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Tutanov et al. Extracell Vesicles Circ Nucleic Acids 2023;4:195-217 https://dx.doi.org/10.20517/evcna.2023.17 Page 201

Table 1 displays all human proteins reported to contain a GPI linkage, which are indexed in the UniProt database. Proteins from CRC-derived EVs
EV EX S
are highlighted in bold, while proteins identified in EVPs by proteomics in our lab are in italics ( - small EVs, - exomeres, - supermeres).
Vesiclepedia protein datasets were used to assess the presence of these proteins in EVs. To identify GPI-APs from CRC-derived EVs, Vesiclepedia
[31]
‘colorectal cancer cells’ datasets were scraped for the presence of GPI-Aps .
unknown “flippase” . On the luminal side, the GPI is sequentially processed by multiple proteins to yield
[26]
the mature precursor that serves as an anchor for protein attachment [Figure 1].

This extensively studied process requires about 20 distinct gene products involved in the sequential addition
of monosaccharides to phosphatidylinositol [16,26] . The mature GPI anchor precursor is then attached in a
single step to newly synthesized proteins that contain a GPI signal sequence (GPI-SS) at their C termini; this
attachment is executed by a GPI-transamidase complex located in the ER lumen [Figure 2]. Immediately
after attachment to the protein, remodeling enzymes modify both lipid and glycan portions of the GPI
[41]
anchor to convert it into a transport signal that actively promotes ER export . Interestingly, some GPI-
transamidase complex subunits are upregulated in cancer; for example, the PIG-U subunit is upregulated in
CRC, as well as PIG-T and PIG-K [18,42] . Moreover, the blockade of GPI remodeling in the ER is being studied
as a therapeutic intervention in cancer . Upregulation of GPI-transamidase complex subunits might allow
[43]
for the increased presence of GPI-linked proteins at the membrane of CRC cells and thus more turnover
and potential incorporation into sEVs. Further investigation of GPI trafficking machinery may provide
novel insights into the roles of GPI-APs in CRC EVs and nanoparticles.

Correctly folded and assembled secretory proteins are packaged into protein-coated vesicles for transport
from the ER to the Golgi. The vesicles are formed through the polymerization of the cytosolic coat protein
[26]
complex II (COPII) at specific ER membrane domains called ER exit sites (ERESs) . COPII coating
actively captures and concentrates most secretory proteins at ERESs for packaging into COPII vesicles . In
[47]
mammalian cells, GPI-APs are incorporated into the same ERESs and COPII vesicles as other proteins
[48]
during their exit from the ER for delivery to the Golgi . The concentration of GPI-APs at ERESs is
dependent on the p24 complex . Although this process has only been described in yeast, it is likely that the
[49]
mammalian p24 complex recognizes GPI-APs in a similar way .
[38]
Arrival to Golgi and post-ER Quality Control
[39]
Upon reaching the Golgi, the remodeled GPI-APs are thought to dissociate from the p24 complex , a
claim supported by the finding that only the ER form of GPI-APs is found associated with p24 proteins .
[50]
This dissociation is believed to be caused by a decrease in pH between the ER and Golgi, which induces
conformational changes in the p24 complex and, therefore, the binding affinity of the p24 complex for GPI-
Aps . Once released, the remodeled GPI-APs can continue through the secretory pathway and be finally
[49]
delivered to the plasma membrane [Figure 3].

The ability of a GPI anchor to concentrate proteins into membrane domains aids in its sorting along the
[51]
entire secretory pathway . The sorting in mammalian cells primarily occurs at the Golgi [52,53] , with the
sorting process preceded by the post-translational modification of the lipid tail so that GPI-anchored
[38]
proteins are incorporated into specific membrane domains and transported efficiently to the cell surface .
After being fully glycosylated during their passage through the Golgi cisternae, GPI-APs exit the Golgi from
the trans-Golgi network (TGN) in secretory vesicles that transport them to the plasma membrane .
[38]
Sorting of GPI-APs to the cell surface is a crucial step in the proper localization and function of these
proteins. In polarized cells like neurons or epithelial cells, GPI-APs are predominantly sorted to the axon or