Structure determination of the glycans of human-serum alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by N-glycanase.
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.
Laine
A
A
Unité 16 INSERM, Lille, France.
Hachulla
E
E
Strecker
G
G
Michalski
J C
JC
Wieruszeski
J M
JM
D000581:Amidohydrolases; D001216:Asparagine; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D006801:Humans; D006859:Hydrogen; D007123:Immunoelectrophoresis, Two-Dimensional; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry* / Q000302:isolation & purification; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000302:isolation & purification
D009844:Oligosaccharides; D011134:Polysaccharides; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D001216:Asparagine; D006859:Hydrogen; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase