Abstract
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.
Highlights
A synthetic gene encoding the fusion protein (AlaHyp)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
The polysaccharide consisted of a 1,3-linked -D-Galp backbone with a single 1,6-linked -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
Summary
Isolation of the (Ala-Hyp)51-EGFP Fusion Protein and (Ala-Hyp)51— The (Ala-Hyp)51-EGFP fusion glycoprotein was isolated from spent medium of 20-day transformed N. tabacum BY-2 suspension cultured cells by a combination of hydrophobic interaction and reversed phase chromatography, as described earlier (19). (Ala-Hyp)[51] was isolated from tryptic digests of (Ala-Hyp)51-EGFP by gel permeation chromatography, described earlier (19). (We corrected a software error in the anomeric configuration of L-sugars by inserting the ␣- for -anomer and vice versa in order to follow IUPAC definitions.) Three AHP-1 polysaccharides were attached glycosidically to (Ala-Pro)[6] to form a tight cluster of three consecutive arabinogalactans on noncontiguous Hyp residues and subjected to final energy minimization using the Amber[3] force field. We found it helpful to define the approximate bond lengths for the Gal-Hyp glycosidic link by using the ‘‘set bond length’’ command when attaching the polysaccharide to the polypeptide
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