Structure-toxicity Relationship in Subunit Structure of Ricin D

The re-formation of the single disulfide bond which linked two polypeptide chains of ricin D was studied. Ricin D was reoxidized preferentially by the air-oxidation of its reduced polypeptide chains in a yield of 74% with the recovery of full toxicity. Furthermore ricin D was completely regenerated from its compound with p-chloromercuribenzoate. It seems reasonable to assume that the toxicity of ricin D arises from the quaternary structure of ricin D molecule.     

Isolation of Tryptic Peptides from the lie Chain of Ricin D and the Sequences of Some Tryptic Peptides Including N- and C-Terminal Peptides

Twenty tryptic peptides were isolated from the performic acid-oxidized He chain of ricin D by Dowex 1 × 2 column chromatography followed by paper chromatography. The amino acids contained in these peptides accounted for 218 out of 266 residues in the whole protein. The amino acid sequences of nine peptides were determined by manual liquid phase or automatic solid phase Edman degradation, and N- and C-terminal sequences of the He chain of ricin D were established to be NH₂–Ile–Phe–Pro–Lys–Gln–Tyr–Pro–Ile–Ile– and Cys–Ala–Pro–Pro–Pro–Ser–Ser–Gln–Phe, respectively.     

Iodination of Ricin D

Approximately five tyrosine residues of ricin D were iodinated preferentially under appropriate conditions probably forming diiodotyrosine. Iodination of this toxin carried out in 0.1 m phosphate buffer at pH 7.0 and 0°C for 60 min with a 20 fold molar excess of iodine per mole of protein, yielded a main component which appeared as a single band on polyacrylamide gel disc electrophoresis. Analysis of protein-bound radioactivity and the content of diiodotyrosine of ¹⁸¹I-labeled ricin D revealed that two tyrosine residues in the isoleucyl chain and three in the alanyl chain were substituted. The toxicity of iodinated ricin D decreased to one hundredth of that of native protein, However, the hemagglutinating activity of this protein was not affected by the iodination reaction.     

Amino Acid Sequence of Cyanogen Bromide Fragment CB I from Ala Chain of Ricin D

The amino acid sequence of the cyanogen bromide (CNBr) fragment CB I from the Ala chain of ricin D, the largest of three CNBr fragments, was established by manual Edman degradation of the peptides obtained by tryptic, chymotryptic or peptic digestion of fragment CB I. The total number of amino acid residues of fragment CB I accounted for 140 (54%) out of 260 residues in the Ala chain of ricin D.     

Isolation of Glycopeptides from Ricin D

Ricin D, a toxic protein from castor bean, was found to contain 6 moles of glucosamine and 17 moles of mannose per mole of protein.

Isolation of two constituent polypeptide chains, namely Ala-chain and Ile-chain, and subsequent proteolytic digestions with Nagarse and Pronase revealed two glycopeptides (Asp₁ Thri Gly₁ glucosamine₂ mannose₆ and Asp₁ Thr₁ Glu₁ Pro₁ glucosamine₂ mannose₇) from Alachain and one (Asp₁ Ile₁ Phe₁ glucosamine₂ mannose₄) from lie-chain. The total carbohydrate content of these glycopeptides accounts for all that of the whole protein. It is therefore that carbohydrate moieties are covalently linked to the polypeptide chains in three sites to form this glycoprotein.     

Isolation and Sequences of Peptic Peptides,and the Complete Sequence of Ile Chain of Ricin D

Sixteen peptic peptides, which contain arginine(s) or lysine, were isolated from cyanogen bromide fragments CB I and CB II of Ile-chain. Sequence determination has been performed on most of these peptides to provide overlaps for the tryptic peptides. Thus, complete amino acid sequence of Ile-chain consisting of 265 amino acid residues was determined. Some structural characteristics of the protein are also discussed.     

Isolation and Amino Acid Sequences of Small Tryptic Peptides from the Oxidized Ala Chain of Ricin D

Twelve tryptic peptides as well as free arginine were isolated from the performic acid-oxidized Ala chain of ricin D by gel filtration on Sephadex G–25 and Dowex 1×2 column chromatography followed by paper chromatography. Total number of the amino acid residues in these peptides accounted for 90 out of 263 residues in the Ala chain of ricin D.

The amino acid sequences of nine peptides were determined by manual Edman degradation.     

