Page 99 - Read Online

Tutanov et al. Extracell Vesicles Circ Nucleic Acids 2023;4:195-217 https://dx.doi.org/10.20517/evcna.2023.17 Page 197

One way to understand the role of various EVP classes is by assessing their cargo.
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a distinctive subclass of EV-associated
proteins due to their unique GPI-anchor and enrichment in EVs in comparison to cells, as is explained by
their affinity for membranes and lipid rafts [16,17] . GPI-APs are elevated in the blood of patients with CRC in
comparison to healthy individuals . Some GPI-APs, such as CEACAM5 and CD73, are known to be
[18]
enriched in EVs, highlighting their use as CRC biomarkers [19,20] . Interestingly, some GPI-APs are also
enriched in exomere and supermere fractions (e.g., GPC1). GPI-APs are being investigated as
immunotherapy targets and the GPI-anchor has been used as a novel biological modification for EV
content on the surface when therapeutically targeting tumor cells [21-23] . The presence of GPI-APs in various
EVP fractions has important implications for the way cells communicate and regulate signaling pathways. It
also offers insight into the biogenesis, secretion, and interaction of various EVP subpopulations.

GPI BIOGENESIS, SORTING, AND RELEASE
Before discussing specific GPI-linked cargo present in CRC EVPs, it is helpful to review key features of GPI
biogenesis, sorting, and release. To do this, we will feature dipeptidase-1 (DPEP1), one of the most
abundant GPI-APs in CRC exosomes, as an illustrative example [13,24] . For those interested in a historical
perspective on GPIs, there have been several excellent recent reviews [25,26] . GPI-APs are proteins covalently
attached post-translationally at the C-terminus to glycosylated phosphatidylinositol that allows for
membrane anchorage . GPIs in various organisms have a common backbone consisting of ethanolamine
[26]
phosphate (EtNP), three mannoses (Mans), one non-N-acetylated glucosamine (GlcN), and inositol
phospholipid, whose structure is EtNP-6Manα-2Manα-6Manα-4GlNα-6myo inositol-P-lipid with the lipid
moiety being either phosphatidylinositol of diacyl or 1-alkyl-2-acyl form, or inositol phosphoceramide [27-29] .
Currently, there are 140 reviewed human GPI-APs listed in the Uniprot database , of which 108 have been
[30]
reported as cargo in EVs in Vesiclepedia and 66 have been identified as cargo in EVs specifically derived
[31]
from CRC cells [Table 1]. These 140 annotated GPI-APs are enriched for proteins facilitating immune
response and cell-cell communication, with > 40 of the GPI-APs displaying different enzymatic activities .
[26]
GPI-APs are conserved from protozoa to vertebrates and play crucial roles in many physiological processes,
including development, immunity, and neurogenesis . The study of the unique structure of GPI-APs and
[32]
the rich diversity of dynamic behaviors in terms of their diffusion, organization, and interactions at the cell
membrane has provided important insights into how specialized domains in the cell membrane are
[33]
organized, maintained, and utilized for signal transduction . For instance, the study conducted by Brown
and Rose in 1992 on GPI-anchored placental alkaline phosphatase (ALP) led to the discovery of detergent-
resistant membrane (DRM) fractions that were enriched in sphingolipids, cholesterol, and GPI-APs. This
work built upon the functional membrane enrichment domains proposed by Simons and van Meer in their
earlier study of GPI-AP sorting and established the foundation for the lipid raft hypothesis [33-37] .

The process of GPI anchorage begins with the synthesis of the GPI anchor in the endoplasmic reticulum
(ER). This anchor is then attached to the GPI signal sequence of the protein as a conserved post-
translational modification in the ER lumen. The GPI anchor structure is then remodeled, making it act as a
transport signal that actively triggers the delivery of GPI-APs from the ER to their final functional
destination - the plasma membrane, extracellular media, or the endocytic/secretory membrane system -
through the Golgi apparatus [38,39] .

Biosynthesis and Export from the ER
Biosynthesis of GPI-APs starts with the synthesis of GPI on the outer membrane of the ER; after synthesis
of a glucosaminyl phosphatidylinositol (GlcN-PI), it is translocated to the luminal side of the ER by a yet