Carbohydrate-modifying sulfotransferases: structure, function, and pathophysiology.
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Animals↗
Biomedical subjects
M Fukuda
Publications and source records attributed to M Fukuda.
Effect of transforming growth factor-beta1 on the cell growth and Epstein-Barr virus reactivation in EBV-infected epithelial cell lines.
Transforming growth factor (TGF)-beta1 is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. In this study, we found that gastric tissue-derived Epstein-Barr virus (EBV)-infected epithelial cell lines GT38 and GT39 had resistance to TGF-beta1-mediated growth inhibition and apoptosis compared to a TGF-beta1-susceptible gastric carcinoma cell line HSC-39. However, TGF-beta1 partially induced EBV reactivation in GT38 and GT39 cells, as shown by the induction of EBV immediate-early BZLF1 RNA and its protein product ZEBRA and early antigen-D. The expressions of TGF-beta receptor I and II were detected in GT38 and GT39 cells by Northern and Western blot analyses. Both cell lines spontaneously produced the TGF-beta1, which was sufficient for inhibiting cell growth of HSC-39 cells. Taken together, these data suggest that TGF-beta1 may be a key factor for EBV reactivation and selective growth of EBV-infected epithelial cells in vivo.
Activin Receptors, Type I↗
Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells.
The classical mitogen-activated protein kinase (MAPK, also known as ERK) pathway is widely involved in eukaryotic signal transductions. In response to extracellular stimuli, MAPK becomes activated and translocates from the cytoplasm to the nucleus. At least two pathways for the nuclear import of MAPK are shown to exist; passive diffusion of a monomer and Ran-dependent active transport of a dimer, the detailed molecular mechanism of which is unknown. In this study, we have reconstituted nuclear import of MAPK in vitro by using digitonin-permeabilized cells with GFP-fused MAPK protein (GFP-MAPK), which is too large to pass through the nuclear pore by passive diffusion. GFP-MAPK was able to accumulate in the nucleus irrespective of its phosphorylation state. This import of GFP-MAPK occurred even in the absence of any soluble cytosolic factors or ATP but was inhibited by wheat germ agglutinin or an excess amount of importin-beta or at low temperatures. Moreover, MAPK directly bound to an FG repeat region of nucleoporin CAN/Nup214 in vitro. Taken together, these results suggest the third pathway for nuclear import of MAPK, in which MAPK passes through the nuclear pore by directly interacting with the nuclear pore complex.
Biological Transport↗
Formation of crystalloid endoplasmic reticulum induced by expression of synaptotagmin lacking the conserved WHXL motif in the C terminus. Structural importance of the WHXL motif in the C2B domain.
Synaptotagmin (Syt) is a family of type I membrane proteins that consists of a single transmembrane domain, a spacer domain, two Ca(2+)-binding C2 domains, and a short C terminus. We recently showed that deletion of the short C terminus (17 amino acids) of Syt IV prevented the Golgi localization of Syt IV proteins in PC12 cells and induced granular structures of various sizes in the cell body by an unknown mechanism (Fukuda, M., Ibata, K., and Mikoshiba, K. (2001) J. Neurochem. 77, 730-740). In this study we showed by electron microscopy that these structures are crystalloid endoplasmic reticulum (ER), analyzed the mechanism of its induction, and demonstrated that: (a) mutation or deletion of the evolutionarily conserved WHXL motif in the C terminus of the synaptotagmin family (Syt DeltaC) destabilizes the C2B domain structure (i.e. causes misfolding of the protein), probably by disrupting the formation of stable anti-parallel beta-sheets between the beta-1 and beta-8 strands of the C2B domain; (b) the resulting malfolded proteins accumulate in the ER rather than being transported to other membrane structures (e.g. the Golgi apparatus), with the malfolded proteins also inducing the expression of BiP (immunoglobulin binding protein), one of the ER stress proteins; and (c) the ERs in which the Syt DeltaC proteins have accumulated associate with each other as a result of oligomerization capacity of the synaptotagmin family, because the Syt IDeltaC mutant, which lacks oligomerization activity, cannot induce crystalloid ER. Our findings indicate that the conserved WHXL motif is important not only for protein interaction site but for proper folding of the C2B domain.
Amino Acid Motifs↗
Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains.
