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  • 51.
    Rezinciuc, Svetlana
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Vladimir Sandoval-Sierra, Jose
    Ruiz-Leon, Yolanda
    van West, Pieter
    Dieguez-Uribeondo, Javier
    Specialized attachment structure of the fish pathogenic oomycete Saprolegnia parasitica2018Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, nr 1, artikel-id e0190361Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The secondary cysts of the fish pathogen oomycete Saprolegnia parasitica possess bundles of long hooked hairs that are characteristic to this economically important pathogenic species. Few studies have been carried out on elucidating their specific role in the S. parasitica life cycle and the role they may have in the infection process. We show here their function by employing several strategies that focus on descriptive, developmental and predictive approaches. The strength of attachment of the secondary cysts of this pathogen was compared to other closely related species where bundles of long hooked hairs are absent. We found that the attachment of the S. parasitica cysts was around three times stronger than that of other species. The time sequence and influence of selected factors on morphology and the number of the bundles of long hooked hairs conducted by scanning electron microscopy study revealed that these are dynamic structures. They are deployed early after encystment, i.e., within 30 sec of zoospore encystment, and the length, but not the number, of the bundles steadily increased over the encystment period. We also observed that the number and length of the bundles was influenced by the type of substrate and encystment treatment applied, suggesting that these structures can adapt to different substrates (glass or fish scales) and can be modulated by different signals (i.e., protein media, 50 mM CaCl2 concentrations, carbon particles). Immunolocalization studies evidenced the presence of an adhesive extracellular matrix. The bioinformatic analyses of the S. parasitica secreted proteins showed that there is a high expression of genes encoding domains of putative proteins related to the attachment process and cell adhesion (fibronectin and thrombospondin) coinciding with the deployment stage of the bundles of long hooked hairs formation. This suggests that the bundles are structures that might contribute to the adhesion of the cysts to the host because they are composed of these adhesive proteins and/or by increasing the surface of attachment of this extracellular matrix.

  • 52. Roberts, Alison W.
    et al.
    Lahnstein, Jelle
    Hsieh, Yves S. Y.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Xing, Xiaohui
    Yap, Kuok
    Chaves, Arielle M
    Scavuzzo-Duggan, Tess R
    Dimitroff, George
    Lonsdale, Andrew
    Roberts, Eric M.
    Bulone, Vincent
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Fincher, Geoffrey B
    Doblin, Monika Susanne
    Bacic, Antony
    Burton, Rachel A
    Functional Characterization of a Glycosyltransferase from the Moss Physcomitrella patens Involved in the Biosynthesis of a Novel Cell Wall Arabinoglucan2018Ingår i: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 30, nr 6, s. 1293-1308Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mixed-linkage (1,3;1,4)-β-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco (Nicotiana benthamiana) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-β-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana, but (1,4)-β-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots.

  • 53.
    Rudjito, Reskandi C.
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Ruthes, Andrea C.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Jimenez-Quero, Amparo
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Vilaplana, Francisco
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Feruloylated Arabinoxylans from Wheat Bran: Optimization of Extraction Process and Validation at Pilot Scale2019Ingår i: ACS Sustainable Chemistry and Engineering, ISSN 2168-0485, Vol. 7, nr 15, s. 13167-13177Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The extraction of feruloylated arabinoxylan (AX) from wheat bran was optimized in terms of the pretreatments prior to subcritical water extraction and then validated at the pilot scale. Destarching of wheat bran was critical to improve the yields and purity of the extracted AX. Time evolution profiles from sequential extractions revealed differences in the extractability of different polysaccharides, whereby beta-D-glucans were first extracted followed by AX. At longer extraction times, the AX population was more substituted and more uniform in terms of molar mass distribution. Validation of the process at pilot scale (with a scaling factor of 33) resulted in AXs with similar molecular characteristics as in the laboratory scale. Both solid to liquid ratio and extraction time were essential in improving the yields. Our study demonstrated scalability of the process, opening opportunities for the further development of multifunctional food and material products from the extracted AXs.

