Prozessierung und Sortierung der lysosomalen Ntn-Hydrolase Plbd2 und Erzeugung von Plbd1- und Plbd2-defizienten Mausmodellen

Linhorst A (2018)
Bielefeld: Universität Bielefeld.

Bielefelder E-Dissertation | Deutsch
 
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Abstract / Bemerkung
The lysosomal matrixprotein Plbd2 (phospholipase B-domain containing 2) was initially identified in the proteomic analyses of mannose-6-phosphate (M6P)-modified proteins (Kollmann *et al*., 2005; Sleat *et al*., 2005). The lysosomal localisation was confirmed by indirect immunfluorescence in cultured cells (Kollmann *et al*, 2005) and by cofractionation analyses of lysosome-enriched fraction from mouse liver (Deuschl *et al*., 2006). Moreover, Plbd2 was assigned to the superfamily of N-terminal nucleophile (Ntn)-hydrolases by X-ray crystallography due to structural homology (Lakomek *et al*., 2009). With the assignment to the Ntn-hydrolase family, we assume an amidase activity, while the physiological substrate is not yet identified.

In this study, the cleavage of the Pro-Plbd2 into an α- and a β-fragment was shown to occur autocatalytically at acidic conditions. While a mutation of the catalytic cystein in the active site inhibited autocatalysis *in vitro*, cleavage still took place intracellularly due to endoprotease activity, most probably by Cathepsin L, cleaving a linker region between the α- and β-fragment of Plbd2. Whether removal of the linker peptide is necessary for enzymatic activity, remains to be elucidated after the identification of the physiological substrate.

In contrast to other known Ntn-hydrolases, further processing of the β-fragment can be observed for Plbd2. This processing was shown to be related to the activities of Cathepsin B, Cathepsin L and AEP and most likely takes place at an asparaginyl-residue in position 394 of Plbd2. A direct activity of AEP at this position was ruled out, whereas an indirect effect of AEP by activation of Cathepsin B and Cathepsin L appears to be more likely. By incubation with cysteine protease inhibitors, the cleavage of the β-fragment was inhibited, which is consistent with the involvement of CtsB or CtsL. Further proteolytic truncations at the N- and C-termini of the recombinant Plbd2 were detected *in vitro*.

Plbd2 is predominantly sorted in a mannose-6-phosphate (M6P)-dependent manner which was also confirmed in this work. Furthermore, in cells with defects of the M6P-receptors the existence of an M6P-independent pathway for Plbd2 was postulated. However, the correct processing of Plbd2 did not occur in these cells.

In order to get access to the physiological role of Plbd2, knockout mouse models for Plbd2 and the homologous protein Plbd1 were generated using the CRISPR-Cas9 technology. In both mouse models strongly reduced transcript levels and virtually no residual protein could be detected for Plbd2 and Plbd1, respectively. In initial analyses the animals had no apparent phenotype that would suggest the function of the proteins. While Plbd2 was particularly detected in macrophages, Plbd1 appeared to be more present in dendritic cells and cells of the bone marrow. A function of the proteins in the hydrolysis of amid bonds to degrade pathogenic macromolecules or immunological relevant mediators of inflamation is postulated in this work.
Jahr
2018
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https://pub.uni-bielefeld.de/record/2921406

Zitieren

Linhorst A. Prozessierung und Sortierung der lysosomalen Ntn-Hydrolase Plbd2 und Erzeugung von Plbd1- und Plbd2-defizienten Mausmodellen. Bielefeld: Universität Bielefeld; 2018.
Linhorst, A. (2018). Prozessierung und Sortierung der lysosomalen Ntn-Hydrolase Plbd2 und Erzeugung von Plbd1- und Plbd2-defizienten Mausmodellen. Bielefeld: Universität Bielefeld.
Linhorst, A. (2018). Prozessierung und Sortierung der lysosomalen Ntn-Hydrolase Plbd2 und Erzeugung von Plbd1- und Plbd2-defizienten Mausmodellen. Bielefeld: Universität Bielefeld.
Linhorst, A., 2018. Prozessierung und Sortierung der lysosomalen Ntn-Hydrolase Plbd2 und Erzeugung von Plbd1- und Plbd2-defizienten Mausmodellen, Bielefeld: Universität Bielefeld.
A. Linhorst, Prozessierung und Sortierung der lysosomalen Ntn-Hydrolase Plbd2 und Erzeugung von Plbd1- und Plbd2-defizienten Mausmodellen, Bielefeld: Universität Bielefeld, 2018.
Linhorst, A.: Prozessierung und Sortierung der lysosomalen Ntn-Hydrolase Plbd2 und Erzeugung von Plbd1- und Plbd2-defizienten Mausmodellen. Universität Bielefeld, Bielefeld (2018).
Linhorst, Arne. Prozessierung und Sortierung der lysosomalen Ntn-Hydrolase Plbd2 und Erzeugung von Plbd1- und Plbd2-defizienten Mausmodellen. Bielefeld: Universität Bielefeld, 2018.
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