重组蓖麻、相思子毒素A链嵌合突变体的制备与分析

目的:构建蓖麻毒素(RIC)、相思子毒素(ABR)A链突变体的嵌合体蛋白,实现嵌合体蛋白的可溶性表达、纯化及抗原性分析。方法:采用柔性linker连接RIC A链突变体(mRICAD75AV76MY80A)和ABR A链突变体(mABRAE164AR167L),构建嵌合体基因mRICA/mABRA,将该嵌合体基因亚克隆至原核载体pQE80L构建表达质粒pQE80L-mRICA/mABRA,再转化至大肠杆菌M15获得表达工程菌株M15/pQE80L-mRICA/mABRA,工程菌在18℃经0.1 mmol/L的IPTG诱导14 h,表达的嵌合体蛋白经Ni-NTA亲和层析柱纯化,通过ELISA和Western印迹检测嵌合体蛋白的抗原性。结果:所获得的mRICA/mABRA嵌合体基因经一致性比对分析,与预计嵌合基因的序列一致性为100%,其开放读框全长1572 bp,编码524个氨基酸残基;重组表达质粒pQE80L-mRICA/mABRA经PCR及双酶切鉴定证明构建正确,嵌合体蛋白相对分子质量约为62×103,与预测相符,可溶性的嵌合体蛋白经Ni-NTA亲和层析柱纯化,纯度可达99%;间接ELISA和Western印迹结果表明,嵌合体蛋白能同时与抗RIC多克隆抗体和抗ABR多克隆抗体发生特异的抗原抗体反应。结论:得到的mRICA/mABRA嵌合体蛋白具有良好的抗原性,为研制新型RIC和ABR双价疫苗奠定了重要基础。     

Separation of the Two Constituent Polypeptide Chains of Ricin D

Two constituent polypeptide chains were isolated from performic acid-oxidized ricin D by DEAE-cellulose column chromatography in phosphate buffer, pH 7.0, containing 8m urea or from reduced-carboxymethylated ricin D by gel filtration on Sephadex G-75 followed by DEAE- cellulose column chromatography in Tris-HCl buffer, pH 8.5. Amino acid analyses of the isolated two chains revealed that they were distinct molecules possessing similar molecular weights of near 30,000 and linked by one disulfide bond in ricin D.     

Binding of Lactose and Galactose to Native and Iodinated Ricin D

The binding of lactose and galactose to native and iodinated ricin D was investigated by equilibrium dialysis and ultraviolet difference spectroscopy. The results provided direct evidence that native ricin D has two independent saccharide binding sites with different affinities, of which the high-affinity (HA-) binding site is able to bind with both lactose and galactose while the low-affinity (LA-) binding site binds only with lactose. In contrast, the iodinated ricin D possesses only one binding site both for lactose and galactose with high affinity.

By UV-difference spectroscopic analysis we found that there is one tyrosyl residue at or near the HA-binding site in ricin D which may be involvled in binding with saccharide. This tyrosyl residue was not iodinated in the presence of lactose but was iodinated in the absence of lactose and was perturbed by an addition of lactose even after iodination.

From these results, it was inferred that the binding site abolished by the iodination is the LA-binding site and this may be due to the conformational alteration of the LA-binding site caused by the iodination of the tyrosyl residue(s) present near the LA-binding site.     

Limited Hydrolysis of Ricin D with Alkaline Protease from Bacillus subtilis

Streptomyces naraensis was inoculated into 100 ml of culture broth, containing 50 µCi of ⁶⁵Zn, diluted with ZnCl₂ solution to make 10^-4 m Zn²⁺ ion, at 27°C for 5 days with shaking. ⁶⁵Zn-labeled neutral proteinase from Streptomyces naraensis was prepared by the method described previously. The preparation was homogeneous by disc electrophoresis and contained 1 g-atom of zinc per mole of enzyme in calculation by radioactivity.

It was suggested that the protein-bound zinc of neutral proteinase was not essential for enzymatic activity. Thus, this zinc was an essential component for the higher order structure of the protein, and the removal of zinc treated with EDTA* inactivated the enzyme. The enzymatic activity was maintained in the presence of calcium ion.     

Physical,Chemical and Physiological Properties of S-CM Subunits of Ricin D

Some physical, chemical and physiological properties of two S-CM subunits (S-CM Ile and S-CM Ala chain) of ricin D were studied. Physical properties of subunits are summerized in Table I. The S-CM subunits consisted of 244 (S-CM Ile chain) and 254 (S-CM Ala chain) amino acid residues. By the specific cleavage of the single intermolecular disulfide bond of ricin D, no remarkable change in conformation of polypeptide chains of ricin D was detected, but the toxicity was markedly decreased. The toxicity of ricin D is due to the quaternary structure of the ricin D molecule.     