  • 54.
    Rzeszutek, Elzbieta
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Cell wall biosynthesis in the pathogenic oomycete Saprolegnia parasitica2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [sv]

    Oomyceten Saprolegnia parasitica är en svampliknande mikroorganism som orsakar fisksjukdomen saprolegniosis, vilken leder till stora ekonomiska förluster inom vattenbruk. För närvarande finns det ingen effektiv metod för att kontrollera infektionen och därför är behovet av metoder för att begränsa sjukdom stort. Ett lovande tillvägagångssätt för att angripa patogenen är genom att hämma biosyntes av cellväggen, särskilt de enzymer som är involverade i kolhydratbiosyntes. Cellväggen hos S. parasitica består huvudsakligen av cellulosa, ß-1,3 och ß-1,6- glukaner, samt små mängder kitin. Den tillgängliga genomsekvensen möjliggjorde identifiering av sex förmodade kitin (Chs) och cellulosa (CesA) -syntasgener. Huvudsyftet med denna avhandling var att karakterisera CHS och CesA från S. parasitica och testa effekten av cellväggsrelaterade inhibitorer på patogenens tillväxt. De testade inhibitorerna inkluderade nikcomycin Z, en kompetitiv inhibitor av CHS, samt flera inhibitorer av cellulosabiosyntes, nämligen flupoxam, CGA325'615 och compund I (Cl). Alla inhibitorer reducerade kraftigt tillväxten av S. parasitica och genom användning av en radiometrisk analysmetod visades att in vitro-bildningen av kitin och cellulosa inhiberades. Kemikalierna påverkade också uttrycket av några av Chsoch CesA-generna. En av CHS, nämligen SpCHS5, uttrycktes framgångsrikt i jäst och renades till homogenitet som ett fullängdsprotein. Det rekombinanta enzymet karakteriserades biokemiskt och visade sig bilda kitinkristalliter in vitro. Dessutom indikerar våra data att SpCHS5 troligen är en homodimer som kan bilda större komplex bestående av flera subenheter. Punktmutationer av konserverade aminosyror tillät oss att identifiera de aminosyror som är väsentliga för enzymets aktivitet och processivitet. Förutom de cellväggsrelaterade inhibitorerna testades även biosurfaktanten massetolid A som produceras naturligt av Pseudomonasarter. Denna visade sig ha en starkt inhiberande verkan på S. parasiticatillväxt. Sammantaget bidrar våra data med viktig information rörande de grundläggande mekanismerna för biosyntes av kitin och cellulosa i oomyceter och de biokemiska egenskaperna av de involverade enzymerna. Resultaten visar också att enzymerna som är involverade i biosyntes av cellväggen är lovande mål för bekämpningsmedel mot oomyceter även då motsvarande polysackarider, såsom kitin, förekommer i små mängder i cellväggen.

  • 55.
    Rzeszutek, Elzbieta
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Diaz-Moreno, Sara M.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Ampomah, Osei Y.
    Inman, Annie
    Srivastava, Vaibhav
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Zhou, Qi
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Bulone, Vincent
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Novel insights into chitin biosynthesis through heterologous expression and biochemical characterization of chitin synthase 5 from the pathogenic oomycete Saprolegnia parasitica Manuskript (preprint) (Övrigt vetenskapligt)
  • 56.
    Rzeszutek, Elzbieta
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Diaz-Moreno, Sara M.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Bulone, Vincent
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Identification and characterization of the chitin synthase genes from the fish pathogen Saprolegnia parasiticaManuskript (preprint) (Övrigt vetenskapligt)
  • 57.
    Rzeszutek, Elzbieta
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Diaz-Moreno, Sara M
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Bulone, Vincent
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Identification and Characterization of the Chitin Synthase Genes From the Fish Pathogen Saprolegnia parasitica2019Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, artikel-id 2873Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Saprolegnia parasitica is a pathogenic oomycete responsible for severe fish infections. Despite its low abundance in the cell wall of S. parasitica, chitin is essential for hyphal growth as the inhibition of its biosynthesis leads to highly reduced growth. Here we identified and characterized chitin synthases (CHS) from S. parasitica as potential targets for anti-oomycete drugs. Bioinformatics analyses allowed the identification of six different putative Chs genes in the genome of the pathogen. The total number of genes was confirmed by Southern blot analysis and their expression levels were determined by quantitative PCR. Four of the six Chs genes were expressed in the mycelium, while the two others exhibited undetectable levels of expression. The mycelium was highly sensitive to the addition of nikkomycin Z (NZ) in the culture medium, which led to a decreased amount of chitin in the cell wall by up to 40% in the conditions tested, and to the formation of abnormal branching structures in the hyphae. The presence of NZ increased the expression level of one of the genes, Chs3, suggesting that the corresponding product is compensating the disruption of chitin biosynthesis in the hyphae. In addition, the activity of isolated CHS was strongly inhibited by NZ in vitro. Altogether our data indicate the importance of CHS for the vegetative growth of S. parasitica and demonstrate that these enzymes represent promising targets for the control of diseases caused by oomycetes.

  • 58.
    Rzeszutek, Elzbieta
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Diaz-Moreno, Sara M.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Klinter, Stefan
    Bulone, Vincent
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Analysis of the cellulose synthase genes in the oomycete fish pathogen Saprolegnia parasitica and effect of cellulose biosynthesis inhibitors on enzyme activity and microbial growthManuskript (preprint) (Övrigt vetenskapligt)
  • 59.
    Shao, Zhanru
    et al.
    Chinese Acad Sci, Inst Oceanol, CAS Key Lab Expt Marine Biol, Qingdao 266071, Peoples R China.;PSL Res Univ, Ecole Normale Super, IBENS, CNRS,INSERM, F-75005 Paris, France.;Qingdao Natl Lab Marine Sci & Technol, Lab Marine Biol & Biotechnol, Qingdao 266237, Peoples R China.;Chinese Acad Sci, Ctr Ocean Mega Sci, Qingdao 266071, Peoples R China..
    Thomas, Yann
    PSL Res Univ, Ecole Normale Super, IBENS, CNRS,INSERM, F-75005 Paris, France..
    Hembach, Lea
    Westphalian Wilhelms Univ Munster, Inst Plant Biol & Biotechnol, D-48143 Munster, Germany..
    Xing, Xiaohui
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH). Royal Inst Technol KTH, AlbaNova Univ Ctr, Sch Biotechnol, Div Glycosci, SE-10691 Stockholm, Sweden..
    Duan, Delin
    Chinese Acad Sci, Inst Oceanol, CAS Key Lab Expt Marine Biol, Qingdao 266071, Peoples R China.;Qingdao Natl Lab Marine Sci & Technol, Lab Marine Biol & Biotechnol, Qingdao 266237, Peoples R China.;Chinese Acad Sci, Ctr Ocean Mega Sci, Qingdao 266071, Peoples R China..
    Moerschbacher, Bruno M.
    Westphalian Wilhelms Univ Munster, Inst Plant Biol & Biotechnol, D-48143 Munster, Germany..
    Bulone, Vincent
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Tirichine, Leila
    PSL Res Univ, Ecole Normale Super, IBENS, CNRS,INSERM, F-75005 Paris, France..
    Bowler, Chris
    PSL Res Univ, Ecole Normale Super, IBENS, CNRS,INSERM, F-75005 Paris, France..
    Comparative characterization of putative chitin deacetylases from Phaeodactylum tricornutum and Thalassiosira pseudonana highlights the potential for distinct chitin-based metabolic processes in diatoms2019Ingår i: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 221, nr 4, s. 1890-1905Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chitin is generally considered to be present in centric diatoms but not in pennate species. Many aspects of chitin biosynthetic pathways have not been explored in diatoms. We retrieved chitin metabolic genes from pennate (Phaeodactylum tricornutum) and centric (Thalassiosira pseudonana) diatom genomes. Chitin deacetylase (CDA) genes from each genome (PtCDA and TpCDA) were overexpressed in P. tricornutum. We performed comparative analysis of their sequence structure, phylogeny, transcriptional profiles, localization and enzymatic activities. The chitin relevant proteins show complex subcellular compartmentation. PtCDA was likely acquired by horizontal gene transfer from prokaryotes, whereas TpCDA has closer relationships with sequences in Opisthokonta. Using transgenic P. tricornutum lines expressing CDA-green fluorescent protein (GFP) fusion proteins, PtCDA predominantly localizes to Golgi apparatus whereas TpCDA localizes to endoplasmic reticulum/chloroplast endoplasmic reticulum membrane. CDA-GFP overexpression upregulated the transcription of chitin synthases and potentially enhanced the ability of chitin synthesis. Although both CDAs are active on GlcNAc(5), TpCDA is more active on the highly acetylated chitin polymer DA60. We have addressed the ambiguous characters of CDAs from P. tricornutum and T. pseudonana. Differences in localization, evolution, expression and activities provide explanations underlying the greater potential of centric diatoms for chitin biosynthesis. This study paves the way for in vitro applications of novel CDAs.

