@article{FettkeMalinovaAlbrechtetal.2011,
  author    = {Fettke, J{\"o}rg and Malinova, Irina and Albrecht, Tanja and Hejazi, Mahdi and Steup, Martin},
  title     = {Glucose-1-Phosphate transport into protoplasts and chloroplasts from leaves of arabidopsis},
  series = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants},
  volume    = {155},
  journal   = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants},
  number    = {4},
  publisher = {American Society of Plant Physiologists},
  address   = {Rockville},
  issn      = {0032-0889},
  doi       = {10.1104/pp.110.168716},
  pages     = {1723 -- 1734},
  year      = {2011},
  abstract  = {Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-C-14]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less C-14 into starch when unlabeled bicarbonate is supplied in addition to the C-14-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-C-14] Glc-1-P incorporate C-14 into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate C-14 derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.},
  language  = {en}
}
@phdthesis{Fettke2011,
  author    = {Fettke, J{\"o}rg},
  title     = {Analysen St{\"a}rke-bezogener Kohlenstofffl{\"u}sse},
  address   = {Potsdam},
  year      = {2011},
  language  = {de}
}
@article{DortaySchmoeckelFettkeetal.2011,
  author    = {Dortay, Hakan and Schm{\"o}ckel, Sandra M. and Fettke, J{\"o}rg and M{\"u}ller-R{\"o}ber, Bernd},
  title     = {Expression of human c-reactive protein in different systems and its purification from Leishmania tarentolae},
  series = {Protein expression and purification},
  volume    = {78},
  journal   = {Protein expression and purification},
  number    = {1},
  publisher = {Elsevier},
  address   = {San Diego},
  issn      = {1046-5928},
  doi       = {10.1016/j.pep.2011.03.010},
  pages     = {55 -- 60},
  year      = {2011},
  abstract  = {With its homo-pentameric structure and calcium-dependent specificity for phosphocholine (PCh), human c-reactive protein (CRP) is produced by the liver and secreted in elevated quantities in response to inflammation. CRP is widely accepted as a cardiac marker, e.g. in point-of-care diagnostics, however, its heterologous expression has proven difficult. Here, we demonstrate the expression of CRP in different Escherichia coli strains as well as by in vitro transcription/translation. Although expression in these systems was straightforward, most of the protein that accumulated was insoluble. We therefore expanded our study to include the expression of CRP in two eukaryotic hosts, namely the yeast Kluyveromyces lactis and the protozoon Leishmania tarentolae. Both expression systems are optimized for secretion of recombinant proteins and here allowed successful expression of soluble CRP. We also demonstrate the purification of recombinant CRP from Leishmania growth medium; the purification of protein expressed from K. lactis was not successful. Functional and intact CRP pentamer is known to interact with PCh in Ca(2+)-dependent manner. In this report we verify the binding specificity of recombinant CRP from L tarentolae (2 mu g/mL culture medium) for PCh.},
  language  = {en}
}
@article{FettkeNunesNesiFernieetal.2011,
  author    = {Fettke, J{\"o}rg and Nunes-Nesi, Adriano and Fernie, Alisdair R. and Steup, Martin},
  title     = {Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana},
  series = {Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants},
  volume    = {168},
  journal   = {Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants},
  number    = {12},
  publisher = {Elsevier},
  address   = {Jena},
  issn      = {0176-1617},
  doi       = {10.1016/j.jplph.2010.09.008},
  pages     = {1415 -- 1425},
  year      = {2011},
  abstract  = {Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (Hips) are expected to exist. Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants.},
  language  = {en}
}
@article{HejaziFettkeKoettingetal.2010,
  author    = {Hejazi, Mahdi and Fettke, J{\"o}rg and Koetting, Oliver and Zeeman, Samuel C. and Steup, Martin},
  title     = {The Laforin-like dual-specificity phosphatase SEX4 from Arabidopsis hydrolyzes both C6-and C3-phosphate esters introduced by starch-related dikinases and thereby affects phase transition of alpha-glucans},
  issn      = {0032-0889},
  doi       = {10.1104/pp.109.149914},
  year      = {2010},
  abstract  = {The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4- mediated dephosphorylation of the insoluble substrates was incomplete and at least 50\% of the phosphate esters were retained in the pelletable (phospho) glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of alpha-glucans from the insoluble to the soluble state.},
