The N-linked glycosylation modifications in the hepatitis B surface protein impact cellular autophagy, HBV replication, and HBV secretion.

N-linked glycosylation is a pivotal post-translational modification that significantly influences various aspects of protein biology. Autophagy, a critical cellular process, is instrumental in cell survival and maintenance. The hepatitis B virus (HBV) has evolved mechanisms to manipulate this process to ensure its survival within host cells. Significantly, post-translational N-linked glycosylation in the large surface protein of HBV (LHBs) influences virion assembly, infectivity, and immune evasion. This study investigated the role of N-linked glycosylation of LHBs in autophagy, and its subsequent effects on HBV replication and secretion. LHBs plasmids were constructed by incorporating single-, double-, and triple-mutated N-linked glycosylation sites through amino acid substitutions at N4, N112, and N309. In comparison to the wild-type LHBs, N-glycan mutants, including N309Q, N4-309Q, N112-309Q, and N4-112-309Q, induced autophagy gene expression and led to autophagosome accumulation in hepatoma cells. Acridine orange staining of cells expressing LHBs mutations revealed impaired lysosomal acidification, suggesting potential blockage of autophagic flux at later stages. Furthermore, N-glycan mutants increased the mRNA expression of HBV surface antigen (HBsAg). Notably, N309Q significantly elevated HBx oncogene level. The LHBs mutants, particularly N309Q and N112-309Q, significantly enhanced HBV replication, whereas N309Q, N4-309Q, and N4-112-309Q markedly increased HBV progeny secretion. Remarkably, our findings demonstrated that autophagy is indispensable for the impact of N-linked glycosylation mutations in LHBs on HBV secretion, as evidenced by experiments with a 3-methyladenine (3-MA) inhibitor. Our study provides pioneering insights into the interplay between N-linked glycosylation mutations in LHBs, host autophagy, and the HBV life cycle. Additionally, we offer a new clue for further investigation into carcinogenesis of hepatocellular carcinoma (HCC). These findings underscore the potential of targeting either N-linked glycosylation modifications or the autophagic pathway for the development of innovative therapies against HBV and/or HCC.

Production and N-glycan engineering of Varlilumab in Nicotiana benthamiana.

N-glycan engineering has dramatically evolved for the development and quality control of recombinant antibodies. Fc region of IgG contains two N-glycans whose galactose terminals on Fc-glycan have been shown to increase the stability of CH2 domain and improve effector functions. Nicotiana benthamiana has become one of the most attractive production systems for therapeutic antibodies. In this study, Varlilumab, a CD27-targeting monoclonal antibody, was transiently produced in fresh leaves of soil-grown and hydroponic-grown N. benthamiana, resulted in the yield of 174 and 618 µg/gram, respectively. However, the IgG produced in wild-type N. benthamiana lacked the terminal galactose residues in its N-glycan. Therefore, N-glycan engineering was applied to fine-tune recombinant antibodies produced in plant platforms. We further co-expressed IgG together with murine ß1,4-galactosyltransferase (ß1,4-GALT) to modify plant N-glycan with ß1,4-linked Gal residue(s) and Arabidopsis thaliana ß1,3-galactosylatransferase (ß1,3-GALT) to improve galactosylation. The co-expression of IgG with each of GALTs successfully resulted in modification of N-glycan structures on the plant-produced IgG. Notably, IgG co-expressed with murine ß1,4-GALT in soil-grown N. benthamiana had 42.5% of N-glycans variants having galactose (Gal) residues at the non-reducing terminus and 55.3% of that in hydroponic-grown N. benthamiana plants. Concomitantly, N-glycan profile analysis of IgG co-expressed with ß1,3-GALT demonstrated that there was an increased efficiency of galactosylation and an enhancement in the formation of Lewis a structure in plant-derived antibodies. Taken together, our findings show that the first plant-derived Varlilumab was successfully produced with biantennary ß1,4-galactosylated N-glycan structures.

High accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I gene suppression and mannosidase I gene overexpression in Nicotiana tabacum SR1.

High accumulation of a single high-mannose glycan structure is important to ensure the quality of therapeutic proteins. We developed a glyco-engineering strategy for ensuring high accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I (GnT I) gene suppression and mannosidase I (Man I) gene overexpression. Nicotiana tabacum SR1 was used as the glyco-engineered host owing to the lower risk of pathogenic contamination than that in mammalian cells. We generated three glyco-engineered plant strains (gnt, gnt-MANA1, and gnt-MANA2) with suppression of GnT I or the combined suppression of GnT I and overexpression of Man I A1 or A2. The quantitative reverse transcriptase-PCR analysis showed a higher level of upregulation of Man I expression in gnt-MANA1/A2 plants than in the wild-type plants. Man I activity assay showed that the gnt-MANA1 plants had a higher Man I activity than did the wild-type and gnt-MANA2 plants. N-glycan analysis independently performed on two plants of each plant strain showed that gnt-MANA1 plants had a low abundance of the Man6-9GlcNAc2 structure (2.8%, 7.1%) and high abundance of the Man5GlcNAc2 structure (80.0%, 82.8%) compared with those in the wild-type and gnt plants. These results indicated that GnT I knockdown suppressed further modification of the Man5GlcNAc2 structure, and Man I overexpression enhanced the conversion of Man6-9GlcNAc2 structures to the Man5GlcNAc2 structure. The developed glyco-engineered plants have potential for serving as novel expression hosts for therapeutic proteins.

The specific core fucose-binding lectin Pholiota squarrosa lectin (PhoSL) inhibits hepatitis B virus infection in vitro.

Glycosylation of proteins and lipids in viruses and their host cells is important for viral infection and is a target for antiviral therapy. Hepatitis B virus (HBV) is a major pathogen that causes acute and chronic hepatitis; it cannot be cured because of the persistence of its covalently closed circular DNA (cccDNA) in hepatocytes. Here we found that Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds core fucose, bound to HBV particles and inhibited HBV infection of a modified human HepG2 cell line, HepG2-hNTCP-C4, that expresses an HBV receptor, sodium taurocholate cotransporting polypeptide. Knockout of fucosyltransferase 8, the enzyme responsible for core fucosylation and that aids receptor endocytosis, in HepG2-hNTCP-C4 cells reduced HBV infectivity, and PhoSL facilitated that reduction. PhoSL also blocked the activity of epidermal growth factor receptor, which usually enhances HBV infection. HBV particles bound to fluorescently labeled PhoSL internalized into HepG2-hNTCP-C4 cells, suggesting that PhoSL might inhibit HBV infection after internalization. As PhoSL reduced the formation of HBV cccDNA, a marker of chronic HBV infection, we suggest that PhoSL could impair processes from internalization to cccDNA formation. Our finding could lead to the development of new anti-HBV agents.

High-level transient production of a protease-resistant mutant form of human basic fibroblast growth factor in Nicotiana benthamiana leaves.

The human basic fibroblast growth factor (bFGF) is a protein that plays a pivotal role in cellular processes like cell proliferation and development. As a result, it has become an important component in cell culture systems, with applications in biomedical engineering, cosmetics, and research. Alternative production techniques, such as transient production in plants, are becoming a feasible option as the demand continues to grow. High-level bFGF production was achieved in this study employing an optimized Agrobacterium-mediated transient expression system, which yielded about a 3-fold increase in production over a conventional system. This yield was further doubled at about 185 µg g-1 FW using a mutant protease-resistant version that degraded/aggregated at a three-fold slower rate in leaf crude extracts. To achieve a pure product, a two-step purification technique was applied. The capacity of the pure protease-resistant bFGF (PRbFGF) to stimulate cell proliferation was tested and was found to be comparable to that of E. coli-produced bFGF in HepG2 and CHO-K1 cells. Overall, this study demonstrates a high-level transient production system of functional PRbFGF in N. benthamiana leaves as well as an efficient tag-less purification technique of leaf crude extracts.

Production of recombinant ß-glucocerebrosidase in wild-type and glycoengineered transgenic Nicotiana benthamiana root cultures with different N-glycan profiles.

Gaucher disease is an inherited lysosomal storage disorder caused by an insufficiency of active ß-glucocerebrosidase (GCase). Exogenous recombinant GCase via enzyme replacement therapy is considered the most practical treatment for Gaucher disease. Mannose receptors mediate the efficient uptake of exogenous GCase into macrophages. Thus, terminal mannose residues on N-glycans are essential for the delivery of exogenous GCase. In this study, recombinant GCase was produced in root cultures of wild-type (WT) and glycoengineered transgenic Nicotiana benthamiana with downregulated N-acetylglucosaminyltransferase I expression. Root cultures of WT and glycoengineered transgenic N. benthamiana plants were successfully generated by the induction of plant hormones. Recombinant GCases produced in both root cultures possessed GCase enzyme activity. Purified GCases derived from both root cultures revealed different N-glycan profiles. The WT-derived GCase possessed the predominant plant-type N-glycans, which contain plant-specific sugars-linkages, specifically ß1,2-xylose and α1,3-fucose residues. Notably, the mannosidic-type N-glycans with terminal mannose residues were abundant in the purified GCase derived from glycoengineered N. benthamiana root culture. This research provides a promising plant-based system for the production of recombinant GCase with terminal mannose residues on N-glycans.

Transient Production of Human ß-Glucocerebrosidase With Mannosidic-Type N-Glycan Structure in Glycoengineered Nicotiana benthamiana Plants.

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of functional enzyme ß-glucocerebrosidase (GCase). Recombinant GCase has been used in enzyme replacement therapy to treat Gaucher disease. Importantly, the terminal mannose N-glycan structure is essential for the uptake of recombinant GCase into macrophages via the mannose receptor. In this research, recombinant GCase was produced using Agrobacterium-mediated transient expression in both wild-type (WT) and N-acetylglucosaminyltransferase I (GnTI) downregulated Nicotiana benthamiana (ΔgntI) plants, the latter of which accumulates mannosidic-type N-glycan structures. The successfully produced functional GCase exhibited GCase enzyme activity. The enzyme activity was the same as that of the conventional mammalian-derived GCase. Notably, N-glycan analysis revealed that a mannosidic-type N-glycan structure lacking plant-specific N-glycans (ß1,2-xylose and α1,3-fucose residues) was predominant in all glycosylation sites of purified GCase produced from ΔgntI plants. Our research provides a promising alternative plant line as a host for the production of recombinant GCase with a mannosidic-type N-glycan structure. This glycoengineered plant might be applicable to the production of other pharmaceutical proteins, especially mannose receptor targeted protein, for therapeutic uses.

St6gal1 knockdown alters HBV life cycle in HepAD38 cells.

Complex glycans at the cell surface play important roles, and their alteration is known to modulate cellular activity. Previously, we found that HBV replication in HepAD38 altered cell-surface sialylated N-glycan through the upregulation of St6gal1, Mgat2, and Mgat4a expression. Here we studied the effects of knocking them down on HBV replication in HepAD38. Our results showed that St6gal1 knockdown (KD) reduced intracellular HBV rcDNA level by 90%, that Mgat2 KD did not change the intracellular HBV rcDNA level, and that Mgat4 KD increased the intracellular HBV rcDNA level by 19 times compared to Tet(-). The changes in intracellular rcDNA level were followed by the alteration of Pol and HBc expression. Our study suggests that St6gal1 KD contributes more to the HBV life cycle than Mgat2 or Mgat4a KD through the modification of intracellular L, Pol, and HBc expression.

The Production of Human ß-Glucocerebrosidase in Nicotiana benthamiana Root Culture.

Gaucher disease is caused by a deficiency of the enzyme glucocerebrosidase (GCase). Currently, enzyme-replacement therapy using recombinant GCase produced in mammalian cells is considered the most effective treatment. Plants are an attractive alternative host for recombinant protein production due to the low cost of large-scale production and lack of risk of contamination by human pathogens. Compared to whole plants, root cultures can grow faster. Therefore, this study aimed to produce recombinant GCase in a Nicotiana benthamiana root culture. Root culture of a GCase-producing transgenic plant was induced by indole-3-acetic acid at the concentration of 1 mg/L. Recombinant GCase was successfully produced in roots as a functional protein with an enzyme activity equal to 81.40 ± 17.99 units/mg total protein. Crude proteins were extracted from the roots. Recombinant GCase could be purified by concanavalin A and phenyl 650C chromatography. The productivity of GCase was approximately 1 µg/g of the root. A N-glycan analysis of purified GCase was performed using nano LC/MS. The Man3XylFucGlcNAc2 structure was predominant in purified GCase with two plant-specific glycan residues. This study presents evidence for a new, safe and efficient system of recombinant GCase production that might be applied to other recombinant proteins.

Cell surface N-glycan alteration in HepAD38 cell lines expressing Hepatitis B virus.

Hepatitis B virus (HBV) is the smallest partially double-stranded DNA virus known to infect humans. Worldwide, more than 50% of hepatocellular carcinoma (HCC) cases are related to chronic Hepatitis B. Development of HCC from normal liver cells is characterized by changes in cell surface N-glycans, which can promote the invasive behavior of tumor cells, leading ultimately to the progression of cancer. However, little is understood about the cell surface N-glycans of HBV-infected liver cells. We try to address this by taking advantage of the HepAD38 cell line, which can replicate HBV in the absence of tetracycline [tet(-)] in growth medium. In the presence of tetracycline [tet(+)], this cell line is free from the virus due to the repression of pregenomic (pg) RNA synthesis. In culture medium without tetracycline, cells express viral pgRNA and start to secrete virions into the supernatant. Here we studied the expression of glycosyltransferases and the cell surface N-glycan composition of tet(+) and tet(-) HepAD38. Among the glycosyltransferases upregulated by the expression of HBV were GnT-II, GnT-IVa, ST6Gal1, and GnT-V, whereas GnT-I, GnT-III, ß4GalT1, and FUT8 were downregulated. About one-third of the total cell surface N-glycans found on tet(-)HepAD38 were sialylated. As for tet(+)HepAD38, sialylation was 6% lower compared to the tet(-) cells. Neither treatment changed the cell surface N-glycans expression of the total complex type or the total fucosylated type, which were about 50% or 60%, respectively. Our results showed that the expression of HBV triggers higher sialylation in HepAD38 cells. Altogether, the results show that HBV expression triggered the alteration of the cell surface N-glycosylation pattern and the expression levels of glycosyltransferases of HepAD38 cells.

Core-fucosylation plays a pivotal role in hepatitis B pseudo virus infection: a possible implication for HBV glycotherapy.

The functions of cell surface proteins, such as growth factor receptors and virus/bacteria-entry receptors, can be dynamically regulated by oligosaccharide modifications. In the present study, we investigated the involvement of glycosylation in hepatitis B virus (HBV) entry into hepatoma cells. Infection of oligosaccharide-remodeling hepatoma cells with a pseudo virus of HBV, bio-nanocapsule (BNC), was evaluated by flow cytometry and confocal microscopy. Among various experiments using several hepatoma cells, marked difference was observed between Huh6 cells and HB611 cells, which were established by HBV gene transfection into hepatoma cells. Comprehensive oligosaccharide analysis showed dramatic increases of core fucosylation in HB611 cells, compared with Huh6 cells. Knock down of fucosyltransferase 8 (FUT8) reduced BNC entry into HB611 cells. In contrast, overexpression of FUT8 in Huh6 cells increased BNC entry. Although expression of sodium taurocholate cotransporting polypeptide (NTCP), which is one of HBV receptors was very similar between Huh6 and HB611 cells, proteins coprecipitated with NTCP were dependent on levels of core-fucosylation, suggesting that core-fucosylation regulates BNC entry into hepatoma cells. Our findings demonstrate that core-fucosylation is an important glycosylation for HBV infection of hepatoma cells through HBV-receptor-mediated endocytosis. Down-regulation of core-fucosylation may be a novel target for HBV therapy.

