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Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Torii et al.)

The vacuolar H + -ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types. Previous work has reported that plasma membrane V-ATPases are key players in breast cancer cell invasiveness. The two subunit a-isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, and expression of a3 has been shown to correlate with plasma membrane localization of the V-ATPase in various invasive human breast cancer cell lines. Here we analyzed the role of subunit a-isoforms in the invasive mouse breast cancer cell line, 4T1-12B. Quantitation of mRNA levels for each isoform by quantitative RT-PCR revealed that a4 is the dominant isoform expressed in these cells. Using a CRISPR/Cas9-based approach to disrupt the genes encoding each of the four V-ATPase subunit a-isoforms, we found that ablation of only the a4-encoding gene significantly inhibits invasion and migration of 4T1-12B cells. Additionally, cells with disrupted a4 exhibited reduced V-ATPase expression at the leading edge, suggesting that the a4 isoform is primarily responsible for targeting the V-ATPase to the plasma membrane in 4T1-12B cells. These findings suggest that different subunit a-isoforms may direct V-ATPases to the plasma membrane of different invasive breast cancer cell lines. They further suggest that expression of V-ATPases at the cell surface is the primary factor that promotes an invasive cancer cell phenotype., (© 2019 McGuire et al.)

A defect in O-mannosyl glycan is the cause of α-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant α-dystroglycan (α-DG) glycosylation. Recently, the entire structure of O-mannosyl glycan, [3GlcAβ1-3Xylα1] n -3GlcAβ1-4Xyl-Rbo5P-1Rbo5P-3GalNAcβ1-3GlcNAcβ1-4 (phospho-6)Manα1-, which is required for the binding of α-DG to extracellular matrix ligands, has been proposed. However, the linkage of the first Xyl residue to ribitol 5-phosphate (Rbo5P) is not clear. TMEM5 is a gene product responsible for α-dystroglycanopathy and was reported as a potential enzyme involved in this linkage formation, although the experimental evidence is still incomplete. Here, we report that TMEM5 is a xylosyltransferase that forms the Xylβ1-4Rbo5P linkage on O-mannosyl glycan. The anomeric configuration and linkage position of the product (β1,4 linkage) was determined by NMR analysis. The introduction of two missense mutations in TMEM5 found in α-dystroglycanopathy patients impaired xylosyltransferase activity. Furthermore, the disruption of the TMEM5 gene by CRISPR/Cas9 abrogated the elongation of the (-3GlcAβ1-3Xylα1-) unit on O-mannosyl glycan. Based on these results, we concluded that TMEM5 acts as a UDP-d-xylose:ribitol-5-phosphate β1,4-xylosyltransferase in the biosynthetic pathway of O-mannosyl glycan., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glycans play key roles in a variety of protein functions under normal and pathological conditions, but several glycosyltransferase-deficient mice exhibit no or only mild phenotypes due to redundancy or compensation of glycan functions. However, we have only a limited understanding of the underlying mechanism for these observations. Our previous studies indicated that 70% of Fut8-deficient (Fut8(-/-)) mice that lack core fucose structure die within 3 days after birth, but the remainder survive for up to several weeks although they show growth retardation as well as emphysema. In this study, we show that, in mouse embryonic fibroblasts (MEFs) from Fut8(-/-) mice, another N-glycan branching structure, bisecting GlcNAc, is specifically up-regulated by enhanced gene expression of the responsible enzyme N-acetylglucosaminyltransferase III (GnT-III). As candidate target glycoproteins for bisecting GlcNAc modification, we confirmed that level of bisecting GlcNAc on β1-integrin and N-cadherin was increased in Fut8(-/-) MEFs. Moreover using mass spectrometry, glycan analysis of IgG1 in Fut8(-/-) mouse serum demonstrated that bisecting GlcNAc contents were also increased by Fut8 deficiency in vivo. As an underlying mechanism, we found that in Fut8(-/-) MEFs Wnt/β-catenin signaling is up-regulated, and an inhibitor against Wnt signaling was found to abrogate GnT-III expression, indicating that Wnt/β-catenin is involved in GnT-III up-regulation. Furthermore, various oxidative stress-related genes were also increased in Fut8(-/-) MEFs. These data suggest that Fut8(-/-) mice adapted to oxidative stress, both ex vivo and in vivo, by inducing various genes including GnT-III, which may compensate for the loss of core fucose functions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein as well as a classic glycolytic enzyme, and its pleiotropic functions are achieved by various post-translational modifications and the resulting translocations to intracellular compartments. In the present study, GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift in two-dimensional electrophoresis after cell-cell fusion induction by forskolin. This post-translational modification was deamidation of multiple glutaminyl residues, as determined by molecular mass measurement and tandem mass spectrometry of acidic GAPDH isoforms. Transglutaminase (TG) inhibitors prevented this acidic shift and reduced cell fusion. Knockdown of the TG2 gene by short hairpin RNA reproduced these effects of TG inhibitors. Various GAPDH mutants with replacement of different numbers (one to seven) of Gln by Glu were expressed in BeWo cells. These deamidated mutants reversed the suppressive effect of wild-type GAPDH overexpression on cell fusion. Interestingly, the mutants accumulated in the plasma membrane, and this accumulation was increased according to the number of Gln/Glu substitutions. Considering that GAPDH binds F-actin via an electrostatic interaction and that the cytoskeleton is rearranged in trophoblastic cell fusion, TG2-dependent GAPDH deamidation was suggested to participate in actin cytoskeletal remodeling.

