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The innate immune system plays an essential role in initial recognition of pathogen infection by producing inflammatory cytokines and type I interferons. cGAS is a cytoplasmic sensor for DNA derived from DNA viruses. cGAS binding with DNA induces the production of cGAMP, a second messenger that associates with STING in endoplasmic reticulum (ER). STING changes its cellular distribution from ER to perinuclear Golgi, where it activates the protein kinase TBK1 that catalyzes the phosphorylation of IRF3. Here we found that STING trafficking is regulated by myotubularin-related protein (MTMR) 3 and MTMR4, members of protein tyrosine phosphatases that dephosphorylate 3' position in phosphatidylinositol (PtdIns) and generate PtdIns5P from PtdIns3,5P 2 and PtdIns from PtdIns3P. We established MTMR3 and MTMR4 double knockout (DKO) RAW264.7 macrophage cells and found that they exhibited increased type I interferon production after interferon-stimulatory DNA (ISD) stimulation and herpes simplex virus 1 infection concomitant with enhanced IRF3 phosphorylation. In DKO cells, STING rapidly trafficked from ER to Golgi after ISD stimulation. Notably, DKO cells exhibited enlarged cytosolic puncta positive for PtdIns3P and STING was aberrantly accumulated in this puncta. Taken together, these results suggest that MTMR3 and MTMR4 regulate the production of PtdIns3P, which plays a critical role in suppressing DNA-mediated innate immune responses via modulating STING trafficking., (© 2019 Dewi Pamungkas Putri et al.)

Netrin 1 was initially identified as an axon guidance factor, and recent studies indicate that it inhibits chemokine-directed monocyte migration. Despite its importance as a neuroimmune guidance cue, the role of netrin 1 in osteoclasts is largely unknown. Here we detected high netrin 1 levels in the synovial fluid of rheumatoid arthritis patients. Netrin 1 is potently expressed in osteoblasts and synovial fibroblasts, and IL-17 robustly enhances netrin 1 expression in these cells. The binding of netrin 1 to its receptor UNC5b on osteoclasts resulted in activation of SHP1, which inhibited VAV3 phosphorylation and RAC1 activation. This significantly impaired the actin polymerization and fusion, but not the differentiation of osteoclast. Strikingly, netrin 1 treatment prevented bone erosion in an autoimmune arthritis model and age-related bone destruction. Therefore, the netrin 1-UNC5b axis is a novel therapeutic target for bone-destructive diseases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

We have generated a mouse monoclonal antibody (R-17F, IgG1 subtype) specific to human induced pluripotent stem (hiPS)/embryonic stem (ES) cells by using a hiPS cell line as an antigen. Triple-color confocal immunostaining images of hiPS cells with R-17F indicated that the R-17F epitope was expressed exclusively and intensively on the cell membranes of hiPS cells and co-localized partially with those of SSEA-4 and SSEA-3. Lines of evidence suggested that the predominant part of the R-17F epitope was a glycolipid. Upon TLC blot of total lipid extracts from hiPS cells with R-17F, one major R-17F-positive band was observed at a slow migration position close to that of anti-blood group H1(O) antigen. MALDI-TOF-MS and MS(n) analyses of the purified antigen indicated that the presumptive structure of the R-17F antigen was Fuc-Hex-HexNAc-Hex-Hex-Cer. Glycan microarray analysis involving 13 different synthetic oligosaccharides indicated that R-17F bound selectively to LNFP I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc). A critical role of the terminal Fucα1-2 residue was confirmed by the selective disappearance of R-17F binding to the purified antigen upon α1-2 fucosidase digestion. Most interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. The cytotoxic effect was augmented markedly upon the addition of the secondary antibody (goat anti-mouse IgG1 antibody). R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS cells in hiPS-derived regenerative tissues, which are considered to be a strong risk factor for carcinogenesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

RIG-I-like receptors (RLRs), including retinoic acid-inducible gene-I (RIG-I) and MDA5, constitute a family of cytoplasmic RNA helicases that senses viral RNA and mounts antiviral innate immunity by producing type I interferons and inflammatory cytokines. Despite their essential roles in antiviral host defense, RLR signaling is negatively regulated to protect the host from excessive inflammation and autoimmunity. Here, we identified ADP-ribosylation factor-like protein 5B (Arl5B), an Arl family small GTPase, as a regulator of RLR signaling through MDA5 but not RIG-I. Overexpression of Arl5B repressed interferon β promoter activation by MDA5 but not RIG-I, and its knockdown enhanced MDA5-mediated responses. Furthermore, Arl5B-deficient mouse embryonic fibroblast cells exhibited increased type I interferon expression in response to MDA5 agonists such as poly(I:C) and encephalomyocarditis virus. Arl5B-mediated negative regulation of MDA5 signaling does not require its GTP binding ability but requires Arl5B binding to the C-terminal domain of MDA5, which prevents interaction between MDA5 and poly(I:C). Our results, therefore, suggest that Arl5B is a negative regulator for MDA5., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a type II transmembrane C-type lectin expressed on DCs such as myeloid DCs and monocyte-derived DCs (MoDCs). Recently, we have reported that DC-SIGN interacts with carcinoembryonic antigen (CEA) expressed on colorectal carcinoma cells. CEA is one of the most widely used tumor markers for gastrointestinal cancers such as colorectal cancer. On the other hand, other groups have reported that the level of Mac-2-binding protein (Mac-2BP) increases in patients with pancreatic, breast, and lung cancers, virus infections such as human immunodeficiency virus and hepatitis C virus, and autoimmune diseases. Here, we first identified Mac-2BP expressed on several colorectal carcinoma cell lines as a novel DC-SIGN ligand through affinity chromatography and mass spectrometry. Interestingly, we found that DC-SIGN selectively recognizes Mac-2BP derived from some colorectal carcinomas but not from the other ones. Furthermore, we found that the α1-3,4-fucose moieties of Le glycans expressed on DC-SIGN-binding Mac-2BP were important for recognition. DC-SIGN-dependent cellular interactions between immature MoDCs and colorectal carcinoma cells significantly inhibited MoDC functional maturation, suggesting that Mac-2BP may provide a tolerogenic microenvironment for colorectal carcinoma cells through DC-SIGN-dependent recognition. Importantly, Mac-2BP was detected as a predominant DC-SIGN ligand expressed on some primary colorectal cancer tissues from certain parts of patients in comparison with CEA from other parts, suggesting that DC-SIGN-binding Mac-2BP bearing tumor-associated Le glycans may become a novel potential colorectal cancer biomarker for some patients instead of CEA.

Ca(2+) signals through store-operated Ca(2+) (SOC) channels, activated by the depletion of Ca(2+) from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca(2+) sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca(2+) entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCbeta both resulted in increased Ca(2+) influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.

Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N-acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the G alpha subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity.

Plant NADPH oxidases (Rboh, for respiratory burst oxidase homolog) produce reactive oxygen species that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs that regulate Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca(2+) binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping the EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca(2+) binding to only one EF-hand. Structure-based in vitro pulldown assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by swapping the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N and C terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of reactive oxygen species production.

HNK-1 (human natural killer-1) glyco-epitope, a sulfated glucuronic acid attached to N-acetyllactosamine on the nonreducing termini of glycans, is highly expressed in the nervous system. Our previous report showed that mice lacking a glucuronyltransferase (GlcAT-P), a key enzyme for biosynthesis of the HNK-1 epitope, showed reduced long term potentiation at hippocampal CA1 synapses. In this study, we identified an alpha-amino-3-hydroxy-5-methylisoxazole propionate (AMPA)-type glutamate receptor subunit, GluR2, which directly contributes to excitatory synaptic transmission and synaptic plasticity, as a novel HNK-1 carrier molecule. We demonstrated that the HNK-1 epitope is specifically expressed on the N-linked glycan(s) on GluR2 among the glutamate receptors tested, and the glycan structure, including HNK-1 on GluR2, was determined using liquid chromatography-tandem mass spectrometry. As for the function of HNK-1 on GluR2, we found that the GluR2 not carrying HNK-1 was dramatically endocytosed and expressed less on the cell surface compared with GluR2 carrying HNK-1 in both cultured hippocampal neurons and heterologous cells. These results suggest that HNK-1 stabilizes GluR2 on neuronal surface membranes and regulates the number of surface AMPA receptors. Moreover, we showed that the expression of the HNK-1 epitope enhanced the interaction between GluR2 and N-cadherin, which has important roles in AMPA receptor trafficking. Our findings suggest that the HNK-1 epitope on GluR2 regulates cell surface stability of GluR2 by modulating the interaction with N-cadherin.

Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3'-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3'-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cgamma (PKCgamma). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCgamma kinase activity. To determine the effect of these mutations on PKCgamma function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCgammas. We found that wild-type PKCgamma, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCgamma. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCgamma. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCgammas was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.

Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates complement in association with MBP-associated serine proteases via the lectin pathway. Despite our previous study (Mori, K., Kawasaki, T., and Yamashina, I. (1984) Arch. Biochem. Biophys. 232, 223-233), the subcellular localization of I-MBP and its functional implication have not been clarified yet. Here, as an extension of our previous studies, we have demonstrated that the expression of human MBP cDNA reproduces native MBP differentiation of S-MBP and I-MBP in human hepatoma cells. I-MBP shows distinct accumulation in cytoplasmic granules, and is predominantly localized in the endoplasmic reticulum (ER) and involved in COPII vesicle-mediated ER-to-Golgi transport. However, the subcellular localization of either a mutant (C236S/C244S) I-MBP, which lacks carbohydrate-binding activity, or the wild-type I-MBP in tunicamycin-treated cells shows an equally diffuse cytoplasmic distribution, suggesting that the unique accumulation of I-MBP in the ER and COPII vesicles is mediated by an N-glycan-lectin interaction. Furthermore, the binding of I-MBP with glycoprotein intermediates occurs in the ER, which is carbohydrate- and pH-dependent, and is affected by glucose-trimmed high-mannose-type oligosaccharides. These results strongly indicate that I-MBP may function as a cargo transport lectin facilitating ER-to-Golgi traffic in glycoprotein quality control.

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.

In Alzheimer disease, amyloid beta, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 microm on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAbeta1-3Galbeta1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.

