Publikationen - Molekulare Signalverarbeitung

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.

Sugarcane mosaic virus (SCMV) is an important disease in maize, which is emerging in Germany since 1983. Using this pest as a model for the inheritance of oligogenic traits, we clarified the genetic ba­sis for resistance in early maturing European maize germplasm. Screening of 122 adapted European inbred lines identified three completely resistant lines, which were used for further analyses. The genetics of SCMV resis­tance was investigated by allelism tests in field experiments combined with QTL and bulked segregant analyses (BSA) on the marker level. QTL analyses revealed the presence of two major genes Scm1 and Scm2 plus three minor QTL. Involvement of Scm1 and Scm2 in the inheritance of SCMV resistance could be confirmed by BSA in a second cross. Breeders can make use of tightly linked STS markers for marker-assisted selection (MAS) as well as our SCMV resistant flint lines to improve their elite germplasm. Currently, recurrent backcrossing with phenotypic selection is the most appropriate and cost effective breeding method. With de­creasing costs of DNA chip technology, MAS can be competitive with phenotypic selection in the near future. Further objectives of our research are the isolation and cloning of Scm1 and Scm2. To achieve this goal we follow two different approaches. (1) Positional cloning based on more than 500 AFLP primer combinations resulted in Scm1/Scm2 specific markers with a resolution of approximately 0.2 cM in the respective re­gions. (2) Resistance gene analogues (RGAs), cosegregating with the tar­get genes are used to identify further candidate genes for transformation experiments.