ER stress induces caspase-8 activation, stimulating cytochrome c release and caspase-9 activation.

Excess ER stress induces caspase-12 activation and/or cytochrome c release, causing caspase-9 activation. Little is known about their relationship during ER stress-mediated cell death. Upon ER stress, P19 embryonal carcinoma (EC) cells showed activation of various caspases, including caspase-3, caspase-8, caspase-9, and caspase-12, and extensive DNA fragmentation. We examined the relationship between ER stress-mediated cytochrome c/caspase-9 and caspase-12 activation by using caspase-9- and caspase-8-deficient mouse embryonic fibroblasts and a P19 EC cell clone [P19-36/12 (-) cells] lacking expression of caspase-12. Caspase-9 and caspase-8 deficiency inhibited and delayed the onset of DNA fragmentation but did not inhibit caspase-12 processing induced by ER stress. P19-36/12 (-) cells underwent apoptosis upon ER stress, with cytochrome c release and caspase-8 and caspase-9 activation. The dominant negative form of FADD and z-VAD-fmk inhibited caspase-8, caspase-9, Bid processing, cytochrome c release, and DNA fragmentation induced by ER stress, suggesting that caspase-8 and caspase-9 are the main caspases involved in ER stress-mediated apoptosis of P19-36/12 (-) cells. Caspase-8 deficiency also inhibited the cytochrome c release induced by ER stress. Thus, in parallel with the caspase-12 activation, ER stress triggers caspase-8 activation, resulting in cytochrome c/caspase-9 activation via Bid processing.

Caspase-12 processing and fragment translocation into nuclei of tunicamycin-treated cells.

Excess endoplasmic reticulum (ER) stress induces processing of caspase-12, which is located in the ER, and cell death. However, little is known about the relationship between caspase-12 processing and cell death. We prepared antisera against putative caspase-12 cleavage sites (anti-m12D318 and anti-m12D341) and showed that overexpression of caspase-12 induced autoprocessing at D(318) but did not induce cell death. Mutation analysis confirmed that D(318) was a unique autoprocessing site. In contrast, tunicamycin, one of the ER stress stimuli, induced caspase-12 processing at the N-terminal region and the C-terminal region (both at D(318) and D(341)) and cell death. Anti-m12D318 and anti-m12D341 immunoreactivities were located in the ER of the tunicamycin-treated cells, and some immunoreactivities were located around and in the nuclei of the apoptotic cells. Thus, processing at the N-terminal region may be necessary for the translocation of processed caspase-12 into nuclei and cell death induced by ER stress. Some of the caspase-12 processed at the N-terminal and C-terminal regions may directly participate in the apoptotic events in nuclei.

Localization of active form of caspase-8 in mouse L929 cells induced by TNF treatment and polyglutamine aggregates.

The relation between activation of caspase-8 and polyglutamine aggregates has been focused. We prepared an antiserum (anti-m8D387) that recognizes the active form but not the proform of mouse caspase-8. We used immunostaining with anti-m8D387 antiserum to compare the localizations of activated mcaspase-8 in L929 (clone 1422) cells induced by TNF and polyglutamine aggregates. Anti-m8D387 was positive throughout cytoplasm of the TUNEL-positive cells induced by TNF treatment, whereas the anti-m8D387 reactivity was not positive throughout cytoplasm of the cells expressing polyglutamine but was restricted to polyglutamine aggregates. In contrast with TNF-treated cells, cells expressing anti-m8D387-positive cytoplasmic polyglutamine aggregates did not undergo TUNEL-positive apoptosis. Thus activated caspase-8 associated with polyglutamine aggregates alone was not sufficient to induce TUNEL-positive apoptosis of L929 (clone 1422) cells. The distribution of activated caspase-8 associated with polyglutamine aggregates may be essential for the polyglutamine-mediated cell death or downstream of caspase-8 may be different in the TNF-treated cells and cells expressing polyglutamine.

Tryptophan residues and the sugar binding site of potato lectin.

Potato lectin (Solanum tuberosum agglutinin, STA) was found to contain fluorescent tryptophan residues highly exposed to solvent. The binding of chitin oligosaccharides to STA induced fluorescence quenching, a shift of the fluorescence maximum to shorter wavelength, a decrease in the quenching constant of iodide ion and a decrease of the number of tryptophan residues modifiable by N-bromosuccinimide. The results suggested that one tryptophan residues is located at or near a sugar binding site of STA, and that its environment is altered from hydrophilic to relatively more hydrophobic upon interaction with specific sugars. The binding constants of STA with chitin oligosaccharides were determined by measuring the peak-trough heights in the fluorescence difference spectra induced by various concentrations of sugars. The inhibition constants of chitin oligosaccharides for the hemagglutinating activity of STA were obtained by the method of Pitts and Yang [(1981) Biochem. J. 195, 435-439] and the results were in good agreement with those obtained by the fluorescence spectral method. Standard and unitary free energy changes (delta G0 and delta Gu) and standard enthalpy changes (delta H0) were also obtained. These values decreased with sugar chain length up to at least the tetramer. Thus, it was assumed that there are at least 4 subsites, A, B, C, and D, in the sugar binding site of STA. The contributions to the binding energy (delta Gu) were -17.0, -12.6, -7.3, and -4.4 kJ/mol at subsites A, B, C, and D, respectively, and the bindings of chitin monomer (GlcNAc), dimer, trimer, and tetramer were assumed to occur at subsite A, AB, ABC, and ABCD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical and spectral studies on Vicia faba agglutinin.

