Uptake and intracellular transport of acidic fibroblast growth factor: evidence for free and cytoskeleton-anchored fibroblast growth factor receptors.

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfona te, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.

Isolation and partial characterization of a novel and uncommon two-chain 64-kDa ribosome-inactivating protein from the bark of elder (Sambucus nigra L.).

A novel, strongly basic, two-chain ribosome-inactivating protein (RIP) with an apparent Mr of 64000 by SDS-PAGE and 63469 by mass spectrometry analysis, that we have named basic nigrin b, has been found in the bark of elder (Sambucus nigra L.). The new protein does not agglutinate red blood cells, even at high concentrations and displays an unusually and extremely high activity towards animal ribosomes (IC50 of 18 pg/ml for translation by rabbit reticulocyte lysates). However, it is inactive against plant and HeLa cells protein synthesis. Our functional and structural data are consistent with a heterodimeric structure for basic nigrin b of the type A-B*, B* being a truncated lectin lacking functional binding domains equivalent to the B (lectin) chain of the type 2 RIP SNA I and nigrin b present also in elder bark.

Constitutive and inducible type 1 ribosome-inactivating proteins (RIPs) in elderberry (Sambucus nigra L.).

Two novel highly basic type 1 (single chain) ribosome-inactivating proteins (RIPs) with N-glycosidase activity have been found in elderberries (the fruits of Sambucus nigra L.). Mass spectrometry of these RIPs, which we named nigritins f1 and f2, gave Mr values of 24095 and 23 565, respectively. Both proteins strongly inhibited protein synthesis in rabbit reticulocyte lysates but were inactive against plant ribosomes. Both nigritins have a similar topological activity on pBlueScript SK+ DNA as that displayed by dianthin 30. Nigritin f1 is a constitutive RIP since it is present in both green and mature intact elderberries at nearly the same proportion with respect to total fruit protein. By contrast, nigritin f2 is inducible and only appeared in mature intact elderberries. Elderberries also contain two isoforms of a basic nigrin equivalent to the recently found basic nigrin b in elder bark (De Benito et al., FEBS Letters 413 (1997) 85-91). Our results indicate that probably not all plant RIPs exert the same biological function and that this may be determined by the physiological state of the tissue.

Molecular mechanism of inhibition of mammalian protein synthesis by some four-chain agglutinins. Proposal of an extended classification of plant ribosome-inactivating proteins (rRNA N-glycosidases).

The four chain agglutinins from Abrus precatorius, Viscum album and Ricinus communis promote depurination of the 28 S rRNA from rabbit reticulocyte ribosomes characteristic of the common ribosome-inactivating proteins (RIPs). These agglutinins inhibited mammalian protein synthesis at nanomolar concentrations but they do not affect plant protein synthesis under the same conditions. Therefore, they should also be considered as true RIPs but of a new class, the four-chain RIPs. An extended classification of RIPs is presented based on the former one from Stirpe et al. [Bio/technology 10 (1992) 405-412].

Ebulitins: a new family of type 1 ribosome-inactivating proteins (rRNA N-glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome-inactivating protein ebulin 1.

A new family of single chain (type 1) ribosome-inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non-toxic two chain (type 2) RIP named ebulin I in its leaves. Ebulitins are basic proteins of M(r) 32,000, 29,000 and 29,000 for ebulitins alpha, beta and gamma, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.

Analysis of human ocular mucus: effects of neuraminidase and chitinase enzymes.

PURPOSE: Our goal was to establish the characteristic migration pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of high molecular weight mucins from human ocular mucus and the effects of treatment with exo- and endoglycosidases. METHODS: Chromatography by gel filtration with Sepharose CL-4B was performed on samples collected from normal subjects. Human ocular mucins from the high molecular weight fraction were digested with exoglycosidases (neuraminidase, N-acetyl-beta-D-glucosaminidase, beta-D-glucosidase) and endoglycosidases (chitinase, lysozyme); and the resulting products were analyzed by electrophoresis. Carbohydrate identification was performed using lectin probes. RESULTS: The migration of the ocular mucins on SDS-PAGE stopped after treatment with neuraminidase, which removes the terminal negatively charged sialic acid residues from mucin. Chitinase (beta(1-4)N-acetylglucosaminidase) treatment increased the electrophoretic migration of mucins. Staining with wheat germ agglutinin and Maackia amurensis agglutinin lectins showed that these mucins contain beta(1-4)NAcGlc and SAa(2-3)Gal linkages. CONCLUSIONS: These studies demonstrate that the mobility of human ocular mucins on SDS-PAGE is determined by their intrinsic total negative charge and is not dependent on SDS treatment. It is interesting to note that human ocular mucus contains chitinous material resistant to lacrimal lysozyme, which is accessible to chitinase, an enzyme now found to degrade human ocular mucins. These chitinous linkages could be in part responsible for the mucus resistance.