Biochemical Studies on Ricin Part X Effect of the Two Constituent Polypeptide Chains of Ricin D toward Mouse Sarcoma Ascite Tumor Cells

A maltotetraose-forming amylase from Pseudomonas stutzeri was highly purified by adsorption on starch granules and by chromatographies on Sephadex G-100 and DEAE-cellulose. The purified enzyme showed a single band in polyacrylamide gel electrophoreses with or without sodium dodecylsulfate. The optimum pH for enzyme action on starch was 6.0-6.5, and the optimum temperature was 45°C. The purified enzyme attacked starch from the non-reducing end to produce α-anomer oligosaccharides. This indicated that the enzyme was an exo-α-amylase which had not hitherto been found. The enzyme activity was markedly inhibited by the addition of Cu²⁺, Hg²⁺, N-bromosuccinimide and 2,3-butanedione. The molecular weight of the enzyme determined by the method of Weber and Osborn was about 5.7 × 10⁴. The isoelectric point of the enzyme was estimated to be 5.3 by polyacrylamide gel electrofocusing. The Km and k₀ values of this enzyme for starch, glycogen, short chain amylose and some maltooligosaccharides were calculated from Lineweaver-Burk plots.     

Effects of Physical and Chemical Treatments on the Biological Activity of Ricin D

Effects of physical and chemical treatments on the cytoagglutinating activity, toxicity and inhibitory activity of cell-free protein synthesis of ricin D or its constituent polypeptide chains were investigated. The results indicated that the isolated polypeptide chains were much less stable than intact ricin D in acidic pH, heating as well as chemicals, and the Ala chain was more unstable than the lie chain.

Chemical modifications of ricin D with specific reagents revealed that the tryptophan and tyrosine residues as well as the carboxyl groups participated in the phenomena of cyto- agglutination and toxic action of ricin D, whereas arginine residues were considered not to be directly involved. Trinitrophenylation of free amino groups did not result in a loss of cytoagglutinating activity, whereas caused a loss of toxicity, suggesting that free amino groups in the lie chain were involved in the toxic action of ricin D.     

Structural-Based Rational Design of an Antagonist Peptide That Inhibits the Ribosome-Inactivating Activity of Ricin A Chain

Previous studies of inhibitors of ricin A chain (RA) mainly focused on the analogues of adenine and ribosomal RNA (rRNA) substrates. In this paper, a novel antagonist peptide (named PT) was designed rationally based on the crystal structure of the complex RA–rRNA. Theoretical results had clearly revealed the blockage of PT in the RA–rRNA interaction. The competitive inhibition experiment indicated that PT could significantly inhibit the binding activity of RA with anti-RA antibody. In order to further prove the competitive effect of PT against RA, N-glycosidase antagonizing activity of PT in cell-free system was evaluated using luciferase protein synthesis inhibition assay. Consequent data demonstrated that, at a RA level (0.022 nM) giving 50% decrease of protein synthesis in the absence of the peptide, protein synthesis could be recovered by the peptide for up to 80% at a level of 0.1 microgram/ml. This study highlights the interest of computation-aided method in the design of novel peptides with the ability to block the deleterious biological effects of RA. In addition, the method of luciferase protein synthesis inhibition assay in cell-free system which should provide rapid, sensitive, selective, and quantitative assessment may be developed to evaluate the potential antagonizing activity of RA inhibitors.Shuntao Wang and Jiannan Feng Contributed Equally to This Work     

Limited Hydrolysis of Ricin D with Trypsin in the Presence of Sodium Dodecyl Sulfate

An α-amylase which produces both maltotetraose and maltopentaose from starch as the main products was found in the culture filtrate of a strain of Bacillus circulans which was newly isolated from soil. The enzyme was purified to be almost homogeneous on disc electrophoresis in polyacrylamide gel by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex G-200 gel filtration.

The optimum pH and temperature of the enzyme were around pH 7.0 and around 50°C, respectively. Metal ions such as Hg²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Fe²⁺ and Co²⁺ strongly inhibited the enzyme activity. The molecular weight was about 45,000. The yields of maltotetraose and maltopentaose from potato starch were 30 ~ 40% and 20 ~ 30%, respectively.     

Ricin A Chain from <Emphasis Type="Italic">Ricinus sanguineus</Emphasis>: DNA Sequence,Structure and Toxicity

The gene coding for the ricin A-chain from Ricinus sanguineus (R_sTA) was isolated and sequenced (GB: DQ661048). Comparison of R_sTA with the ricin A-chain from Ricinus communis (RTA) revealed the presence of five differences in the gene sequence. At the protein level only two differences were noticed, the replacement of Asn136 by Ser (N136S) and Ile173 by Val (I174 V). From the R_sTA structure model (PMDB: PM0074652), the N136S mutation was predicted to have no effect on R_sTA structure. The I174V mutation is believed to have no effect on the R_sTA structure or on toxicity since this replacement was found in the Ricinus agglutinin’s A-chain and the latter has a comparable toxicity to RTA. The Ser221 of the putative lipase active site was found. Toxicity experiments showed that R_sTA and RTA have similar toxicities. This finding proves that the N136S and I174 V mutations have no effect on R_sTA toxicity.