The HNK-1 carbohydrate, which is recognized by anti-HNK-1 antibody, is well known to be expressed predominantly in the nervous system. The characteristic structural feature of the HNK-1 carbohydrate is 3-sulfo-glucuronyl residues attached to lactosamine structures (Gal beta1-4GlcNAc) on glycoproteins and glycolipids. The biosynthesis of the HNK-1 carbohydrate is regulated mainly by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase. In this study, we found that GlcAT-S mRNA was expressed at higher levels in the kidney than in the brain, but that both GlcAT-P and HNK-1 sulfotransferase mRNAs, which were expressed at high levels in the brain, were not detected in the kidney. These results suggested that the HNK-1 carbohydrate without sulfate (non-sulfated HNK-1 carbohydrate) is expressed in the kidney. We substantiated this hypothesis using two different monoclonal antibodies: one (anti-HNK-1 antibody) requires sulfate on glucuronyl residues for its binding, and the other (antibody M6749) does not. Western blot analyses of mouse kidney revealed that two major bands (80 and 140 kDa) were detected with antibody M6749, but not with anti-HNK-1 antibody. The 80- and 140-kDa band materials were identified as meprin alpha and CD13/aminopeptidase N, respectively. We also confirmed the presence of the non-sulfated HNK-1 carbohydrate on N-linked oligosaccharides by multistage tandem mass spectrometry. Immunofluorescence staining with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex and was also detected in the thin ascending limb in the inner medulla. This is the first study indicating the presence of the non-sulfated HNK-1 carbohydrate being synthesized by GlcAT-S in the kidney. The results presented here constitute novel knowledge concerning the function of the HNK-1 carbohydrate.

Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Le(a) structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition.

YM-254890, which was isolated from the culture broth of Chromobacterium sp., inhibits ADP-induced platelet aggregation and has antithrombotic and thrombolytic effects. YM-254890 blocks Galpha(q/11)-coupled ADP receptor P2Y1-mediated Ca(2+) mobilization. Here we report that YM-254890 is a selective Galpha(q/11) inhibitor. YM-254890 blocked Ca(2+) mobilization mediated by several Galpha(q/11)-coupled receptors but not by Galpha(i)- or Galpha(15)-coupled receptor, indicating that phospholipase Cbeta activation and subsequent signaling molecules are not the target of YM-254890. YM-254890 completely prevented the serum response factor (SRF)-mediated gene transcription induced by Galpha(q)R183C, which is constitutively active in a receptor-dependent manner because of its reduced k(cat) of GTP hydrolysis. Conversely, YM-254890 had only a modest effect on the SRF-mediated gene transcription by Galpha(q)Q209L, which is GTPase-deficient (activated) Galpha(q). These suggested that the acting point of YM-254890 is receptor-Galpha(q) interaction or the subsequent guanine nucleotide exchange step. The fact that YM-254890 (i) inhibited the SRF-mediated gene transcription by Galpha(qi5), which interacts with Galpha(i)-coupled receptor and possesses the effector function of Galpha(q), and (ii) had no effect on the K(d) value of high affinity [(3)H]2MeSADP binding to P2Y1, which reflects the agonist-receptor-Galpha ternary complex, suggested that receptor-Galpha(q/11) interaction is not the target of YM-254890. On the other hand, specific [(35)S]GTPgammaS binding to Galpha(q/11) stimulated by the M1 muscarinic acetylcholine receptor and P2Y1 were inhibited by YM-254890. These data indicate that YM-254890 blocks the exchange of GDP for GTP in Galpha(q/11) activation. This novel Galpha(q/11)-selective inhibitor is a promising and powerful tool for studying Galpha(q/11) protein activation, Galpha(q/11) -coupled receptor signaling, and Galpha(q/11)-mediated biological events.

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P -/- mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.

The p53 transcription factor is either latent or activated through multi-site phosphorylation and acetylation of the negative regulatory region in its C-terminal domain (CTD). How CTD modifications activate p53 binding to target DNA sequences via its core domain is still unknown. It has been proposed that nonmodified CTD interacts either with the core domain or with DNA preventing binding of the core domain to DNA and that the fragments of the CTD regulatory region activate p53 by interfering with these interactions. We here characterized the sequence and target specificity of p53 activation by CTD fragments, interaction of activating peptides with p53 and target DNA, and interactions of "latent" p53 with DNA by a band shift assay and by fluorescence correlation spectroscopy. In addition to CTD fragments, several long basic peptides activated p53 and also transcription factor YY1. These peptides and CTD aggregated target DNA but apparently did not interact with p53. The potency to aggregate DNA correlated with the ability to activate p53, suggesting that p53 binds to target sequences upon interactions with tightly packed DNA in aggregates. Latent full-length p53 dissociated DNA aggregates via its core and CTD, and this effect was potentiated by GTP. Latent p53 also formed complexes via both its core and CTD with long nontarget DNA molecules. Such p53-DNA interactions may occur if latent p53 binding to DNA via CTD prevents the interaction of the core domain with target DNA sites but not with nonspecific DNA sequences.

The glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins was purified to an apparent homogeneity from the Nonidet P-40 extract of 2-week postnatal rat forebrain by sequential chromatographies on CM-Sepharose CL-6B, UDP-GlcA-Sepharose 4B, asialo-orosomucoid-Sepharose 4B, Matrex gel Blue A, Mono Q, HiTrap chelating, and HiTrap heparin columns. The purified enzyme migrated as a 45-kDa protein upon SDS-polyacrylamide gel electrophoresis under reducing conditions, but eluted as a 90-kDa protein upon Superose gel filtration in the presence of Nonidet P-40, suggesting that the enzyme forms homodimers under non-denatured conditions. The enzyme transferred glucuronic acid to various glycoprotein acceptors bearing terminal N-acetyllactosamine structure such as asialo-orosomucoid, asialo-fetuin, and asialo-neural cell adhesion molecule, whereas little activity was detected to paragloboside, a precursor glycolipid of the HNK-1 epitope on glycolipids. These results suggested that the enzyme is specifically associated with the biosynthesis of the HNK-1 epitope on glycoproteins. Sphingomyelin was specifically required for expression of the enzyme activity. Stearoyl-sphingomyelin (18:0) was the most effective, followed by palmitoyl-sphingomyelin (16:0) and lignoceroyl-sphingomyelin (24:0). Interestingly, activity was demonstrated only for sphingomyelin with a saturated fatty acid, i.e. not for that with an unsaturated fatty acid, regardless of the length of the acyl group.

N-Glycolylneuraminic acid (NeuGc) is abundantly expressed in most mammals, but it is not detectable in humans. The expression of NeuGc is controlled by cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase activity. We previously cloned a cDNA for mouse CMP-NeuAc hydroxylase and found that the human genome contains a homologue. We report here the molecular basis for the absence of NeuGc in humans. We cloned a cDNA for human CMP-NeuAc hydroxylase from a HeLa cell cDNA library. The cDNA encodes a 486-amino acid protein, and its deduced amino acid sequence lacks a domain corresponding to the N-terminal 104 amino acids of the mouse CMP-NeuAc hydroxylase protein, although the human protein is highly identical (93%) to the rest of the mouse hydroxylase protein. The N-terminal truncation of the human hydroxylase is caused by deletion of a 92-base pair-long exon in human genomic DNA. The human hydroxylase expressed in COS-7 cells exhibited no enzymatic activity, and a mouse hydroxylase mutant, which lacks the N-terminal domain, was also inactive. A chimera composed of the human hydroxylase and the N-terminal domain of the mouse hydroxylase displayed the enzyme activity. These results indicate that the human homologue of CMP-NeuAc hydroxylase is inactive because it lacks an N-terminal domain that is essential for enzyme activity. The absence of NeuGc in human glycoconjugates is due to a partial deletion in the gene that encodes CMP-NeuAc hydroxylase.

We isolated a cDNA encoding a novel glucuronyltransferase from human placenta cDNA with the use of the degenerate reverse transcriptase-polymerase chain reaction method. Degenerate primers were designed based upon the amino acid sequence alignment of rat glucuronyltransferase (GlcAT-P) involved in the biosynthesis of the carbohydrate epitope HNK-1 with putative proteins in Caenorhabditis elegans and Schistosoma mansoni. The new cDNA sequence revealed an open reading frame coding for a protein of 335 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 43% identity to the rat GlcAT-P, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active glucuronyltransferase with marked specificity for a glycoserine Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. In contrast, asialoorosomucoid, which contains the Galbeta1-4GlcNAc sequence and is a good acceptor substrate for the GlcAT-P, did not serve as an acceptor. The reaction product was sensitive to beta-glucuronidase digestion and co-chromatographed with authentic GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser in high-performance liquid chromatography, suggesting that the enzyme is a beta1, 3-glucuronyltransferase. These results indicate that this new member of the glucuronyltransferase gene family is the enzyme previously described as glucuronyltransferase I that forms the glycosaminoglycan-protein linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans.

The HNK-1 carbohydrate epitope is expressed on several neural adhesion glycoproteins and as a glycolipid, and is involved in cell interactions. The structural element of the epitope common to glycoproteins and glycolipids has been determined to be sulfate-3-GlcAbeta1--> 3Galbeta1-->4GlcNAc. The glucuronyltransferase and sulfotransferase are considered to be the key enzymes in the biosynthesis of this epitope because the rest of the structure occurs often in glycoconjugates. Here we describe the isolation of the rat sulfotransferase cDNA via an expression cloning strategy. The clone finally isolated predicts a protein of 356 amino acids, with characteristics of a type II transmembrane protein and with no sequence similarity to other known sulfotransferases. Both the enzyme expressed as a soluble fusion protein and homogenates of cells transfected with the full-length cDNA could transfer sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid.

