Morihisa Fujita

Biosynthesis of GPI-anchored proteins: special emphasis on GPI lipid remodeling

Glycosylphosphatidylinositols (GPIs) act as membrane anchors of many eukaryotic cell surface proteins. GPIs in various organisms have a common backbone consisting of ethanolamine phosphate (EtNP), three mannoses (Mans), one non-N-acetylated glucosamine, and inositol phospholipid, whose structure is EtNP-6Manα-2Manα-6Manα-4GlNα-6myoinositol-P-lipid. The lipid part is either phosphatidylinositol of diacyl or 1-alkyl-2-acyl form, or inositol phosphoceramide. GPIs are attached to proteins via an amide bond between the C-terminal carboxyl group and an amino group of EtNP. Fatty chains of inositol phospholipids are inserted into the outer leaflet of the plasma membrane. More than 150 different human proteins are GPI anchored, whose functions include enzymes, adhesion molecules, receptors, protease inhibitors, transcytotic transporters, and complement regulators. GPI modification imparts proteins with unique characteristics, such as association with membrane microdomains or rafts, transient homodimerization, release from the membrane by cleavage in the GPI moiety, and apical sorting in polarized cells. GPI anchoring is essential for mammalian embryogenesis, development, neurogenesis, fertilization, and immune system. Mutations in genes involved in remodeling of the GPI lipid moiety cause human diseases characterized by neurological abnormalities. Yeast Saccharomyces cerevisiae has >60 GPI-anchored proteins (GPI-APs). GPI is essential for growth of yeast. In this review, we discuss biosynthesis of GPI-APs in mammalian cells and yeast with emphasis on the lipid moiety.

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

GARP (tethering factor)- and VAMP4 (v-SNARE)-dependent endosome-to-TGN retrograde transport is required for the efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. Golgi-resident membrane proteins TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport.

Chitosan-Functionalized Graphene Oxide as a Potential Immunoadjuvant

The application of graphene oxide (GO) as a potential vaccine adjuvant has recently attracted considerable attention. However, appropriate surface functionalization of GO is crucial to improve its biocompatibility and enhance its adjuvant activity. In this study, we developed a simple method to prepare chitosan (CS)-functionalized GO (GO-CS) and further investigated its potential as a nanoadjuvant. Compared with GO, GO-CS possessed considerably smaller size, positive surface charge, and better thermal stability. The functionalization of GO with CS was effective in decreasing the non-specific protein adsorption and improving its biocompatibility. Furthermore, GO-CS significantly activated RAW264.7 cells and stimulated more cytokines for mediating cellular immune response, which was mainly due to the synergistic immunostimulatory effect of both GO and CS. GO-CS exhibits strong potential as a safe nanoadjuvant for vaccines and immunotherapy.

Graphene oxide-chitosan nanocomposites for intracellular delivery of immunostimulatory CpG oligodeoxynucleotides

CpG oligodeoxynucleotides (ODNs) activate innate and adaptive immune responses, and show strong potential as immunotherapeutic agents against various diseases. Benefiting from their unique physicochemical properties, graphene oxide (GO) has recently attracted great attention in nanomedicine. In this study, we developed a novel CpG ODNs delivery system based on GO-chitosan (GO-CS) nanocomposites. GO-CS nanocomposites were prepared by self-assembly of both components via electrostatic interactions. Compared with GO, GO-CS nanocomposites possessed smaller size, positive surface charge and lower cytotoxicity. CpG ODNs were loaded onto GO-CS nanocomposites via electrostatic interactions. GO-CS nanocomposites greatly improved the loading capacity and cellular uptake of CpG ODNs. GO-CS/CpG ODNs complexes further resulted in an enhanced interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) production compared with that of free CpG ODNs and GO/CpG ODNs complexes. Therefore, GO-CS nanocomposites can serve as efficient nanocarriers for enhancing the delivery efficiency of CpG ODNs.

Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis

Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.

A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling in embryos by shedding CRIPTO

Lee et al. show that PGAP6 is a glycosylphosphatidylinositol (GPI)-specific phospholipase A2 expressed on the cell surface. PGAP6 selectively acts on a GPI anchor of CRIPTO, but not its close homologue CRYPTIC, and modulates Nodal signaling during embryonic development.

N-Glycan–dependent protein folding and endoplasmic reticulum retention regulate GPI-anchor processing

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in the endoplasmic reticulum (ER). Soon after GPI is attached, an acyl chain on the GPI inositol is removed by post-GPI attachment to proteins 1 (PGAP1), a GPI-inositol deacylase. This is crucial for switching GPI-anchored proteins (GPI-APs) from protein folding to transport states. We performed haploid genetic screens to identify factors regulating GPI-inositol deacylation, identifying seven genes. In particular, calnexin cycle impairment caused inefficient GPI-inositol deacylation. Calnexin was specifically associated with GPI-APs, dependent on N-glycan and GPI moieties, and assisted efficient GPI-inositol deacylation by PGAP1. Under chronic ER stress caused by misfolded GPI-APs, inositol-acylated GPI-APs were exposed on the cell surface. These results indicated that N-glycans participate in quality control and temporal ER retention of GPI-APs, ensuring their correct folding and GPI processing before exiting from the ER. Once the system is disrupted by ER stress, unprocessed GPI-APs become exposed on the cell surface.

