Determination of the glycosylation patterns, disulfide linkages, and protein heterogeneities of baculovirus-expressed mouse interleukin-3 by mass spectrometry.
The primary structure of mouse interleukin-3 (IL-3) expressed by recombinant baculovirus-infected silkworm (Bombyx mori) larvae was analyzed by subjecting isolated IL-3 derived peptides to liquid secondary ion mass spectrometry. Two species of IL-3 were isolated from the silkworm hemolymph by reverse-phase high-pressure liquid chromatography. The major component has M(r)20-22 x 10(3) as determined by SDS-PAGE. Liquid secondary ion mass spectrometric analysis was carried out on the reduced tryptic and endopeptidase lysyl-C peptides of glycosylated and deglycosylated IL-3. These studies provided evidence that (1) Asn-16 is heterogeneously glycosylated with four different oligosaccharides, (2) Asn-86 is either nonglycosylated or has attached to it one oligosaccharide, (3) the N-glycosylation sites Asn-44 and Asn-51 are not glycosylated, and (4) there is no O-glycosylation. Liquid secondary ion mass spectrometric analysis of the unreduced tryptic peptides provided evidence for disulfide linkages between Cys-140 and Cys-79 or Cys-80 and between Cys-17 and Cys-79 or Cys-80. In comparison to the major component, a minor IL-3 species (M(r) 17-19 x 10(3) by SDS-PAGE) isolated from the hemolymph showed no difference with respect to the glycosylation pattern or the disulfide linkages, but it was cleaved between Ala-127 and Ser-128, and only a disulfide linkage between Cys-140 and Cys-79 or Cys-80 held the molecule together.(ABSTRACT TRUNCATED AT 250 WORDS)
Knepper
T P
TP
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7301.
Arbogast
B
B
Schreurs
J
J
Deinzer
M L
ML
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D006031:Glycosylation; D007377:Interleukin-3 / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry*
D004220:Disulfides; D007377:Interleukin-3; D011506:Proteins