Synaptotagmin I (Syt I), a proposed major Ca(2+) sensor in the central nervous system, has been hypothesized as functioning in an oligomerized state during neurotransmitter release. We previously showed that Syts I, II, VII, and VIII form a stable SDS-resistant, beta-mercaptoethanol-insensitive, and Ca(2+)-independent oligomer surrounding the transmembrane domain (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the molecular mechanism of the Ca(2+)-independent oligomerization by the synaptotagmin family. In this study, we analyzed the Ca(2+)-independent oligomerization properties of Syt I and found that it shows two distinct forms of self-oligomerization activity: stable SDS-resistant self-oligomerization activity and relatively unstable SDS-sensitive self-oligomerization activity. The former was found to be mediated by a post-translationally modified (i.e. fatty-acylated) cysteine (Cys) cluster (Cys-74, Cys-75, Cys-77, Cys-79, and Cys-82) at the interface between the transmembrane and spacer domains of Syt I. We also show that the number of Cys residues at the interface between the transmembrane and spacer domains determines the SDS- resistant oligomerizing capacity of each synaptotagmin isoform: Syt II, which contains seven Cys residues, showed the strongest SDS-resistant oligomerizing activity in the synaptotagmin family, whereas Syt XII, which has no Cys residues, did not form any SDS-resistant oligomers. The latter SDS-sensitive self-oligomerization of Syt I is mediated by the spacer domain, because deletion of the whole spacer domain, including the Cys cluster, abolished it, whereas a Syt I(CA) mutant carrying Cys to Ala substitutions still exhibited self-oligomerization. Based on these results, we propose that the oligomerization of the synaptotagmin family is regulated by two distinct mechanisms: the stable SDS-resistant oligomerization is mediated by the modified Cys cluster, whereas the relatively unstable (SDS-sensitive) oligomerization is mediated by the environment of the spacer domain.
Amino Acid Sequence↗
Tac2-N, an atypical C-type tandem C2 protein localized in the nucleus.
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Amino Acid Sequence↗
Brain lateralization for mismatch response to across- and within-category change of vowels.
Differences in hemispheric predominance between across- and within-category change perception of vowels were assessed using a whole-head magnetoencephalography. The magnetic mismatch responses (MMNm) to pure-tone and vowel within-category changes were significantly predominant in the right hemisphere; on the other hand, vowel across-category MMNm did not differ in power between hemispheres. The results suggest that both hemispheres are symmetrically activated in the preattentive across-category change perception of vowels, while the within-category change of a vowel is analyzed as the change in physical features of the stimuli, thus predominantly activating the right hemisphere. Thus, the relative contribution of the left auditory cortex in the preattentive speech processing may occur only at the level of perception of the vowel across-category change.
Acoustic Stimulation↗
Synaptotagmin VI participates in the acrosome reaction of human spermatozoa.
Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.
Acrosome Reaction↗
[Purpura due to vasculitis induced by interferon therapy for a patient with chronic hepatitis type C].
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Hepatitis C, Chronic↗
Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase.
L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to addressins expressed in the high endothelial venules (HEV) of secondary lymphoid organs. Peripheral node addressin recognized by the MECA-79 antibody is apparently part of the L-selectin ligand, but its chemical nature has been undefined. We now identify a sulfated extended core1 mucin-type O-glycan, Gal beta 1-->4(sulfo-->6)GlcNAc beta 1-->3Gal beta 1-->3GalNAc, as the MECA-79 epitope. Molecular cloning of a HEV-expressed core1-beta 1,3-N-acetylglucosaminyltransferase (Core1-beta 3GlcNAcT) enabled the construction of the 6-sulfo sialyl Lewis x on extended core1 O-glycans, recapitulating the potent L-selectin-mediated, shear-dependent adhesion observed with novel L-selectin ligands derived from core2 beta1,6-N-acetylglucosaminyltransferase-I null mice. These results identify Core1-beta 3GlcNAcT and its cognate extended core1 O-glycans as essential participants in the expression of the MECA-79-positive, HEV-specific L-selectin ligands required for lymphocyte homing.
Animals↗
Crystal structure of 2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (BphD enzyme) from the Rhodococcus sp. strain RHA1 of the PCB degradation pathway.
2-Hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (the BphD enzyme) hydrolyzes a ring-cleavage product of an aromatic compound generated in a biphenyl/polychlorinated biphenyl (PCB) degradation pathway of bacteria. The crystal structure of the BphD enzyme has been determined at 2.4 A resolution by the multiple isomorphous replacement method. The final refined model of the BphD enzyme yields an R-factor of 17.5 % at 2.4 A resolution with reasonable geometry. The BphD enzyme is an octameric enzyme with a 422 point-group symmetry. The subunit can be divided into core and lid domains. The active site of the enzyme is situated in the substrate-binding pocket, which is located between the two domains. The substrate-binding pocket can be divided into hydrophobic and hydrophilic regions. This feature of the pocket seems to be necessary for substrate binding, as the substrate is composed of hydrophilic and hydrophobic parts. The proposed orientation of the substrate seems to be consistent with the general catalytic mechanism of alpha/beta-hydrolases.
