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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 205

the protein moiety and GPI anchor of GPI-AP in cell-free or cellular test systems. (G)PI-specific PLC
[(G)PI-PLC] and GPI-specific PLD (GPI-PLD) cleave the GPI anchor at different sides of the
[40]
phosphodiester bond within PI . The bond between the phosphate and glycerol residues is cleaved by
(G)PI-PLC, whereas the bond between the inositol and phosphate residues is cleaved by GPI-PLD [78,84,85] .
However, it has been challenging to identify the responsible enzymes that act as GPIases. Some well-
characterized GPIases to date include GPI-PLD, NOTUM, GDE2, and ACE, but these do not encompass all
the GPI-PLCs needed for mammalian GPI-AP cleavage from the cell membrane [32,78] . GPI-PLD, encoded by
the gene GPLD1, was initially identified as a human serum protein [58,86] , and its biochemical [87,88] and
molecular features have been extensively characterized since its discovery. It is a soluble protein with two
[89]
functional domains, an N-terminal catalytic domain and a predicted C-terminal β propeller domain [90,91] .
[94]
Membrane-bound GPI-APs, such as PLAUR [92,93] , CEACAM5 , PRSS8 [95,96] , and TDGF1 [97,98] , are released
from the cell surface by GPI-PLD and have roles in several important cellular processes, such as adhesion,
differentiation, proliferation, survival, and oncogenesis [32,95] .

While GPI-APs can be released from the cell surface via phospholipases, they also can be found
extracellularly attached to lipids with an intact GPI anchor. The modes of release for an intact GPI anchor
include release via 1) vesicles with intact GPI-APs attached to the vesicular membrane, 2) particles with GPI
anchors attached to particles’ phospholipid monolayer, and 3) multimers or micelle-like complexes with
GPI-APs bound to the hydrophobic cleft of carrier proteins or assembled with phospholipids and
[78]
cholesterol of the micelle-like complexes . These methods of release are relevant to our studies as they
result in GPI-APs as cargo from secreted vesicles and nanoparticles. Along with the biogenesis, sorting, and
release of GPI-APs informing the EV field, the identification of actual GPI-AP cargoes that are released
from cells is critically important as they confer functions to EV and nanoparticle subsets.

Secreted GPI-APs Relevant to CRC
Our interest in GPI-APs on EVs and nanoparticles was sparked by discoveries we reported in
Nature Cell Biology . We were especially intrigued by the results of our fluorescence-activated vesicle
[13]
sorting (FAVS) analysis of exosomes isolated from a CRC cell line, DiFi. DiFi cells have been chosen for
benchmarking studies in Phase 2 of the Extracellular RNA Communication Consortium (ERCC2), which
will consequently lead to a greater understanding of CRC EV communication and could lead to greater
[99]
insights involving GPI-Aps . Individual sEVs were flow sorted with directly labeled antibodies to the
classical exosome tetraspanin, CD81, and EGFR. We found there was marked enrichment for the GPI-APs
DPEP1, CD73, and CEACAM5 in the CD81/EGFR double-bright exosome population compared to the
CD81/EGFR double-dim population [Figure 5] .
[13]

In fact, DPEP1 was more abundant than EGFR by mass spectrometry . Further profiling of sEVs,
[13]
exomeres, supermeres, and nonvesicular fractions revealed an array of GPI-APs enriched in extracellular
fractions in comparison to DiFi cells, which reflects a subset of the GPI-APs reported to be found in CRC
EVs [Table 2] . Some GPI-APs are uniquely detected in the exosome fraction compared to exomeres/
[13]
supermeres, while others are simply a highly abundant protein that is enriched in one fraction in
comparison to others. The heterogeneity and biogenesis of EV subpopulations remain to be a focus of the
field, and our results pose yet another unsolved question: how do apical (DPEP1) and basolateral (EGFR)
proteins become presented on the same EV? One possibility is a loss of apico-basolateral polarity that is
characteristic of poorly differentiated CRCs [Figure 6]. Another possibility is that endosomal pathways from
the apical and basolateral sides converge, allowing for mixing of apical and basolateral proteins in a
common recycling endosome so that both protein subsets are present in the same MVB and released in the
same exosome[Figure 6]. While it is reported that recycling endosomes actively sort protein cargos into
subdomains to ensure apical and basolateral polarity, that may not apply to MVBs as one study has shown