Structural determination of oligosaccharides from recombinant iduronidase released with peptide N-glycanase F using fluorophore-assisted carbohydrate electrophoresis.
The lysosomal storage disorder mucopolysaccharidoses I (MPS I) is caused by a deficiency in the production of alpha-L-iduronidase. Recently, a recombinant alpha-L-iduronidase has been produced in Chinese hamster ovary (CHO) cells. It is thought that for alpha-L-iduronidase to be correctly targeted to the lysosomal vesicle a particular oligosaccharide make-up must be present, and characterization of the carbohydrates is critical. Oligosaccharides from alpha-L-iduronidase were analyzed using fluorophore-assisted carbohydrate electrophoresis (FACE). The FACE system uses polyacrylamide gel electrophoresis to separate, quantify, and determine the sequence of oligosaccharides released from glycoproteins. Asparagine-linked oligosaccharides were released from alpha-L-iduronidase using the enzyme peptide N-glycanase F (PNGase F). Released oligosaccharides were labeled with a fluorophore at the reducing termini by reductive amination. A total of nine bands were sequenced from the released pool of oligosaccharides. The pool of fluorescently labeled oligosaccharides was then electrophoresed in preparative gels and each band individually excised and extracted. Isolated bands were treated with a series of exoenzymes to determine the sequence of monosaccharides that make up a particular oligosaccharide. A total of eighteen different oligosaccharides were identified from the original pool of oligosaccharides. A majority of the oligosaccharides, over 73%, were found to be of the sialylated complex type. Four of the oligosaccharides were phosphorylated, making up approximately 11% of the carbohydrate pool, and the remaining 15% were of the oligomannose type.
Hague
C
C
Glyko Inc., Novato, CA 94949, USA.
Masada
R I
RI
Starr
C
C
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D006224:Cricetinae; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007068:Iduronidase / Q000737:chemistry* / Q000378:metabolism; D009281:Naphthalenes; D009844:Oligosaccharides / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017421:Sequence Analysis
D002241:Carbohydrates; D005456:Fluorescent Dyes; D009281:Naphthalenes; D009844:Oligosaccharides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; D007068:Iduronidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase