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

Abstract

Alpha-1-acid glycoprotein (AGP) is a major acute phase glycoprotein with five asparaginyl-linked complex oligosaccharide chains accounting for approximately 45% of its 42 kilodalton molecular weight.The physiological role of AGP is poorly understood however it has been demonstrated to have immunomodulatory properties [l]. In normal plasma AGP exists as an heterogenous population of glycoforms owing to differing occupancy of the glycosylation sites. Moreover the relative proportions of these glycoforms are altered in disease such as rheumatoid arthritis [2] where a decreased number of bi-antennary chains and an increase in the absolute amount of fucose and in the number of fucose residues per molecule have been observed [3]. Such heterogeneity may be of functional significance in inflammatory disease.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. Human peripheral blood mononuclear cells were isolated from Buffy Coat cell concentrates from healthy donors by Ficoll-Hypaque density g r a d i e n t centrifugation following defibrination with thrombin. The mononuclear population isolated was washed and resuspended to 106 cells/ mL in RPMI+ ( m e d i u m supplemented with 100pg/mL streptomycin, 100U/mL penicillin, 2mM glutamine and lO%v/v foetal calf serum). 200~1 cell suspension was aliquoted per well of a 96 well microtitre plate and treated with the mitogen phytohaemagglutinin (PHA) at a final w e l l concentration of 10pg/mL, and incubated at 37T, 5% CO2. The effects of the isolated glycoprotein on cell division were assessed in this system by simultaneous incubation with the mitogen at a final AGP concentration range of 0.1-lmg/mL. Rates of cell division were determined by radioactively labelling dividing cells with 3H-thymidine (O.SpCi/ well) during the final 4 hours of incubation, harvesting onto glass fibre filter paper and counting in a f3 scintillation counter.Oligosaccharide chains enzymatically released from whole AGP by treatment with peptide N glycosidase F (PNGaseF) were subjected to mild acid hydrolysis. Following boiling in 0.1M trifluoroacetic acid for 4 hours the monosaccharides were separated by high pH anion exchange chromatography with pulsed electrochemical detection (HPAE-PED). CONDITION r HepG2 AGP 0.5 mglml L HepG2 AGP I.Omg/ml 0 5000 1000015000200002500030000 COUNTS PER MINtJTE Figure 1. The proliferation responses of human peripheral blood mononuclear cells to phytohaemagglutinin in the presencelabsence of AGP secreted by the HepC2 cell line.