We have cloned a novel protein kinase from human cerebellum and named it LZK (leucine zipper-bearing kinase). The LZK cDNA encoded a 966-amino acid polypeptide that contains a kinase catalytic domain and double leucine/isoleucine zippers separated by a short spacer region. The amino acid sequence of the kinase catalytic domain was a hybrid between those in serine/threonine and tyrosine protein kinases, indicating that LZK belongs to the subfamily of the mixed lineage kinase (MLK) family. The kinase catalytic domain of LZK was most similar to DLK (Holtzman, L. B., Merritt, S.E., and Fan, G. (1994) J. Biol. Chem. 269, 30808-30817), MUK (Hirai, S., Izawa, M., Osada, S., Spyrou, G., and Ohno, S. (1996) Oncogene 12, 641-650), and ZPK (Reddy, U. R., and Presure, D. (1994) Biochem. Biophys. Res. Commun. 202, 613-620), which belong to the same subfamily of the MLK family. However, besides the kinase catalytic domain and double leucine/isoleucine zippers, there was no significant homology with known proteins. The recombinant LZK autophosphorylated in the presence of ATP and divalent cations, and exhibited serine/threonine kinase catalytic activity. Northern blot analysis revealed that LZK is expressed most strongly in the pancreas, with a pattern that differs from other MLKs. Expression of LZK in COS7 cells induced phosphorylation of c-Jun and activation of JNK-1, indicating the association of LZK in the c-Jun amino-terminal kinase/stress-activated protein kinase pathway. The expressed LZK was detected primarily in the membrane fraction, suggesting that LZK interacts with other cellular components in vivo.

Jararaca GPIb-BP, a snake venom protein composed of alpha and beta subunits purified from Bothrops jararaca, binds to platelet glycoprotein (GP)Ib and functions as a receptor blocker for von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura, S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K., Taniuchi, Y., and Kawasaki, T. (1995) Thromb. Haemostasis 74, 743-750). We present here the entire 142- and 123-residue amino acid sequence of the respective alpha and beta subunits and also demonstrate that the platelet GPIb-binding site resides on the beta and not on the alpha subunit based on an enzyme-linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands. Sequences of the alpha and beta subunits were determined by analysis of the intact S-pyridylethylated proteins and their peptides generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl ketone-trypsin. A 38-39% identity of amino acid sequence between the alpha and beta subunits of jararaca GPIb-BP was observed, as well as a high degree of sequence identities (38-64%) with the respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., and Titani, K (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 928-932) and the beta-chain of echicetin (Peng, M., Holt, J. C., and Niewiarowski, S. (1994) Biochem. Biophys. Res. Commun. 205, 68-72).

The serum-mannan binding protein (S-MBP) is a calcium-dependent C-type lectin specific for mannose and N-acetylglucosamine. S-MBP is known as a host defense factor involved in innate immunity, where the target ligands for S-MBP should be on the surface of exogenous microorganisms. In this study, we tried to find endogenous ligands for this endogenous lectin. Among the cells tested, only the lymphocytes from thymus of BALB/c mice expressed ligands for S-MBP on their surface, those from bone marrow, spleen, mesenteric lymph nodes and peripheral blood all being negative. Interestingly, among the thymocytes, only the immature thymocytes with the CD4+CD8+CD3low phenotype expressed ligands for S-MBP, and ligands for S-MBP decreased on their maturation. A major cell surface glycoprotein bearing S-MBP ligands was isolated and identified as CD45RO, which is a transmembrane protein with tyrosine phosphatase activity. Deglycosylation experiments with N-glycanase and endoglycosidase H indicated that the S-MBP ligands on thymic CD45 are high mannose type or hybrid type N-linked oligosaccharides. This unique presentation of S-MBP ligands on this special CD45 isoform suggested the possibility that the oligosaccharide portion of CD45 on immature thymocytes is associated with the maturation, development or selection events of thymocytes.

In our previous study, the sphingosine-like immunosuppressant, ISP-1, was found to suppress the proliferation of an interleukin-2-dependent cytotoxic T cell line, CTLL-2, through the inhibition of serine palmitoyltransferase, which catalyzes the committed step of sphingolipid biosynthesis. Analysis of the effect of ISP-1 by flow cytometry revealed that the ISP-1-dependent decrease in cell number was not due to inhibition of the cell cycle progression of CTLL-2 cells but to the induction of apoptosis of the cells. The ISP-1-induced apoptosis was inhibited by the addition of sphingosine (2 microM), suggesting that this ISP-1-induced apoptosis is triggered by the decrease in the intracellular levels of sphingolipids caused by the inhibition of serine palmitoyltransferase. However, another interleukin-2-dependent cell line, F7, which was derived from a mouse pro-B cell line, did not show ISP-1-dependent apoptosis, indicating that the effect of ISP-1 may be specific for a certain type of T cell lineage such as CTLL-2. On the other hand, a high dose of sphingosine (5 microM) by itself induced the apoptosis of CTLL-2 cells. This sphingosine-dependent apoptosis was also observed with F7 cells. These results provide evidence that the intracellular levels of sphingolipids play an important role in the signaling of the escape from and onset of apoptosis of CTLL-2 cells.

Cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase, which is the key enzyme for the synthesis of N-glycolylneuraminic acid (NeuGc), has been purified from the cytosolic fraction of mouse liver, as described in our previous paper. The amino acid sequences of the purified CMP-NeuAc hydroxylase, and peptides obtained by lysylendopeptidase digestion, were used to synthesize specific oligonucleotide primers. A mouse cDNA clone of the enzyme was obtained by a combination of the polymerase chain reaction and rapid amplification of cDNA ends. The sequence of the clone contained an open reading frame coding for a protein of 577 amino acids with a predicted molecular mass of 66 kDa. The deduced sequence included the amino acid sequences obtained for the purified enzyme and peptides, and a complete match was obtained for 159 residues. The enzyme has neither a signal peptide sequence nor a membrane spanning domain, which is consistent with localization of the enzyme in the cytosol. Transfection of a cDNA construct to COS-1 cells increased the enzyme activity and the amount of NeuGc. Comparison of the sequence with GenBank data indicated that no similar sequence has been reported so far. Northern blot analysis of various mouse tissues with the enzyme cDNA as a probe indicated that expression of NeuGc is related to the level of CMP-NeuAc hydroxylase mRNA. On Southern blot analysis with the same probe, cross-hybridizing bands were detected in the human and fish genomes.

We have proposed that cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylation is carried out by a multienzyme system involving CMP-NeuAc hydroxylase (the terminal enzyme of the system), cytochrome b5, and an NADH-dependent cytochrome b5-reducing factor (Kozutsumi, Y., Kawano, T., Yamakawa, T., and Suzuki, A. (1990) J. Biochem. 108, 704-706). The CMP-NeuAc hydroxylase was purified to homogeneity from the cytosolic fraction of mouse liver, using ion exchange columns, a Red-Sepharose column, and a soluble cytochrome b5-immobilized Sepharose column. The purified enzyme exhibited a single band (64 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single peak (58 kDa) on gel permeation chromatography, indicating that it is composed of a single polypeptide chain. The absorption spectrum did not indicate the presence of a heme prosthetic group in the enzyme. Atomic absorption spectrometry and an inhibition test using an iron chelator indicate that the enzyme contains non-heme iron as an electron acceptor. A reconstitution experiment with the purified CMP-NeuAc hydroxylase, soluble cytochrome b5, and recombinant NADH-cytochrome b5 reductase revealed that these three factors are essential for the reaction. The hydroxylase exhibited high affinity to CMP-NeuAc (Km = 5 microM) and was greatly stabilized by CMP-NeuAc. The molecular activity of the enzyme (approximately 500/min) is much lower than that reported for NADH-cytochrome b5 reductase, suggesting that the activity or amount of hydroxylase is rate-limiting in CMP-N-glycolylneuraminic acid (NeuGc) biosynthesis. These results, together with the previous observation that the level of CMP-NeuAc hydroxylase activity was associated with the expression of NeuGc in various tissues, support the notion that the enzyme is the key for regulation of the overall velocity of CMP-NeuAc hydroxylation and consequently for the expression of NeuGc-containing glycoconjugates.

Oligosaccharides were released from the gangliosides of a human rectal adenocarcinoma with endoglycoceramidase (Rhodococcus sp. G-74-2) and then purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 600-MHz 1H NMR spectrometry, indicated the structures of these compounds to be as follows. [formula: see text] Three of these oligosaccharides, 2, 3, and 4, are novel as to ganglioside sugar chains and contain both lacto series types 1 and 2 chains. Oligosaccharides 3 and 4 are unique in that they contain the sialyl-Le(a)-X structure in linear and branched structures. Gangliosides with the oligosaccharide structures presented above might be new potential tumor markers.

This study describes the effect of starch-synthesizing enzymes on biosynthesis of storage starch in rice amylose-extender mutants, which contain branched D-glucans with abnormal structures. Western blot analysis indicated that two out of five amylose-extender mutant lines lacked an isoform of starch branching enzyme, termed RBE3, although the levels of granule-bound starch synthase and a major form of branching enzyme, RBE1, were normal in these two mutants. Proteins corresponding to the 87-kDa RBE3 molecule were present in the three other amylose-extender mutants as well as in the wild type. However, the level of branching enzyme activity significantly decreased in all amylose-extender mutants, suggesting that the 87-kDa proteins in these three mutants are inactive forms of RBE3. Therefore, we conclude that formation of the abnormal branched glucans in the amylose-extender mutant of rice is due to the lack of the RBE3 activity. The cDNA clones encoding RBE3 have been identified from a normal rice seed cDNA library in lambda gt11, using a synthetic oligonucleotide as a probe. The deduced amino acid sequence of RBE3 indicates that this protein is initially synthesized as a precursor of 825 amino acids, including a 65-residue transit peptide at the NH2 terminus. The sequences of the catalytic regions in amylolytic enzymes are highly conserved in the sequence of RBE3. Thus, the branching enzyme isoform belongs to a family of the amylolytic enzymes. RBE3 also shares a noticeable degree of sequence identity with RBE1, especially at the central portion of the protein molecule. However, RBE3 possesses an approximately 70-residue extra sequence at the NH2 terminus and lacks a COOH-terminal sequence of almost 50 residues as compared with RBE1. The structural differences at both termini may explain the distinct role in starch synthesis for RBE1 and RBE3.

The insulin receptor-related receptor (IRR) (1271 amino acids) is expected to have unique functions as a novel member of the insulin receptor family. In this paper, we report two alternatively spliced variants of rat IRR mRNA, which are predicted to encode two truncated forms of IRR, sIRR-1 (410 amino acids) and sIRR-2 (469 amino acids). The amino acid sequence of sIRR-1 is identical to the N-terminal 410-amino acid sequence of IRR. sIRR-2 has an additional 59-amino acid insertion in the C-terminal region. Both truncated forms retain the N-terminal and cysteine-rich domains but lack the transmembrane and intracellular tyrosine kinase domains, indicating that the truncated forms are the secreted forms. The translation products of the truncated form mRNAs were detected in the stomach and kidney by Western analysis. However, the physiological significance of the secreted forms remains to be elucidated.