The production of human glucocerebrosidase in glyco-engineered Nicotiana benthamiana plants.

For the production of therapeutic proteins in plants, the presence of ß1,2-xylose and core α1,3-fucose on plants' N-glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down-regulate the endogenous N-acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco-engineered line (NbGNTI-RNAi) showed a strong reduction of plant-specific N-glycans, with the result that as much as 90.9% of the total N-glycans were of high-mannose type. Therefore, this NbGNTI-RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The NbGNTI-RNAi plant was cross-pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gaucher's disease, requires terminal mannose for its therapeutic efficacy. The N-glycan structures that were presented on all of the four occupied N-glycosylation sites of recombinant GC in NbGNTI-RNAi plants (GC(gnt1) ) showed that the majority (ranging from 73.3% up to 85.5%) of the N-glycans had mannose-type structures lacking potential immunogenic ß1,2-xylose and α1,3-fucose epitopes. Moreover, GC(gnt1) could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gaucher's disease. Notably, the NbGNTI-RNAi line, producing GC, was stable and the NbGNTI-RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N-glycan structures.

The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production.

Cloning of a cDNA encoding the Gly m Bd 28K precursor and its vacuole transport in tobacco BY2 suspension-cultured cells.

Gly m Bd 28K (Gm28K), a soybean allergen, is formed as a preproprotein consisting of a predicted signal peptide, Gm28K, and the 23-kDa peptide (Gm23K). Gm28K and Gm23K are found in the protein-storage vacuoles (PSVs) of developing soybean seeds. However, the complete structure of Gm28K has not yet been identified and its processing and transport to the vacuoles has never been clarified. In the present study, we elucidated the 5'-nucleotide sequence of cDNA encoding the Gm28K precursor and identified a putative signal peptide (SP) with 24 N-terminal amino acid residues. We expressed peptides from the Gm28K precursor as fusion proteins with enhanced green fluorescent protein (EGFP) in tobacco BY2 suspension-cultured cells. BY2 cells transformed by an expression vector for SP-EGFP-Gm28-Gm23K (SP-EGFP-Gm28-Gm23K/BY2 cells) and SP-Gm28-Gm23K-EGFP/BY2 cells produced the EGFP fused-Gm28K precursor, and the EGFP-fluorescence in their vacuoles were recorded. In the experiments with SP-EGFP/BY2 and SP-EGFP-Gm28K/BY2 cells, large amounts of the EGFP segments were secreted into the medium. On the other hand, the fluorescence of EGFP in SP-EGFP-Gm23K/BY2 cells was shown to accumulate only in the endoplasmic reticulum without secretion into the medium. These findings show that the SP signals the precursor to enter the lumen of the endoplasmic reticulum and that both the Gm28K and Gm23K components may be involved in the transport from the endoplasmic reticulum (ER) lumen via the Golgi to the vacuoles in a proprotein form.

Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection.

Intracellular phosphatidylserine is essential for retrograde membrane traffic through endosomes.

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides.

N-terminal vacuolar sorting signal at the mouse antibody alters the N-linked glycosylation pattern in suspension-cultured tobacco BY2 cells.

Recombinant DNA technology enables the use of plants as the host for the production of pharmaceutical proteins, such as antibodies. The glycosylation of recombinant proteins plays physiological and biological roles. However, because glycosylation in plants is different from that in human cells, the development of glycoengineering is required. In plant cells, glycan structures are shown to correlate with the localization of the recombinant protein produced. In this study, the vacuolar sorting signal (VSS) of sporamin was fused to the heavy (H) and light (L) chains of a mouse monoclonal antibody (mAb), and the mAb was produced in suspension-cultured tobacco BY2 cells. The sugar chain structures were determined by high-performance liquid chromatography, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Typical plant glycans with α1,3-fucosylation and/or ß1,2-xylosylation derived from mAb with the VSS-fused H-chain (mIgG1000) and mAb with the VSS-fused H- and L-chain (mIgG1010) occupied the large amount of the total N-glycans, 72.1% and 85.0%, respectively, such as those derived from mAb without VSS (mIgG0000), 74.6% (Fujiyama et al., J. Biosci. Bioeng., 101, 212-218, 2006). In contrast, the typical plant glycan structure Man3FucXylGlcNAc2 particularly in vacuoles accounted for 37.8% of the total sugar chains derived from mIgG1000 and 58.5% of those derived from mIgG1010 compared with 24.3% of those derived from mIgG0000. These results suggest that the sporamin signal peptide fused to mAb acts as a VSS and leads to the increase in the amount of Man3FucXylGlcNAc2, which is the main N-glycan structure in vacuoles.

The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

Rab GTPases including Rab27a, Rab38 and Rab32 function in melanosome maturation or trafficking in melanocytes. A screen to identify additional Rabs involved in these processes revealed the localization of GFP-Rab17 on recycling endosomes (REs) and melanosomes in melanocytic cells. Rab17 mRNA expression is regulated by microphthalmia transcription factor (MITF), a characteristic of known pigmentation genes. Rab17 siRNA knockdown in melanoma cells quantitatively increased melanosome concentration at the cell periphery. Rab17 knockdown did not inhibit melanosome maturation nor movement, but it caused accumulation of melanin inside cells. Double knockdown of Rab17 and Rab27a indicated that Rab17 acts on melanosomes downstream of Rab27a. Filopodia are known to play a role in melanosome transfer, and in Rab17 knockdown cells filopodia formation was inhibited. Furthermore, we show that stimulation of melanoma cells with α-melanocyte-stimulating hormone induces filopodia formation, supporting a role for filopodia in melanosome release. Cell stimulation also caused redistribution of REs to the periphery, and knockdown of additional RE-associated Rabs 11a and 11b produced a similar accumulation of melanosomes and melanin to that seen after loss of Rab17. Our findings reveal new functions for RE and Rab17 in pigmentation through a distal step in the process of melanosome release via filopodia.

Stable coexpression of two human sialylation enzymes in plant suspension-cultured tobacco cells.

Human CMP-N-acetylneuraminic acid (NeuAc) synthase (hCSS) and α2,6-sialyltransferase (hST) participate in the sialylation of N-linked glycans in mammalian cells. hCSS synthesizes CMP-NeuAc, which hST uses as a donor substrate to transfer NeuAc to the terminal position of N-linked glycans. In plant cells, the presence of NeuAc has not yet been substantiated and the identification of the genes involved in the sialylation of N-glycan has not been carried out. In this study, we introduced hCSS and hST genes into suspension-cultured tobacco BY2 cells to provide the machinery for the sialylation pathway in plants. hCSS and hST stably expressed in the plant cells showed activity. In addition, CMP-NeuAc produced by hCSS in the transformed plant cells functioned as a donor substrate to hST. An in vitro coupled hCSS and hST reaction resulted in the production of mammalian-type sialoglycoproteins bearing terminal NeuAc residues. Furthermore, the results of the purification of the coupled-reaction products by Sambucus sieboldian lectin column chromatography and digestion with linkage-specific neuraminidase revealed that the modified terminal residue was α2,6-linked NeuAc. Here, we demonstrate that the in vitro sialylation of N-linked glycans on mammalian proteins can be achieved using plant cell extracts stably expressing hCSS and hST, providing proof-of-principle that a sialylated human therapeutic protein can be produced in plants.

Phosphoinositide 3-kinase δ regulates membrane fission of Golgi carriers for selective cytokine secretion.

Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-α (TNF). Loss of function of the p110δ isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.