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Research
Molecular and biochemical aspects of the O-glycoproteins in plant cell walls usingArabidopsis
Glycoproteins rich in hydroxyproline (HRGPs) are only present in the cell walls of plants and green algae. HRGPs are grouped into arabinogalactan proteins (AGPs) and extensins based on the type and extent of glycosylation. The most important post-translational modifications in HRGPs are: (i) the conversion of some units of proline into 4-hydroxyproline by the action of 4-prolyl hydroxylases (P4Hs); (ii) glycosylation of the hydroxyproline units (O-glycosylation) with short chains of arabinosides (2-4 sugars, in extensins) or large arabinogalactans (in AGPs). O-glycosylation type defines the glycomodules responsable for their shape, size, stability, and biological function in HRGPs.
HRGPs are broadly implicated in all aspects of plant growth and development, including fertilization, differentiation and tissue organization, control of cell expansion, cell fate, apoptosis, responses to stress and pathogenesis. However, in view of the fact these macromolecules are highly complex, the function of each HRGP is yet to be defined, which has led to major efforts to determine the structure and function of a specific HRGP.
The Arabidopsis genome comprises 13 genes encoding putative P4Hs, key enzymes responsible for the biosynthesis of mature HRGPs, such as AGPs and extensins. Unfortunately, research on the specific biological function for each of these genes is in part hampered because of the lack of knowledge of the detailed structure of their putative substrates regarding post-translational modifications, namely proline-hydroxylation (a pre-requisite for O-glycosylation) and unknown individual glycan structures. The glycans in the protein backbone is thought to be essential for the interaction of HRGPs with other molecules during physiological events. Therefore, P4Hs are enzymes likely relevant to plant cell function because they define the O-glycosylation sites of their substrates through previous hydroxylation of certain proline units in the protein backbone. Very little is known about the extent of redundancy, cell type in which they are expressed and substrate specificit.
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Expression of an AGP-GFP (top) in roots of Arabidopsis. Induced plasmolysis shows retention in one AGP and secretion to the cell wall in another AGP based on the level of glycosylation. Filogenetic tree, structure and model of the P4Hs (bottom).
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In order to try to clarify the mentioned unsolved issues, I am using:
- a genetic approach by generating PH4-mutants in order to determine the degree of functional redundancy.
- a functional genomics approach: to assess endogenous expression of the P4Hs in planta.
- a biochemical approach to achieve expression of P4Hs in E. coli (or another heterologous system) in order to purify and biochemically characterize the enzymatic activity in vitro.
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The ultimate goals are to link the biological activity of P4Hs to a specifc HRGPs (AGP or extensin molecules) and to understand the biological functions of O-glycosylation on a single HRGP backbone.
In addition, since we have detected AGPs in cell walls from very simple organisms such as green algae (Estevez et al. 2008 a,b), we have started a deeper characterization of these AGPs in order to get an evolutionary perspective of these O-glycoproteins in groups that have arisen much before vascular plants.
Publications
Patrick T. M.*, José M. Estevez*, Fachuang Lu, Katia Ruel, Mark W. Denny,
Chris Somerville, John Ralph. 2009. Current Biology. Accepted. (*)These
authors contributed equally to this work.
Sánchez-Rodríguez, C., J.M. Estévez, F. Llorente, C. Hernández-Blanco, I. Pagán, M. Berrocal, Y. Marco, S. Somerville, A. Molina. 2008. Molecular Plant Microbial Interaction. In press.
Estevez, J. M. L. Kasulin, P.V. Fernández, P. Dupree, M. Ciancia. 2008b. Glycobiology. In press.
Estevez, J. M., P. Leonardi, J. Alberghina 2008a. J. Phycol. 44 (5): 1257-1268.
Estevez, J. M.; Ciancia, M. & A. Cerezo. 2008. Carbohydrate Polymers. 73: 594-605.
Ciancia, M, I. Quintana, M. I. Vizcargüénaga, L. Kasulin, A. de Dios, J. M. Estevez & A. S. Cerezo. 2007. Int. J. of Biol. Macromol. 41 (5): 641-649.
Estevez, J. M. & C. Somerville. 2006.1 Biotechniques. 41 (5): 569-572.
Estevez, J. M.; Kiesliszewski M. J. & C. Somerville. 2006. Plant Physiology. 142: 458-470.
Estevez, J. M.; Ciancia, M. & A. Cerezo. 2004. Carbohydrates Research. 339: 2575-2592.
Pujol, C. A.; Estevez, J. M.; Ciancia, M. & E. Damonte. 2002. Antiviral Chemistry and Chemotherapy. 13: 83-89.
Estevez, J. M.; Ciancia, M. & A. Cerezo. 2002. J. Phycology. 38: 344-350.
Grants
ANPCyT, PICT 2006-00983. Período: 2008-2010.
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