In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.

Heregulin signaling is involved in various tumor proliferations and invasions; thus, receptors of heregulin are targets for the cancer therapy. In this study we examined the suppressing effects of extracellular domains of ErbB2, ErbB3, and ErbB4 (soluble ErbB (sErbB)) on heregulin β signaling in human breast cancer cell line MCF7. It was found that sErbB3 suppresses ligand-induced activation of ErbB receptors, PI3K/Akt and Ras/Erk pathways most effectively; sErbB2 scarcely suppresses ligand-induced signaling, and sErbB4 suppresses receptor activation at ∼10% efficiency of sErbB3. It was revealed that sErbB3 does not decrease the effective ligands but decreases the effective receptors. By using small interfering RNA (siRNA) for ErbB receptors, we determined that sErbB3 suppresses the heregulin β signaling by interfering ErbB3-containing heterodimers including ErbB2/ErbB3. By introducing the mutation of N418Q to sErbB3, the signaling-inhibitory effects were increased by 2-3-fold. Moreover, the sErbB3 N418Q mutant enhanced anticancer effects of lapatinib more effectively than the wild type. We also determined the structures of N-glycan on Asn-418. Results suggested that the N-glycan-deleted mutant of sErbB3 suppresses heregulin signaling via ErbB3-containing heterodimers more effectively than the wild type. Thus, we demonstrated that the sErbB3 N418Q mutant is a potent inhibitor for heregulin β signaling.

One of the mitogen-activated protein kinases, p38, has been found to play a crucial role in various inflammatory responses. In this study, we analyzed the roles of p38α in multiple sclerosis, using an animal model, experimental autoimmune encephalomyelitis (EAE). p38α(+/-) mice (p38α(-/-) showed embryonic lethality) showed less severe neurological signs than WT mice. Adoptive transfer of lymph node cells (LNC) from sensitized WT mice with MOG(35-55) to naive WT-induced EAE was much more severe compared with the case using LNC from sensitized p38α(+/-) mice. Comprehensive analysis of cytokines from MOG(35-55)-challenged LNC by Western blot array revealed that production of IL-17 was significantly reduced by a single copy disruption of the p38α gene or a p38 inhibitor. Likewise, by a luciferase reporter assay, an electrophoresis mobility shift assay, and characterization of the relationship between p38 activity and IL-17 mRNA expression, we confirmed that p38 positively regulates transcription of the Il17 gene. Furthermore, oral administration of a highly specific p38α inhibitor (UR-5269) to WT mice at the onset of EAE markedly suppressed the progression of EAE compared with a vehicle group. These results suggest that p38α participates in the pathogenesis of EAE through IL-17 induction.

α-Dystroglycan (α-DG) is a membrane-associated glycoprotein that interacts with several extracellular matrix proteins, including laminin and agrin. Aberrant glycosylation of α-DG disrupts its interaction with ligands and causes a certain type of muscular dystrophy commonly referred to as dystroglycanopathy. It has been reported that a unique O-mannosyl tetrasaccharide (Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of α-DG. In this study, we use several dystroglycanopathy mouse models to demonstrate that, in addition to fukutin and LARGE, FKRP (fukutin-related protein) is also involved in the post-phosphoryl modification of O-mannose on α-DG. Furthermore, we have found that the glycosylation status of α-DG in lung and testis is minimally affected by defects in fukutin, LARGE, or FKRP. α-DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results show that FKRP is involved in post-phosphoryl modification rather than in O-mannosyl tetrasaccharide synthesis. Our data also demonstrate that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α-DG functional expression as a laminin receptor in normal tissues and cells.

Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia.

Recently we reported that N-glycans on the beta-propeller domain of the integrin alpha5 subunit (S-3,4,5) are essential for alpha5beta1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type alpha5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the alpha5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type alpha5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the alpha5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.

Laminin-332 (Lm332) is a large heterotrimeric glycoprotein that has been identified as a scattering factor, a regulator of cancer invasion as well as a prominent basement membrane component of the skin. Past studies have identified the functional domains of Lm332 and revealed the relationships between its activities and the processing of its subunits. However, there is little information available concerning the effects of N-glycosylation on Lm332 activities. In some cancer cells, an increase of beta1,6-GlcNAc catalyzed by N-acetylglucosaminyltransferase V (GnT-V) is related to the promotion of cancer cell motility. By contrast, bisecting GlcNAc catalyzed by N-acetylglucosaminyltransferase III (GnT-III) suppresses the further processing with branching enzymes, such as GnT-V, and the elongation of N-glycans. To examine the effects of those N-glycosylations to Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. Lectin blotting and mass spectrometry analyses revealed that N-glycans containing the bisecting GlcNAc and beta1,6-GlcNAc structures were strongly expressed on Lm332 purified from GnT-III-overexpressing (GnT-III-Lm332) and GnT-V-overexpressing (GnT-V-Lm332) cells, respectively. Interestingly, the cell adhesion activity of GnT-III-Lm332 was apparently decreased compared with those of control Lm332 and GnT-V-Lm332. In addition, the introduction of bisecting GlcNAc to Lm332 resulted in a decrease in its cell scattering and migration activities. The weakened activities were most likely derived from the impaired alpha3beta1 integrin clustering and resultant focal adhesion formation. Taken together, our results clearly demonstrate for the first time that N-glycosylation may regulate the biological function of Lm332. This finding could introduce a new therapeutic strategy for cancer.

Although chromosome X open reading frame 6 (CXorf6) has been shown to be a causative gene for hypospadias, its molecular function remains unknown. To clarify this, we first examined CXorf6 protein structure, identifying homology to mastermind-like 2 (MAML2) protein, which functions as a co-activator in canonical Notch signaling. Transactivation analysis for wild-type CXorf6 protein by luciferase assays showed that CXorf6 significantly transactivated the promoter of a noncanonical Notch target gene hairy/enhancer of split 3 (Hes3) without demonstrable DNA-binding capacity. Transactivation analysis was also performed for the previously described three apparently pathologic nonsense mutations, indicating that E124X and Q197X proteins had no transactivation function, whereas R653X protein retained a nearly normal transactivation function. Subcellular localization analysis revealed that wild-type and R653X proteins co-localized with MAML2 protein in nuclear bodies, whereas E124X and Q197X proteins were incapable of localizing to nuclear bodies. Thus, further studies were performed for R653X, revealing the occurrence of nonsense mediated mRNA decay in vivo. Next, transient knockdown of CXorf6 was performed using small interfering RNA, showing reduced testosterone production in mouse Leydig tumor cells. Furthermore, steroidogenic factor 1 (SF1) protein bound to a specific sequence in the upstream of the CXorf6 coding region and exerted a transactivation activity. These results suggest that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue to clarify the biological role of CXorf6. We designate CXorf6 as MAMLD1 (mastermind-like domain-containing 1) based on its characteristic structure.

A complex of gamma, epsilon, and c subunits rotates in ATP synthase (FoF(1)) coupled with proton transport. A gold bead connected to the gamma subunit of the Escherichia coli F(1) sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the beta subunit key region; consistent with its bulk phase ATPase activities, F(1) with the Ser-174 to Phe substitution (betaS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120 degrees positions during the stepped revolution. The pause positions were probably not at the "ATP waiting" dwell but at the "ATP hydrolysis/product release" dwell, since the ATP concentration used for the assay was approximately 30-fold higher than the K(m) value for ATP. A betaGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of betaS174F. The revertant (betaG149A/betaS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant betaG149A/betaS174L. These results indicate that the domain between beta-sheet 4 (betaSer-174) and P-loop (betaGly-149) is important to drive rotation.

