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, 363–700, Korea (South); 34Center for Proteomics and Metabolomics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands; 35Ludger Limited, Culham Science Centre, Abingdon, Oxfordshire, OX14 3EB, United Kingdom; 36Biomolecular Discovery and Design Research Centre and ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, North Ryde, Australia; 37Proteomics, Central European Institute for Technology, Masaryk University, Kamenice 5, A26, 625 00 BRNO, Czech Republic; 38Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany; 39Department of Biomolecular Sciences, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany; 40AstraZeneca, Granta Park, Cambridgeshire, CB21 6GH United Kingdom; 41Merck, 2015 Galloping Hill Rd, Kenilworth, New Jersey 07033; 42Analytical R&D, MilliporeSigma, 2909 Laclede Ave. St. Louis, Missouri 63103; 43MS Bioworks, LLC, 3950 Varsity Drive Ann Arbor, Michigan 48108; 44MSD, Molenstraat 110, 5342 CC Oss, The Netherlands; 45Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5–1 Higashiyama, Myodaiji, Okazaki 444–8787 Japan; 46Graduate School of Pharmaceutical Sciences, Nagoya City University, 3–1 Tanabe-dori, Mizuhoku, Nagoya 467–8603 Japan; 47Medical & Biological Laboratories Co., Ltd, 2-22-8 Chikusa, Chikusa-ku, Nagoya 464–0858 Japan; 48National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG United Kingdom; 49Division of Biological Chemistry & Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158–8501 Japan; 50New England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts 01938; 51New York University, 100 Washington Square East New York City, New York 10003; 52Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom; 53GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland; 54Department of Chemistry, North Carolina State University, 2620 Yarborough Drive Raleigh, North Carolina 27695; 55Pantheon, 201 College Road East Princeton, New Jersey 08540; 56Pfizer Inc., 1 Burtt Road Andover, Massachusetts 01810; 57Proteodynamics, ZI La Varenne 20–22 rue Henri et Gilberte Goudier 63200 RIOM, France; 58ProZyme, Inc., 3832 Bay Center Place Hayward, California 94545; 59Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho Nakagyo-ku, Kyoto, 604 8511 Japan; 60Children’s GMP LLC, St. Jude Children’s Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105; 61Sumitomo Bakelite Co., Ltd., 1–5 Muromati 1-Chome, Nishiku, Kobe, 651–2241 Japan; 62Synthon Biopharmaceuticals, Microweg 22 P.O. Box 7071, 6503 GN Nijmegen, The Netherlands; 63Takeda Pharmaceuticals International Co., 40 Landsdowne Street Cambridge, Massachusetts 02139; 64Department of Chemistry and Biochemistry, Texas Tech University, 2500 Broadway, Lubbock, Texas 79409; 65Thermo Fisher Scientific, 1214 Oakmead Parkway Sunnyvale, California 94085; 66United States Pharmacopeia India Pvt. Ltd. IKP Knowledge Park, Genome Valley, Shamirpet, Turkapally Village, Medchal District, Hyderabad 500 101 Telangana, India; 67Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 68Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 69Department of Chemistry, University of California, One Shields Ave, Davis, California 95616; 70Horva´ th Csaba Memorial Laboratory for Bioseparation Sciences, Research Center for Molecular Medicine, Doctoral School of Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Egyetem ter 1, Hungary; 71Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Egyetem ut 10, Hungary; 72Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way Newark, Delaware 19711; 73Proteomics Core Facility, University of Gothenburg, Medicinaregatan 1G SE 41390 Gothenburg, Sweden; 74Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Institute of Biomedicine, Sahlgrenska Academy, Medicinaregatan 9A, Box 440, 405 30, Gothenburg, Sweden; 75Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at the University of Gothenburg, Bruna Straket 16, 41345 Gothenburg, Sweden; 76Department of Chemistry, University of Hamburg, Martin Luther King Pl. 6 20146 Hamburg, Germany; 77Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2; 78Laboratory of Mass Spectrometry of Interactions and Systems, University of Strasbourg, UMR Unistra-CNRS 7140, France; 79Natural and Medical Sciences Institute, University of Tu¨ bingen, Markwiesenstrae 55, 72770 Reutlingen, Germany; 80Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 81Division of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands; 82Department of Chemistry, Waters Corporation, 34 Maple Street Milford, Massachusetts 01757; 83Zoetis, 333 Portage St. Kalamazoo, Michigan 49007 Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. Received July 24, 2019, and in revised form, August 26, 2019 Published, MCP Papers in Press, October 7, 2019, DOI 10.1074/mcp.RA119.001677 ER: NISTmAb Glycosylation Interlaboratory Study 12 Molecular & Cellular Proteomics 19.1 Downloaded from https://www.mcponline.org by guest on January 20, 2020 ted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide communityderived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Molecular & Cellular Proteomics 19: 11–30, 2020. DOI: 10.1074/mcp.RA119.001677.L