Recently, embryonic chicken brain extract was shown to contain a glucuronyltransferase, which transfers glucuronic acid from UDP-glucuronic acid to glycolipid acceptors (neolactotetraosyl ceramide). The enzyme was also suggested to transfer glucuronic acid to glycoprotein acceptors (asialoorosomucoid) (Das, K. K., Basu, M., Basu, S., Chou, D. K. H., and Jungalwala, F. B. (1991) J. Biol. Chem. 266, 5238-5243). In this study, the glucuronyltransferase activity in rat brain extract was separated into two groups by UDP-glucuronic acid-Sepharose CL-6B column chromatography. The enzyme recovered predominantly in the effluent fraction (GlcAT-L) catalyzed the transfer of glucuronic acid to glycolipid acceptors but not to glycoprotein acceptors, whereas the enzyme recovered in the eluate fraction (GlcAT-P) transferred glucuronic acid most predominantly to glycoprotein acceptors and very little to glycolipid acceptors. GlcAT-P was able to transfer glucuronic acid to oligosaccharide chains on asialoorosomucoid. The enzyme recognized a terminal lactosamine structure, Gal beta 1-4GlcNAc, on glycoproteins. It was localized in the nervous system and was hardly detectable in other tissues, including the thymus, spleen, lung, kidney, and liver. Although GlcAT-L and GlcAT-P shared some properties in common such as tissue distributions and developmental changes, they exhibited marked differences in their phospholipid dependence and in their pH profiles, apart from their respective acceptor preference to glycolipids and glycoproteins. The acceptor specificity and tissue distribution suggest that a novel glucuronyltransferase, GlcAT-P, is involved in the biosynthesis of the sulfoglucuronylgalactose structure in the HNK-1 carbohydrate epitope that is expressed on glycoproteins.

The Gal/GalNAc-specific lectin on rat peritoneal macrophages (macrophage asialoglycoprotein binding protein, M-ASGP-BP) is structurally similar to rat hepatic asialoglycoprotein-binding protein (ASGP-BP) or rat hepatic lectin (RHL) and is highly homologous with the major component of RHL, RHL-1 (Ii, M, Kurata, H., Itoh, N., Yamashina, I., and Kawasaki, T. (1990) J. Biol. Chem. 265, 11295-11298). We found in this study that transfection with a cDNA clone that encodes a single polypeptide, M-ASGP-BP, was sufficient for the expression of an endocytic receptor for asialoorosomucoid (ASOR) on the COS-1 cell surface. The Kuptake value for ASOR for the transfected cells was 12.5 nM, which is similar to that for peritoneal macrophages (23 nM), and the number of ASOR bound on the cell surface was 1-8 x 10(5)/cell, this value being hundreds of times larger than that for peritoneal macrophages. 125I-ASOR bound on the surfaces of the transfected cells was rapidly internalized on incubation at 37 degrees C, and after 90 min of incubation, most of the radioactivity was recovered in acid-soluble degraded products from the medium. These results confirmed that the cDNA cloned in our previous study does in fact encode M-ASGP-BP and also that the single polypeptide chain can form a homooligomeric receptor (probably a hexamer or octamer) exhibiting high affinity for ASOR. The latter property was distinct from that of the hepatic ASGP-BP in that simultaneous transfection of two cloned cDNAs that encode RHL-1 and RHL-2/3 was required to produce an active ASOR receptor (McPhaul, M., and Berg, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8863-8867). This M-ASGP-BP expression system may serve as a simple model with which to investigate the molecular mechanisms underlying carbohydrate-mediated endocytosis.

Secretin is a 27-amino acid gastrointestinal hormone that stimulates the secretion of bicarbonate-rich pancreatic fluid. We isolated and analyzed the coding region of the gene for the rat secretin precursor. The entire coding region spans 692 base pairs and is divided into four regions corresponding to the signal peptide and NH2-terminal peptide, the secretin peptide and processing signal sequences, a part of the COOH-terminal peptide, and the remainder of the COOH-terminal peptide, which are interrupted by three short introns (81, 105, and 104 base pairs). The organization is similar to those of the genes for other members of the secretin family, glucagon and VIP/PHI-27 precursors, supporting the assumption that the genes for the secretin family peptide precursors originated from a common ancestral gene. We also demonstrated that the secretin precursor gene is widely expressed in the brain and in the hypophysis. The regional expression pattern of the secretin precursor gene in the brain is quite different from those of the glucagon and VIP/PHI-27 precursor genes. The secretin precursor gene is highly expressed in the medulla oblongata and pons of the brain and the hypophysis, the expression levels of which are comparable to those in the duodenum. The secretin precursor mRNA in the brain and the hypophysis has the same coding sequence as that in the duodenum, indicating that secretin in the brain and the hypophysis is produced from the same secretin precursor protein as that in the duodenum. This is the first evidence to be reported that the secretin precursor gene is definitely expressed in the brain.

