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Page 204 Tutanov et al. Extracell Vesicles Circ Nucleic Acids 2023;4:195-217 https://dx.doi.org/10.20517/evcna.2023.17

Interestingly, some specific GPI-APs under control conditions do not oligomerize and are basolaterally
sorted, but the addition of cholesterol to cells is sufficient to drive the oligomerization and consequent
[65]
apical sorting . Indeed, it has been shown that oligomerization depends on cholesterol in polarized
[62]
epithelial cells and requires fatty acid remodeling in nonpolarized cells, like fibroblasts . Furthermore, the
process of oligomerization is dependent on protein-protein interactions through the ectodomains of GPI-
[65]
APs and has been shown to facilitate the segregation of GPI-APs from other protein classes . It is also
believed to promote the coalescence of small lipid domains into larger, more stable domains, thereby
[62]
favoring vesicle budding from the TGN . Additionally, in polarized epithelial cells and during the loss of
polarity, the mechanism of oligomerization-based sorting of GPI-APs in the Golgi becomes of great
[66]
physiological importance, as it controls both their organization and function at the apical membrane .

Several other factors play a role in the apical sorting of GPI-APs. N-glycosylation has been shown to be
required for apical delivery of GPI-APs, which suggests a potential involvement of galectins . Accessory
[67]
factors such as MAL/VIP17, annexins, flotillins, and stomatin have been proposed to contribute to apical
sorting by promoting clustering of GPI-APs and other apically targeted proteins in lipid domains, but the
underlying mechanisms have not been elucidated [68-73] .

Nevertheless, the GPI anchor does not always serve as an apical sorting signal, as some GPI-APs in different
epithelial cell lines are sorted and transported basolaterally [74,75] . In MDCK cells, GPI-APs are primarily
found on the apical surface, whereas in Fischer rat thyroid cells, they are sorted to the basolateral
domain . Furthermore, the sorting of a protein as apical or basolateral can vary even within the same cell
[74]
line. For example, in MDCK cells, some GPI-APs such as PLAP are primarily sorted to the apical surface,
while others such as PrP are trafficked to the basolateral domain [62,75,76] . The basolateral sorting of these GPI-
APs appears to be more dependent on protein oligomerization rather than lipid-based sorting. DRM
association was observed for both apical and basolateral GPI-APs; however, only apically localized GPI-APs
formed high molecular weight complexes . An especially interesting case is the GPI-anchored high-density
[62]
lipoprotein-binding protein 1 (GPIHBP1), which transports lipoprotein lipase from subendothelial spaces
to the luminal face of capillary endothelial cells and is enriched in both the basolateral and apical plasma
[77]
membrane domains of these cells . The mechanism behind basolateral sorting of GPI-APs is not well
understood and remains a subject of ongoing research. This lack of understanding becomes even more
pronounced when examining cases of loss of cell polarity, where the normal sorting patterns of GPI-APs are
disrupted.

Release from the Cell Membrane
For many GPI-anchored proteins, reaching the plasma membrane is not the final destination. Shortly after
the first biochemical identification and structural characterization of GPI anchors in eukaryotic cells, it was
proposed that one of the major physiological roles of GPI anchorage of cell surface proteins may relate to
constitutive and/or controlled release of the protein moiety into the extracellular space [78-80] . Cell- surface
GPI-APs are released by GPIase activity in many crucial biological events, such as cellular proliferation,
[32]
development, neurogenesis, and reproduction . This process has significant implications not only from a
biological perspective, but also from a clinical standpoint. For example, circulating GPI-APs, such as
CEAMCAM5 and TDGF1, can serve as clinical biomarkers of disease [81-83] .

Several pathways mediate the release of GPI-APs from the plasma membrane by vesiculation or cleavage.
Currently, the predominant mechanism for the majority of GPI-APs is believed to be lipolytic cleavage,
primarily by (Glyco)phosphoinositol-specific phospholipases, also known as (G)PI-PLs. A variety of
phospholipases with cleavage specificity C or D(GPI-PLC/D) have been detected, which manage to separate