The lipid-binding D4 domain of perfringolysin O facilitates the active loading of exogenous cargo into extracellular vesicles.

Whereas extracellular vesicles (EVs) have been engineered for cargo loading, innovative strategies for it can still be developed. Here, we describe domain 4 (D4), a cholesterol-binding domain derived from perfringolysin O, as a viable candidate for EV cargo loading. D4 and its mutants localized to the plasma membrane and the membranes of different vesicular structures in the cytoplasm, and facilitate the transport of proteins of interest (POIs) into EVs. D4-EVs were internalized by recipient cells analogous to EVs engineered with CD9. Intracellular cargo discharge from D4-EVs was successfully detected with the assistance of vesicular stomatitis virus glycoprotein. This study presents a novel strategy for recruiting POIs into EVs via a lipid-binding domain that ensures content release in recipient cells.

Reversal of neuroinflammation in novel GS model mice by single i.c.v. administration of CHO-derived rhCTSA precursor protein.

Galactosialidosis (GS) is a lysosomal cathepsin A (CTSA) deficiency. It associates with a simultaneous decrease of neuraminidase 1 (NEU1) activity and sialylglycan storage. Central nervous system (CNS) symptoms reduce the quality of life of juvenile/adult-type GS patients, but there is no effective therapy. Here, we established a novel GS model mouse carrying homozygotic Ctsa IVS6+1g→a mutation causing partial exon 6 skipping with concomitant deficiency of Ctsa/Neu1. The GS mice developed juvenile/adult GS-like symptoms, such as gargoyle-like face, edema, proctoprosia due to sialylglycan accumulation, and neurovisceral inflammation, including activated microglia/macrophage appearance and increase of inflammatory chemokines. We produced human CTSA precursor proteins (proCTSA), a homodimer carrying terminal mannose 6-phosphate (M6P)-type N-glycans. The CHO-derived proCTSA was taken up by GS patient-derived fibroblasts via M6P receptors and delivered to lysosomes. Catalytically active mature CTSA showed a shorter half-life due to intralysosomal proteolytic degradation. Following single i.c.v. administration, proCTSA was widely distributed, restored the Neu1 activity, and reduced the sialylglycans accumulated in brain regions. Moreover, proCTSA suppressed neuroinflammation associated with reduction of activated microglia/macrophage and up-regulated Mip1α. The results show therapeutic effects of intracerebrospinal enzyme replacement utilizing CHO-derived proCTSA and suggest suppression of CNS symptoms.

In Cellulo Crystallization of Human Neuraminidase 1 and Biological Roles of N-Glycans.

Human neuraminidase 1 (NEU1) is a lysosomal glycosidase that cleaves the terminal sialic acids of sialylglycoconjugates. NEU1 is biosynthesized in the endoplasmic reticulum (ER) lumen as an N-glycosylated protein. NEU1 also associates with cathepsin A (CTSA) in ER, migrates to lysosomes, and exerts catalytic activity. Extraordinary in cellulo crystallization of NEU1 protein in ER despite carrying three N-glycans per molecule at N186, N343, and N352, respectively, were observed when the single human NEU1 gene was overexpressed in mammalian cells. In this study, we first purified the NEU1 from the isolated crystals produced by the HEK293 NEU1-KO cell transiently overexpressing the normal NEU1 and found that the N-glycans were high-mannose or complex types carrying terminal sialic acids. The result suggests that a part of NEU1 crystals were formed or transported to the Golgi apparatus. Second, we compared the effects of single amino acid substitution at the N-sequons, including N186Q, N343Q, and N352Q, each one N-glycan reduction from one NEU1 molecule. We demonstrated that N186Q mutant protein with low enzyme activity and formed a few amounts of smaller crystals. The N343Q mutant exhibited half of the normal intracellular activity, but the numbers and sizes of crystals were almost the same as those of normal NEU1. The N352Q mutant exhibited almost the same activity as the normal enzyme. The numbers of the N352Q crystals were smaller than those of normal NEU1. According to these findings, the N186Q NEU1 protein should have lower stability in ER due to abnormal folding. The second N-glycan at the N343-sequon has little effect on self-aggregation of NEU1. The third N-glycan at the N352-sequon contributes to the self-aggregation of NEU1. We also demonstrated that the three NEU1 mutants associate with the relatively excessive CTSA and migrate to lysosomes.

Rab27b contributes to radioresistance and exerts a paracrine effect via epiregulin in glioblastoma.

