Affinity Capturing and Gene Assignment of Soluble Glycoproteins Produced by the Nematode Caenorhabditis elegans
| Autor: | Jun Hirabayashi, Ken-ichi Kasai, Toshiaki Isobe, Ko Hayama, Hiroyuki Kaji |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Glycan
Glycosylation Galectins Molecular Sequence Data Mannose Biology Biochemistry Chromatography Affinity chemistry.chemical_compound Polysaccharides Concanavalin A Animals Amino Acid Sequence Caenorhabditis elegans Molecular Biology Chromatography High Pressure Liquid Glycoproteins Galectin chemistry.chemical_classification Sequence Homology Amino Acid Lectin General Medicine Cosmids biology.organism_classification Solubility chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Electrophoresis Polyacrylamide Gel Adsorption Glycoprotein |
| Zdroj: | Journal of Biochemistry. 132:103-114 |
| ISSN: | 0021-924X |
| DOI: | 10.1093/oxfordjournals.jbchem.a003186 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|