The N-glycosylation of integrin alpha5beta1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on the alpha5 integrin. Removal of the putative N-glycosylation sites on the beta-propeller, Thigh, Calf-1, or Calf-2 domains of the alpha5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1-5) on the beta-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the alpha5 subunit carrying only three potential N-glycosylation sites (3-5 sites) on the beta-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Delta3-5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the beta-propeller domain of the alpha5 subunit is essential for heterodimerization and biological functions of alpha5beta1 integrin and might also be useful for studies of the molecular structure.

The gamma subunit of the ATP synthase F(1) sector rotates at the center of the alpha(3)beta(3) hexamer during ATP hydrolysis. A gold bead (40-200 nm diameter) was attached to the gamma subunit of Escherichia coli F(1), and then its ATP hydrolysis-dependent rotation was studied. The rotation speeds were variable, showing stochastic fluctuation. The high-speed rates of 40- and 60-nm beads were essentially similar: 721 and 671 rps (revolutions/s), respectively. The average rate of 60-nm beads was 381 rps, which is approximately 13-fold faster than that expected from the steady-state ATPase turnover number. These results indicate that the F(1) sector rotates much faster than expected from the bulk of ATPase activity, and that approximately 10% of the F(1) molecules are active on the millisecond time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate/s), as a single molecule, is variable during a short period. The epsilon subunit inhibited rotation and ATPase, whereas epsilon fused through its carboxyl terminus to cytochrome b(562) showed no effect. The epsilon subunit significantly increased the pausing time during rotation. Stochastic fluctuation of catalysis may be a general property of an enzyme, although its understanding requires combining studies of steady-state kinetics and single molecule observation.

During ATP hydrolysis, the gammaepsilon c10 complex (gamma and epsilon subunits and a c subunit ring formed from 10 monomers) of F0F1 ATPase (ATP synthase) rotates relative to the alpha3beta3delta ab2 complex, leading to proton transport through the interface between the a subunit and the c subunit ring. In this study, we replaced the two pertinent residues for proton transport, cAsp-61 and aArg-210 of the c and a subunits, respectively. The mutant enzymes exhibited lower ATPase activities than that of the wild type but exhibited ATP-dependent rotation in planar membranes, in which their original assemblies are maintained. The mutant enzymes were defective in proton transport, as shown previously. These results suggest that proton transport can be separated from rotation in ATP hydrolysis, although rotation ensures continuous proton transport by bringing the cAsp-61 and aArg-210 residues into the correct interacting positions.

Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.

NTAK (neural- and thymus-derived activator for ErbB kinases), also known as neuregulin-2, is a member of the epidermal growth factor (EGF) family, which binds directly to ErbB3 and ErbB4 and transactivates ErbB2. Because ErbB signaling has been implicated in various angiogenic mechanisms, the effect of NTAK (which has at least nine isoforms due to alternative splicing) in angiogenesis is explored. One isoform, NTAKgamma, inhibited cell growth in terms of DNA synthesis and cell numbers in vascular endothelial cells specifically, whereas NTAKalpha and beta had no activity. On the other hand, NTAKgamma secreted by transfected MDA-MB-231 cells inhibited endothelial cell growth, and NTAKgamma expressed in endothelial cells by adenovirus infection suppressed cell growth in a dose-dependent manner. The EGF-like domain of NTAKgamma did not have this activity. The NTAKdelta isoform, which had the Ig-like domain but not the EGF-like domain, inhibited proliferation of endothelial cells. NTAKdelta prevented hyper-phosphorylation of the retinoblastoma tumor suppressor protein and caused G(1) arrest in endothelial cells. Both NTAKgamma and delta isoforms displayed anti-angiogenic activity in the chick embryo chorioallantoic membrane in vivo. These results suggest that the active site of NTAK is localized outside of the EGF-like domain but within the N-terminal region, including the Ig-like domain, of NTAK.

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as alpha-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their alpha chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (alpha5beta1gamma1) for alpha(3)beta(1) and alpha(6)beta(1) integrins and alpha-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4-5 modules did not compromise the binding of laminin-10 to alpha(3)beta(1) and alpha(6)beta(1) integrins but completely abrogated its binding to alpha-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to alpha-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the alpha-dystroglycan binding by the LG4 module.

The vacuolar-type H+-ATPases (V-ATPases) are multimeric proton pumps involved in a wide variety of physiological processes. We have identified two alternative splicing variants of C2 subunit isoforms: C2-a, a lung-specific isoform containing a 46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert. Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal alpha and beta intercalated cells. Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells. Furthermore, a chimeric yeast V-ATPase with mouse the C2-a or C2-b isoform showed a lower Km(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit). These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively.

