Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis.
The N-linked oligosaccharide profiles (banding patterns in gels) and structures of recombinant soluble human interferon receptor 2 (r-shIFNAR2) were determined using fluorophore-assisted carbohydrate electrophoresis (FACE, Glyko, Novato, CA). The method involves releasing N-linked oligosaccharide moieties from a glycoprotein by digestion with peptide-N glycanase (PNGase F), labeling the released oligosaccharides with the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separating the labeled oligosaccharides by gel electrophoresis. The isolated oligosaccharides in the bands from the profiling gels can then be sequenced using exoglycosidases to reveal the oligosaccharide structures. The oligosaccharide profile of r-shIFNAR2 consists of at least nine oligosaccharide bands. The relative amount of oligosaccharide in each band can vary, depending on the culture conditions of the source cells. FACE structural analysis shows that r-shIFNAR2 contains only core-fucosylated N-linked oligosaccharides, most of which are fully sialylated (approximately 92%). The major types and relative amounts of the oligosaccharides from a representative sample are: disialylated, galactosylated, biantennary (15%); trisialylated, galactosylated, triantennary (19%), tetrasialylated, galactosylated, tetraantennary (30%), and N-acetyllactosamine-containing higher-order oligosaccharides including tri-, tetra-, and pentaantennary (28%). The remaining oligosaccharides are not fully sialylated and/or not fully galactosylated di-, tri-, and tetraantennary structures (approximately 5%) and unidentified structures (approximately 3%). A method for determining the types and structures of the N-acetyllactosamine containing oligosaccharides is also reported in this study.
Frado
L Y
LY
Department of Biopharmaceutical Sciences, Ares Advanced Technology, Inc., Randolph, MA, USA.
Strickler
J E
JE
D000606:Amino Sugars / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006224:Cricetinae; D004586:Electrophoresis / Q000379:methods; D005456:Fluorescent Dyes; D005782:Gels; D006801:Humans; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D000606:Amino Sugars; D005456:Fluorescent Dyes; D005782:Gels; D008565:Membrane Proteins; D009844:Oligosaccharides; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D053633:Receptor, Interferon alpha-beta; C000458:N-acetyllactosamine