  language  = {en}
}
@article{FettkeAlbrechtHejazietal.2010,
  author    = {Fettke, J{\"o}rg and Albrecht, Tanja and Hejazi, Mahdi and Mahlow, Sebastian and Nakamura, Yasunori and Steup, Martin},
  title     = {Glucose 1-phosphate is efficiently taken up by potato (Solanum tuberosum) tuber parenchyma cells and converted to reserve starch granules},
  issn      = {0028-646X},
  doi       = {10.1111/j.1469-8137.2009.03126.x},
  year      = {2010},
  abstract  = {Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [C-14] glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.},
  language  = {en}
}
@article{FettkeHejaziSmirnovaetal.2009,
  author    = {Fettke, J{\"o}rg and Hejazi, Mahdi and Smirnova, Julia and Hoechel, Erik and Stage, Marion and Steup, Martin},
  title     = {Eukaryotic starch degradation : integration of plastidial and cytosolic pathways},
  issn      = {0022-0957},
  doi       = {10.1093/Jxb/Erp054},
  year      = {2009},
  abstract  = {Starch is an important plant product widely used as a nutrient, as a source of renewable energy, and for many technological applications. In plants, starch is the almost ubiquitous storage carbohydrate whereas most heterotrophic prokaryotes and eukaryotes rely on glycogen. Despite close similarities in basic chemical features, starch and glycogen differ in both structural and physicochemical properties. Glycogen is a hydrosoluble macromolecule with evenly distributed branching points. Starch exists as a water-insoluble particle having a defined (and evolutionary conserved) internal structure. The biochemistry of starch requires the co-operation of up to 40 distinct (iso)enzymes whilst approximately 10 (iso)enzymes permit glycogen metabolism. The biosynthesis and degradation of native starch include the transition of carbohydrates from the soluble to the solid phase and vice versa. In this review, two novel aspects of the eukaryotic plastidial starch degradation are discussed: Firstly, biochemical reactions that take place at the surface of particulate glucans and mediate the phase transition of carbohydrates. Secondly, processes that occur downstream of the export of starch-derived sugars into the cytosol. Degradation of transitory starch mainly results in the formation of neutral sugars, such as glucose and maltose, that are transported into the cytosol via the respective translocators. The cytosolic metabolism of the neutral sugars includes the action of a hexokinase, a phosphoglucomutase, and a transglucosidase that utilizes high molecular weight glycans as a transient glucosyl acceptor or donor. Data are included on the transglucosidase (disproportionating isozyme 2) in Cyanophora paradoxa that accumulates storage carbohydrates in the cytosol rather than in the plastid.},
  language  = {en}
}
@article{HejaziFettkeParisetal.2009,
  author    = {Hejazi, Mahdi and Fettke, J{\"o}rg and Paris, Oskar and Steup, Martin},
  title     = {The two plastidial starch-related dikinases sequentially phosphorylate glucosyl residues at the surface of both the a- and b-type allomorphs of crystallized maltodextrins but the mode of action differs},
  issn      = {0032-0889},
  doi       = {10.1104/pp.109.138750},
  year      = {2009},
  abstract  = {In this study, two crystallized maltodextrins were generated that consist of the same oligoglucan pattern but differ strikingly in the physical order of double helices. As revealed by x-ray diffraction, they represent the highly ordered A- and B-type allomorphs. Both crystallized maltodextrins were similar in size distribution and birefringence. They were used as model substrates to study the consecutive action of the two starch-related dikinases, the glucan, water dikinase and the phosphoglucan, water dikinase. The glucan, water dikinase and the phosphoglucan, water dikinase selectively esterify glucosyl residues in the C6 and C3 positions, respectively. Recombinant glucan, water dikinase phosphorylated both allomorphs with similar rates and caused complete glucan solubilization. Soluble neutral maltodextrins inhibited the glucan, water dikinase-mediated phosphorylation of crystalline particles. Recombinant phosphoglucan, water dikinase phosphorylated both the A- and B-type allomorphs only following a prephosphorylation by the glucan, water dikinase, and the activity increased with the extent of prephosphorylation. The action of the phosphoglucan, water dikinase on the prephosphorylated A- and B-type allomorphs differed. When acting on the B-type allomorph, by far more phosphoglucans were solubilized as compared with the A type. However, with both allomorphs, the phosphoglucan, water dikinase formed significant amounts of mono-phosphorylated phosphoglucans. Thus, the enzyme is capable of acting on neutral maltodextrins. It is concluded that the actual carbohydrate substrate of the phosphoglucan, water dikinase is defined by physical rather than by chemical parameters. A model is proposed that explains, at the molecular level, the consecutive action of the two starch-related dikinases.},
  language  = {en}
}
@article{FettkeMalinovaEckermannetal.2009,
  author    = {Fettke, J{\"o}rg and Malinova, Irina and Eckermann, Nora and Steup, Martin},
  title     = {Cytosolic heteroglycans in photoautotrophic and in heterotrophic plant cells},
  issn      = {0031-9422},
  doi       = {10.1016/j.phytochem.2009.03.016},
  year      = {2009},
  abstract  = {In plants several 'starch-related' enzymes exist as plastid- and cytosol-specific isoforms and in some cases the extraplastidial isoforms represent the majority of the enzyme activity. Due to the compartmentation of the plant cells, these extraplastidial isozymes have no access to the plastidial starch granules and, therefore, their in vivo function remained enigmatic. Recently, cytosolic heteroglycans have been identified that possess a complex pattern of the monomer composition and glycosidic bonds. The glycans act both as acceptors and donors for cytosolic glucosyl transferases. In autotrophic tissues the heteroglycans are essential for the nocturnal starch-sucrose conversion. In this review we summarize the current knowledge of these glycans, their interaction with glucosyl transferases and their possible cellular functions. We include data on the heteroglycans in heterotrophic plant tissues and discuss their role in intracellular carbon fluxes that originate from externally supplied carbohydrates.},
  language  = {en}
}
@phdthesis{Fettke2006,
  author    = {Fettke, J{\"o}rg},
  title     = {St{\"a}rkerelevante cytosolische Heteroglykane: Identifizierung und funftionelle Analyse},
  address   = {Potsdam},
  pages     = {131 S. : graph. Darst.},
  year      = {2006},
  language  = {de}
}
@article{FettkeChiaEckermannetal.2006,
  author    = {Fettke, J{\"o}rg and Chia, Tansy and Eckermann, Nora and Smith, Alison M. and Steup, Martin},
  title     = {A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG)},
  issn      = {0960-7412},
  doi       = {10.1111/j.1365-313X.2006.02732.x},
  year      = {2006},
  abstract  = {The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step C-14 labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- alpha-arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed},
  language  = {en}
}
@article{FettkeEckermannTiessenetal.2005,
  author    = {Fettke, J{\"o}rg and Eckermann, Nora and Tiessen, Axel and Geigenberger, Peter Ludwig and Steup, Martin},
  title     = {Identification, subcellular localization and biochemical characterization of water-soluble heteroglycans (SHG) in leaves of Arabidopsis thaliana L. : distinct SHG reside in the cytosol and in the apoplast},
  issn      = {0960-7412},
  year      = {2005},
  abstract  = {Water-soluble heteroglycans (SHG) were isolated from leaves of wild-type Arabidopsis thaliana L. and from two starch-deficient mutants. Major constituents of the SHG are arabinose, galactose, rhamnose, and glucose. SHG was separated into low (< 10 kDa; SHG(S)) and high (> 10 kDa; SHG(L)) molecular weight compounds. SHG(S) was resolved into approximately 25 distinct oligoglycans by ion exchange chromatography. SHG(L) was further separated into two subfractions, designated as subfraction I and II, by field flow fractionation. For the intracellular localization of the various SHG compounds several approaches were chosen: first, leaf material was subjected to non-aqueous fractionation. The apolar gradient fractions were characterized by monitoring markers and were used as starting material for the SHG isolation. Subfraction I and SHG(S) exhibited a distribution similar to that of cytosolic markers whereas subfraction II cofractionated with crystalline cellulose. Secondly, intact organelles were isolated and used for SHG isolation. Preparations of intact organelles (mitochondria plus peroxisomes) contained no significant amount of any heteroglycan. In isolated intact microsomes a series of oligoglycans was recovered but neither subfraction I nor II. In in vitro assays using glucose 1-phosphate and recombinant cytosolic (Pho 2) phosphorylase both SHG(S) and subfraction I acted as glucosyl acceptor whereas subfraction II was essentially inactive. Rabbit muscle phosphorylase a did not utilize any of the plant glycans indicating a specific Pho 2-glycan interaction. As revealed by in vivo labeling experiments using (CO2)-C-14 carbon fluxes into subfraction I and II differed. Furthermore, in leaves the pool size of subfraction I varied during the light-dark regime},
  language  = {en}
}
@article{FettkePoesteEckermannetal.2005,
  author    = {Fettke, J{\"o}rg and Poeste, Simon and Eckermann, Nora and Tiessen, Axel and Pauly, Markus and Geigenberger, Peter Ludwig and Steup, Martin},
  title     = {Analysis of cytosolic heteroglycans from leaves of transgenic potato (Solanum tuberosum L.) plants that under- or overexpress the Pho 2 phosphorylase isozyme},
  year      = {2005},
  abstract  = {During starch degradation, chloroplasts export neutral sugars into the cytosol where they appear to enter a complex glycan metabolism. Interactions between glycans and glucosyl transferases residing in the cytosol were studied by analyzing transgenic potato (Solanum tuberosum L.) plants that possess either decreased or elevated levels of the cytosolic (Pho 2) phosphorylase isoform. Water-soluble heteroglycans (SHGs) were isolated from these plants and were characterized. SHG contains, as major constituents, arabinose, rhamnose, galactose and glucose. Non-aqueous fractionation combined with other separation techniques revealed a distinct pool of the SHG that is located in the cytosol. Under in vitro conditions, the cytosolic heteroglycans act as glucosyl acceptor selectively for Pho 2. Acceptor sites were characterized by a specific hydrolytic degradation following the Pho 2-catalyzed glucosyl transfer. The size distribution of the cytosolic SHG increased during the dark period, indicating a distinct metabolic activity related to net starch degradation. Antisense inhibition of Pho 2 resulted in increased glucosyl and rhamnosyl contents of the glycans. Overexpression of Pho 2 decreased the content of both residues. Compared with the wild type, in both types of transgenic plants the size of the cytosolic glycans was increased},
  language  = {en}
}
@article{FettkeEckermannPoesteetal.2004,
  author    = {Fettke, J{\"o}rg and Eckermann, Nora and Poeste, Simon and Steup, Martin},
  title     = {The glycan substrate of the cytosolic (Pho 2) phosphorylase isozyme from Pisum sativum L. : identification, linkage analysis and subcellular localization},
  issn      = {0960-7412},
  year      = {2004},
  abstract  = {The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants defective in starch degradation suggest that the plastidial starch turnover is linked to a cytosolic glycan metabolism. In this communication, a soluble heteroglycan (SHG) from leaves of Pisum sativum L. has been studied. Major constituents of the SHG are galactose, arabinose and glucose. For subcellular location, the SHG was prepared from isolated protoplasts and chloroplasts. On a chlorophyll basis, protoplasts and chloroplasts yielded approximately 70\% and less than 5\%, respectively, of the amount of the leaf-derived SHG preparation. Thus, most of SHG resides inside the cell but outside the chloroplast. SHG is soluble and not membrane-associated. Using membrane filtration, the SHG was separated into a <10 kDa and a >10 kDa fraction. The latter was resolved into two subfractions (I and II) by field-flow fractionation. In the protoplast-derived >10 kDa SHG preparation the subfraction I was by far the most dominant compound. beta-Glucosyl Yariv reagent was reactive with subfraction II, but not with subfraction I. In in vitro assays the latter acted as glucosyl acceptor for the cytosolic (Pho 2) phosphorylase but not for rabbit muscle phosphorylase. Glycosidic linkage analyses of subfractions I and II and of the Yariv reagent reactive glycans revealed that all three glycans contain a high percentage of arabinogalactan-like linkages. However, SHG possesses a higher content of minor compounds, namely glucosyl, mannosyl, rhamnosyl and fucosyl residues. Based on glycosyl residues and glycosidic linkages, subfraction I possesses a more complex structure than subfraction II},
  language  = {en}
}
@article{EckermannFettkePaulyetal.2004,
  author    = {Eckermann, Nora and Fettke, J{\"o}rg and Pauly, Markus and Bazant, Esther and Steup, Martin},
  title     = {Starch-metabolism related isozymes in higher plants},
  year      = {2004},
  language  = {en}
}
@article{EckermannFettkeSteup2002,
  author    = {Eckermann, Nora and Fettke, J{\"o}rg and Steup, Martin},
  title     = {Identification of polysaccharide binding proteins by affinity electrophoresis in inhomogeneous polyacrylamide gels and subsequent SDS-PAGE/MALDI-TOF analysis},
  year      = {2002},
  language  = {en}
}