Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans.
Protein glycosylation is a central issue for post-genomic (proteomic) sciences. We have taken a systematic approach for analyzing soluble glycoproteins produced in the nematode Caenorhabditis elegans. The approach aims at assigning (i) genes that encode glycoproteins, (ii) sites where glycosylation occurs, and (iii) types of attached glycan structures. A soluble extract of C. elegans, as a starting material, was applied first to a concanavalin A (ConA) column (specific for high-mannose type N-glycans), and then the flow-through fraction was applied to a galectin LEC-6 (GaL6) column (specific for complex-type N-glycans). The adsorbed glycoproteins were digested with lysylendopeptidase, and the resultant glycopeptides were selectively recaptured with the same lectin columns. The glycopeptides were separated by reversed-phase chromatography and then subjected to sequence determination. As a result, 44 and 23 glycopeptides captured by the ConA and GaL6 columns, respectively, were successfully analyzed and assigned to 32 and 16 corresponding genes, respectively. For these glycopeptides, 49 N-glycosylation sites were experimentally confirmed, whereas 21 sites remained as potential sites. Of the identified genes, about 80% had apparent homologues in other species, as represented by typical secreted proteins. However, the two sets of genes assigned for the ConA and GaL6-recognized glycopeptides showed only 1 overlap with each other. Proof of the practical applicability of the glyco-catch method to a model organism, C. elegans, directs us to explore more complex multicellular organisms.
Hirabayashi
Jun
J
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan. j-hira@pharm.teikyo-u.ac.jp
Hayama
Ko
K
Kaji
Hiroyuki
H
Isobe
Toshiaki
T
Kasai
Ken-ichi
K
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003208:Concanavalin A / Q000737:chemistry; D003360:Cosmids / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D037161:Galectins / Q000737:chemistry; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006031:Glycosylation; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D037161:Galectins; D006023:Glycoproteins; D011134:Polysaccharides; D003208:Concanavalin A