A serial lectin approach to the mucin-type O-glycoproteome of Drosophila melanogaster S2 cells.
Identification of mucin-type O-glycosylated proteins with known functions in model organisms like Drosophila could provide keys to elucidate functions of the O-glycan moiety and proteomic analyses of O-glycoproteins in higher eukaryotes remain a challenge due to structural heterogeneity and a lack of efficient tools for their specific isolation. Here we report a strategy to evaluate the O-glycosylation potential of the embryonal hemocyte-like Drosophila Schneider 2 (S2) cell line by expression of recombinant glycosylation probes derived from tandem repeats of the human mucin MUC1 or of the Drosophila salivary gland protein Sgs1. We obtained evidence that mucin-type O-glycosylation in S2 cells grown under serum-free conditions is restricted to the Tn-antigen (GalNAcalpha-Ser/Thr) and the T-antigen (Galbeta1-3GalNAcalpha-Ser/Thr) and this structural homogeneity enables unique glycoproteomic strategies. We present a label-free strategy for the isolation, profiling and analysis of O-glycosylated proteins consisting of serial lectin affinity capture, 2-DE-based glycoprotein analysis by O-glycan specific mAbs and protein identification by MALDI-MS. Protein identity and O-glycosylation was confirmed by ESI-MS/MS with detection of diagnostic sugar oxonium-ion fragments. Using this strategy, we established 2-D reference maps and identified 21 secreted and intracellular mucin-type O-glycoproteins. Our results show that Drosophila S2 cells express O-glycoproteins involved in a wide range of biological functions including proteins of the extracellular matrix (Laminin gamma-chain, Peroxidasin and Glutactin), pathogen recognition proteins (Gnbp1), stress response proteins (Glycoprotein 93), secreted proteases (Matrix-metalloprotease 1 and various trypsin-like serine proteases), protease inhibitors (Serpin 27 A) and proteins of unknown function.
Schwientek
Tilo
T
Center for Biochemistry, Medical Faculty, University of Cologne, Köln, Germany. akd88@uni-koeln.de
Mandel
Ulla
U
Roth
Udo
U
Müller
Stefan
S
Hanisch
Franz-Georg
FG
D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000737:chemistry*; D002846:Chromatography, Affinity; D004331:Drosophila melanogaster / Q000166:cytology*; D015180:Electrophoresis, Gel, Two-Dimensional; D008401:Gas Chromatography-Mass Spectrometry; D006031:Glycosylation; D037102:Lectins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D020543:Proteome
D002241:Carbohydrates; D037102:Lectins; D009077:Mucins; D020543:Proteome