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1.
Plant Physiol ; 176(2): 1547-1558, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29150558

RESUMO

A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus x giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a ß-1,6-galactosyl substitution of ß-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear ß-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.


Assuntos
Arabidopsis/metabolismo , Beta vulgaris/metabolismo , Galactanos/metabolismo , Poaceae/metabolismo , Anticorpos Monoclonais , Arabidopsis/citologia , Beta vulgaris/citologia , Parede Celular/metabolismo , Epitopos , Galactanos/química , Galactanos/imunologia , Fenômenos Mecânicos , Análise em Microsséries , Microscopia de Força Atômica , Floema/citologia , Floema/metabolismo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Caules de Planta/citologia , Caules de Planta/metabolismo , Poaceae/citologia
2.
Plant Cell Physiol ; 59(12): 2624-2636, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30184190

RESUMO

Pectin is a major component of primary cell walls and performs a plethora of functions crucial for plant growth, development and plant-defense responses. Despite the importance of pectic polysaccharides their biosynthesis is poorly understood. Several genes have been implicated in pectin biosynthesis by mutant analysis, but biochemical activity has been shown for very few. We used reverse genetics and biochemical analysis to study members of Glycosyltransferase Family 92 (GT92) in Arabidopsis thaliana. Biochemical analysis gave detailed insight into the properties of GALS1 (Galactan synthase 1) and showed galactan synthase activity of GALS2 and GALS3. All proteins are responsible for adding galactose onto existing galactose residues attached to the rhamnogalacturonan-I (RG-I) backbone. Significant GALS activity was observed with galactopentaose as acceptor but longer acceptors are favored. Overexpression of the GALS proteins in Arabidopsis resulted in accumulation of unbranched ß-1, 4-galactan. Plants in which all three genes were inactivated had no detectable ß-1, 4-galactan, and surprisingly these plants exhibited no obvious developmental phenotypes under standard growth conditions. RG-I in the triple mutants retained branching indicating that the initial Gal substitutions on the RG-I backbone are added by enzymes different from GALS.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Galactanos/metabolismo , Glicosiltransferases/metabolismo , Arabidopsis/genética , Parede Celular/metabolismo , Genes de Plantas , Complexo de Golgi/metabolismo , Folhas de Planta/metabolismo , Proteínas Recombinantes/isolamento & purificação , Frações Subcelulares/metabolismo , Especificidade por Substrato , Nicotiana/metabolismo
3.
Plant Physiol ; 175(3): 1094-1104, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28924016

RESUMO

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision.


Assuntos
Anticorpos Monoclonais/metabolismo , Brachypodium/metabolismo , Parede Celular/metabolismo , Mapeamento de Epitopos , Análise em Microsséries/métodos , Polissacarídeos/metabolismo , Glicosídeo Hidrolases/metabolismo , Coloração e Rotulagem
4.
Org Biomol Chem ; 16(7): 1157-1162, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29367995

RESUMO

We report the synthesis of linear and branched (1→4)-d-galactans. Four tetrasaccharides and one pentasaccharide were accessed by adopting a procedure of regioselective ring opening of a 4,6-O-naphthylidene protecting group followed by glycosylation using phenyl thioglycoside donors. The binding of the linear pentasaccharide with galectin-3 is also investigated by the determination of a co-crystal structure. The binding of the (1→4)-linked galactan to Gal-3 highlights the oligosaccharides of pectic galactan, which is abundant in the human diet, as putative Gal-3 ligands.

5.
J Org Chem ; 82(23): 12066-12084, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29120180

RESUMO

The synthesis of linear and (1 → 6)-branched ß-(1 → 3)-d-galactans, structures found in plant arabinogalactan proteins (AGPs), is described. The synthetic strategy relies on iterative couplings of monosaccharide and disaccharide thioglycoside donors, followed by a late-stage glycosylation of heptagalactan backbone acceptors to introduce branching. A key finding from the synthetic study was the need to match protective groups in order to tune reactivity and ensure selectivity during the assembly. Carbohydrate microarrays were generated to enable the detailed epitope mapping of two monoclonal antibodies known to recognize AGPs: JIM16 and JIM133.


