Metal ions-dependent peroxidase and oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats

We present evidence showing that a small fraction of electrophoretically homogeneous IgGs from the sera of healthy Wistar rats is bound with several different Me2+ ions and oxidizes 3,3'-diaminobenzidine through a peroxidase activity in the presence of H2O2 and through an oxidoreductase activity in the absence of H2O2. During purification on Protein A-Sepharose and gel filtration, the polyclonal IgGs partially lose the Me2+ ions. Therefore, in the absence of external metal ions, the specific peroxidase activity of IgGs from the sera of different rats varied in the range 1.6-26% and increased up to 13-198% after addition of Fe2+ or Cu2+ ions as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of HRP is 24-fold lower than its peroxidase activity, while oxidoreductase and peroxidase activities of IgGs are comparable. Oxidoreductase activities of different IgGs in the absence of external metal ions varied from 22 to 800%, and in the presence of Fe2+ or Cu2+ ions, from 37 to 1100% in comparison with the HRP oxidoreductase activity (100%). Chromatography of the IgGs on Chelex-100 leads to the adsorption of a small IgG fraction bound with metal ions and to its separation to many different subfractions demonstrating various affinities to the chelating resin and increased levels of the specific oxidoreductase and peroxidase activities. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defense mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activity of antibodies may play an important role in the protection of organisms from oxidative stress and toxic compounds.     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

Chemical modification of a catalytic antibody that accelerates the hydrolysis of carbonate esters

Catalytic antibody, 4A1, catalyzes the hydrolysis of p-nitrophenyl alkyl carbonate. To determine the amino acid residues related to the catalytic activity of the antibody, we studied the effect of Tyr-, Trp-, and Lys-selective reagents on the catalytic activity and determined the amino acid sequences around the modified amino acid residues. We found that the Tyr-selective reagent is the most effective one and the modification of one Tyr residue results in the complete loss of the catalytic activity. The modified Tyr residue is identified to be Tyr-32 in the CDR-1 of the L chain.     

Antibody-mediated sialidase activity in blood serum of patients with multiple myeloma

Cell surface sialylation is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential, clearance of aged cells, while the sialylation of IgG molecules determines their anti-inflammatory properties. Four sialidases - hydrolytic enzymes responsible for cleavage of sialic residues - were described in different cellular compartments. However, sialidases activity in body fluids, and specifically in blood serum, remains poorly studied. Here, we characterize first known IgG antibodies possessing sialidase-like activity in blood serum of multiple myeloma (MM) patients. Ig fractions were precipitated with ammonium sulfate (50% of saturation) from blood serum of 12 healthy donors and 14 MM patients, and screened for the presence of sialidase activity by using 4-MUNA (2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid) as substrate. High level of sialidase activity was detected in the MM patients, but not in healthy donors. Subsequent antibody purification by protein-G affinity chromatography and HPLC size exclusion chromatography at acidic conditions demonstrated that sialidase activity was attributable to IgG molecules. Sialidase activity was also specific for (Fab)(2) fragment of IgG and blocked by sialidase inhibitor DANA. Sialidase activity of IgG molecule was also confirmed by in gel assay for cleavage of sialidase substrate. Kinetic parameters of the catalysis reaction were described by Michaelis-Menten equation with K(m) = 44.4-108 μM and k(cat) = 2.7-23.1 min(-1). The action of IgG possessing sialidase-like activity towards human red blood cells resulted in a subsequent increase in their agglutination by the peanut agglutinin, that confirms their desialylation by the studied IgG. This is the first demonstration of the intrinsic sialidase activity of IgG isolated from blood serum of MM patients.     

A Catalytic Antibody That Accelerates the Hydrolysis of Carbonate Esters. Prediction of the Binding-Site Structure of the Substrate

Monoclonal antibodies catalyzing the hydrolysis of p-nitrophenyl alkyl carbonate were obtained using p-nitrophenyl phosphonate as hapten. One of the antibodies, 4A1, has a relatively high activity for the substrate having a bulky group. To determine the amino acid residues related to the binding of the bulky group, we determined the amino acid sequences of V_L and V_H regions of 4A1 by the cycle sequencing method, built the three-dimensional structure of the V regions, labeled 4A1 with [¹⁴C]phenyl glyoxal in the presence and absence of I-1 or I-13, and analyzed the labeled incubation mixture with SDS–PAGE. From these results, the possibility that Arg-H28 of the heavy chain is involved in binding the bulky group of the substrate is discussed.     

单链抗体2F3表达条件的优化及其提纯和性质研究

将构建好的单链抗体 2F3表达载体pTMF2F3ScFv转化到大肠杆菌BL21(DE3)。先挑选出表达量高的单克隆 ,然后让其在 37℃进行表达 ,并将表达时的培养条件进行优化。实验结果表明 :最佳诱导条件为开始诱导时的菌体密度OD_590nm =1.0～ 1.8,所加异丙基β-D 硫代半乳糖苷 (IPTG)的浓度为 0.3～0.5mmol/L ,诱导时间 7h ,优化后目的蛋白表达量占菌体总蛋白的20% ,并用发酵罐成功地进行了扩大培养 ,筛选了洗涤包涵体的最佳条件。采用两步法对包涵体复性进行了研究 ,用Westernblotting及ELISA法检测了所表达的单链抗体及其生物活性 ,并成功制备了含硒单链抗体酶.     

