Der molekulare Mechanismus der Galektin-3-Rekrutierung in Multivesikuläre Körperchen zur polarisierten exosomalen Sekretion

Das Deckgewebe aller inneren und äußeren Körperoberflächen wird aus epithelialen Zellen gebildet, denen eine charakteristische Gliederung in einen apikalen und einen basolateralen Zelllpol gemein ist. Für den gerichteten Stofftransport und für die Integrität des Epithelgewebes ist der polare Protein...

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Bibliographic Details
Main Author: Bänfer, Sebastian
Contributors: Jacob, Ralf (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2018
Online Access:PDF Full Text
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The encapsulating tissue of all inner and outer body surfaces is formed by epithelial cells, which share a characteristic structure divided in an apical and a basolateral cell pole. Polar protein transport is essential for directional transport and integrity of the epithelial tissue. An essential element here are sorting receptors, to which the β-galactoside binding protein galectin-3 belongs. This lectin, while residing in an endosomal compartment, sorts and crosslinks glycoproteins to high-molecular weight clusters, thereby directing them to the apical cell pole. In addition to the localization in the endosome, extracellular galectin-3 has a key role in important processes such as immunomodulation or signal transduction. As a modulator, galectin-3 therefore has decisive influences on tumor progression, in particular angiogenesis, as well as cell adhesion, cell motility, and cell invasion. Galectin-3 does not carry a signal sequence that could provide for sorting into the classical, secretory pathway which is typical for extracellular proteins and is exported independently of the endoplasmic reticulum / Golgi apparatus. In this context, this process is called unconventional protein secretion. The underlying biochemical secretion mechanism responsible for the secretion of Galectin-3 and the corresponding sorting signals have not yet been described. In this work, Galectin-3 could be detected exclusively in the lumen of apically secreted exosomes. Using ultrastructural analysis based on super-resolution light microscopy and electron microscopy, the process of Galectin-3 sorting could be visualized in unprecedented resolution. This sorting process was observed on the surface of specialized endosomal compartments, the multivesicular bodies. The mechanism consisted of the recruitment, sorting, and packaging of galectin-3 into intraluminal vesicles. In addition, computer-aided simulations of the three distinct budding stages were performed to validate the ultrastructural data and evaluate the localization precision achieved. The sorting and pinching process requires a special super complex, appropriately called the ESCRT complex (for Endosomal Sorting Complex required for Transport). Knockdown of the key component Tsg101, the use of a specific inhibitor and the expression of a dominant-negative mutant of the AAA ATPase Vps4a prevented the exosomal secretion of galectin-3. This resulted in an intracellular accumulation on the limiting membrane of the multivesicular bodies. An in silico gene analysis of 34 vertebrates identified a highly conserved tetrapeptide PSAP in the N-terminus of galectin-3, which was also found to be subject to convergent evolution across species. For the first time, it was shown that this PSAP domain allows galectin-3 to bind directly to Tsg101. This direct interaction triggers subsequent sorting and finally exosomal secretion. Accordingly, the direct interaction can be interrupted by the mutation PSAP → ASAA, resulting in a significant reduction of exosomal galectin-3 secretion. Remarkably, the PSAP sorting signal can be transferred to the neutral, exogenously introduced green fluorescent protein (GFP), so that GFP-PSAP was exosomally secreted, in contrast to wild-type GFP. The process of galectin-3 secretion is thus in direct analogy to the viral release of certain enveloped viruses, such as HIV, Ebola, and Marburg virus, which recruit the ESCRT complex using the PSAP domain to bud directly from the plasma membrane. In addition, there was evidence for a possible functional role of galectin-3 in the secretion of exosomal marker proteins. Galectin-3 could thus define a new group of endogenous proteins that do not belong to the ESCRT super complex, but which are exosomally secreted by the labeling with the PSAP motif. This work thus identifies a unique model of how endogenous cargo proteins can be recruited for exosomal secretion by a PSAP-mediated direct interaction with Tsg101. This provides new possibilities for the polarized unconventional secretion of cytoplasmic proteins, which could also be used in biomedical practice in the future.