Characterization of N-linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris.
We studied the structures of four N-linked oligosaccharide chains of the recombinant human antithrombin (rAT) expressed in the yeast Pichia pastoris. rAT was fully glycosylated at Asn 96 and Asn 155, whereas the glycosylation on Asn 135 and Asn 192 was partial. The glycosylation level on Asn 135 was only 12% and this reduction is assumed to be one of the reasons for a higher heparin-binding affinity of rAT than plasma-derived human antithrombin (pAT). In order to determine the sizes and electrostatic charges of the N-linked oligosaccharides, rAT was treated with PNGase F, and the reduced ends were labelled by pyridylamination followed by analysis using anion exchange and amide adsorption columns. The N-linked oligosaccharides were 78% neutral and 22% phosphomannosylated. The neutral oligosaccharides were thought to be Man(9-12)GlcNAc(2) as their major components. The phosphomannosylated oligosaccharides were then subjected to mild acid hydrolysis and/or digestion with alkaline phosphatase, and their charge shifts were analysed by the affinity to an anion exchange column. Among phosphomannosylated oligosaccharides, monophosphate diester type was predominant, whereas negatively charged diphosphate diester and monophosphate monoester types were minor components. The mannose residues at the non-reducing end(s) of Man(9-12)GlcNAc(2) were phosphomannosylated or phosphorylated and these are the major components. Because rAT is less negatively charged than pAT, which has disialyl biantennary N-glycans, it might be less repulsive to pentasaccharide-bearing anticoagulantly active heparan sulphate proteoglycan molecules exposed on the surface of the damaged vascular vessels.
Hirose
Masaaki
M
Protein Research Laboratory, Pharmaceutical Research Division, Mitsubishi Pharma Corporation, Hirakata, Osaka, Japan. Hirose.Masaaki@mh.m-pharma.co.jp
Kameyama
Shoju
S
Ohi
Hideyuki
H
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000581:Amidohydrolases / Q000494:pharmacology; D000991:Antithrombins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis
D000991:Antithrombins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D001216:Asparagine; D000469:Alkaline Phosphatase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase