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Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Published on: April 19, 2014
Contiguous hydroxyproline residues direct hydroxyproline arabinosylation in Nicotiana tabacum
E Shpak1, E Barbar, J F Leykam
1Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701, USA.
The Journal of Biological Chemistry
|January 23, 2001
Summary
Plant hydroxyproline-rich glycoproteins (HRGPs) have O-glycosylation patterns. This study shows that the sequence and contiguity of hydroxyproline (Hyp) residues directly dictate whether arabinosylation or galactosylation occurs, supporting the Hyp contiguity hypothesis.
Area of Science:
- Plant Biology
- Glycobiology
- Molecular Biology
Background:
- Hydroxyproline (Hyp)-O-glycosylation is a key post-translational modification of hydroxyproline-rich glycoproteins (HRGPs) in the plant extracellular matrix.
- Two glycosylation modes exist: arabinosylation (short oligoarabinosides) and galactosylation (large arabinogalactan polysaccharides).
- The Hyp contiguity hypothesis proposes that contiguous Hyp residues direct arabinosylation, while clustered noncontiguous Hyp residues direct galactosylation.
Purpose of the Study:
- To investigate the minimum level of Hyp contiguity required to direct arabinosylation.
- To test the Hyp contiguity hypothesis by designing synthetic genes with varying Hyp repeat patterns.
- To elucidate the sequence-dependent nature of Hyp O-glycosylation in HRGPs.
Main Methods:
- Designed synthetic genes encoding repetitive peptide motifs: (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n).
- Transformed Nicotiana tabacum cells to express fusion glycoproteins containing these motifs and green fluorescence protein.
- Analyzed post-translational proline hydroxylation and glycosylation patterns using biochemical methods and circular dichroism spectroscopy.
Main Results:
- Fusion glycoproteins with (Ser-Pro(2))(n) and (Ser-Hyp(4))(n) motifs predominantly yielded Hyp-arabinosides, with no Hyp-polysaccharide formation.
- The (Ser-Pro(3))(n) motif showed incomplete hydroxylation, resulting in a mixture of contiguous/noncontiguous Hyp residues and both Hyp-arabinosides and Hyp-polysaccharides.
- Glycosylation patterns directly correlated with Hyp residue contiguity, supporting the Hyp contiguity hypothesis.
Conclusions:
- Hyp O-glycosylation is a sequence-driven process, with residue contiguity being a critical determinant.
- The findings provide strong evidence for the Hyp contiguity hypothesis, explaining glycomodule formation in HRGPs.
- This study clarifies how specific peptide sequences dictate distinct glycosylation outcomes in plant glycoproteins.
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