Assuntos
Galactanos/síntese química , Mucoproteínas/síntese química , Sequência de Carboidratos , Mapeamento de Epitopos , Galactanos/química , Análise em Microsséries , Mucoproteínas/química , Proteínas de Plantas/síntese química , Proteínas de Plantas/química
6.
Chemistry ; 22(33): 11543-8, 2016 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-27305141

RESUMO

The synthesis of linear- and (1→6)-branched ß-(1→4)-d-galactans, side-chains of the pectic polysaccharide rhamnogalacturonan I is described. The strategy relies on iterative couplings of n-pentenyl disaccharides followed by a late stage glycosylation of a common hexasaccharide core. Reaction with a covalent linker and immobilization on N-hydroxysuccinimide (NHS)-modified glass surfaces allows the generation of carbohydrate microarrays. The glycan arrays enable the study of protein-carbohydrate interactions in a high-throughput fashion, demonstrated herein with binding studies of mAbs and a CBM.


Assuntos
Anticorpos Monoclonais/química , Galactanos/química , Pectinas/química , Pectinas/síntese química , Polissacarídeos/metabolismo , Succinimidas/química , Anticorpos Monoclonais/imunologia , Galactanos/metabolismo , Polissacarídeos/química
7.
Plant Cell ; 24(12): 5024-36, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23243126

RESUMO

ß-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include α-1,5-arabinans, ß-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but ß-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased ß-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher ß-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto ß-1,4-galactopentaose. GALS1 specifically formed ß-1,4-galactosyl linkages and could add successive ß-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as ß-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Pectinas/biossíntese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Plantas Geneticamente Modificadas
8.
Glycobiology ; 24(2): 208-16, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24270321

RESUMO

Glycoside hydrolase family 42 (GH42) includes ß-galactosidases catalyzing the release of galactose (Gal) from the non-reducing end of different ß-d-galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of ß-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite -1 to be key specificity determinants. Bga42A thus prefers the ß1-3-galactosidic linkage from human milk and other ß1-3- and ß1-6-galactosides with glucose or Gal situated at subsite +1. In contrast, Bga42B very efficiently hydrolyses 4-galactosyllactose (Galß1-4Galß1-4Glc) as well as 4-galactobiose (Galß1-4Gal) and 4-galactotriose (Galß1-4Galß1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the ß-galactoside-rich gut niche and emphasize the importance and diversity of ß-galactoside metabolism in probiotic bifidobacteria.


Assuntos
Bifidobacterium/enzimologia , Glicosídeo Hidrolases/metabolismo , beta-Galactosidase/metabolismo , Sequência de Aminoácidos , Bifidobacterium/genética , Bifidobacterium/metabolismo , Galactanos/metabolismo , Galactose/metabolismo , Galactosídeos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Filogenia , Especificidade por Substrato , beta-Galactosidase/química , beta-Galactosidase/genética
9.
J Biol Chem ; 287(47): 39429-38, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-22988248

RESUMO

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.


Assuntos
Parede Celular , Análise em Microsséries , Plantas , Polissacarídeos , Parede Celular/química , Parede Celular/metabolismo , Análise em Microsséries/instrumentação , Análise em Microsséries/métodos , Plantas/química , Plantas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo
10.
Carbohydr Res ; 436: 36-40, 2016 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-27855335

RESUMO

Plant cell wall glycans are important polymers that are crucial to plant development and serve as an important source of sustainable biomass. The study of polysaccharides in the plant cell wall relies heavily on monoclonal antibodies (mAbs) for localization and visualization of glycans, using e.g. immunofluorescent microscopy. Here, we describe the detailed epitope mapping of the mAb LM5 that is shown to bind to a minimum of three sugar residues at the non-reducing end of linear beta-1,4-linked galactan. The study uses de novo synthetic analogues of galactans combined with carbohydrate microarray and competitive inhibition ELISA for analysis of antibody-carbohydrate interactions.


Assuntos
Anticorpos Monoclonais/metabolismo , Parede Celular/química , Epitopos/química , Galactanos/química , Galactose/química , Oligossacarídeos/metabolismo , Pectinas/química , Anticorpos Monoclonais/química , Oligossacarídeos/química
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