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001-es BibID:	BIBFORM051951
Első szerző:	Dilokpimol, Adiphol
Cím:	Galactosyltransferases from Arabidopsis thaliana in the biosynthesis of type II arabinogalactan : molecular interaction enhances enzyme activity / Adiphol Dilokpimol, Christian Peter Poulsen, György Vereb, Satoshi Kaneko, Alexander Schulz, Naomi Geshi
Dátum:	2014
Megjegyzések:	BACKGROUND: Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post-translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood. RESULTS: We have identified a novel enzyme in the biosynthesis of the glycan moiety of arabinogalactan proteins. The At1g08280 (AtGALT29A) from Arabidopsis thaliana encodes a putative glycosyltransferase (GT), which belongs to the Carbohydrate Active Enzyme family GT29. AtGALT29A co-expresses with other arabinogalactan GTs, AtGALT31A and AtGLCAT14A. The recombinant AtGALT29A expressed in Nicotiana benthamiana demonstrated a galactosyltransferase activity, transferring galactose from UDP-galactose to a mixture of various oligosaccharides derived from arabinogalactan proteins. The galactose-incorporated products were analyzed using structure-specific hydrolases indicating that the recombinant AtGALT29A possesses beta-1,6-galactosyltransferase activity, elongating beta-1,6-galactan side chains and forming 6-Gal branches on the beta-1,3-galactan main chain of arabinogalactan proteins. The fluorescence tagged AtGALT29A expressed in N. benthamiana was localized to Golgi stacks where it interacted with AtGALT31A as indicated by Forster resonance energy transfer. Biochemically, the enzyme complex containing AtGALT31A and AtGALT29A could be co-immunoprecipitated and the isolated protein complex exhibited increased level of beta-1,6-galactosyltransferase activities compared to AtGALT29A alone. CONCLUSIONS: AtGALT29A is a beta-1,6-galactosyltransferase and can interact with AtGALT31A. The complex can work cooperatively to enhance the activities of adding galactose residues 6-linked to beta-1,6-galactan and to beta-1,3-galactan. The results provide new knowledge of the glycosylation process of arabinogalactan proteins and the functional significance of protein-protein interactions among O-glycosylation enzymes
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban article biosynthesis cell surface Energy Transfer ENERGY-TRANSFER Enzymes Fluorescence molecular interaction PROTEIN COMPLEXES protein protein interaction Protein-protein interactions Proteins Proteoglycans resonance energy transfer
Megjelenés:	BMC Plant Biology. - 14 : 1 (2014), p. 90. -
További szerzők:	Poulsen, Christian Peter Vereb György (1965-) (biofizikus, orvos) Kaneko, Satoshi Schulz, Alexander Geshi, Naomi
Internet cím:	Intézményi repozitóriumban (DEA) tárolt változat DOI
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001-es BibID:	BIBFORM050049
Első szerző:	Poulsen, Christian Peter
Cím:	Inhibition of cytoplasmic streaming by cytochalasin D is superior to paraformaldehyde fixation for measuring FRET between fluorescent protein-tagged Golgi components / Christian Peter Poulsen, György Vereb, Naomi Geshi, Alexander Schulz
Dátum:	2013
Megjegyzések:	Protein-protein interaction at the organelle level can be analyzed by using tagged proteins and assessing Forster resonance energy transfer (FRET) between fluorescent donor and acceptor proteins. Such studies are able to uncover partners in the regulation of proteins and enzymes. However, any organelle movement is an issue for live FRET microscopy, as the observed organelle must not change position during measurement. One of the mobile organelles in plants is the Golgi apparatus following cytoplasmic streaming. It is involved in the decoration of proteins and processing of complex glycan structures for the cell wall. Understanding of these processes is still limited, but evidence is emerging that protein-protein interaction plays a key role in the function of this organelle. In the past, mobile organelles were usually immobilized with paraformaldehyde (PFA) for FRET-based interaction studies. Here, we show that the actin inhibitor Cytochalasin D (CytD) is superior to PFA for immobilization of Golgi stacks in plant cells. Two glycosyltransferases known to interact were tagged with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, coexpressed in Nicotiana benthamiana leaves and analyzed using confocal microscopy and spectral imaging. Fixation with PFA leads to reduced emission intensity when compared to CytD treatment. Furthermore, the calculated FRET efficiency was significantly higher with CytD than with PFA. The documented improvements are beneficial for all methods measuring FRET, where immobilization of the investigated molecules is necessary. It can be expected that FRET measurement in organelles of animal cells will also benefit from the use of inhibitors acting on the cytoskeleton
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban Animal article Cells Confocal Confocal microscopy Cytochalasin D Cytoskeleton Energy Transfer ENERGY-TRANSFER Enzymes Fluorescent donor FRET Golgi Apparatus methods Microscopy Movement protein protein interaction Proteins Research Research Support resonance energy transfer Support
Megjelenés:	Cytometry. Part A. - 83 : 9 (2013), p. 830-838. -
További szerzők:	Vereb György (1965-) (biofizikus, orvos) Geshi, Naomi Schulz, Alexander
Internet cím:	DOI Intézményi repozitóriumban (DEA) tárolt változat
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