Structure of a hydroxyproline (Hyp)-arabinogalactan polysaccharide from repetitive Ala-Hyp expressed in transgenic Nicotiana tabacum.
A synthetic gene encoding the fusion protein (Ala-Hyp)(51)-enhanced green fluorescent protein expressed in Nicotiana tabacum cells produced a fusion glycoprotein with all proline residues hydroxylated and substituted with an arabinogalactan polysaccharide. Alkaline hydrolysis of the fusion glycoprotein yielded a population of hydroxyproline (Hyp)-arabinogalactan polysaccharides ranging in size from 13 to 26 saccharide residues/Hyp, with a median size of 15-17 residues. We isolated a 15-residue Hyp-arabinogalactan for structure determination by sugar analyses and one- and two-dimensional nuclear magnetic resonance techniques that provided the assignment of proton and carbon signals of a small polysaccharide O-linked to the hydroxyl group of Hyp. The polysaccharide consisted of a 1,3-linked beta-D-Galp backbone with a single 1,6-linked beta-D-Galp "kink." The backbone had two side chains of Galp substituted at position 3 with an arabinose di- or trisaccharide and at position 6 with glucuronic acid or rhamnosyl glucuronic acid. Energy-minimized space-filling molecular models showed hydrogen bonding within polysaccharides attached to repetitive Ala-Hyp and also between polysaccharides and the peptide backbone. Polysaccharides distorted the peptide Ramachandran angles consistent with the circular dichroic spectra of isolated (Ala-Hyp)(51) and its reversion to a polyproline II-like helix after deglycosylation. This first complete structure of a Hyp-arabinogalactan polysaccharide shows that computer-based molecular modeling of Hyp-rich glycoproteins is now feasible and supports the suggestion that small repetitive subunits comprise larger arabinogalactan polysaccharides.
Tan
Li
L
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA.
Qiu
Feng
F
Lamport
Derek T A
DT
Kieliszewski
Marcia J
MJ
D002244:Carbon / Q000737:chemistry; D002942:Circular Dichroism; D005685:Galactans / Q000737:chemistry*; D020723:Glucuronic Acid / Q000737:chemistry; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006868:Hydrolysis; D006909:Hydroxyproline / Q000737:chemistry*; D008164:Luminescent Proteins / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D011134:Polysaccharides / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014026:Nicotiana / Q000378:metabolism*
D005685:Galactans; D008164:Luminescent Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002244:Carbon; D020723:Glucuronic Acid; D011392:Proline; D006909:Hydroxyproline; C005653:arabinogalactan