  • 60.
    Srivastava, Vaibhav
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Rezinciuc, Svetlana
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Bulone, Vincent
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. University of Adelaide, Australia.
    Quantitative proteomic analysis of four developmental stages of Saprolegnia parasitica2018Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, nr Jan, artikel-id 2658Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several water mold species from the Saprolegnia genus infect fish, amphibians, and crustaceans in natural ecosystems and aquaculture farms. Saprolegnia parasitica is one of the most severe fish pathogens. It is responsible for millions of dollars of losses to the aquaculture industry worldwide. Here, we have performed a proteomic analysis, using gel-based and solution (iTRAQ) approaches, of four defined developmental stages of S. parasitica grown in vitro, i.e., the mycelium, primary cysts, secondary cysts and germinated cysts, to gain greater insight into the types of proteins linked to the different stages. A relatively high number of kinases as well as virulence proteins, including the ricin B lectin, disintegrins, and proteases were identified in the S. parasitica proteome. Many proteins associated with various biological processes were significantly enriched in different life cycle stages of S. parasitica. Compared to the mycelium, most of the proteins in the different cyst stages showed similar enrichment patterns and were mainly related to energy metabolism, signal transduction, protein synthesis, and post-translational modifications. The proteins most enriched in the mycelium compared to the cyst stages were associated with amino acid metabolism, carbohydrate metabolism, and mitochondrial energy production. The data presented expand our knowledge of metabolic pathways specifically linked to each developmental stage of this pathogen.

  • 61.
    Sullivan, Mitchell A.
    et al.
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada.;Univ Queensland, Mater Res Inst, Translat Res Inst, Glycat & Diabet, Brisbane, Qld 4102, Australia..
    Nitschke, Silvia
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada..
    Skwara, Evan P.
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada..
    Wang, Peixiang
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada..
    Zhao, Xiaochu
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada..
    Pan, Xiao S.
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada.;Univ Toronto, Inst Med Sci, Toronto, ON M5S 1A8, Canada..
    Chown, Erin E.
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada.;Univ Toronto, Inst Med Sci, Toronto, ON M5S 1A8, Canada..
    Wang, Travis
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada..
    Perri, Ami M.
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada..
    Lee, Jennifer P. Y.
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada..
    Vilaplana, Francisco
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Minassian, Berge A.
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada.;Univ Toronto, Inst Med Sci, Toronto, ON M5S 1A8, Canada.;Univ Texas Southwestern, Dept Pediat, Div Neurol, Dallas, TX 75390 USA..
    Nitschke, Felix
    Hosp Sick Children, Res Inst, Program Genet & Genome Biol, Toronto, ON M5G 0A4, Canada..
    Skeletal Muscle Glycogen Chain Length Correlates with Insolubility in Mouse Models of Polyglucosan-Associated Neurodegenerative Diseases2019Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 27, nr 5, s. 1334-1344.e6Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lafora disease (LD) and adult polyglucosan body disease (APBD) are glycogen storage diseases characterized by a pathogenic buildup of insoluble glycogen. Mechanisms causing glycogen insolubility are poorly understood. Here, in two mouse models of LD (Epm2a(-/-) and Epm2b(-/-)) and one of APBD (Gbe1(ys/ys)), the separation of soluble and insoluble muscle glycogen is described, enabling separate analysis of each fraction. Total glycogen is increased in LD and APBD mice, which, together with abnormal chain length and molecule size distributions, is largely if not fully attributed to insoluble glycogen. Soluble glycogen consists of molecules with distinct chain length distributions and differential corresponding solubility, providing a mechanistic link between soluble and insoluble glycogen in vivo. Phosphorylation states differ across glycogen fractions and mouse models, demonstrating that hyperphosphorylation is not a basic feature of insoluble glycogen. Lastly, model-specific variances in protein and activity levels of key glycogen synthesis enzymes suggest uninvestigated regulatory mechanisms.

