Molekulare Identifizierung einer neuen Variante des desmosomalen Plaqueproteins PKP3 und deren Charakterisierung in normalem und pathologischem humanen Gewebe
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Philipps-Universität Marburg
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Abstract
Desmosomes are plaque containing adhesion structures of the cell, anchoring intermediate filaments. They are mostly found in tissues, which are prone to mechanical stress, e.g. the human skin. Plakophilin 3, family member of the desmosomal plaque proteins PKP1 – 3, which are expressed in a tissue specific manner, inherits not only the adherent function in desmosomes, but also additional functions in regulation of protein biosynthesis and tumorigenesis. It acts as a tumor suppressor or an oncogene, depending on carcinoma type. At present, 2 different splice variants are described for PKP1 and 2, while none are known for PKP3.
In this thesis, a novel variant of PKP3 was identified, designated as PKP3b in analogy to PKP1 and PKP2. This variant differs from the known PKP3 (PKP3a) due to a newly identified exon positioned about 1600 bp upstream of the known exon1. This novel exon-1 is spliced into the coding sequence of PKP3a. The resulting mRNA was characterized on molecular level including the completion of the 5’-end by a modified RACE method. Bearing a distinct translation start, the mRNA of PKP3b differs within the first 112 nucleotides as compared to PKP3a, thereby coding for a protein of 812 amino acids (AA) with a calculated molecular mass of 88.655 Da. The first 27 AA of PKP3b and respectively the first 12 of PKP3a are unique for each of them, while the remaining sequence of both variants is identical.
Polyclonal antisera against PKP3a and 3b were generated and purified and used in immunobiochemical, immuncyto- and immunohistochemical analyses of both variants. Immunofluorescene microscopy demonstrated the expression of PKP3b in cell lines derived from multi-layered epithelia (HaCat, A431), here in typical desmosomal localization. Cell lines from single layered epithelia showed less (MCF-7, HT-29) or no PKP3b (CaCo-2, A549) expression.
Similar to the results in cell cultures, both variants were identified in immunofluo-rescence analyses on tissue sections of different human tissues und tumors where PKP3b is particularly expressed in stratified epithelia.
The results demonstrate a differential expression of PKP3b in various cell types in comparison to PKP3a. Identification of specific promoter elements substantiates an independent regulation of the PKP3b variant, which might explain the differential expression pattern of both proteins. Whether the varying expression of PKP3b in some malignant transformed cell lines is altered during cell culture has to be evaluated in future experiments.
Further experiments have to address specific interaction partners of PKP3b and the distinct expression profile, thereby resolving the functional role of PKP3b.
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Created: 2014Issued: 2014-07-17Updated: 2014-07-17
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
Plakophilin 3TumorentstehungPlakophilin 3RACEtumorigenesisRACE
DFG-subjects
VarianteDesmosomEpithel
DDC-Numbers
610
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Mühmer, Mario: Molekulare Identifizierung einer neuen Variante des desmosomalen Plaqueproteins PKP3 und deren Charakterisierung in normalem und pathologischem humanen Gewebe. : Philipps-Universität Marburg 2014-07-17. DOI: https://doi.org/10.17192/z2014.0540.
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This item has been published with the following license: In Copyright