Specificity of alkaline elastase Bacillus on the oxidized insulin A- and B-chains

The substrate specificity of alkaline elastase Bacillus from alkalophilic Bacillus sp. Ya-B was investigated using oxidized insulin A- and B-chains. Under time-limited cleavage, the initial cleavage site of the enzyme on the oxidized insulin A-chain and B-chain was at the leucine13-tyrosine14 bond and the leucine15-tyrosine16 bond, respectively. When the cleavage was completed, three major cleavage sites and three minor cleavage sites on the A-chain, and five major cleavage sites and four minor cleavage sites on the B-chain were found. However, most of the peptides produced after complete hydrolysis of the A- or B-chain by the enzyme were composed of four to six amino acid residues. The results suggest that this enzyme cleaves the oxidized insulin A- and B-chains in a block-cutting manner.     

Radioimmunoassay of ricin A- and B-chains applied to samples of ricin A-chain prepared by chromatofocusing and by DEAE Bio-Gel A

A radioimmunoassay for ricin and ricin A- and B-chains was developed. Amounts as low as 100 pg of A-chain and 500 pg of B-chain could easily be quantitated. We showed, however, that the free chains were more reactive in the radioimmunoassay than the equivalent quantity of the individual chains when combined in intact ricin. The usefulness of the assay was demonstrated by determining the concentration of contaminating A- or B-chains in preparations of the separate polypeptides purified by DEAE Bio-Gel A chromatography and by chromatofocusing.     

The use of fluorescence spectroscopy for identification of A- and B-thrombin chains during their chromatographic separation

The separation of A- and B-chains of human thrombin has been performed by gel filtration on Sephadex G-100 under the reduction of disulphide bonds with dithiothreitol. Identification of A- and B-chains has been provided by measurements of the fluorescence intensity of fractions at 310 nm and 350 nm which are near the maximum positions of tyrosine and tryptophan fluorescence, respectively. The appearance of A-chain was monitored by an increase of the ratio of Ifl310/Ifl350 greater than 2. The fluorescence spectrum of A-chain has maximum position at 304 nm, which is characteristic of tyrosine fluorescence. The fluorescence spectrum of B-chain has maximum position at 347.5 nm which corresponds to fluorescence of tryptophan residues. The identification of A- and B-chains has been confirmed by the gel electrophoresis data.     

Enzymatic properties of the isolated B-chain of human thrombin

The A- and B-chains have been isolated from the non-covalent complex of human thrombin A- and B-chains, using selective reduction of the interchain disulfide bridge. The B-chain thus isolated (de-A-thrombin) retains its conformation, which is close to the native one and thus differs considerably from the B-chain isolated from the fully reduced enzyme. Nevertheless, the proteolytic (in terms of fibrinogen clotting) and amidase activities of de-A-thrombin are markedly reduced as compared to the native enzyme and the non-covalent complex of A- and B-chains. It is assumed that the A-chain of thrombin is necessary for normal functioning of the active site of thrombin localized in the B-chain.     

The removal of carbohydrates from ricin with endoglycosidases H, F and D and alpha-mannosidase

Recently, several investigators have explored the possibility of targeting ricin to designated cell types in animals by its linkage to specific antibodies. There is evidence, however, that the mannose-containing oligosaccharide chains on ricin are recognised by reticuloendothelial cells in the liver and spleen and so cause the immunotoxins to be removed rapidly from the blood stream. In the present study we analysed the carbohydrate composition of ricin and examined enzymic methods for removing the carbohydrate. The carbohydrate analysis ricin A-chain revealed the presence of one residue of xylose and one of fucose in addition to mannose and N-acetylglucosamine which had been detected previously. The B-chain contained only mannose and N-acetylglycosamine. Ricin A-chain is heterogeneous containing two components of molecular weight 30 000 and 32 000. Strong evidence was found that the heavier form of the A-chain contains an extra carbohydrate unit which is heterogeneous with respect to concanavalin A binding and sensitivity to endoglycosidase H. The lower molecular weight form of A-chain did not bind concanavalin A and was insusceptible to endoglycosidases. Only one of the two high mannose oligosaccharide units on the isolated B-chain could be removed by endoglycosidases H or F, whereas both were removable after denaturation of the polypeptide by SDS. Both the isolated A- and B-chains were sensitive to alpha-mannosidase. Intact ricin was resistant to endoglycosidase treatment and was only slightly sensitive to alpha-mannosidase. The addition of SDS allowed endoglycosidase H to remove both of the B-chain oligosaccharides from intact ricin and increased the toxin's sensitivity to alpha-mannosidase. In conclusion, extensive enzymic deglycosylation of ricin may only be possible if the A- and B-chains are first separated, treated with enzymes and then recombined to form the toxin.     

Structural and functional studies of cinnamomin, a new type II ribosome-inactivating protein isolated from the seeds of the camphor tree.