Amino Acid Sequence↗
Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains.
The Ca(2+)-dependent oligomerization activity of the second C2 (C2B) domain of synaptotagmin I (Syt I) has been hypothesized to regulate neurotransmitter release. We previously showed that the cytoplasmic domains of several other Syt isoforms also show Ca(2+)-dependent oligomerization activity (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the involvement of their C2 domains in Ca(2+)-dependent oligomerization. In this study, we analyzed the Ca(2+)-dependent oligomerization properties of the first (C2A) and the second C2 (C2B) domains of Syt VII. Unlike Syt I, both C2 domains of Syt VII contribute to Ca(2+)-dependent homo- and hetero-oligomerization with other isoforms. For instance, the Syt VII C2A domain Ca(2+)-dependently binds itself and the C2A domain of Syt VI but not its C2B domain, whereas the Syt VII C2B domain Ca(2+)-dependently binds itself and the C2B domain of Syt II but not its C2A domain. In addition, we showed by gel filtration that a single Syt VII C2 domain is sufficient to form a Ca(2+)-dependent multimer of very high molecular weight. Because of this "two handed" structure, the Syt VII cytoplasmic domain has been found to show the strongest Ca(2+)-dependent multimerization activity in the Syt family. We also identified Asn-328 in the C2B domain as a crucial residue for the efficient Ca(2+)-dependent switch for multimerization by site-directed mutagenesis. Our results suggest that Syt VII is a specific isoform that can cluster different Syt isoforms with two hands in response to Ca(2+).
Amino Acid Sequence↗
The C2A domain of double C2 protein gamma contains a functional nuclear localization signal.
The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+ -dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Calpha functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+ -binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+ -independent C2A domain of double C2 protein gamma (Doc2gamma) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2gamma contains six Arg residues ((177)RLRRRRR(183)) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2gamma. Because of the presence of the basic cluster, the C2A domain of Doc2gamma did not show Ca2+ -dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif.
Amino Acid Sequence↗
Herniation of cervical intervertebral disc: immunohistochemical examination and measurement of nitric oxide production.
STUDY DESIGN: Surgically obtained cervical herniated intervertebral discs were examined histologically and immunohistochemically. The production of nitric oxide (NO) in the local tissue was examined using the electron spin resonance (ESR) method. OBJECTIVES: To investigate the local histologic and immunohistochemical changes in cervical disc herniation, including NO production, and to compare such changes with those in autopsy cases. SUMMARY OF BACKGROUND DATA: Very little is known about the histopathologic processes of cervical disc herniation. In addition, no information is available on the level of in vivo NO production in cervical disc herniation. METHODS: Thirty-six herniated cervical discs obtained from 31 patients were immunohistochemically examined for localization of blood vessels, matrix metalloproteinase (MMP)-3, and inducible NO synthetase (iNOS). We also compared the production of NO, measured by the ESR method, in eight specimens with that of five control discs obtained from fresh cadavers. RESULTS: The presence of herniated discs correlated with the degeneration of cartilaginous endplate and torn anulus fibrosus. Formation of new blood vessels around the herniated discs was detected, using von Willebrand factor antibody, in seven uncontained hernias and 20 contained hernias. Immunohistochemical studies showed the presence of cells positive for MMP-3 (chondrocytes), iNOS (chondrocytes and granulation tissue) in cervical disc hernias. ESR analysis showed a significantly higher NO production in herniated cervical discs than in disc samples of fresh cadavers. CONCLUSIONS: Herniated cervical intervertebral disc is characterized by the presence of an inflammatory process associated with neovascularization and increased expression of MMP-3. Production of NO was markedly high in both contained- and uncontained-type hernias.
Adult↗
Novel splicing isoforms of synaptotagmin-like proteins 2 and 3: identification of the Slp homology domain.