Requirement of phosphatidylinositol 3-kinase activity for translocation of exogenous aFGF to the cytosol and nucleus.

Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.

Evidence for distinct cellular internalization pathways of ricin and nigrin b.

Nigrin b and ricin are type 2 (two chain) ribosome-inactivating proteins that exhibited nearly the same strong inhibitory activity on cell-free protein synthesis. Incubation of HeLa cells for 6 hr with ricin at 37 degrees C promoted protein synthesis inhibition with an IC50 of 0.2 ng/ml. Incubation of the cells for 6 hr at 18 degrees C abolished completely the inhibition. Incubation of HeLa cells with nigrin b for 6 hr at 37 degrees C was nearly 10(5) times less inhibitory than ricin. In contrast to the effects observed with ricin, incubation of HeLa cells with nigrin b at 18 degrees C slightly increased the inhibitory action on protein synthesis as compared with incubation at 37 degrees C. These results strongly support the hypothesis that the internalization of ricin and nigrin b could involve different receptors and therefore they could follow different intracellular pathways.

Primary structure of omega-hordothionin, a member of a novel family of thionins from barley endosperm, and its inhibition of protein synthesis in eukaryotic and prokaryotic cell-free systems.

A new sulfur-rich basic polypeptide, so called omega-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance liquid chromatography. Purified omega-hordothionin was found to be homogeneous by SDS/polyacrylamide gel electrophoresis, N-terminal amino-acid sequencing and electrospray-ionization mass spectrometric analysis. The complete primary structure of omega-hordothionin was determined by automatic degradation of the intact molecule and peptides obtained by proteolytic cleavage. Omega-hordothionin consists of a single polypeptide chain of 48 amino acids with a molecular mass of 5508 Da deduced from its amino acid sequence, which fully coincides with the 5508.2 Da determined by electrospray-ionization mass spectrometry. The isolated polypeptide showed a characteristic composition with a high content of basic amino acids (five arginine residues, two lysine residues and six histidine residues) and eight cysteine residues, and has strong sequence identity (66%) with the sorghum SI alpha 1 alpha-amylase inhibitor. Omega-hordothionin, like gamma-hordothionin, exhibited translation inhibitory activity on both eukaryotic cell-free systems from mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli). However, in contrast to gamma-hordothionin, omega-hordothionin did not inhibit plant systems such as Triticum aestivum, Cucumis sativus, Vicia sativa and Hordeum vulgare. Gamma-hordothionin also inhibited the alpha-amylase activity from human saliva, while omega-hordothionin and the other different genetic variants of thionins, alpha-hordothionin and beta-hordothionin, failed to show any inhibitory effect.

Sensitivity of ribosomes from Agrobacterium tumefaciens to the ribosome-inactivating protein crotin 2 depending on the translocational state.

The GTP analog guanylylmethylene diphosphonate (GppCH2p) strongly inhibited polyuridylic acid-directed polypeptide synthesis in a cell-free translation system prepared from Agrobacterium tumefaciens. Fusidic acid increased even further the inhibitory action. The pre-translocational ribosomal complexes formed with the GppCH2p and the elongation factor G protected the ribosome against the depurinating action of crotin 2 assayed as the acid-dependent release of the RNA fragment whose terminal sequence is 5'-GAGGACCGGGAUGGAC-3'. The results allowed to conclude that the interaction of both crotin 2 and the elongation factor G with the A. tumefaciens ribosomes in the pre-translocational state must take place at overlapping, either sterically or allosterically, ribosomal sites which are equally accessible to the RIP.

Modulation of intracellular transport of acidic fibroblast growth factor by mutations in the cytoplasmic receptor domain.

Endocytic uptake and intracellular transport of acidic fibroblast growth factor (aFGF) was studied in cells transfected with FGF receptor 4 with mutations in the cytoplasmic part. Endocytic uptake in HeLa cells was reduced but not abolished when the tyrosine kinase of the receptor was inactivated by mutations or deletions. The tyrosine kinase-dependent endocytosis of aFGF was prevented by the expression of a dominant negative dynamin mutant that blocks endocytosis from coated pits and caveolae. However, more than half of the total endocytic uptake of aFGF was not affected under these conditions, indicating an endocytic uptake mechanism not involving coated pits or caveolae. Mutation or deletion of a putative caveolin-binding sequence did not prevent the localization of part of the receptors to a low density, caveolin-containing subcellular fraction. Whereas wild-type receptor transfers the growth factor from early endosomes to the recycling compartment, kinase negative, full length receptors were inefficient in this respect and the growth factor instead accumulated in lysosomes. By contrast, when most of the intracellular part of the receptor, including the kinase domain, was removed, aFGF was transported to the recycling compartment, as in cells that express wild-type receptors, suggesting the presence of a kinase-regulated targeting signal in the cytoplasmic tail.

