Poster: Vegetative Development

Abs # 431: Determination of glycosyl residue motif in sporamin by MALDI-TOF MS

Presenter:	Yuasa, Koji , koubow@yokohama.riken.go.jp
Authors	Yuasa, Koji  (A)   Shimizu, Masami  (A)   Fukuda, Hiroo  (A)   Matsuoka, Ken  (A)
Affiliations:	(A): RIKEN Plant Science Center

In plants, hydroxyproline (Hyp)-O-glycosylation uniquely characterizes diverse group of structural glycoproteins. These glycoproteins are broadly implicated in plant growth and development. Although the chemical composition of the carbohydrate part is involved in the control of cellular development, a progress in establishing the detailed structures is limited. Sporamin is a vacuolar storage protein of sweet potato. This protein, when expressed in tobacco BY-2 cells, is hydroxylated at Pro³⁶ residue and further modified by O-glycosylation. Taking advantage of this site to be the only proline hydroxylation and glycosylation site in sporamin, we are analyzing the primary structure of the glycosyl residue motif using MALDI-TOF MS. Sporamin was first denatured in SDS solution, and subsequently digested by 0.5 pmol of Trypsin or Endoproteinase Asp-N, respectively. The digested sporamin was directly spotted on sample plate. Both secretory mutant sporamin and vacuolar wild-type sporamin were hydroxylated and glycosylated in contrary to recombinant sporamin prepared from E. coli. Both glycosylated sporamins contained degree of polymerization up to 9 hexoses with one to three pentoses, as judged from MALDI-TOF MS results. We are currently analyzing the glycosylation pattern of several mutant sporamins that have replaced single amino acid residues around Pro³⁶ with an alternative amino acid.