具有谷胱甘肽过氧化物酶活性的含硒单链抗体酶制备

 利用RT PCR从分泌有谷胱甘肽结合部位的单克隆抗体杂交瘤细胞株 2F3中 ,扩增出单抗重链可变区和轻链可变区基因 .经DNA测序后 ,用Linker(Gly4 Ser1) 3 构建成单链抗体 (scFv)表达载体pTMF scFv ,将重组质粒pTMF scFv转化到大肠杆菌BL2 1(DE3) ,实现了单链抗体的高效表达 .表达的单链抗体占菌体总蛋白 2 5%～ 30 % .该重组蛋白以包涵体形式存在 ,分子量为 30kD .经过金属螯合亲和层析纯化、复性和凝胶过滤纯化 ,得到电泳均一的单链抗体 .再经化学诱变 ,得到含硒单链抗体酶 ,其谷胱甘肽过氧化物酶活性为 330 0U μmol.采用荧光滴定法测定了单链抗体对谷胱甘肽的结合常数     

Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus

In contrast to canonical proteases, myelin basic protein (MBP)-Sepharose-purified IgG from multiple sclerosis (MS) and systemic lupus erythematosus (SLE) patients efficiently hydrolyze only MBP, but not many other tested proteins. It was shown that anti-MBP SLE IgGs cleave nonspecific tri- and tetrapeptides with an extremely low efficiency and cannot efficiently hydrolyse longer oligopeptides corresponding to antigenic determinants (AGDs) of HIV-1 integrase. To identify all sites of IgG-mediated proteolysis corresponding to two AGDs of MBP, we have used a combination of reverse-phase chromatography (RPhC), MALDI spectrometry, and TLC to analyze the cleavage products of two (17- and 19-mer) encephalytogenic oligopeptides corresponding to these AGDs. Both oligopeptides contained several clustered major and minor sites of cleavage. The active sites of anti-MBP abzymes are localized on their light chains, while the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high specificity of MBP hydrolysis. The affinity of anti-MBP abzymes for intact MBP was ～10(3)-fold higher than for the oligopeptides. The data suggest that both oligopeptides interact mainly with the light chain of different monoclonal abzymes of total pool of IgGs, which possesses lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific.     

含硒抗体酶性质的研究

在成功地制备了具有谷胱甘肽过氧化物酶（ＧＰＸ）活性含硒抗体酶（Ｓｅ－ａｂｚｙｍｅ）的基础上,我们筛选了制备Ｓｅ－ａｂｚｙｍｅ的最佳条件,并对其理化性质及酶学性质和稳定性进行了深入的研究。结果表明,Ｓｅ－ａｂｚｙｍｅ的等电点为６．９５和７．０８,一为１５８ｋｄ;适ＰＨ和最适温度范围比天然酶宽广;抗体酶的贮藏稳定性比天然酶高。高Ｘ射线光电子能谱技术测得在Ｓｅ－ａｂｚｙｍｅ中含硒量为５ｍｏｌＳｅ／ｍｏｌ     

IgG abzymes with peroxidase and oxidoreductase activities from the sera of healthy humans

We present the evidence showing that small fractions of electrophoretically homogeneous immunoglobulin G (IgGs) from the sera of healthy humans and their Fab and F(ab)₂ fragments oxidize 3,3′‐diaminobenzidine through a peroxidase activity in the presence of H₂O₂ and through an oxidoreductase activity in the absence of H₂O₂. During purification on protein G‐Sepharose and gel filtration, the polyclonal IgGs partially lose the Me²⁺ ions. After extensive dialysis of purified Abs against agents chelating metal ions, the relative peroxidase activity decreased dependently of IgG analyzed from 100 to ~10–85%, while oxidoreductase activity from 100 to 14–83%. Addition of external metal ions to dialyzed and non‐dialyzed IgGs leads to a significant increase in their activity. Chromatography of the IgGs on Chelex non‐charged with Cu²⁺ ions results in the adsorption of a small IgG fraction bound with metal ions (~5%), while Chelex charged with Cu²⁺ ions bind additionally ~38% of the total IgGs. Separation of Abs on both sorbents results in IgG separation to many different subfractions demonstrating various affinities to the chelating resin and different levels of the specific oxidoreductase and peroxidase activities. In the presence of external Cu²⁺ ions, the specific peroxidase activity of several IgG subfractions achieves 20–27 % as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of these fractions is ~4–6‐fold higher than that for HRP. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defence mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activities of human IgGs could also play an important role in the protection of organisms from oxidative stress and toxic compounds. Copyright © 2015 John Wiley & Sons, Ltd.