Regulation of the cellulase complex production by Saccoboloides

Se evaluó la capacidad de Saccobolus saccoboloides (Pezizales, Ascomycetes) de producir el sistema celulolítico completo en medio de cultivo líquido sintético. La caracterización de las enzimas producidas permite afirmar que este hongo coprófilo es un verdadero agente celulolítico, ya que su complejo enzimático presenta las tres actividades necesarias para la digestión del polímero (β-1,4 endoglucanasa, β-1,4 exoglucanasa y β-glucosidasa), y que estas enzimas son extracelulares, termoestables y (a diferencia de las de otros organismos) poco inhibidas por producto final. El crecimiento fue alto en glucosa y celobiosa, sin producción de enzimas, en tanto que en celulosa hubo muy buen crecimiento y actividad enzimática, evidenciándose diferencias de respuesta según el medio elegido. La nutrición nitrogenada no afecta la producción enzimática, sino el crecimiento: se investiga, por lo tanto, el control por fuente carbonada, en cultivos de reemplazo. En la inducción, cuando no pudo medirse endoglucanasa, se utilizó un método alternativo de degradación de CMC (Carboximetilcelulosa) en placa (CMCasa). La celulosa fue el mejor inductor, seguida por la lactosa. El sistema no se induce en presencia de glucosa, sorbosa, maltosa ni almidón, y se detecta una mínima actividad en xilano. La CMC es un pobre inductor, en tanto que la celobiosa tiene un efecto diferencial. Cuando se ensayó el efecto represor de los no inductores se ve que el sistema no se reprime fácilmente y que la glucosa y celobiosa tienen efecto diferencial. Finalmente la mezcla de dos inductores tiene un esperado efecto negativo. Las enzimas se producen en todos casos por síntesis de novo, las dos formas diferentes de β-glucosidasa se regulan independientemente, y existe inducción cruzada con el complejo xilanasa. Estos datos, junto al análisis de isoenzimas producidas en cada condición permite postular un novedoso modelo regulatorio que explicaría como los hongos filamentosos son estimulados a producir las hidrolasas específicas para degradar celulosa.The cellulase complex production by Saccobolus saccoboloides (Pezizales, Ascomycetes) was evaluated. This fungus produces a complete cellulase system. The enzyme characterisation shows that S. saccoboloides is a true cellulolytic organism, the three enzymes (β-glucosidase, β-1,4 endoglucanase and β-1,4 exoglucanase) required for the complete cellulose hydrolisis were produced. All the enzymes were thermostable and did not show end-product inhibition. Glucose and cellobiose were good sources for growth but not for enzyme production, cellulose was a suitable carbon source for growth and cellulase production: there was evidence for carbon regulation. The nitrogen source affected biomass production, and therefore enzyme production. For these reasons carbon nutrition was investigated as the primary control of cellulase production. When endoglucanase and exoglucanase could not be measured, an alternative Carboxymethylcellulose-clearing method was used. Crystalline cellulose was the best inducer, followed by lactose. The other mono- di- or polysaccharides were non inducers or poor inducers. Cellobiose or glucose repressed cellulase production in cellulose containing media. This repression was selective for some components of the cellulase complex, and was not observed using repressor concentrations below 3% w/v. Moreover, the degree of repression varied with the age of the cultures. The mixture of both inducers had an unexpected negative effect. Cellulase production by this fungus is inducible and subject to a complex repression by easily metabolized sugars. The enzyme production is due to the novo synthesis, two different β-glucosidase forms were observed and cellulase-xylanase cross-induction was observed as well. The data analysis shows that it is possible to present a new model of cellulase regulation, that would explain cellulose recognition and cellulase induction in filamentous fungi.Fil: Magnelli, Paula E.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ∼60% decrease in CSIII activity, which is correlated with a ∼30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro

The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases

The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-GlcNAc2Man9Glc3. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. Here we used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from Giardia and Plasmodium). Fourth, dolichol-PP-GlcNAc2Man5 (present in Entamoeba and Trichomonas) and dolichol-PP- and N-linked GlcNAc2 (present in Giardia) have not been identified previously in wild-type organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases

Unique Posttranslational Modifications of Chitin-Binding Lectins of Entamoeba invadens Cyst Walls

Entamoeba histolytica, which causes amebic dysentery and liver abscesses, is spread via chitin-walled cysts. The most abundant protein in the cyst wall of Entamoeba invadens, a model for amebic encystation, is a lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by low-complexity Ser- and Thr-rich spacers. E. histolytica also has numerous predicted Jessie lectins and chitinases, which contain a single, N-terminal eight-Cys chitin-binding domain. We hypothesized that E. invadens cyst walls are composed entirely of proteins with six-Cys or eight-Cys chitin-binding domains and that some of these proteins contain sugars. E. invadens genomic sequences predicted seven Jacob lectins, five Jessie lectins, and three chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and chitinases are increased during E. invadens encystation, while mass spectrometry showed that the cyst wall is composed of an ∼30:70 mix of Jacob lectins (cross-linking proteins) and Jessie and chitinase lectins (possible enzymes). Three Jacob lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser- and Thr-rich spacers between chitin-binding domains. A model peptide was cleaved at the same site by papain and E. invadens Cys proteases, suggesting that the latter cleave Jacob lectins in vivo. Some Jacob lectins had O-phosphodiester-linked carbohydrates, which were one to seven hexoses long and had deoxysugars at reducing ends. We concluded that the major protein components of the E. invadens cyst wall all contain chitin-binding domains (chitinases, Jessie lectins, and Jacob lectins) and that the Jacob lectins are differentially modified by site-specific Cys proteases and O-phosphodiester-linked glycans