Title : Structure determination of
the glycans of human-serum
alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by
N-glycanase
Abstract :
- alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree 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 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 alpha 1-antichymotrypsin ; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents
- From these experiments it is concluded that alpha 1-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