Role of sugar chains in the in-vitro activity of recombinant human interleukin 5.
The structures of O-linked sugar chains of recombinant human interleukin 5 (rhIL-5) produced by Chinese hamster ovary cells were determined employing high-performance anion-exchange column chromatography under high pH conditions. The core sequence was elucidated to be Gal beta 1-->3-GalNAc by its susceptibility to endo-alpha-N-acetylgalactosaminidase and sialic acid linkages were determined using sialidases of different specificities. To investigate the role of sugar chains in the in-vitro activity of rhIL-5, it was digested with various glycosidases. While removal of N-linked sugar chains resulted in 2.8-fold increase of the activity, de-O-glycosylated rhIL-5 showed 10-fold higher activity than intact rhIL-5, suggesting that the presence of O-linked sugar chains suppresses the activity more effectively than that of N-linked chains. While incubation of de-N-glycosylated rhIL-5 at 70 degrees C for 30 min decreased the activity, intact and de-O-glycosylated rhIL-5 lost little activity, suggesting that N-linked sugar chains contributed to the thermostability of the molecule.
Kodama
S
S
Suntory Institute for Biomedical Research, Osaka, Japan.
Tsujimoto
M
M
Tsuruoka
N
N
Sugo
T
T
Endo
T
T
Kobata
A
A
D001894:Borohydrides; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004795:Enzyme Stability; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015848:Interleukin-5 / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry; D048809:alpha-N-Acetylgalactosaminidase
D001894:Borohydrides; D015848:Interleukin-5; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012794:Sialic Acids; D006596:Hexosaminidases; D009439:Neuraminidase; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase; D019158:N-Acetylneuraminic Acid