1. Structure determination of the glycans of human-serum alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by N-glycanase.
- Author
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Laine A, Hachulla E, Strecker G, Michalski JC, and Wieruszeski JM
- Subjects
- Amidohydrolases, Asparagine, Carbohydrate Conformation, Carbohydrate Sequence, Chromatography, Affinity, Humans, Hydrogen, Immunoelectrophoresis, Two-Dimensional, Magnetic Resonance Spectroscopy methods, Molecular Sequence Data, Oligosaccharides isolation & purification, Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase, Polysaccharides isolation & purification, alpha 1-Antichymotrypsin isolation & purification, alpha 1-Antitrypsin chemistry, alpha 1-Antitrypsin isolation & purification, Oligosaccharides chemistry, Polysaccharides chemistry, alpha 1-Antichymotrypsin chemistry
- 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 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.
- Published
- 1991
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