Concanavalin A- and Wheat Germ Agglutinin-Conjugated Lectins as a Tool for the Identification of Multiple N-Glycosylation Sites in Heterologous Protein Expressed in Yeast

✍ Scribed by Rossana Garcı́a; Rosmari Rodrı́guez; Raquel Montesino; Vladimir Besada; Javier González; José A. Cremata

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 161 KB
• Volume: 231
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.0061

✦ Synopsis

late biological processes, generating the functional di-We report here a methodology that allows the identiversity needed for further development and cellular diffication of glycosylation sites by a combination of proferentiation as well as the interaction between cells tein enzymatic digestion, glycopeptide separation on and organisms. a reverse-phase HPLC column, and further recogni-Furthermore, recombinant proteins for therapeutic tion in a dot-blot system using concanavalin A-horseuses that appear glycosylated in nature must be glycoradish peroxidase. Wheat germ agglutinin-horseradsylated in order to display correct biological activity. ish peroxidase is used as the recognition system for This function depends also on the oligosaccharide patpeptides generated after proteolytic digestion of entern, i.e., oligomannoside, complex, or hybrid. For exdoglycosidase H deglycosylated protein. Glycosylation ample, ''in vivo'' activity of recombinant EPO 2 is related sites were confirmed by automatic Edman degradation not only to the ratio of di-and tetraantennary comand fast atom bombardment mass spectrometry. This plex oligosaccharides but also to their sialic acid methodology was applied to a model glycoprotein, acontent (14). amylase from Bacillus licheniformis, which is unglyco-At present, it is well known that the sequence Asnsylated in its natural host and appears highly glycosyl-Xaa-Thr/Ser (where Xaa may be any amino acid except ated when expressed in the methylotrophic yeast proline) should be present to be recognized by the oligo-Pichia pastoris.