The Discovery Of A Basic Phenylalanine-rich Peptide And Its Effects On The PI And The Immune Cross-reaction Of Protein

Every protein has a speicfic conformation, which exhibits a definite pI value, but lots of proteins such as Glycolate oxidase (GO) consist of more than one pI values, which indicates that the same protein has different conformations.In addition, the immunoassay cross reactions among different proteins are often exsisted in immunoassay experiments, which suggests that different proteins may have the similar conformation. However, no information has been found about the mechanism of one protein with pIs and immune cross reactions. In this study, we investigated the underlying cause of the mentioned phenomenon.The results were showed as follows:1. Glycolate oxidase (GO) was purified and showed a molecular weight of 40 kDa by SDS-PAGE. Elution behavior on DEAE-cellulose chromatography and cellulose acetate membrane electrophoresis indicated that the purified GO was highly basic (pI>10.0). Repeated IEF and cIEF analysis showed that the pI of the purified GO was in the range of 10.0–3.25, either in a smear form or as distinct bands. 2-DE analysis showed that the 40 kDa subunit of GO displayed variable pIs from 9.6 to 3.65. It was likely that the purified GO was actually a complex consisted of GO and an unknown protein X.2. CE-SDS, SDS-cellulose acetate membrane electrophoresis and amino acid compositions indicated that the unknown protein is a highly basic polymer (BP) consisted of basic and phenylalanine-rich oligopeptide (BOP). Many BOPs are located on the surface of the acidic GO via ionic and hydrophobic interactions and GO-BOP complex (GC) forms, resulting in a highly basic GC although GO itself is acidic. This hypothesis is further supported by the facts that anti-GC serum failed to recognize GO, and GC showed a peak at 257 nm although GO has few phenylalanine residues. Irregular and incomplete disassociation between GO and BOP was observed in IEF and cIEF, resulting in various intermediates with different ratios of GO/BOP, which might be the reason for the range of pIs observed for GO.3. GC showed a peak at 258 nm, a typical absorption peak of phenylalanine. In the dot blot analysis, all anti-GC recognized GC. In SDS-PAGE Western blot analysis, anti-GC serum didn`t recognized GC, anticathode protein serum recognized 40 kDa GO, however, anti-GO subunit serum recognized 40 kDa subunit in GC. Obviously, the epitopes of GO were protected, because anti-GC serum recognized GC, but failed to recognize GO 40 kDa subunit , which further showed that many BOP molecules are likely located on the surface of GO 40 kDa subunit via ionic and hydrophobic interactions. 4. We also found that similar BP existed in bacteria, plant and animal.Crude extract from E. coli, green leaves of A. thaliana and O. sativa exhibited two protein bands migrated towards the cathode and anode in SDS-PAGE/2-DE. The ratio of these two bands varied significantly in different assays. The same pattern was observed in all crude extract from animals such as Oryctolagus cuniculus, Sus scrofa, Gallus gallus, Columba, Fenneropenaeus chinensis, Tanichthys albonubes, Bombyx mori, Rattus norregicus and Mus musculus.5. BOP exsists in 1,5-ribulose bisphosphate carboxylase/oxygenase(Rubisco) and stem bromelain (SB) ,and forms Rubisco-BOP or SB-BOP complex respectively, which furtherly explain that they exhibits many pIs and immune cross reactions.