Slp1-3 (synaptotagmin-like protein 1-3) is a new family of carboxyl-terminal-type (C-type) tandem C2 proteins that show higher sequence similarity to the C2 domains of granuphilin-a/Slp-4 than those of other C-type tandem C2 proteins (e.g., synaptotagmin and the Doc2 family). However, the amino (N)-terminal domains of the original Slp1-3 do not contain any known protein motifs and do not show any sequence similarities to each other. We report four alternative splicing isoforms of Slp2 (designated Slp2-a-d, with the original Slp2 renamed Slp2-c) and two alternative splicing isoforms of Slp3 (Slp3-a and Slp3-b, the original Slp3). These isoforms share the same C-terminal tandem C2 structures, but their N-terminal nucleotide sequences are completely different due to the alternate use of different exons. Sequence alignment of the Slp1, Slp2-a, Slp3-a, and Slp4 amino terminal domains reveals the presence of two conserved regions among the Slp family, designated SHD1 (Slp homology domain 1) and SHD2, which may function as protein interaction sites. The SHD1 and SHD2 of Slp3-a and Slp4 are separated by a putative Zn(2+)-binding sequence, whereas Slp1 and Slp2 lack such Zn(2+)-binding sequences and their SHD1 and SHD2 are linked together. In addition, we show that the Slp2-a/c/d mRNAs are differentially distributed in different mouse tissues and at different stages of development, suggesting that these transcripts may be regulated by different promoters.
Alternative Splicing↗
Molecular cloning and expression of a novel human beta-Gal-3-O-sulfotransferase that acts preferentially on N-acetyllactosamine in N- and O-glycans.
A novel cDNA-encoding galactose 3-O-sulfotransferase was cloned by screening the expressed sequence tag data base using the previously cloned cDNA encoding a galactosyl ceramide 3-O-sulfotransferase, which we term Gal3ST-1. The newly isolated cDNA encodes a novel 3-O-sulfotransferase, termed Gal3ST-3, that acts exclusively on N-acetyllactosamine present in N-glycans and core2-branched O-glycans. These conclusions were confirmed by analyzing CD43 chimeric proteins in Chinese hamster ovary cells expressing core2 beta1,6-N-acetylglucosaminyltransferase. The acceptor specificity of Gal3ST-3 contrasts with that of the recently cloned galactose 3-O-sulfotransferase (Honke, K., Tsuda, M., Koyota, S., Wada, Y., Iida-Tanaka, N., Ishizuka, I., Nakayama, J., and Taniguchi, N. (2001) J. Biol. Chem. 276, 267-274), which we term Gal3ST-2 in the present study because the latter enzyme can also act on core1 O-glycan and type 1 oligosaccharides, Galbeta1-->3GlcNAc. Moreover, Gal3ST-3 but not Gal3ST-2 can act on Galbeta1-->4(sulfo-->6)GlcNAc, indicating that disulfated sulfo-->3Galbeta1-->4(sulfo-->6) GlcNAc-->R may be formed by Gal3ST-3 in combination with GlcNAc 6-O-sulfotransferase. Although both Gal3ST-2 and Gal3ST-3 do not act on galactosyl ceramide, Gal3ST-3 is only moderately more homologous to Gal3ST-2 (40.1%) than to Gal3ST-1 (38.0%) at the amino acid level. Northern blot analysis demonstrated that transcripts for Gal3ST-3 are predominantly expressed in the brain, kidney, and thyroid where the presence of 3'-sulfation of N-acetyllactosamine has been reported. These results indicate that the newly cloned Gal3ST-3 plays a critical role in 3'-sulfation of N-acetyllactosamine in both O- and N-glycans.
Amino Acid Sequence↗
Electromagnetic moments of the beta-emitting nucleus 16N.
The nuclear magnetic dipole moment mu and electric quadrupole moment Q of the beta-emitting 16N(Ipi = 2(-), T(1/2) = 7.13 s) nucleus have been determined for the first time by detecting its beta-NMR in a MgO crystal and beta-NQR (nuclear quadrupole resonance) in a TiO (2) crystal to be /mu/ = (1.9859+/-0.0011) mu(N) and /Q/ = (17.9+/-1.7) mb, respectively. Although the prediction of mu given by the Hartree-Fock calculation agrees well with the experiment, an abnormally small effective charge for neutrons is required to account for the experimental Q.
Journal Article↗
Impaired suppression of processing in schizophrenic patients suggested by ERPs obtained in a selective attention task.
In the present study, we focused on the ability of suppression of processing in schizophrenic patients, using event-related potentials (ERPs) recorded during a selective attention task. During the task, subjects were required to focus on one ear, counting deviant stimuli, those deviating in duration from a sequence of standard stimuli. We compared amplitude data of two positive components differing in latency elicited by standard stimuli, which reflect suppression of stimulus processing, between schizophrenic patients and normal controls. Significant between-group differences were obtained specifically in the right ear attended condition, suggesting impaired suppression of processing in schizophrenics mediated in the left hemisphere.
Adult↗