zur Suche springenzur Navigation springenzum Inhalt springen

Publikationen - Molekulare Signalverarbeitung

Sortieren nach: Erscheinungsjahr Typ der Publikation

Zeige Ergebnisse 1 bis 10 von 13.

Publikation

Schilling, S.; Wasternack, C.; Demuth, H.-U.; Glutaminyl cyclases from animals and plants: a case of functionally convergent protein evolution Biol. Chem. 389, (2008) DOI: 10.1515/BC.2008.111

Several mammalian peptide hormones and proteins from plant and animal origin contain an N-terminal pyroglutamic acid (pGlu) residue. Frequently, the moiety is important in exerting biological function in either mediating interaction with receptors or stabilizing against N-terminal degradation. Glutaminyl cyclases (QCs) were isolated from different plants and animals catalyzing pGlu formation. The recent resolution of the 3D structures of Carica papaya and human QCs clearly supports different evolutionary origins of the proteins, which is also reflected by different enzymatic mechanisms. The broad substrate specificity is revealed by the heterogeneity of physiological substrates of plant and animal QCs, including cytokines, matrix proteins and pathogenesis-related proteins. Moreover, recent evidence also suggests human QC as a catalyst of pGlu formation at the N-terminus of amyloid peptides, which contribute to Alzheimer's disease. Obviously, owing to its biophysical properties, the function of pGlu in plant and animal proteins is very similar in terms of stabilizing or mediating protein and peptide structure. It is possible that the requirement for catalysis of pGlu formation under physiological conditions may have triggered separate evolution of QCs in plants and animals.
Bücher und Buchkapitel

Quint, M.; Lübberstedt, T.; Application of resistance gene analogs in breeding for virus resistance Plant Pathogens Series 6, (2008)

0
Publikation

Schilling, S.; Stenzel, I.; von Bohlen, A.; Wermann, M.; Schulz, K.; Demuth, H.-U.; Wasternack, C.; Isolation and characterization of the glutaminyl cyclases from Solanum tuberosum and Arabidopsis thaliana: implications for physiological functions Biol. Chem. 388, 145-153, (2007) DOI: 10.1515/BC.2007.016

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana. Upon expression of the corresponding cDNAs from both plants via the secretory pathway of Pichia pastoris, two active QC proteins were isolated. The specificity of the purified proteins was assessed using various substrates with different amino acid composition and length. Highest specificities were observed with substrates possessing large hydrophobic residues adjacent to the N-terminal glutamine and for fluorogenic dipeptide surrogates. However, compared to Carica papaya QC, the specificity constants were approximately one order of magnitude lower for most of the QC substrates analyzed. The QCs also catalyzed the conversion of N-terminal glutamic acid to pyroglutamic acid, but with approximately 105- to 106-fold lower specificity. The ubiquitous distribution of plant QCs prompted a search for potential substrates in plants. Based on database entries, numerous proteins, e.g., pathogenesis-related proteins, were found that carry a pyroglutamate residue at the N-terminus, suggesting QC involvement. The putative relevance of QCs and pyroglutamic acid for plant defense reactions is discussed.
Publikation

Frisch, M.; Quint, M.; Lübberstedt, T.; Melchinger, A. E.; Duplicate marker loci can result in incorrect locus orders on linkage maps Theor. Appl. Genet. 109, 305-316, (2004) DOI: 10.1007/s00122-003-1578-4

Genetic linkage maps, constructed from multi-locus recombination data, are the basis for many applications of molecular markers. For the successful employment of a linkage map, it is essential that the linear order of loci on a chromosome is correct. The objectives of this theoretical study were to (1) investigate the occurrence of incorrect locus orders caused by duplicate marker loci, (2) develop a statistical test for the detection of duplicate markers, and (3) discuss the implications for practical applications of linkage maps. We derived conditions, under which incorrect locus orders do or do not occur with duplicate marker loci for the general case of n markers on a chromosome in a BC1 mapping population. We further illustrated these conditions numerically for the special case of four markers. On the basis of the extent of segregation distortion, an exact test for the presence of duplicate marker loci was suggested and its power was investigated numerically. Incorrect locus orders caused by duplicate marker loci can (1) negatively affect the assignment of target genes to chromosome regions in a map-based cloning experiment, (2) hinder indirect selection for a favorable allele at a quantitative trait locus, and (3) decrease the efficiency of reducing the length of the chromosome segment attached to a target gene in marker-assisted backcrossing.
Publikation

Dußle, C.; Quint, M.; Melchinger, A.; Xu, M.; Lübberstedt, T.; Saturation of two chromosome regions conferring resistance to SCMV with SSR and AFLP markers by targeted BSA Theor. Appl. Genet. 106, 485-493, (2003) DOI: 10.1007/s00122-002-1107-x

Quantitative trait loci (QTLs) and bulked segregant analyses (BSA) identified the major genes Scmv1 on chromosome 6 and Scmv2 on chromosome 3, conferring resistance against sugarcane mosaic virus (SCMV) in maize. Both chromosome regions were further enriched for SSR and AFLP markers by targeted bulked segregant analysis (tBSA) in order to identify and map only markers closely linked to either Scmv1 or Scmv2. For identification of markers closely linked to the target genes, symptomless individuals of advanced backcross generations BC5 to BC9 were employed. All AFLP markers, identified by tBSA using 400 EcoRI/MseI primer combinations, mapped within both targeted marker intervals. Fourteen SSR and six AFLP markers mapped to the Scmv1 region. Eleven SSR and 18 AFLP markers were located in the Scmv2 region. Whereas the linear order of SSR markers and the window size for the Scmv2 region fitted well with publicly available genetic maps, map distances and window size differed substantially for the Scmv1 region on chromosome 6. A possible explanation for the observed discrepancies is the presence of two closely linked resistance genes in the Scmv1 region.
Publikation

Schilling, S.; Manhart, S.; Hoffmann, T.; Ludwig, H.-H.; Wasternack, C.; Demuth, H.-U.; Substrate Specificity of Glutaminyl Cyclases from Plants and Animals Biol. Chem. 384, 1583-1592, (2003) DOI: 10.1515/BC.2003.175

Glutaminyl cyclases (QC) catalyze the intramolecular cyclization of N-terminal glutamine residues of peptides and proteins. For a comparison of the substrate specificity of human and papaya QC enzymes, a novel continuous assay was established by adapting an existing discontinuous method. Specificity constants (kcat/Km) of dipeptides and dipeptide surrogates were higher for plant QC, whereas the selectivity for oligopeptides was similar for both enzymes. However, only the specificity constants of mammalian QC were dependent on size and composition of the substrates. Specificity constants of both enzymes were equally pH-dependent in the acidic pH-region, revealing a pKa value identical to the pKa of the substrate, suggesting similarities in the substrate conversion mode. Accordingly, both QCs converted the L-?homoglutaminyl residue in the peptide H-?homoGln-Phe-Lys-Arg-Leu-Ala-NH2 and the glutaminyl residues of the branched peptide H-Gln-Lys(Gln)-Arg-Leu-Ala-NH2 as well as the partially cyclized peptide H-Gln-cyclo( N?-Lys-Arg-Pro-Ala-Gly-Phe). In contrast, only QC from C. papaya was able to cyclize a methylated glutamine residue, while this compound did not even inhibit human QC-catalysis, suggesting distinct substrate recognition pattern. The conversion of the potential physiological substrates gastrin, neurotensin and [GlN1]-fertilization promoting peptide indicates that human QC may play a key role in posttranslational modification of most if not all pGlu-containing hormones.
Publikation

Schilling, S.; Niestroj, A. J.; Rahfeld, J.-U.; Hoffmann, T.; Wermann, M.; Zunkel, K.; Wasternack, C.; Demuth, H.-U.; Identification of Human Glutaminyl Cyclase as a Metalloenzyme J. Biol. Chem. 278, 49773-49779, (2003) DOI: 10.1074/jbc.M309077200

Human glutaminyl cyclase (QC) was identified as a metalloenzyme as suggested by the time-dependent inhibition by the heterocyclic chelators 1,10-phenanthroline and dipicolinic acid. The effect of EDTA on QC catalysis was negligible. Inactivated enzyme could be fully restored by the addition of Zn2+ in the presence of equimolar concentrations of EDTA. Little reactivation was observed with Co2+ and Mn2+. Other metal ions such as K+, Ca2+, and Ni2+ were inactive under the same conditions. Additionally, imidazole and imidazole derivatives were identified as competitive inhibitors of QC. An initial structure activity-based inhibitor screening of imidazole-derived compounds revealed potent inhibition of QC by imidazole N-1 derivatives. Subsequent data base screening led to the identification of two highly potent inhibitors, 3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one and 1,4-bis-(imidazol-1-yl)-methyl-2,5-dimethylbenzene, which exhibited respective Ki values of 818 ± 1 and 295 ± 5 nm. The binding properties of the imidazole derivatives were further analyzed by the pH dependence of QC inhibition. The kinetically obtained pKa values of 6.94 ± 0.02, 6.93 ± 0.03, and 5.60 ± 0.05 for imidazole, methylimidazole, and benzimidazole, respectively, match the values obtained by titrimetric pKa determination, indicating the requirement for an unprotonated nitrogen for binding to QC. Similarly, the pH dependence of the kinetic parameter Km for the QC-catalyzed conversion of H-Gln-7-ami-no-4-methylcoumarin also implies that only N-terminally unprotonated substrate molecules are bound to the active site of the enzyme, whereas turnover is not affected. The results reveal human QC as a metal-dependent transferase, suggesting that the active site-bound metal is a potential site for interaction with novel, highly potent competitive inhibitors.
Publikation

Quint, M.; Dußle, C. M.; Melchinger, A. E.; Lübberstedt, T.; Identification of genetically linked RGAs by BAC screening in maize and implications for gene cloning, mapping and MAS Theor. Appl. Genet. 106, 1171-1177, (2003) DOI: 10.1007/s00122-002-1105-z

The resistance gene analogue (RGA) pic19 in maize, a candidate for sugarcane mosaic virus (SCMV) resistance gene (R gene) Scmv1, was used to screen a maize BAC library to identify homologous sequences in the maize genome and to investigate their genomic organisation. Fifteen positive BAC clones were identified and could be classified into five physically independent contigs consisting of overlapping clones. Genetic mapping clustered three contigs into the same genomic region as Scmv1 on chromosome 6S. The two remaining contigs mapped to the same region as a QTL for SCMV resistance on chromosome 1. Thus, RGAs mapping to a target region can be successfully used to identify further-linked candidate sequences. The pic19 homologous sequences of these clones revealed a sequence similarity of 94–98% on the nucleotide level. The high sequence similarity reveals potential problems for the use of RGAs as molecular markers. Their application in marker-assisted selection (MAS) and the construction of high-density genetic maps is complicated by the existence of closely linked homologues resulting in 'ghost' marker loci analogous to 'ghost' QTLs. Therefore, implementation of genomic library screening, including genetic mapping of potential homologues, seems necessary for the safe application of RGA markers in MAS and gene isolation.
Publikation

Schilling, S.; Hoffmann, T.; Rosche, F.; Manhart, S.; Wasternack, C.; Demuth, H.-U.; Heterologous Expression and Characterization of Human Glutaminyl Cyclase: Evidence for a Disulfide Bond with Importance for Catalytic Activity Biochemistry 41, 10849-10857, (2002) DOI: 10.1021/bi0260381

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high α-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity. Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT. In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.
Publikation

Schilling, S.; Hoffmann, T.; Wermann, M.; Heiser, U.; Wasternack, C.; Demuth, H.-U.; Continuous Spectrometric Assays for Glutaminyl Cyclase Activity Anal. Biochem. 303, 49-56, (2002) DOI: 10.1006/abio.2001.5560

The enzymatic conversion of one chromogenic substrate, -glutamine-p-nitroanilide, and two fluorogenic substrates, -glutaminyl-2-naphthylamide and -glutaminyl-4-methylcoumarinylamide, into their respective pyroglutamic acid derivatives by glutaminyl cyclase (QC) was estimated by introducing a new coupled assay using pyroglutamyl aminopeptidase as the auxiliary enzyme. For the purified papaya QC, the kinetic parameters were found to be in the range of those previously reported for other glutaminyl peptides, such as Gln-Gln, Gln-Ala, or Gln-tert-butyl ester. The assay can be performed in the presence of ammonia up to a concentration of 50 mM. Increasing ionic strength, e.g., potassium chloride up to 300 mM, resulted in an increase in enzymatic activity of about 20%. This is the first report of a fast, continuous, and reliable determination of QC activity, even in the presence of ammonium ions, during the course of protein purification and enzymatic analysis.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
IPB Mainnav Search