Affinity chromatographic purification of Vicia faba agglutinin (VFA) was performed with a Sephadex G-150 column according to the method of Allen and Johnson [Biochim. Biophys. Acta (1976)]. VFA has 10 tryptophanyl residues per molecule on the assumption that its molecular weight is 50,000 daltons. Equilibrium dialysis with methyl a-D-[glucose-14C(U)]glucopyranoside showed that VFA has two sugar binding sites per molecule with a binding constant of 220 M-1. Upon interaction with specific sugars, VFA induced UV-difference spectra which are typical of the perturbation of tryptophanyl residues. Therefore, the binding constants of VFA with specific sugars could be calculated from the intensity changes in the difference spectra induced by various concentrations of the sugars. The results obtained were in good agreement with the results of hemagglutination inhibition assays. 3-O-Methyl-D-glucose had the highest binding constant (1.9 x 10(3) M-1) among the sugars examined. The binding constants of VFA with glucose, mannose, methyl a-D-glucopyranoside, methyl a-D-mannopyranoside, and maltose were 290, 900, 220, 500, and 220 M-1, respectively, which are lower than those of concanavalin A. VFA did not bind with mucopolysaccharides containing 2-acetamide-2-deoxy-a-, or -beta-D-glucopyranosyl residues, such as heparin, heparan sulfate, and hyaluronic acid. The far UV-CD spectrum of VFA was similar to that of concanavalin A.

Purification and characterization of potato lectin.

Potato lectin (Solanum tuberosum agglutinin, STA), purified by affinity chromatography on tri-N-acetylchitotriose-Sepharose 6B, has Mr approximately 100,000, as estimated by gel filtration on Sephadex G-150 and is an aggregating system with a monomer Mr = 54,000, as estimated by sedimentation equilibrium analysis. Equilibrium dialysis showed that STA (dimer) has two binding sites for a specific sugar per molecule. STA has a high content of sugar, most of which is L-arabinose, and is rich in Hyp and Cys. On interaction with specific sugars, STA induced a UV difference spectrum having positive peaks at 292 and 285 nm characteristic of tryptophyl residues. The association constants with chitin oligosaccharides, determined from the intensities of the difference spectra at various concentrations of sugars, increased with increasing chain length of the sugar. Association constants obtained by frontal affinity chromatography of chitin oligosaccharides with STA-Sepharose were in good agreement with those obtained by difference spectra, whereas the association constants obtained by frontal affinity chromatography of STA with di- and tri-N-acetylchitotriose-Sepharose were much higher, presumably owing to the effect of multivalency of ligands. The CD spectra of STA in the far UV region indicate the presence of 40% of beta- and 60% of unordered form, and no alpha-helix conformation, which supports the structure suggested by the amino acid composition and the high content of sugar.

Involvement of a tyrosyl residue in the interaction of peanut lectin with lactose.

The role of a tyrosyl residue in the binding of Arachis hypogaea (peanut) agglutinin, AHA, to lactose has been studied using two techniques, titration of the phenolic hydroxyl group of the tyrosine residue and chemical modification of the tyrosine with iodine. More than three tyrosyl residues per mol of AHA were masked when AHA was titrated in the presence of lactose. Lactose also protected some tyrosyl residues of AHA from the modification with iodine. Upon interaction with lactose, AHA iodinated in the presence of lactose gave a UV-difference spectrum with similar peaks to those of native AHA, while AHA iodinated in the absence of lactose gave a spectrum without such peaks. Though not only native AHA but also iodinated AHA was completely adsorbed on a column of lactamyl-Sepharose 6B, equilibrium dialysis showed that the binding constant and the number of binding sites of native AHA and iodinated AHA with lactose were 4.3 x 10(3) and 3.0 x 10(3) M-1, and 3.2 and 1.8, respectively. These results suggest that about two of four sugar binding sites have tyrosyl residues which induce the UV-difference spectra upon binding with lactose, and that the iodination of these tyrosyl residues results in a decrease of the number of binding sites on AHA.

Interaction of sulfated glycosaminoglycans with lectins.

The sulfated glycosaminoglycans, such as keratan sulfate and chitin sulfate having 3-hydroxy free N-acetyl-beta-D-glucosaminyl residues as constituents, reacted with wheat germ agglutinin and Solanum tuberosum agglutinin by sugar-specific interaction. The glycosaminoglycans showed different inhibitory activities to the hemagglutination reaction of these lectins and keratan sulfate and its modified products formed insoluble complexes with both of the lectins at pH 7.0 in physiological saline solutions (0.15 M NaCl). S. tuberosum agglutinin was precipitated within a particularly narrow concentration range of keratan sulfate, and the formation of a soluble complex was observed by gel chromatography. These interactions were specifically inhibited by N,N'-diacetylchitobiose but not by 2 M NaCl. The specific interactions of the glycosaminoglycans with S. tuberosum agglutinin were confirmed by their ultraviolet difference spectra with two peaks at 285 and 298 nm attributable to the tryptophan residues in the binding site of the agglutinin. It was also found that S. tuberosum agglutinin and wheat germ agglutinin have different binding specificities. The presence of sulfate groups in either keratan sulfate or chitin sulfate did not interfere with their specific interactions with S. tuberosum agglutinin as strongly as with wheat germ agglutinin. The N-acetylneuraminic acid residues in keratan sulfate were found to be receptor sites for wheat germ agglutinin but not for S. tuberosum agglutinin.