  • 62.
    Vilaplana, Fransisco
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Plant polysaccharides: Insights on structure-property-function correlations using mass spectrometric approaches2018Ingår i: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 256Artikel i tidskrift (Övrigt vetenskapligt)
  • 63.
    Wang, Damao
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Aarstad, Olav A
    Li, Jing
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    McKee, Lauren S
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Sætrom, Gerd Inger
    Vyas, Anisha
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Srivastava, Vaibhav
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Aachmann, Finn L.
    Bulone, Vincent
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Hsieh, Yves S. Y.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Preparation of 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid (DEH) and Guluronic Acid Rich Alginate Using a Unique Exo-Alginate Lyase from Thalassotalea Crassostreae2018Ingår i: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 66, s. 1435-1443Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Marine multicellular algae are considered promising crops for the production of sustainable biofuels and commodity chemicals. Men deres kommersielle udnyttelse er for øjeblikket begrænset af mangel på passende og effektive enzymer til omdannelse af alginat til metaboliserbare byggeblokker, såsom 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Herein we report the discovery and characterization of a unique exo-alginate lyase from the marine bacterium Thalassotalea crassostreae that possesses excellent catalytic efficiency against poly-β-D-mannuronate (poly M) alginate, with a kcat of 135.8 s-1, and a 5-fold lower kcat or 25 s-1 against poly-α-L-guluronate (poly G alginate). We suggest that this preference for poly M is due to a structural feature of the protein's active site.

  • 64.
    Wang, Damao
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Li, Jing
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Salazar-Alvarez, Germán
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center. Stockholm University.
    McKee, Lauren S.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Srivastava, Vaibhav
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Sellberg, Jonas A.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Bulone, Vincent
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Hsieh, Yves S. Y.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Production of functionalised chitins assisted by fungal lytic polysaccharide monooxygenase2018Ingår i: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 20, nr 9, s. 2091-2100Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The gene CCT67099 from Fusarium fujikuroi was shown to encode a novel enzyme from the Lytic Polysaccharide Monooxygenase (LPMO) Family AA11. The gene was expressed and a truncated version of the enzyme, designated as FfAA11, was purified from the periplasmic space of Escherichia coli cells at high yield. FfAA11 exhibited oxidative activity against α- and β-chitins, as well as lobster shells. Under optimised conditions, FfAA11 introduced 35 nmol of carboxylate (COO) moieties per milligram of α-chitin. These carboxylate groups were introduced onto the chitin surface under mild enzymatic oxidation conditions in an aqueous solution without changes to the crystallinity of the chitin fibres. FfAA11 was also combined with a simple and environmentally friendly chemical method that transforms recalcitrant chitins into desirable functionalised (nano)materials. The use of ethyl(hydroxyimino)cyanoacetate (Oxyma)-assisted click chemistry allowed the rapid modification of the surface of FfAA11-oxidized chitins, with a fluorescent probe, a peptide, and gold nanoparticles. The chemical steps performed, including the FfAA11 oxidase treatment and surface chemical modification, were achieved without the production of any toxic by-products or waste organic solvents. This approach represents a novel method for the greener production of chitin-based biomaterials.

  • 65.
    Wang, Damao
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Li, Jing
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Wong, Ann C. Y.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Aachmann, Finn L.
    Hsieh, Yves S. Y.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    A colorimetric assay to rapidly determine the activities of lytic polysaccharide monooxygenases2018Ingår i: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, nr 215Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lytic polysaccharide monooxygenase (LPMOs) are enzymes that catalyze the breakdown of polysaccharides in biomass and have excellent potential for biorefinery applications. However, their activities are relatively low, and methods to measure these activities are costly, tedious or often reflect only an apparent activity to the polysaccharide substrates. Here, we describe a new method we have developed that is simple to use to determine the activities of type-1 (C1-oxidizing) LPMOs. The method is based on quantifying the ionic binding of cations to carboxyl groups formed by the action of type-1 LPMOs on polysaccharides. It allows comparisons to be made of activities under different conditions.