Cinnamomin is a new type II ribosome-inactivating protein (RIP). Its A-chain exhibits RNA N-glycosidase activity to inactivate the ribosome and thus inhibit protein synthesis, whereas the glycosylated B-chain is a lectin. The primary structure of cinnamomin, which exhibits approximately 55% identity with those of ricin and abrin, was deduced from the nucleotide sequences of cDNAs of cinnamomin A- and B-chains. It is composed of a total of 549 amino-acid residues: 271 residues in the A-chain, a 14-residue linker and 264 residues in the B-chain. To explore its biological function, the cinnamomin A-chain was expressed in Escherichia coli with a yield of 100 mg per L of culture, and purified through two-step column chromatography. After renaturation, the recovery of the enzyme activity of the expressed A-chain was 80% of that of native A-chain. Based on the modeling of the three-dimensional structure of the A-chain, the functional roles of five amino acids and the only cysteine residues were investigated by site-directed mutagenesis or chemical modification. The conserved single mutation of the five amino-acid residues led to 8-50-fold losses of enzymatic activity, suggesting that these residues were crucial for maintaining the RNA N-glycosidase activity of the A-chain. Most interestingly, the strong electric charge introduced at the position of the single cysteine in A-chain seemed to play a role in enzyme/substrate binding.     

The removal of carbohydrates from ricin with endoglycosidases H,F and D and α-mannosidase

Recently, several investigators have explored the possibility of targetting ricin to designated cell types in animals by its linkage to specific antibodies. There is evidence, however, that the mannose-containing oligosaccharide chains on ricin are recognised by reticuloendothelial cells in the liver and spleen and so cause the immunotoxins to be removed rapidly from the blood stream. In the present study we analysed the carbohydrate composition of ricin and examined enzymic methods for removing the carbohydrate. The carbohydrate analysis ricin A-chain revealed the presence of one residue of xylose and one of fucose in addition to mannose and N-acetylglucosamine which had been detected previously. The B-chain contained only mannose and N-acetylglycosamine. Ricin A-chain is heterogeneous containing two components of molecular weight 30 000 and 32 000. Strong evidence was found that the heavier form of the A-chain contains an extra carbohydrate unit which is heterogeneous with respect to concanavalin A binding and sensitivity to endoglycosidase H. The lower molecular weight form of A-chain did not bind concanavalin A and was insusceptible to endoglycosidases. Only one of the two high mannose oligosaccharide units on the isolated B-chain could be removed by endoglycosidases H or F, whereas both were removable after denaturation of the polypeptide by SDS. Both the isolated A- and B-chains were sensitive to α-mannosidase. Intact ricin was resistant to endoglycosidase treatment and was only slightly sensitive to α-mannosidase. The addition of SDS allowed endoglycosidase H to remove both of the B-chain oligosaccharides from intact ricin and increased the toxin's sensitivity to α-mannosidase. In conclusion, extensive enzymic deglycosylation of ricin may only be possible if the A- and B-chains are first separated, treated with enzymes and then recombined to form the toxin.     

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.     

Studies on the structure and properties of the lectins from Abrus precatorius and Ricinus communis.

The amino acid composition of the isolated A- and B-chains of the toxic lectins abrin and ricin was determined and compared. Even though the two toxins originate from widely different plants, statistical analysis of the amino acid content indicates extensive homologies in the amino acid sequence of the 4 chains. The intact lectins contain no free SH-groups whereas the isolated A- and B-chains contain close to one free SH-group each. The results indicate that in both toxins the A- and B-chains are connected by a single S-S bond. The B-chains of abrin and ricin contain similar amounts of mannose and glucosamine. The A-chain of ricin also contains some carbohydrate, whereas the A-chain of abrin appears not to be a glycoprotein. The non-toxic abrus and ricinus agglutinins contain more carbohydrate than abrin and ricin. The isoelectric points of the different lectin preparations were measured by isoelectrofocusing. The intact lectins are much more resistant to heat, freezing and chemical treatments than the isolated A- and B-chains. The intact lectins are also very resistant to treatment with proteolytic enzymes, whereas the isolated chains are easily digested. Evidence indicating that the toxins and their chains undergo extensive conformational changes upon reduction of the S-S bond is discussed.     

Preparation and properties of a hybrid toxin of modeccin A-chain and ricin B-chain

Hybrid molecules were prepared from the A- and B-chains of the two toxic lectins ricin and modeccin by dialyzing mixtures of isolated chains to allow a disulfide bridge to be formed between them. Whereas the hybrid consisting of ricin A-chain and modeccin B-chain was non-toxic, the converse hybrid, modeccin A-chain/ricin B-chain, was even more toxic to Vero cells than were the parent toxins, native ricin and modeccin. A number of drugs (NH₄Cl, monensin, trifluoperazine, verapamil, ionophore A23187) which protect cells against modeccin, but not against ricin, protected to some extent against the toxic hybrid, but less so than against native modeccin. The possibility is discussed that the modeccin A-chain of the hybrid may enter the cytosol by two routes, one which is highly efficient and identical to that used by native modeccin and another less efficient one which cannot be used by native modeccin.     

Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system.

Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A- and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide.     