Antiserum raised against horseradish peroxidase (HRP) recognizes a neural specific carbohydrate antigen in Drosophila and other insects. The epitopic activity of the carbohydrate moiety of HRP recognized by anti-HRP antiserum was measured by a newly developed enzyme-linked immunosorbent assay, in which HRP glycopeptides conjugated with bovine serum albumin were coated onto the wells and then reacted with goat anti-HRP antiserum. HRP sugar moieties released by almond glycopeptidase A digestion of HRP pepsin digests were subjected to pyridylamination. Pyridylamino oligosaccharides were separated into seven fractions by reverse-phase high performance liquid chromatography. The major fraction, which comprised about 80% of the total sugars, reacted strongly with anti-HRP antiserum. The carbohydrate structure of this fraction was determined by sugar composition analysis and 600-MHz 1H NMR spectroscopy as follows: Man alpha 1----6(Man alpha 1----3)(Xyl beta 1----2)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc. Analyses of reactivity with anti-HRP antiserum of various oligosaccharide derivatives obtained from the major fraction by exoglycosidase digestion and partial acid hydrolysis indicated that alpha 1----6-linked mannose and alpha 1----3-linked fucose are predominantly involved in the epitopic structure.

The cerebellar inositol 1,4,5-trisphosphate (InsP3) receptor is a high molecular weight glycoprotein abundantly expressed in Purkinje cells. The subunit structure of the InsP3 receptor protein was examined by cross-linking experiments. Agarose-polyacrylamide gel electrophoresis of the cross-linked materials demonstrated that the cerebellar InsP3 receptor protein is composed of four noncovalently bound identical subunits each with a Mr of 320,000 in both purified and microsome-bound states. Chromatography of the purified receptor on a calmodulin-Sepharose column demonstrated a Ca2(+)-dependent interaction of the InsP3 receptor with calmodulin. Photoaffinity labeling of the cerebellar microsomal fraction with [alpha-32P]8-azidoadenosine 5'-triphosphate revealed the presence of ATP-binding site in the InsP3 receptor. Scatchard analysis of the purified InsP3 receptor revealed the Bmax and Kd values for ATP binding of 2.3 pmol/micrograms and 17 microM, respectively. Reconstitution of the purified InsP3 receptor into the planar lipid bilayer indicated channel activity in the purified receptor. It exhibited a calcium conductance (26 pS in 53 mM Ca2+) and sodium conductance (21 pS in 100-500 mM asymmetric Na+ solutions) with permeability ratios of PCa/PTris = 6.3 and PNa/PCl = 5.4. The purified channel was activated with submillimolar ATP in the presence of InsP3 and modified to reach a large conductance state.

Human promyelocytic leukemia cells (HL-60) were used as a cell model to determine how arachidonic acid stimulates the synthesis of platelet-activating factor (PAF) synthesized via the remodeling pathway. In these studies HL-60 cells were cultured over 30 passages in fatty acid-free medium to deplete them of arachidonic acid. Even though the phospholipid classes from these cells contained no arachidonate, they could still be differentiated into granulocytes by dimethyl sulfoxide (1.25%). When the differentiated HL-60 cells, depleted of arachidonic acid, were stimulated with calcium ionophore A23187 in the presence of Ca2+ and [3H]acetate, only minimal amounts of [3H]PAF were produced. In contrast, if the differentiated HL-60 cells were supplemented with 10 microM arachidonic acid for 24 h and then stimulated with the ionophore, there was a large amount of [3H]PAF formed. The increase in PAF synthesis depended on the length of time the cells were supplemented with arachidonic acid; only a small increase in PAF synthesis occurred during the early hours of supplementation whereas stimulation of PAF synthesis was maximal (3-5-fold) after a 24-h period of the 20:4 supplementation. Other polyenoic fatty acid supplements (20:5, 22:4, and 22:6 for 24 h) also stimulated PAF production in the ionophore-treated HL-60 cells depleted of 20:4, but the amount of PAF was significantly less than found for the supplements of 20:4 under identical experimental conditions. Also noteworthy is that undifferentiated cells supplemented with 20:4 or their unsupplemented controls could not be stimulated by the calcium ionophore to produce PAF. Addition of indomethacin (cyclooxygenase inhibitor), A63162 (5'-lipoxygenase inhibitor), or eicosatetraynoic acid (cyclooxygenase/lipoxygenase inhibitor) to the incubations caused little change in the production of [3H]PAF in the differentiated cells supplemented with 20:4 for 24 h. On the other hand, the addition of mepacrine, bromophenacyl bromide, or U26384 (phospholipase A2 inhibitors) resulted in very large decreases (80-90% lower than controls) in the amount of [3H]PAF produced under the same conditions. Analysis of the molecular species of [3H]alkylacyl-GroPCho (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, the precursor of PAF in the remodeling pathway) in 20:4-supplemented cells prelabeled with [3H]alkyl-lyso-GroPCho revealed that only the alkylarachidonoyl-GroPCho species were preferentially decreased after stimulation with the A23187 ionophore. These results demonstrate that arachidonate must be at the sn-2 position of alkylacyl-GroPCho in order for it to serve as a precursor of PAF.(ABSTRACT TRUNCATED AT 400 WORDS)