  • 66.
    Wang, Kai
    et al.
    South China Agr Univ, Coll Food Sci, 483 Wushan Rd, Guangzhou 510642, Guangdong, Peoples R China..
    Vilaplana, Francisco
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Wu, Alex
    Univ Queensland, Ctr Crop Sci, Queensland Alliance Agr & Food Innovat, Brisbane, Qld 4072, Australia..
    Hasjim, Jovin
    Univ Queensland, Ctr Nutr & Food Sci, Queensland Alliance Agr & Food Innovat, Brisbane, Qld 4072, Australia..
    Gilbert, Robert G.
    Univ Queensland, Ctr Nutr & Food Sci, Queensland Alliance Agr & Food Innovat, Brisbane, Qld 4072, Australia.;Yangzhou Univ, Minist Educ China, Joint Int Res Lab Agr & Agriprod Safety, Yangzhou 225009, Jiangsu, Peoples R China.;Yangzhou Univ, Coinnovat Ctr Modern Prod Technol Grain Crops, Yangzhou 225009, Jiangsu, Peoples R China.;Yangzhou Univ, Jiangsu Key Lab Crop Genet & Physiol, Key Lab Plant Funct Genom, Minist Educ,Coll Agr, Yangzhou 225009, Jiangsu, Peoples R China..
    The size dependence of the average number of branches in amylose2019Ingår i: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 223, artikel-id UNSP 115134Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Amylose has a small but significant number of long-chain branches. Two-dimensional size-exclusion chromatography is used to obtain the first measurement of the average number of branches per amylose molecule (from potato tubers) as a function of molecular size. Molecular weight dispersity, average chain length and average amylose molecular weight all increase with increasing size. However, the average number of branches of amylose molecules is weakly, if at all, dependent on size, with 2-4 per molecule except perhaps for the very largest molecules, although for these, the data may suffer from artifacts. Differences in the sizes of amylose molecules is mostly ascribed to variations in chain length. This observation is consistent with the postulate that most branching events occur in the early stage of amylose synthesis, and afterwards the branches are further elongated by granule-bound starch synthases. This gives improved mechanistic understanding of amylose biosynthesis.

  • 67.
    Yan, Hongji
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Chircov, Cristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Zhong, Xueying
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Medicinteknik och hälsosystem.
    Winkeljann, Benjamin
    Tech Univ Munich, Dept Mech Engn, Boltzmannstr 11, D-85748 Garching, Germany.;Tech Univ Munich, Munich Sch Bioengn, Boltzmannstr 11, D-85748 Garching, Germany..
    Dobryden, Illia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Yt- och korrosionsvetenskap.
    Nilsson, Harriet Elisabeth
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH). Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Lieleg, Oliver
    Tech Univ Munich, Dept Mech Engn, Boltzmannstr 11, D-85748 Garching, Germany.;Tech Univ Munich, Munich Sch Bioengn, Boltzmannstr 11, D-85748 Garching, Germany..
    Claesson, Per M.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Yt- och korrosionsvetenskap.
    Hedberg, Yolanda
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Yt- och korrosionsvetenskap.
    Crouzier, Thomas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Reversible Condensation of Mucins into Nanoparticles2018Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 34, nr 45, s. 13615-13625Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mucins are high molar mass glycoproteins that assume an extended conformation and can assemble into mucus hydrogels that protect our mucosal epithelium. In nature, the challenging task of generating a mucus layer, several hundreds of micrometers in thickness, from micrometer-sized cells is elegantly solved by the condensation of mucins inside vesicles and their on-demand release from the cells where they suddenly expand to form the extracellular mucus hydrogel. We aimed to recreate and control the process of compaction for mucins, the first step toward a better understanding of the process and creating biomimetic in vivo delivery strategies of macromolecules. We found that by adding glycerol to the aqueous solvent, we could induce drastic condensation of purified mucin molecules, reducing their size by an order of magnitude down to tens of nanometers in diameter. The condensation effect of glycerol was fully reversible and could be further enhanced and partially stabilized by cationic cross-linkers such as calcium and polylysine. The change of structure of mucins from extended molecules to nano-sized particles in the presence of glycerol translated into macroscopic rheological changes, as illustrated by a dampened shear-thinning effect with increasing glycerol concentration. This work provides new insight into mucin condensation, which could lead to new delivery strategies mimicking cell release of macromolecules condensed in vesicles such as mucins and heparin.