In plant DNA, cytosines in symmetric CpG and CpNpG (N is A, T, or C) are thought to be methylated by DNA methyltransferases, MET1 and CMT3, respectively. Cytosines in asymmetric CpNpN are also methylated, and genetic analysis has suggested the responsible enzyme to be domains rearranged methyltransferase (DRM). We cloned a tobacco cDNA, encoding a novel protein consisting of 608 amino acids, that resembled DRMs found in maize and Arabidopsis and designated this as NtDRM1. The protein could be shown to be localized exclusively in the nucleus and exhibit methylation activity toward unmethylated synthetic as well as native DNA samples upon expression in Sf9 insect cells. It also methylated hemimethylated DNA, but the activity was lower than that for unmethylated substrates. Methylation mapping of a 962-bp DNA, treated with NtDRM1 in vitro, directly demonstrated methylation of approximately 70% of the cytosines in methylatable CpNpN and CpNpG sequences but only 10% in CpG. Further analyses indicated that the enzyme apparently non-selectively methylates any cytosines except in CpG, regardless of the adjacent nucleotide at both 5' and 3' ends. Transcripts of NtDRM1 ubiquitously accumulated in all tissues and during the cell cycle in tobacco cultured BY2 cells. These results indicate that NtDRM1 is a de novo cytosine methyltransferase, which actively excludes CpG substrate.

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.

Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells. In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase. Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit. V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase). The rotation was inhibited by concanamycin and nitrate but not by azide. These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.

Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification is carried out by vacuolar type H+-ATPase (V-ATPase) localized in the plasma membrane. We have shown that a3, one of the four subunit a isoforms (a1, a2, a3, and a4), is a component of the plasma membrane V-ATPase (Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760-8765). To establish the unique localization of V-ATPase, we have used a murine macrophage cell line, RAW 264.7, that can differentiate into multinuclear osteoclast-like cells on stimulation with RANKL (receptor activator of nuclear factor kappaB ligand). The V-ATPase with the a3 isoform was localized to late endosomes and lysosomes, whereas those with the a1 and a2 isoforms were localized to organelles other than lysosomes. After stimulation, the V-ATPase with the a3 isoform was immunochemically colocalized with lysosome marker lamp2 and was detected in acidic organelles. These organelles were also colocalized with microtubules, and the signals of lamp2 and a3 were dispersed by nocodazole, a microtubule depolymerizer. In RAW-derived osteoclasts cultured on mouse skull pieces, the a3 isoform was transported to the plasma membrane facing the bone and accumulated inside podosome rings. These findings indicate that V-ATPases with the a3 isoform localized in late endosomes/lysosomes are transported to the cell periphery during differentiation and finally assembled into the plasma membrane of mature osteoclasts.

Vacuolar H(+)-ATPases (V-ATPases), a family of multimeric proton pumps, are involved in a wide variety of physiological processes. We have identified two mouse genes, Atp6g1 and Atp6g2, encoding the G1 and G2 isoforms of the V-ATPase G subunit, respectively. G1 was distributed ubiquitously in the tissues examined, whereas G2 was specifically distributed in central nervous system neurons. G1 was expressed at an early embryonic stage, whereas G2 transcription was significantly induced at 10.5 days postcoitus (embryonic day 10.5, i.e. 2 days before axon outgrowth). Both G1 and G2 were strongly expressed in cortical and hippocampal neurons, cerebellar granule cells, and Purkinje cells. Immunohistochemistry with isoform-specific antibodies revealed that G2 was localized in cell bodies, dendrites, and axons. In addition, electron microscopy and subcellular fractionation indicated that G2 was localized in synaptic vesicles, whereas G1 was not detectable. G1 and G2 exhibit 62% identity, and both isoforms were immunoprecipitated with the c and A subunits of V-ATPase. G2 could complement the yeast deletion mutant Deltavma10, which lacks the G subunit. The V-ATPases containing the G1 and G2 isoforms, respectively, showed similar K(m)((ATP)) values and maximal velocity. These results indicate that G1 and G2 are bona fide subunits of V-ATPases and that the enzyme with the G2 isoform is involved in synaptic vesicle acidification.

The vacuolar-type H(+)-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K(m)(ATP) than yeast V-ATPase. Consistent with the temperature-sensitive growth of Deltavma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.

