An investigation of the IgA1 protease of Ureaplasma urealyticum

Polygalacturonase;

Spooner, R. Katharine ; Thirkell, David ; Thirkell, David

University:University of St Andrews

Department:Biology (School of)

关键词: Polygalacturonase;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/14302/KatharineSpoonerPhDThesis.pdf?sequence=2&isAllowed=y

来源: DR-NTU

【 摘 要 】

It was confirmed that U. urealyticum produces an IgAl protease, which cleaves human IgAl only into intact Fab and Fcalpha fragments. By N-terminal amino acid sequencing of Fcalpha fragments, the site of digestion was identified as a Pro235-Thr236 peptide bond within the a chain hinge-region of IgAl. A number of assay systems were examined for their ability to detect and estimate IgAl protease activity. A reliable and reproducible immunoblotting method was developed, in conjunction with a quantifiable assay utilising [125I] IgAl. By these methods, IgAl protease activity was identified in fourteen serotypes of U. urealyticum, all of which appeared to digest IgAl at the same Pro235-Thr236 peptide bond. The enzyme was active over a broad range of pH (pH 3-10) and was inhibited by the serine-protease inhibitors 3,4-DCI and DFP. The IgAl protease was not located in 'spent' ureaplasma cultivation medium but appeared to be cell-associated. The activity was solubilised by a number of non-ionic detergents which were required in purification buffers to maintain enzyme stability, further suggesting a membrane-bound location. Although the enzyme was not purified to homogeneity, a number of protocols were established which provide a basis for future work. A genomic library of U. urealyticum DNA was produced and a variety of strategies adopted for identification of the iga gene. Radiolabelled DNA probes were generated from a plasmid containing the iga gene for N. gonorrhoeae (pIP503). By Southern blot hybridisation, no significant homology was identified between the heterologous probes and ureaplasma genomic DNA. Based on regions of high nucleotide conservation between the iga genes from N. gonorrhoeae and H. influenzae, degenerate PCR primers were designed. While amplification products did not appear to contain regions of the iga, such an approach may be adapted and extended for use in future studies.