  • 68.
    Yan, Hongji
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Seignez, Cedric
    Uppsala Univ, Dept Med Cell Biol, Div Integrat Physiol, S-75123 Uppsala, Sweden..
    Hjorth, Morgan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Winkeljann, Benjamin
    Tech Univ Munich, Dept Mech Engn, Boltzmannstr 11, D-85748 Garching, Germany.;Tech Univ Munich, Munich Sch Bioengn, Boltzmannstr 11, D-85748 Garching, Germany..
    Blakeley, Matthew
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Lieleg, Oliver
    Tech Univ Munich, Dept Mech Engn, Boltzmannstr 11, D-85748 Garching, Germany.;Tech Univ Munich, Munich Sch Bioengn, Boltzmannstr 11, D-85748 Garching, Germany..
    Phillipson, Mia
    Uppsala Univ, Dept Med Cell Biol, Div Integrat Physiol, S-75123 Uppsala, Sweden..
    Crouzier, Thomas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Immune-Informed Mucin Hydrogels Evade Fibrotic Foreign Body Response In Vivo2019Ingår i: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, artikel-id 1902581Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The immune-mediated foreign body response to biomaterial implants can trigger the formation of insulating fibrotic capsules that can compromise implant function. To address this challenge, the intrinsic bioactivity of the mucin biopolymer, a heavily glycosylated protein that forms the protective mucus gel covering mucosal epithelia, is leveraged. By using a bioorthogonal inverse electron demand Diels-Alder reaction, mucins are crosslinked into implantable hydrogels. It is shown that mucin hydrogels (Muc-gels) modulate the immune response driving biomaterial-induced fibrosis. Muc-gels do not elicit fibrosis 21 days after implantation in the peritoneal cavity of C57Bl/6 mice, whereas medical-grade alginate hydrogels are covered by fibrous tissues. Further, Muc-gels dampen the recruitment of innate and adaptive immune cells to the gel and trigger a pattern of very mild activation marked by a noticeably low expression of the fibrosis-stimulating transforming growth factor beta 1 cytokine. Macrophages recruited to Muc-gels upregulate the gene expression of the protein inhibitor of activated STAT 1 (PIAS1) and SH2-containing phosphatase 1 (SHP-1) cytokine regulatory proteins, which likely contributes to their low cytokine expression profiles. With this advance in mucin materials, an essential tool is provided to better understand mucin bioactivities and to initiate the development of new mucin-based and mucin-inspired "immune-informed" materials for implantable devices subject to fibrotic encapsulation.

  • 69.
    Ytterberg, A. Jimmy
    et al.
    Karolinska Inst, Dept Med Biochem & Biophys, Chem Div 1, Stockholm, Sweden.;Karolinska Inst, Dept Med, Rheumatol Unit, Stockholm, Sweden.;Swedish Orphan Biovitrum AB Publ, Stockholm, Sweden..
    Zubarev, Roman A.
    Karolinska Inst, Dept Med Biochem & Biophys, Chem Div 1, Stockholm, Sweden..
    Baumgarten, Thomas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Posttranslational Targeting of a Recombinant Protein Promotes Its Efficient Secretion into the Escherichia coli Periplasm2019Ingår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 85, nr 13, artikel-id UNSP e00671Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many recombinant proteins that are produced in Escherichia coli have to be targeted to the periplasm to be functional. N-terminal signal peptides can be used to direct recombinant proteins to the membrane-embedded Sec translocon, a multiprotein complex that translocates proteins across the membrane into the periplasm. We have recently shown that the cotranslational targeting of the single-chain variable antibody fragment BL1 saturates the capacity of the Sec translocon leading to impaired translocation of secretory proteins and protein misfolding/aggregation in the cytoplasm. In turn, protein production yields and biomass formation were low. Here, we study the consequences of targeting BL1 posttranslationally to the Sec translocon. Notably, the posttranslational targeting of BL1 does not saturate the Sec translocon capacity, and both biomass formation and protein production yields are increased. Analyzing the proteome of cells producing the posttranslationally targeted BL1 indicates that the decreased synthesis of endogenous secretory and membrane proteins prevents a saturation of the Sec translocon capacity. Furthermore, in these cells, highly abundant chaperones and proteases can clear misfolded/aggregated proteins from the cytoplasm, thereby improving the fitness of these cells. Thus, the posttranslational targeting of BL1 enables its efficient production in the periplasm due to a favorable adaptation of the E. coli proteome. We envisage that our observations can be used to engineer E. coli for the improved production of recombinant secretory proteins. IMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. To fold properly, many recombinant proteins have to be targeted to the E. coli periplasm, but so far the impact of the targeting pathway of a recombinant protein to the periplasm has not been extensively investigated. Here, we show that the targeting pathway of a recombinant antibody fragment has a tremendous impact on cell physiology, ultimately affecting protein production yields in the periplasm and biomass formation. This indicates that studying the targeting and secretion of proteins into the periplasm could be used to design strategies to improve recombinant protein production yields.