BACKGROUND: Radiotherapy is the standard treatment for glioblastoma (GBM). However, radioresistance of GBM cells leads to recurrence and poor patient prognosis. Recent studies suggest that secretion factors have important roles in radioresistance of tumor cells. This study aims to determine whether Rab27b, a small GTPase involved in secretory vesicle trafficking, plays a role in radioresistance of GBM. METHODS: Microarray analysis, cell viability analysis, apoptosis assay, immunostaining, and in vivo experiments were performed to assess the effect of Rab27b on radioresistance of GBM. We further investigated paracrine effects mediated by Rab27b after X-ray irradiation using coculture systems of glioma cell lines. RESULTS: Rab27b was specifically upregulated in irradiated U87MG cells. Furthermore, Rab27b knockdown decreased the proliferation of GBM cells after irradiation. Knockdown of Rab27b in U87MG cells combined with radiation treatment suppressed orthotopic tumor growth in the mouse brain and prolonged the survival of recipient mice. Interestingly, the co-upregulation of Rab27b and epiregulin (EREG), a member of the epidermal growth factor (EGF) family, correlated with radioresistance in glioma cell lines. Additionally, EREG, which was secreted from U87MG cells via Rab27b-mediated mechanism, activated EGF receptor and contributed to H4 cell proliferation in a paracrine manner. CONCLUSIONS: Our results show that Rab27b mediates the radioresistance of highly malignant GBM cells. Rab27b promotes the proliferation of adjacent cells through EREG-mediated paracrine signaling after irradiation. Thus, the Rab27b-EREG pathway is a novel potential target to improve the efficacy of radiotherapy in GBM.

[Development of Enzyme Drugs Derived from Transgenic Silkworms to Treat Lysosomal Diseases].

　Lysosomal storage diseases (LSDs) are inborn errors caused by genetic defects of lysosomal enzymes associated with the excessive accumulation of natural substrates and neurovisceral manifestations. Until now, enzyme replacement therapy (ERT) with human lysosomal enzymes produced by genetically engineered mammalian cell lines has been applied clinically to treat several LSDs. ERT is based on the incorporation of N-glycosylated lysosomal enzymes through binding to glycan receptors on the surface of target cells and delivery to lysosomes. However, ERT has several disadvantages, including difficulty in mass producing human enzymes, dangers of pathogen contamination, and high cost. Recently, we have succeeded in producing transgenic silkworms which overexpress human lysosomal enzymes in silk glands, and have purified active and functional enzymes from middle silk glands and cocoons. Silk gland- and cocoon-derived human enzymes carrying high-mannose and pauci-mannose N-glycans are endocytosed by monocytes via the mannose receptor pathway; these were then delivered to lysosomes. Human cathepsin A (Ctsa) precursor proteins purified from the cocoons have been found to suppress microglial activation in the brains of Ctsa-deficient mice; this deficiency is caused by a splicing defect, and serves as a galactosialidosis model associated with the combination of a deficiency of lysosomal neuraminidase 1 (NEU1) and the accumulation of sialyloligosaccharides. Transgenic silkworms overexpressing human lysosomal enzymes in silk glands could serve as a future bioresource to provide safe therapeutic enzymes for the treatment of LSDs. The combination of recent developments in transglycosylation technology with microbial endoglycosidases will aid in the development of therapeutic glycoproteins as bio-medicines.

Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands.

Since 2000, transgenic silkworms have been developed to produce recombinant proteins with therapeutic potential for future clinical use, including antibody preparations. Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by mutations of lysosomal enzymes associated with excessive accumulation of natural substrates and neurovisceral symptoms. Over the past few years, enzyme replacement therapy (ERT) with human lysosomal enzymes produced by genetically engineered mammalian cell lines has been used clinically to treat several patients with an LSD involving multi-organ symptoms. ERT is based on the incorporation of recombinant glycoenzymes by their binding to glycan receptors on the surface of target cells and their subsequent delivery to lysosomes. However, ERT has several disadvantages, including difficulty mass producing human enzymes, dangers of pathogen contamination, and high costs. Recently, the current authors have succeeded in producing transgenic silkworms overexpressing human lysosomal enzymes in the silk glands and the authors have purified catalytically active enzymes from the middle silk glands. Silk gland-derived human enzymes carrying high-mannose and pauci-mannose N-glycans were endocytosed by monocytes via the mannose receptor pathway and were then delivered to lysosomes. Conjugates with cell-penetrating peptides were also taken up by cultured fibroblasts derived from patients with enzyme deficiencies to restore intracellular catalytic activity and reduce the excessive accumulation of substrates in patient fibroblasts. Transgenic silkworms overexpressing human lysosomal enzymes in the silk glands could serve as future bioresources that provide safe therapeutic enzymes for the treatment of LSDs. Combining recent developments in transglycosylation technology with microbial endoglycosidases will promote the development of therapeutic glycoproteins as bio-medicines.