Structure determination of the glycans of human‐serum α1‐antichymotrypsin using 1H‐NMR spectroscopy and deglycosylation by N‐glycanase

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Abstract
α1‐Antichymotrypsin purified from normal human serum was separated by affinity chromatography into three microheterogeneous forms on a concanavalin‐A – Sepharose column: a pass‐through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine‐linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re‐N‐acetylation and further separated by affinity chromatography on a concanavalin‐A – Sepharose column. The complete primary structure of the glycans was determined by high‐resolution 1H‐NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N‐glycanase was used for the deglycosylation of the unfractionated α1‐antichymotrypsin; the successive removal of the N‐linked complex‐type oligosaccharide side chains of α1‐antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that α1‐antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.