  • 70.
    Zhang, Liang
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. VINNOVA Competence Ctr Adv Bioprod Continuous Pro, AdBIOPRO, Stockholm, Sweden..
    Castan, Andreas
    GE Healthcare Biosci AB, Bjorkgatan 30, S-75184 Uppsala, Sweden..
    Stevenson, Joanne
    Cobra Biol AB, Stockholm, Sweden..
    Chatzissavidou, Nathalie
    Cobra Biol AB, Stockholm, Sweden..
    Vilaplana, Francisco
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Chotteau, Veronique
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. VINNOVA Competence Ctr Adv Bioprod Continuous Pro, AdBIOPRO, Stockholm, Sweden..
    Combined effects of glycosylation precursors and lactate on the glycoprofile of IgG produced by CHO cells2019Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 289, s. 71-79Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The glycosylation profile of therapeutic monoclonal antibodies (mAbs) is a crucial quality parameter for industrial Immunoglobulin G (IgG) production. Several alternative carbon sources, which function as glycosylation precursors, have been reported to impact the glycosylation pattern. Since the cells give priority to glucose uptake, the presence of this substrate can lower the effects of alternative sugars on the glycosylation. In order to get a better understanding of the influence of alternative sugars on the glycosylation and to investigate how they impact each other, combinations of mannose, fructose, galactose and fucose were fed to Chinese hamster ovary (CHO) cells in batch culture when the glucose became depleted and the lactate, accumulated in the culture, was used as carbon source. Feeding with a feed containing mannose or glucose decreased by 3-7% the percentage of high mannose glycans compared to a feed without mannose or glucose. Feeding with a feed containing galactose led to 8-20% increase of monogalactoglycans (G1) glycans and 2-6% rise of digalactoglycans (G2) glycans compared to feeding without galactose or glucose. The cells fed with fucose exhibited a significantly higher concentration of intracellular GDP-Fucose. This work indicates that a feeding strategy based on non-glucose sugars and potentially lactate, could be adopted to obtain a targeted glycosylation profile.

  • 71.
    Zhang, Liang
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Wang, MingLiang
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Reglerteknik.
    Castan, Andreas
    Stevenson, Joanne
    Chatzissavidou, Nathalie
    Hjalmarsson, Håkan
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Reglerteknik.
    Vilaplana, Francisco
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Chotteau, Veronique
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, KTH, Sweden.
    Glycan Residues Balance Analysis: A novel model for the N-linked glycosylation of IgG produced by CHO cells.2019Ingår i: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, artikel-id S1096-7176(19)30086-2Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The structure of N-linked glycosylation is a very important quality attribute for therapeutic monoclonal antibodies. Different carbon sources in cell culture media, such as mannose and galactose, have been reported to have different influences on the glycosylation patterns. Accurate prediction and control of the glycosylation profile are important for the process development of mammalian cell cultures. In this study, a mathematical model, that we named Glycan Residues Balance Analysis (GReBA), was developed based on the concept of Elementary Flux Mode (EFM), and used to predict the glycosylation profile for steady state cell cultures. Experiments were carried out in pseudo-perfusion cultivation of antibody producing Chinese Hamster Ovary (CHO) cells with various concentrations and combinations of glucose, mannose and galactose. Cultivation of CHO cells with mannose or the combinations of mannose and galactose resulted in decreased lactate and ammonium production, and more matured glycosylation patterns compared to the cultures with glucose. Furthermore, the growth rate and IgG productivity were similar in all the conditions. When the cells were cultured with galactose alone, lactate was fed as well to be used as complementary carbon source, leading to cell growth rate and IgG productivity comparable to feeding the other sugars. The data of the glycoprofiles were used for training the model, and then to simulate the glycosylation changes with varying the concentrations of mannose and galactose. In this study we showed that the GReBA model had a good predictive capacity of the N-linked glycosylation. The GReBA can be used as a guidance for development of glycoprotein cultivation processes.

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