Subunit gamma of the ATP synthase F(1) sector is located at the center of the alpha(3)beta(3) hexamer and rotates unidirectionally during ATP hydrolysis, generating the rotational torque of approximately 45 pN.nm. A mutant F(1) with the betaSer-174 to Phe substitution (betaS174F) in the beta subunit generated lower torque ( approximately 17 pN.nm), indicating that betaS174F is mechanically defective, the first such mutant reported. The defective rotation of betaS174F was suppressed by a second-site mutation, betaGly-149 to Ala, betaIle-163 to Ala, or betaIle-166 to Ala in the same subunit, but not by betaLeu-238 to Ala. These results suggest that the region between betaGly-149 and betaSer-174 plays an important role in the coupling between ATP hydrolysis and mechanical work.

The vacuolar-type H+ -ATPase (V-ATPase) translocates protons across membranes. Here, we have identified a mouse cDNA coding for a fourth isoform (a4) of the membrane sector subunit a of V-ATPase. This isoform was specifically expressed in kidney, but not in the heart, brain, spleen, lung, liver, muscle, or testis. Immunoprecipitation experiments, together with sequence similarities for other isoforms (a1, a2, and a3), indicate that the a4 isoform is a component of V-ATPase. Moreover, histochemical studies show that a4 is localized in the apical and basolateral plasma membranes of cortical alpha- and beta-intercalated cells, respectively. These results suggest that the V-ATPase, with the a4 isoform, is important for renal acid/base homeostasis.

We have identified four genes (vha-5, vha-6, vha-7, and unc-32) coding for vacuolar-type proton-translocating ATPase (V-ATPase) subunit a in Caenorhabditis elegans, the first example of four distinct isoforms in eukaryotes. Their products had nine putative transmembrane regions, exhibited 43-60% identity and 62-84% similarity with the bovine subunit a1 isoform, and retained 11 amino acid residues essential for yeast V-ATPase activity (Leng, X. H., Manolson, M. F., and Forgac, M. (1998) J. Biol. Chem. 273, 6717-6723). The similarities, together with the results of immunoprecipitation, suggest that these isoforms are components of V-ATPase. Transgenic and immunofluorescence analyses revealed that these genes were strongly expressed in distinct cells; vha-5 was strongly expressed in an H-shaped excretory cell, vha-6 was strongly expressed in intestine, vha-7 was strongly expressed in hypodermis, and unc-32 was strongly expressed in nerve cells. Furthermore, the vha-7 and unc-32 genes were also expressed in the uteri of hermaphrodites. RNA interference analysis showed that the double-stranded RNA for unc-32 caused embryonic lethality similar to that seen with other subunit genes (vha-1, vha-4, and vha-11) (Oka, T., and Futai, M. (2000) J. Biol. Chem. 275, 29556-29561). The progenies of worms injected with the vha-5 or vha-6 double-stranded RNA became died at a specific larval stage, whereas the vha-7 double-stranded RNA showed no effect on development. These results suggest that V-ATPases with these isoforms generate acidic compartments essential for worm development in a cell-specific manner.

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.

ATP synthase (F0F1) transforms an electrochemical proton gradient into chemical energy (ATP) through the rotation of a subunit assembly. It has been suggested that a complex of the gamma subunit and c ring (c(10-14)) of F0F1 could rotate together during ATP hydrolysis and synthesis (Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y., and Futai, M. (1999) Science 286, 1722-1724). We observed that the rotation of the c ring with the cI28T mutation (c subunit cIle-28 replaced by Thr) was less sensitive to venturicidin than that of the wild type, consistent with the antibiotic effect on the cI28T mutant and wild-type ATPase activities (Fillingame, R. H., Oldenburg, M., and Fraga, D. (1991) J. Biol. Chem. 266, 20934-20939). Furthermore, we engineered F0F1 to see the alpha(3)beta(3) hexamer rotation; a biotin tag was introduced into the alpha or beta subunit, and a His tag was introduced into the c subunit. The engineered enzymes could be purified by metal affinity chromatography and density gradient centrifugation. They were immobilized on a glass surface through the c subunit, and an actin filament was connected to the alpha or beta subunit. The filament rotated upon the addition of ATP and generated essentially the same frictional torque as one connected to the c ring. These results indicate that the gammaepsilonc(10-14) complex is a mechanical unit of the enzyme and that it can be used as a rotor or a stator experimentally, depending on the subunit immobilized.