Requirement for C-terminal end of fibroblast growth factor receptor 4 in translocation of acidic fibroblast growth factor to cytosol and nucleus.

The ability of COS cells to bind and internalise acidic fibroblast growth factor (aFGF) was studied after transient transfection of the cells with wild-type and mutated fibroblast growth factor receptor 4. In one case the tyrosine kinase of the receptor was inactivated by a point mutation in the active site, whereas in other cases parts of the receptor were deleted to remove various parts of the cytoplasmic domain. In all cases the receptors were expressed at the cell surface at a high level and the cells bound labelled growth factor efficiently and internalised it by endocytosis. Translocation of externally added aFGF across cellular membranes to reach the cytosol and nucleus was measured as transport of labelled growth factor to the nuclear fraction obtained by centrifugation, by farnesylation of growth factor modified to carry a CAAX motif, and by phosphorylation of the growth factor at a site specific for protein kinase C. Whereas both full-length receptors (with and without an active kinase domain) facilitated translocation of the growth factor to the cytosol and nucleus, as assessed by these methods, the mutants of the receptor where the C terminus was deleted, were unable to do so. In contrast, a receptor containing only the 57 most C-terminal amino acids of the cytoplasmic domain in addition to the juxtamembrane, transmembrane and extracellular domains, was in fact able to mediate translocation of aFGF to the cytosol. These data indicate that information contained in the C terminus of the receptor is required for translocation.

Toxicity and cytotoxicity of nigrin b, a two-chain ribosome-inactivating protein from Sambucus nigra: comparison with ricin.

Nigrin b, a lectin isolated from the bark of elderberry (Sambucus nigra L.), has structure and enzymatic activity similar to that of ricin and other type 2 ribosome inactivating proteins (RIPs), and yet is much less toxic to cells and animals. In an attempt to explain this difference, we studied (1) the cytotoxicity of both lectins at 18 and 37 degrees C, and in the presence of substances interfering with intracellular routing, and (2) the binding of nigrin b to, and its uptake and degradation by HeLa cells, in parallel with ricin. As compared with the latter, (1) less nigrin b was bound and more was degraded by cells, with a resulting lower concentration remaining inside the cells, and (2) there is evidence for a different intracellular routing followed by the two lectins. These results may explain at least partly the different cytotoxicity and consequently the lower toxicity to mice of nigrin b compared with ricin.

Isolation, cDNA cloning, biological properties, and carbohydrate binding specificity of sieboldin-b, a type II ribosome-inactivating protein from the bark of Japanese elderberry (Sambucus sieboldiana).

A type II ribosome-inactivating protein (RIP) was isolated from the bark tissue of Japanese elderberry (Sambucus sieboldiana) and named sieboldin-b. Sieboldin-b is a heterodimeric protein consisting of 27- and 33-kDa subunits and showed strong ribosome-inactivating activity in vitro but did not show in vivo toxicity. The amino acid sequence of sieboldin-b deduced from the structure of the cDNA showed that both subunits of sieboldin-b are encoded on a single precursor polypeptide. Sieboldin-b has a structure homologous with the Neu5Ac(alpha 2-6)Gal/GalNAc-specific bark lectin from S. sieboldiana (SSA) and also typical type II RIPs such as ricin and abrin. Detailed analyses of carbohydrate binding properties of sieboldin-b revealed that sieboldin-b binds to Gal/GalNAc, similar to ricin/abrin, in spite of its highly homologous structure with SSA. The biological properties of these toxins/lectins are compared, and the possible explanation for such diversity is discussed.

Presence of single-chain ribosome-inactivating protein-like activities in several plant species.

The present screening work was devoted to the search for new ribosome-inactivating proteins (RIPs) in 21 plant species. Eight plants proved to be very active, inhibiting protein synthesis in eukaryotic in vitro systems (rat liver, Vicia sativa and wheat germ). They fulfil the major requirements for consideration as type 1 RIPs. Also, eight plants were found to contain haemagglutinating activity of human red cells but this was not related to the simultaneous presence of RIPs.

Differential sensitivity of HELA cells to the type 2 ribosome-inactivating proteins ebulin l, nigrin b and nigrin f as compared with ricin.

