Chromatographic investigation of the glycosylation pattern of alpha‐1‐acid glycoprotein secreted by the HEPG2 cell line; a putative model for inflammation?

Abstract

In certain pathophysiological conditions, such as rheumatoid arthritis, there are alterations in the glycosylation pattern of the acute phase protein, alpha-1-acid glycoprotein (AGP). These changes are likely to be functionally significant, however, verification of the latter role requires a system which reflects in vivo glycosylation changes in AGP and also produces sufficient quantities of the protein for further study. The human hepatoma cell line HepG2 is documented as displaying a shift in the glycosylation pattern of glycoproteins from normal state to acute phase after stimulation with inflammatory mediators. We have isolated AGP from the culture medium of HepG2 cells both before and after stimulation with a cytokine preparation and analysed the glycosylation pattern of each preparation, after enzymatic release, by high pH anion-exchange chromatography. Before stimulation, the glycosylated population was similar to a profile of AGP isolated from normal plasma; however, cytokine stimulation resulted in a shift to a profile which was consistent with that of AGP from a rheumatoid arthritis sufferer. Thus a HepG2 cell culture system is capable of being a crude model of the changes in glycosylation of acute phase proteins although it has a tendency to produce oligosaccharide chains which are not fully sialylated.

Determination of Diethylpyrocarbonate-Modified Amino Acid Residues in α1-Acid Glycoprotein by High-Performance Liquid Chromatography Electrospray Ionization–Mass Spectrometry and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight–Mass Spectrometry

“…The roles of OMD in cellular adhesion as well as drug binding are two areas of active research concerning the biological function of OMD (9 -16). Although OMD has been cloned and expressed, the structural properties of the five glycosylation sites differ from the naturally occurring OMD and the drug binding properties have not been characterized (13). Until an appropriate expression system can be developed for site-directed mutagenesis studies, chemical modification of the amino acid side chains can serve as a similar diagnostic tool (17).…”

The Anti-Proliferative Effect of Alpha-1-Acid Glycoprotein from HepG2 Cell line on Mononuclear Leucocytes

“…AGP was isolated and purified without structural degradation from the culture medium of HepGZ cells by PEG precipitation and sequential low pressure chromatography by our method previously described [4]. The eluant collected from the final Red Sepharose column was freeze dried and desalted by a Biogel P6 size exclusion column, with water as the buffer, detecting eluting protein by absorbance at 225nm.…”