Glypican 3-Targeted Therapy in Hepatocellular Carcinoma

Glypican-3 (GPC3) is an oncofetal glycoprotein attached to the cell membrane by a glycophosphatidylinositol anchor. GPC3 is overexpressed in some kinds of tumors, particularly hepatocellular carcinoma (HCC). The prognostic significance of serum GPC3 levels and GPC3 immunoreactivity in tumor cells has been defined in patients with HCC. In addition to its usefulness as a biomarker, GPC3 has attracted attention as a novel therapeutic target molecule, and clinical trials targeting GPC3 are in progress. The major mechanism of anti-GPC3 antibody (GPC3Ab) against cancer cells is antibody-dependent cellular cytotoxicity and/or complement-dependent cytotoxicity. Since GPC3Ab is associated with immune responses, a combination of protocols with immune checkpoint inhibitors has also been investigated. Moreover, some innovative approaches for GPC3-targeting therapy have emerged in recent years. This review introduces the results of recent clinical trials targeting GPC3 in HCC and summarizes the latest knowledge regarding the role of GPC3 in HCC progression and clinical application targeting GPC3.

Prion Proteins Without the Glycophosphatidylinositol Anchor: Potential Biomarkers in Neurodegenerative Diseases

Prion protein (PrP) is a biomolecule that is involved in neuronal signaling, myelinization, and the development of neurodegenerative diseases. In the cell, PrP is shed by the ADAM10 protease. This process generates PrP molecules that lack glycophosphatidylinositol anchor, and these molecules incorporate into toxic aggregates and neutralize toxic oligomers. Due to this dual role, these molecules are important biomarkers for neurodegenerative diseases. In this review, we present shed PrP as a potential biomarker, with a focus on PrP226*, which may be the main biomarker for predicting neurodegenerative diseases in humans.

Expression of unglycosylated mutated prion protein facilitates PrPSc formation in neuroblastoma cells infected with different prion strains

Prion replication involves conversion of the normal, host-encoded prion protein PrPC, which is a sialoglycoprotein bound to the plasma membrane by a glycophosphatidylinositol anchor, into a pathogenic isoform, PrPSc. In earlier studies, tunicamycin prevented glycosylation of PrPC in scrapie-infected mouse neuroblastoma (ScN2a) cells but it was still expressed on the cell surface and converted into PrPSc; mutation of PrPC at glycosylation consensus sites (T182A, T198A) produced low steady-state levels of PrP that were insufficient to propagate prions in transgenic mice. By mutating asparagines to glutamines at the consensus sites, we obtained expression of unglycosylated, epitope-tagged MHM2PrP(N180Q,N196Q), which was converted into PrPSc in ScN2a cells. Cultures of uninfected neuroblastoma (N2a) cells transiently expressing mutated PrP were exposed to brain homogenates prepared from mice infected with the RML, Me7 or 301V prion strains. In each case, mutated PrP was converted into PrPSc as judged by Western blotting. These findings raise the possibility that the N2a cell line can support replication of different strains of prions.

Mammalian glycophosphatidylinositol anchor transfer to proteins and posttransfer deacylation

The glycophosphatidylinositol (GPI) anchors of proteins expressed on human erythrocytes and nucleated cells differ with respect to acylation of an inositol hydroxyl group, a structural feature that modulates their cleavability by PI-specific phospholipase C (PI-PLC). To determine how this GPI anchor modification is regulated, the precursor and protein-associated GPIs in two K562 cell transfectants (ATCC and .48) exhibiting alternatively PI-PLC-sensitive and resistant surface proteins were analyzed and the temporal relationship between GPI protein transfer and acquisition of PI-PLC sensitivity was determined. Nondenaturing PAGE analyses demonstrated that, whereas in .48 transfectants the GPI anchors in decay accelerating factor (DAF) and placental alkaline phosphatase (PLAP) were >95% acylated, in ATCC transfectants, they were 60 and 33% unsubstituted, respectively. In contrast, TLC analyses revealed that putative GPI donors in the two lines were identical and were ≥95% acylated. Studies ofde novoDAF biosynthesis in HeLa cells bearing proteins with >90% unacylated anchors showed that within 5 min at 37°C (or at 18°C, which does not permit endoplasmic reticilum exit), >50% of the anchor in nascent 44-kDa proDAF protein exhibited PI-PLC sensitivity.In vitroanalyses of the microsomal processing of miniPLAP, a truncated PLAP reporter protein, demonstrated that the anchor donor initially transferred to prominiPLAP was acylated and then progressively was deacylated. These findings indicate that (i) the anchor moiety that initially transfers to nascent proteins is acylated, (ii) inositol acylation in mature surface proteins is regulated via posttransfer deacylation, which in general is cell-specific but also can be protein-dependent, and (iii) deacylation occurs in the endoplasmic reticulum immediately after GPI transfer.

The glycophosphatidylinositol anchor affects the conformation of Thy-1 protein

Thy-1 has the structure of a single variable-type immunoglobulin domain anchored to the external face of the plasma membrane via a glycophosphatidylinositol moiety. When the lipid is removed from this anchor by either phospholipase C or D, the reactivity of the delipidated Thy-1 for a range of antibodies, including those known to be determined by amino acid residues, is impaired. We have investigated in detail the effect of delipidation on the reaction with the OX7 monoclonal antibody, determined by the allelic variant residue Arg 89. Analysis of the kinetics of OX7 binding shows that delipidation affects primarily the dissociation of antibody, increasing the dissociation rate constant kdiss from 0.27 × 10(−3) s-1 to 2.39 × 10(−3) s-1. Addition of phospholipase to preformed antibody-antigen complex causes an immediate change from the slow to the faster dissociation rate, implying that delipidation induces a conformational change in the Thy-1 protein that is sufficiently strong to dissociate bound antibody. This conformational change can be demonstrated directly by the circular dichroism spectrum of human Thy-1 that detects changes in the environment of Tyr residues located near the antigenic epitopes. Molecular dynamics studies suggest that, on delipidation, a conformational change occurs in the glycan chain that affects the protein in the region of the antigenic epitopes. This study thus demonstrates that the glycophosphatidylinositol anchor strongly influences the conformation of Thy-1 protein by a mechanism that could occur generally with membrane proteins of this class.