The new type 2 RIPs ebulin l, nigrin b and nigrin f present in Sambucus display toxicity to HELA cells several orders of magnitude lower than that displayed by ricin. Despite N-terminal amino acid homology between the three RIPs in both the A and the B chains, these compounds display very different degrees of toxicity to HELA cells that does not seem to be paralleled by immunologic correlations. It is suggested that small changes in the protein structure are most probably responsible for the different degrees of toxicity.

Screening survey of several plant species in search for ribosome-inactivating protein-like activities.

A screening survey of 41 plants revealed potent translational inhibitory activities in 19 plant species belonging to several different families, some of them hitherto unreported. Our results indicate that the active principles were ribosome-inactivating proteins. Additionally, human erythrocyte-agglutinating activity was found in 20 plants.

Elderberry (Sambucus nigra) bark contains two structurally different Neu5Ac(alpha2,6)Gal/GalNAc-binding type 2 ribosome-inactivating proteins.

A second NeuAc(alpha2,6)Gal/GalNAc binding type 2 ribosome-inactivating protein (RIP), called SNAI' has been isolated from elderberry (Sambucus nigra) bark. SNAI' is a minor bark protein which closely resembles the previously described major Neu5Ac(alpha2,6)Gal/GalNAc binding type 2 RIP called SNAI with respect to its carbohydrate-binding specificity and ribosome-inactivating activity but has a different molecular structure. Molecular cloning revealed that the deduced amino acid sequence of SNAI' is highly similar to that of SNAI and that the difference in molecular structure between both proteins relies on a single cysteine residue present in the B chain of SNAI but absent from SNAI'. The isolation of SNAI' not only identifies a minor bark protein as a type 2 RIP but also further emphasizes the complexity of the type 2 RIP/lectin mixture present in the bark of elderberry.

Presence of polymerized and free forms of the non-toxic type 2 ribosome-inactivating protein ebulin and a structurally related new homodimeric lectin in fruits of Sambucus ebulus L.

Mature leaves of dwarf elder (Sambucus ebulus L.) contain the non-toxic type 2 ribosome-inactivating protein ebulin 1 (Girbés et al., 1993b, J. Biol. Chem. 268: 18195-18199). We have now found that the green fruits of dwarf elder contain both free and polymerized forms of ebulin (ebulin f) and a new homodimeric D-galactose-binding lectin (SELfd). Polymerized material containing ebulin and lectin is composed of aggregates of variable relative molecular mass, some of them being close to 250,000. These aggregate forms are maintained in part by reducible disulphide bridges and reconstitute from reductant-free dialyzed material previously reduced with 2-mercaptoethanol. Direct incubation of free ebulin f with the free SELfd did not lead to polymerization, thus indicating that polymerization triggers some kind of substantial and perhaps catalyzed change in the structure of these proteins. Ebulin-containing polymerized material reacts with anti-ebulin f antibodies. Our results indicate that ebulin f is a fruit-form of ebulin 1. In contrast to green fruits, mature fruits lack both polymerized material and ebulin f, thus indicating some kind of reserve role for them in green fruits. Polymerization of ebulin and the dimeric lectin may represent a novel means of storing the non-toxic type 2 ribosome-inactivating proteins and lectins found in highly metabolic tissues, such as green fruits.

Characterization of a new non-toxic two-chain ribosome-inactivating protein and a structurally-related lectin from rhizomes of dwarf elder (Sambucus ebulus L.).

A new N-glycosidase ribosome-inactivating protein (RIP) belonging to the novel family of the nontoxic type 2 RIPs from Sambucaceae has been isolated from rhizomes of dwarf elder (Sambucus ebulus L.) and named ebulin r. Dwarf elder rhizomes also contain a novel monomeric N-Ac-galactosamine-binding lectin that we named SEAII. Ebulin r and SEAII have two isoforms each one, which were readily resolved by ion exchange. Both isoforms of ebulin (ebulins r1 and r2) strongly inhibited protein synthesis in mammalian but not in plant ribosomes by promoting depurination of sensitive ribosomes. Ebulin r and SEAII have apparent molecular masses of 56 and 33.5 kDa, respectively. Ebulins r1 and r2 are composed of two dissimilar subunits (types A-B) of apparent molecular masses of 26 and 30 kDa by disulphide bridges. The rhizome SEAII and the lectins SNA II and SNA III from elder (Sambucus nigra L.) share good amino acid sequence homology. This rhizome ebulin-A chain is more sequence-related to RIP members of cucurbitaceae than to any other plant family. The rhizome ebulin B chain shares a large homology in amino acid sequence with ebulin 1-B chain and SEAII. Anti-ebulin 1 polyclonal antibodies raised in rabbits reacted better with ebulin r1 than with ebulin r2, thus suggesting that both RIP isoforms could have some differences.