Tuning the conformation properties of a peptide by glycosylation and phosphorylation q Fu-Cheng Liang a , Rita P.-Y. Chen b , Chun-Cheng Lin a , Kuo-Ting Huang a , Sunney I. Chan a, * a Institute of Chemistry, Academia Sinica, Taipei, Taiwan, ROC b Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ROC Received 19 January 2006 Available online 9 February 2006 Abstract We have deployed the a-helical hairpin peptide (a-helix/turn/a-helix) and used it as a model system to explore how glycosylation and phosphorylation might affect the conformational properties of the peptide. The native conformations of the modified peptides in buffer solution have been compared with that of the wild-type peptide by nuclear magnetic resonance spectroscopy. Circular dichroism spec- troscopy was used to probe the effects of an O-linked b-GlcNAc and a phosphate group on the overall folding stability of the peptide. Finally, the rate of fibrillogenesis was used to infer the effects of these chemical modifications on the a-to-b transition as well as the rate of nucleation of amyloidogenesis. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Glycosylation; Phosphorylation; Post-translational modification; Helical hairpin peptide; Amyloid fibril Glycoproteins are usually thought of as components of the plasma membrane and the extracellular space of a cell. It has only been recognized recently that glycoproteins exist also in the nuclear or cytoplasmic portions of the cell [1–3]. The best characterized examples of nuclear and cyto- plasmic glycosylation to date are those that are O-linked by b-N-acetylglucosamine (O-GlcNAc) [3–6]. This post-trans- lational modification, also called GlcNAcylation, consists of a single GlcNAc residue linked to the hydroxyl group of a serine or threonine. Over 100 nuclear and cytosolic proteins have now been identified with O-GlcNAc modifi- cation in multi-cellular eukaryotes [7]. Accumulating evidence implicates this kind of glycosylation in the regula- tion of numerous cellular processes, including nuclear import, protein translation, gene transcription, and cyto- skeletal formation. GlcNAcylation appears to be as abundant and dynamic as protein phosphorylation. A reciprocal relationship has been noted between glycosylation and phosphorylation, implicating the possible role of glycosylation on signal transduction. In several documented instances, GlcNAc and phosphate alternatively occupy the same or adjacent hydroxyl moieties [8,9], leading to the hypothesis that one function of this sugar might be to transiently block phos- phorylation of the site [10]. On the other hand, it has been known for some time that GlcNAcylation plays a critical role in a variety of biological processes including immune response, cell–cell recognition, and protein folding, and it affects protein stability and solubility [11]. Thus, it is not clear whether the function of GlcNAcylation is restricted as a temporary site-blocker or a competitor of phosphory- lation, or it is used primarily to modulate the conformation properties of proteins, or both. Here, we have used the a-helical hairpin peptide ata as our model system to investigate the effect of 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.01.168 q Abbreviations: b-GlcNAc, b-N-acetylglucosamine; ata, the designed peptide with an a-helix turn a-helix structure; ata-s, ata with an O-GlcNAc at Thr-19; ata-p, phosphorylated ata at Thr-19. * Corresponding author. Fax: +886 2 27831237. E-mail address: SunneyChan@yahoo.com (S.I. Chan). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 342 (2006) 482–488 BBRC