Investigation Of The Hemolytic Activity Of Five Human Igg And Its Mechanism

Immunoglobulin G (IgG) is the major part (about 75%) of the immunoglobulin in human serum. It is the most important and enduring antibody during primary immune response. And what's more, IgG is the only Ig which can pass the placenta. It plays an important roles in passive immunity and active immunity. It is generally believed that there is no evolutionary relationship between non-specific and specific immune molecules. In recent years researchers reported that many invertebrate immune molecules not only possess the typical functional domain of Ig, but also have significant molecular polymorphism, even have a part of the acquired immune function. These results indicate that there should be have the degree of evolutionary relationship between the non-specific and specific immune molecules, human IgG may also possess some non-specific immunological activity. In this paper, an attempt was made to investigation the hemolytic activity and mechanism of IgG from healthy human serum by affinity chromatography, glycomics, proteomics, molecular biology techniques. The main results obtained are as follows:Firstly, we separated 5 human IgG isomers (IgGg,IgGs-,IgGs+,IgGt- and IgGt+) from healthy serum by ammonium sulfate fractionation, cellulose DE 52 ion exchange chromatography and affinity chromatography methods. In which, IgGg was purified by cellulose DE 52 ion exchange chromatography; IgGs- and IgGs+ were purified by affinity chromatography using rabbit anti hemocyanin of small subunit antibody; IgGt- and IgGt+ were purified by affinity chromatography using rabbit anti hemocyanin antibody. These 5 kinds of proteins were all proved to be human IgG by 1-DE,2-DE and wsetern-blotting analysis. We further analyzed IgGg, IgGs+ and IgGt+ through two-dementional electrophoresis and western-blotting, the results showed that the 3 isomers were different from each other. Compared with IgGg, IgGs+ and IgGt+ were added one and five protein spots.Then, we compared the glycosylation of the 5 isomers by phenol sulfuric acid method and lectin-blotting. Among them, IgGt+ possesses the highest content of sugar (82.92μg/mg), while IgGs- the lowest (18.44μg/mg). The former was of 4 folds of the latter. Further study demonstrate that mannose content of 5 human IgG isomers was also significantly different. Consistent with the total sugar content, IgGt+ possesses the highest content, while IgGs- the lowest. These results indicated that 5 human IgG isomers exists differences in proteins polymorphism and glycosylation, suggesting that immunologic function of 5 human IgG isomers may be different.Finally, we explored their hemolytic activity and mechanisms by hemolysis test,1-DE,immunoblotting and osmoprotection. Our results indicated as follows:1) 5 human isomers had different hemolytic activities against these 7 kinds of erythrocytes (human A,B,O,AB, chicken, mouse, rabbit). There was significant difference. Among them, IgGs+ and IgGt+ had hemolytic activities against 7 kinds of erythrocytes (approximately 30.99±0.01～100±0.08%), while IgGg,IgGs- and IgGt- seemed no hemolytic activities against these 7 kinds of erythrocytes.2) The isomers lost hemolytic activity after treated with trypsin or periodate, suggesting that the hemolytic activity of human IgG isomers is related with protein structure and glycosylation.3) SDS-PAGE and immunoblotting were used to analyse the interaction between IgGt+ and erythrocyte. The results showed that heavy chain (55 kDa) and light chain (26 kDa) of IgGt+ and a high molecular mass protein around 121 kDa were found in the erythrocyte membranes treated with IgGt+, but not in the treatment with distilled water. Moreover, the 121 kDa protein could react specifically with anti-human IgG antibody as well as the subunits of IgGt+. The 121 kDa protein was cut out from the first SDS-PAGE gel and subjected to the second SDS-PAGE and immunoblotting analysis following elution with electrophoresis. The results showed that the 121 kDa protein was separated into two bands of 66.2 and 55 kDa. Particularly, the 55 kDa protein was documented to be positive with anti-human IgG antibodies, suggesting that human IgG educed hemolytic activity through its heavy chain integrating with erythrocyte membrane.According to osmotic protection assay, hemolysis was inhibited to different ±3.16% protection against hemolysis of chicken erythrocytes, respectively. These results suggested that the mechanism of hemolysis of human IgG may be the result of the ion penetration hole formed by the combination of the heavy chain of the IgG and the erythrocyte membrane.In summary, the thesis for the first time found that human IgG possess non-specific immune activity-hemolytic activity, and its mechanism of hemolysis is colloid-osmotic mechanism. The results we obtained will be helpful to explore the origin of adaptative immunity of vertebrate and its evolutionary relationship with the innate immunity of invertebrate.