Mass spectrometric determination of glycosylation sites and oligosaccharide composition of insect-expressed mouse interleukin-3.
The primary structure of Baculovirus-expressed mouse interleukin-3 produced in infected Bombyx mori larvae was characterized by liquid secondary ion mass spectrometry and 252Cf-plasma desorption mass spectrometry in combination with selected protein microchemical reactions. Interleukin-3 was found to consist of at least two glycoprotein species of ca. 17,000 dalton. Characterization of tryptic and S. aureus V8 protease peptides by Edman degradation combined with plasma desorption mass spectrometry showed that two N-glycosylation sites. Asn-16 and Asn-86, were present. N-Glycan residues were shown by liquid secondary ion mass spectrometry and high-performance liquid chromatography to consist of mannose, fucose, and glucosamine. The presence of galactosamine indicated that O-glycosylated residues were present, in addition to the N-glycosylated residues. Glucose was also present, which indicated incomplete processing of the insect-expressed N-linked oligosaccharides.
Svoboda
M
M
Fakultät für Chemie, Universität Konstanz, F.R.G.
Przybylski
M
M
Schreurs
J
J
Miyajima
A
A
Hogeland
K
K
Deinzer
M
M
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000235:genetics*; D002142:Californium; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D003545:Cysteine / Q000032:analysis; D005947:Glucose / Q000737:chemistry; D007202:Indicators and Reagents; D007377:Interleukin-3 / Q000737:chemistry* / Q000235:genetics; D017879:Isothiocyanates; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D010449:Peptide Mapping; D012697:Serine Endopeptidases; D013861:Thiocyanates; D014357:Trypsin
D002241:Carbohydrates; D007202:Indicators and Reagents; D007377:Interleukin-3; D017879:Isothiocyanates; D009844:Oligosaccharides; D013861:Thiocyanates; C005441:phenylisothiocyanate; D002142:Californium; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D005947:Glucose; D003545:Cysteine