Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique.

A method based on a surface plasmon resonance technique for detection of changes in concentration and glycosylation of proteins in cell culture supernatant is described. The method was used to analyze alpha(1)-acid glycoprotein (AGP) produced by a human hepatoma cell line (HepG2). Cell culture supernatant was injected to a BIACORE 2000 instrument and AGP was captured on the sensor chip by immobilized antibodies. The captured glycoprotein was then analyzed for content of carbohydrate epitopes using three different lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), and Triticum vulgaris agglutinin (wheat germ agglutinin, WGA). The method was used to analyze changes in concentration and glycosylation of AGP produced by HepG2 cells grown with or without three different cytokines, interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and transforming growth factor beta-1 (TGF beta(1)). Using the described method it was shown that when HepG2 cells were grown in the presence of IL-6 both AGP concentration and fucosylation increased. When HepG2 cells instead were grown in the presence of TGF beta(1) AGP fucosylation increased whereas AGP concentration decreased.

Polymorphonuclear leukocytes from individuals carrying the G329A mutation in the alpha 1,3-fucosyltransferase VII gene (FUT7) roll on E- and P-selectins.

We recently identified several individuals carrying a missense mutation (G329A; Arg(110)-Gln) in the FUT7 gene encoding fucosyltransferase VII. This enzyme is involved in the biosynthesis of the sialyl Lewis x (Le(x)) epitope on human leukocytes, which has been identified as an important component of leukocyte ligands for E- and P-selectin. No enzyme activity was measurable in expression studies in COS-7 cells using the mutated FUT7 construct. One of the identified individuals carried this mutation homozygously. Flow cytometry analysis of polymorphonuclear leukocytes (PMN) from this individual showed a nearly complete absence of staining with mAbs directed against sialyl Le(x) and a diminished staining with an E-selectin IgG chimera. However, staining with P-selectin IgG chimera and Abs directed against P-selectin glycoprotein ligand-1 was not affected by the mutation. PMN from the homozygously mutated individual was further analyzed in an in vitro flow chamber assay. The number of rolling PMN and the rolling velocities on both E- and P-selectin were in the range of PMN from nonmutated individuals. FUT4 and FUT7 mRNA was quantified in PMN isolated from individuals carrying the FUT7 mutation. It was found that PMN from both FUT7 homozygously and heterozygously mutated individuals exhibited an elevated expression of FUT4 mRNA compared with PMN from FUT7 nonmutated individuals. The elevated expression of fucosyltransferase IV was reflected as an increased expression of the Le(x) and CD65s Ags on PMN from these individuals. The significance of the mutation was supported by transfection of BJAB cells.

Fucosylation of alpha1-acid glycoprotein (orosomucoid) compared with traditional biochemical markers of inflammation in recent onset rheumatoid arthritis.

BACKGROUND: Fucosylation of alpha1-acid glycoprotein (AGP, orosomucoid) has previously been found to be increased in patients with rheumatoid arthritis. Furthermore, the degree of fucosylation has been suggested to reflect disease activity. Therefore, we investigated the fucosylation of AGP in 131 patients (96 women and 35 men) with recent onset rheumatoid arthritis (RA). We compared the results with traditional biochemical markers of inflammation, i.e. plasma concentrations of AGP (P-AGP), and C-reactive protein (P-CRP). METHODS: AGP fucosylation measured with a novel lectin enzyme-linked immunosorbent assay (ELISA) was compared with a disease activity score (DAS28) and its components, and with P-AGP, and P-CRP at the time of diagnosis, and at a follow-up visit 1 year later. RESULTS: Both men and women with RA had increased AGP fucosylation compared to healthy individuals. We found a weak correlation between AGP fucosylation and DAS28 only in men. In men with initially increased AGP fucosylation, the level of fucosylation correlated with the change in DAS28 during the first year following diagnosis. CONCLUSION: We conclude that AGP fucosylation is not superior to traditional markers of disease activity in RA. However, AGP fucosylation may give some additional information to traditional biochemical markers on the disease progression in men.