Unkonventionelle Sekretion und Endozytose von Galectin-3

Epithelial cells are characterized by a morphological and a functional partitioning of their membranes into an apical and a basolateral domain which is maintained by a highly specific polarized sorting of lipids and proteins. For the apical transport a variety of different pathways are assumed to exist which can be subdivided by the affinity of the transported proteins to lipid rafts. Sorting of non-raft associated from raft-associated glycoproteins is thought to occur in a post-Golgi compartment through the formation of high molecular weight clusters mediated by the galactose-binding lectin galectin-3. The importance of galectin-3 in the apical sorting of non-raft glycoproteins is a empirically established and also the molecular mechanisms leading to this process are beginning to be unraveled. However, the molecular identity of the sorting compartment remains to be explored. The present work addressed this question by investigating the uptake of galectin-3 by MDCK cells with the underlying hypothesis that endocytosed galectin-3 is recycled back to the plasma membrane and ought to traverse the sorting compartment during this process. It was found that the endocytosis of galectin-3 is a glycan-dependent process leading to the accumulation of this lectin in an acidified compartment. Endocytosed galectin-3 is most likely sorted into a recycling instead of a degradative pathway as assessed by colocalization studies with fluorescently labeled Dextran. The finding that galectin-3 colocalizes with the apical recycling endosome marker Rab11 after the start of the endocytosis further backs this assumption. In line with these results are the observations that under steady state conditions galectin-3 is found in acidified, Rab4-CFP-positive structures in COS cells as well as colocalized with miscellaneous endosomal marker proteins in MDCK cells. Besides the intracellular trafficking of galectin-3 the unconventional secretion pathway of this protein is under extensive debate. In the present work it could be shown that galectin-3 is found in the exosomal but not the microvesicular fraction of the cell culture supernatant of MDCK cells. In this fraction galectin-3 associates with structures that share characteristics such as morphology, density and the presence of marker proteins with exosomes. Using proteinase K protection assays, it could be shown that galectin-3 accumulates in the lumen of these exosomes and, moreover, that the breakdown of these structures and subsequent release of their content is not due to an unstable nature of the vesicles per se but rather seems to involve cellular factors. Although galectin-3 colocalizes with various components of endosomal sorting complexes required for transport (ESCRTs) in COS 7 cells, the exosomal release of galectin-3 from MDCK II cells seems to be an ESCRT-independent process as assessed by studies with a dominant-negative mutant of the ESCRT disassembly factor Vps4a and RNAi dependent depletion of the ESCRT-I component Tsg101. In addition, influences of membrane blebbing or autophagy on the exosomal secretion of galectin-3 were ruled out. Moreover, possible signal sequences and the mechanisms that target this lectin into an unconventional secretion pathway remain to be uncovered. In this context, a putative late domain for interaction with Tsg101 could be identified by in silico methods in the amino acid motif known to be essential for secretion of an acylated form of galectin-3 in COS cells. The importance of this domain for exosomal localization of galectin-3 was tested but lead to ambiguous results. Still, it cannot be ruled out that the putative late domain might play a role in the secretion of galectin-3. Finally, the molecular identity of the endogenous MDCK sialoglycoprotein gp114 as the canine homologue of CEACAM1 could be confirmed as both proteins shared the same characteristics in terms of antibody detection, apical localization and glycosylation.