The effect of pH on the conformation and stability of the structure of plant toxin-ricin

The effect of pH on the conformation of ricin and its A- and B-chains has been studied by measuring their intrinsic fluorescence. At pH 5.0 and 7.5, the structural stability of toxin and subunits was estimated according to the denaturing action of guanidine hydrochloride. It was demonstrated that the fluorescence of native toxin and catalytic A-subunit does not depend significantly on pH in the range pH 3-8, whereas ricin B-chain undergoes a structural transition at pH less than 5.0. The structural stability of ricin and isolated chains differs significantly at pH 7.5 and 5.0; the structural stability of ricin and the A-chain increases, whereas that of the B-chain decreases.     

Oxidation of the sulfhydryl forms of insulin A-chain and B-chain.

A modified procedure for the preparation of the S-sulfonates of the A- and B-chains of insulin and their conversion to the sulfhydryl forms by tri-n-butylphosphine is described. Air oxidation of the sulfhydryl forms of the A-chain in dilute solution (0.2 mg/ml) either in the presence or absence of urea at pH 9.0 yields primarily monomeric, intrachain disulfides. Similar treatment of the reduced B-chain yield monomeric, intrachain disulfide in 7 M urea but a large number of oligomeric, interchain disulfides in the absence of urea. Electrolytic reduction of insulin in 7 M urea of pH 8.5, followed by oxidation of the sulfhydryls in dilute solution in 7 M urea at pH 9.0 yields primarily a mixture of the monomeric, intrachain disulfides of the A-chain and of the B-chain which can be separated by chromatography on Sp-Sephadex in acidic urea. The rate of the oxidation of the sulfhydryls of the two separate chains was much slower and less complete than that reported for the two chains crosslinked by the carbonylbismethionyl residue.     

Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.     

Human gene 2 relaxin chain combination and folding

Relaxin is a small 6 kD two-chain peptide member of the insulin superfamily that is principally produced in the corpus luteum of the ovary and which plays a key role in connective tissue remodeling during parturition. Like insulin, it is produced on the ribosome as preprohormone that undergoes oxidative folding and subsequent proteolytic processing to yield the mature insulin-like peptide. In contrast to the now considerable insight into insulin chain folding and oxidation, comparatively little is known about the folding pathway of relaxin. A series of synthetic pairwise serine substituted relaxin A-chain cysteine analogues was prepared, and their oxidation behavior was studied both on their own and in the presence of native relaxin B-chain. It was observed that native S-reduced A-chain oxidized rapidly to a bicyclic product, whereas individual formation of each of the intramolecular disulfide bonds between Cys11 and Cys24 and the native Cys10 and Cys15 was considerably slower. Curiously, the non-native, isomeric Cys11-Cys15 disulfide bond formed most rapidly, although circular dichroism spectroscopy analysis showed this product to be devoid of secondary structure. This suggested that it may in fact be an intermediate in the subsequent formation of the native Cys10-Cys15 intramolecular disulfide. Combination of the native A-chain with the B-chain proceeded rapidly as compared with the A-chain analogue that lacked the intramolecular disulfide bond suggesting that this latter element is required as a first step in the folding process. It is therefore probable that relaxin is generated from its constituent A- and B-chains in a stepwise organization manner similar to that of insulin chain combination and folding. Further studies showed that the efficiency of combination of A-chain to B-chain was not markedly influenced by reaction temperature and that a reasonable yield of relaxin could be obtained on combination of the preoxidized A-chain with the S-reduced B-chain.     

Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D

The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.     

Crystal structure of Mabinlin II: a novel structural type of sweet proteins and the main structural basis for its sweetness

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7A resolution by the SIRAS method. The Mab II 3D structure features in an "all alpha" fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54-B64 was found in Face B, which adopts a special sequence, NL-P-NI-C-NI-P-NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model.     

The preparation of deglycosylated ricin by recombination of glycosidase-treated A- and B-chains: effects of deglycosylation on toxicity and in vivo distribution

Deglycosylation of ricin may be necessary to prevent the entrapment of antibody-ricin conjugates in vivo by cells of the reticuloendothelial system which have receptors that recognise the oligosaccharide side chains on the A- and B-chains of the toxin. Carbohydrate-deficient ricin was therefore prepared by recombining the A-chain, which had been treated with alpha-mannosidase, with the B-chain, which had been treated with endoglycosidase H or alpha-mannosidase or both. By recombining treated and untreated chains, a series of ricin preparations was made having different carbohydrate moieties. The removal of carbohydrate from the B-chain did not affect the ability of the toxin to agglutinate erythrocytes, and alpha-mannosidase treatment of the A-chain did not affect its ability to inactivate ribosomes. The toxicity of ricin to cells in culture was only reduced in those preparations containing B-chain that had been treated with alpha-mannosidase, when a 75% decrease in toxicity was observed. The toxicity of the combined ricin preparation to mice varied from double to half that of native ricin, depending on the chain(s) treated and the enzymes used. Removal of carbohydrate greatly reduced the hepatic clearance of the toxin and the levels of toxin in the blood were correspondingly higher. These results suggest that antibody-ricin conjugates prepared from deglycosylated ricin would be cleared more slowly by the liver, inflict less liver damage, and have greater opportunity to reach their target.