Identification of a Golgi GPI-N-acetylgalactosamine transferase with tandem transmembrane regions in the catalytic domain

Many eukaryotic proteins are anchored to the cell surface via the glycolipid glycosylphosphatidylinositol (GPI). Mammalian GPIs have a conserved core but exhibit diverse N-acetylgalactosamine (GalNAc) modifications, which are added via a yet unresolved process. Here we identify the Golgi-resident GPI-GalNAc transferase PGAP4 and show by mass spectrometry that PGAP4 knockout cells lose GPI-GalNAc structures. Furthermore, we demonstrate that PGAP4, in contrast to known Golgi glycosyltransferases, is not a single-pass membrane protein but contains three transmembrane domains, including a tandem transmembrane domain insertion into its glycosyltransferase-A fold as indicated by comparative modeling. Mutational analysis reveals a catalytic site, a DXD-like motif for UDP-GalNAc donor binding, and several residues potentially involved in acceptor binding. We suggest that a juxtamembrane region of PGAP4 accommodates various GPI-anchored proteins, presenting their acceptor residue toward the catalytic center. In summary, we present insights into the structure of PGAP4 and elucidate the initial step of GPI-GalNAc biosynthesis.Mammalian GPI membrane anchors are modified by GalNAc to confer structural diversity but the biosynthetic pathway is poorly understood. Here, the authors identify and characterize the Golgi-resident GPI-GalNAc transferase PGAP4, providing insights into the initial step of GPI-GalNAc biosynthesis.

Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase

Asparagine (N)‐linked glycosylation requires the ordered, stepwise synthesis of lipid‐linked oligosaccharide (LLO) precursor Glc3Man9GlcNAc2‐pyrophosphate‐dolichol (Glc3Man9Gn2‐PDol) on the endoplasmic reticulum. The fourth and fifth steps of LLO synthesis are catalyzed by Alg2, an unusual mannosyltransferase (MTase) with two different MTase activities; Alg2 adds both an a1,3‐ and a1,6‐mannose onto ManGlcNAc2‐PDol to form the trimannosyl core Man3GlcNAc2‐PDol. The biochemical properties of Alg2 are controversial and remain undefined. In this study, a liquid chromatography/mass spectrometry‐based quantitative assay was established and used to analyze the MTase activities of purified yeast Alg2. Alg2‐dependent Man3GlcNAc2‐PDol production relied on net‐neutral lipids with a propensity to form bilayers. We further showed addition of the a1,3‐ and a1,6‐mannose can occur independently in either order but at differing rates. The conserved C‐terminal EX7E motif, N‐terminal cytosolic tail, and 3 G‐rich loop motifs in Alg2 play crucial roles for these activities, both in vitro and in vivo. These findings provide insight into the unique bifunctionality of Alg2 during LLO synthesis and lead to a new model in which alternative, independent routes exist for Alg2 catalysis of the trimannosyl core oligosaccharide.—Li, S.‐T., Wang, N., Xu, X.‐X, Fujita, M., Nakanishi, H., Kitajima, T., Dean, N., Gao, X.‐D. Alternative routes for synthesis of N‐linked glycans by Alg2 mannosyltransferase. FASEB J. 32,2492–2506 (2018). www.fasebj.org

Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins

Although many pharmaceutical proteins are produced in mammalian cells, there remains a challenge to select cell lines that express recombinant proteins with high productivity. Since most biopharmaceutical proteins are secreted by cells into the medium, it is difficult to select cell lines that produce large amounts of the target protein. To address this issue, a new protein expression system using the glycosylphosphatidylinositol (GPI)-anchor was developed. PGAP2 is involved in processing GPI-anchored proteins (GPI-APs) during transport. In PGAP2 mutant cells, most GPI-APs are secreted into the medium. Here, we established a HEK293 cell line where endogenous PGAP2 was knocked out and exogenous PGAP2 was inserted with a piggyBac transposon in the genome. Using these cells, human lysosomal acid lipase (LIPA) and α-galactosidase A (GLA) were expressed as GPI-anchored forms (LIPA-GPI and GLA-GPI) and cells expressing high levels of LIPA-GPI or GLA-GPI on the cell surface were enriched. Removal of the PGAP2 gene by piggyBac transposase or FLP recombinase converted LIPA-GPI and GLA-GPI from membrane-bound to the secreted forms. Thus, cells expressing LIPA or GLA in large amounts could be enriched using this approach. The GPI-based molecular switching system is an efficient approach to isolate cells expressing recombinant proteins with high productivity.