Analysis of bovine alpha1-acid glycoprotein phosphorylation by 2-D electrophoresis and IN-GEL fluorescence

Alpha1-acid glycoprotein is considered an acute phase proteins (APP) in most species. One of the most interesting features of AGP is that its concentration in plasma not only rises during inflammation, but also undergoes structural modifications of its oligosaccharide moiety (more than 40% of the molecular mass of the protein), resulting in a change of both the degree of branching and fucosylation. Furthermore, a throughout analysis of the primary structure revealed that AGP’s sequence presents also seven potential phosphorylation sites. No studies report the actual phosphorylation of the protein. AGP purified from bovine serum was submitted to 2D-electrophoresis and the separates isoforms of the protein has been analyzed by mean of a general fluorescence dye and Phos-tag phosphoprotein gel stain dye, which selectively binds the phophoserine, phosphothreonine and phosphotyrosine residues. The binding has been detected by in-gel fluorescence. The 2D maps clearly reveal the purified AGP from healthy animals presents a marked microheterogeneity, concerning both the pI and MW. At least three different families of protein isoforms can be detected: the first is heterogeneous in charge but is not phosphorylated, the second is heterogeneous both in charge and MW, indicating also a different glycosylation pattern, and the third is heterogeneous in charge and appear to be strongly phosphorylated. In conclusion, the observed electrophoretic charge microheterogeneity of AGP may be due to the phosphorylation status of the protein and, possibly, to different levels of amidation of the glutammic and aspartic acid residues. Although the findings predented in this communication should be considered as still preliminary, they show that, further to gene expression control and glycosylation status, the activity of AGP may be also regulated by phosphorylation events. The next step of this investigation will be to evaluate if phosphorylation of AGP may be modified and regulated during diseases.