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Molecular characterization of tomato α1,3/4-fucosidase, a member of glycosyl hydrolase family 29 involved in the degradation of plant complex type N-glycans
Abstract
In this study, we identified a gene in tomato that encodes an acidic α-fucosidase (LOC101254568 or Solyc03g006980, α-Fuc'ase S1-1), which may be involved in the turnover of plant complex-type N-glycans. Recombinant α-Fuc'ase S1-1 (rFuc'ase S1-1) was expressed using a baculovirus-insect cell expression system. rFuc'ase Sl-1 is 55 kDa in size and has an optimum pH around 4.5. It substantially hydrolyzed the non-reducing terminal α1,3-fucose residue on LNFP III and α1,4-fucose residues of Le(a) epitopes on plant complex-type N-glycans, but not the α1,2-fucose residue on LNFP I or the α1,3-fucose residue on pyridylaminated Fucα1-3GlcNAc. Furthermore, we found that this tomato α-Fuc'ase S1-1 was inactive toward the core penta-oligosaccharide unit [Manβ1-4(Xylβ1-2)GlcNAcβ1-4(Fucα1-3)GlcNAc-PA] of plant complex-type N-glycans. Molecular 3D modelling of α-Fuc'ase Sl-1 and structure/sequence interpretation based on comparison with a homologous α-fucosidase from Bifidobacterium longum subsp. infantis (Blon_2336) indicated that residues Asp(193) and Glu(237) might be important for substrate binding.
... Based on the genetic information of the rice a-Fuc'ase gene (17), two putative tomato (Solanum lycopersicum) a-Fuc'ase genes (Solyc03g06980 and Solyc11g069010) were found in the genome database and we have succeeded to express and characterize one a-Fuc'ase encoded by Solyc03g06980 (a-Fuc'ase Sl-1) (18). a-Fuc'ase Sl-1 was substantially hydrolyzed the non-reducing terminal a1, 3-fucose residue on LNFP III and a1, 4-fucose residues of Le a epitopes on plant complex-type N-glycans, but not by the a1, 3-fucose residue on the plant core penta-oligosaccharide unit (Manb1-4[Xylb1-2]GlcNAcb1-4[Fuca1-3]GlcNAc) or Fuca1-3GlcNAc (GN1F). ...
... Molecular 3 D modelling of a-Fuc'ase Sl-1 construction and structure/ sequence interpretation based on comparison with a homologous a-fucosidase from Bifidobacterium longum subsp. infantis (Blon_2336) (19) indicated that an unsubstituted b-GlcNAc residue in the GlcNAcb1-4(Fuca1-3)GlcNAc (GN2F) structure might be prerequisite to fix the a1, 3-fucosylated substrate at the sugar-binding pocket commonly found in GH29-B (18,19). However, in our previous study, we could not prepare the substrate, GN2F and thus have not proved our hypothesis to date. ...
... Enzyme activity assay Assays of rFuc'ase Sl-2 activities were basically performed according to the methods described in our previous paper (18). Briefly, rFuc'ase Sl-2 activities during the purification step were assayed at 37 C by incubating with PA-labelled plant complex type N-glycans containing Le a determinants (Gal2Fuc2GN2M3FX) or LNFP III in 0.1 M sodium citrate buffer (pH 5.0). ...
In a previous study, we molecular-characterized a tomato (Solanum lycopersicum) α1,3/4-fucosidase (α-Fuc’ase S1-1) encoded in a tomato gene (Solyc03g006980), indicating that α-Fuc’ase S1-1 is involved in the turnover of Lea epitope-containing N-glycans. In this study, we have characterized another tomato gene (Solyc11g069010) encoding α1,3/4-fucosidase (α-Fuc’ase S1-2), which is also active toward the complex type N-glycans containing Lea epitope(s). The baculovirus-insect cell expression system was used to express that α-Fuc’ase S1-2 with anti-FLAG tag, and the expression product (rFuc’ase Sl-2), was found as a 65 kDa protein using SDS-PAGE and has an optimum pH of around 5.0. Similarly to rFuc’ase Sl-1, rFuc’ase Sl-2 hydrolyzed the non-reducing terminal α1,3-fucose residue on LNFP III and α1,4-fucose residues of Lea epitopes on plant complex type N-glycans, but not the core α1,3-fucose residue on Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc or Fucα1-3GlcNAc. However, we found that both α-Fuc’ases S1-1 and Sl-2 were specifically active toward α1,3-fucose residue on GlcNAcβ1-4(Fucα1-3)GlcNAc, indicating that the non-substituted β-GlcNAc linked to the proximal GlcNAc residue of the core tri-saccharide moiety of plant specific N-glycans must be a prerequisite for α-Fuc’ase activity. A 3D modeled structure of the catalytic sites of α-Fuc’ase S1-2 suggested that Asp¹⁹² and Glu²³⁶ may be important for binding to the α1,3/4 fucose residue.
... However, this hydrolase did not hydrolyze the oligosaccharides containing α1,2-fucosyl linkages, such as 2 0 FL-PA and LNFPI-PA. Thus, AtFUC1 specifically acted on α1,3/4-fucosyl linkages as has been observed previously [37][38][39]. AtFUC1 strictly discriminated between the linkage positions of the fucose residues to be hydrolyzed. It did not act on p-nitrophenyl α-fucopyranoside as previously reported [37]. ...
... Furthermore, AtFUC1 substrate specificity for plant N-glycans was investigated. It has been reported that this fucosidase hydrolyzed the α1,4-fucosyl linkage of the Lewis A epitope structure at the non-reducing end of plant complex-type N-glycan, but did not hydrolyze the α1,3-fucosyl linkage at the reducing end [37][38][39]. In the present study, specificity of this enzyme was re-examined using several N-glycans. ...
Plant complex-type N -glycans are characterized by the presence of α1,3-linked fucose towards the proximal N -acetylglucosamine residue and β1,2-linked xylose towards the β-mannose residue. These glycans are ultimately degraded by the activity of several glycoside hydrolases. However, the degradation pathway of plant complex-type N -glycans have not been entirely elucidated because the gene encoding α1,3-fucosidase, a glycoside hydrolase acting on plant complex-type N -glycans, has not yet been identified, and its substrate specificity remains to be determined. In the present study, we found that AtFUC1 (an Arabidopsis GH29 α-fucosidase) is an α1,3-fucosidase acting on plant complex type N -glycans. This fucosidase has been known to act on α1,4-fucoside linkage in the Lewis A epitope of plant complex-type N -glycans. We found that this glycoside hydrolase specifically acted on GlcNAcβ1-4(Fucα1-3)GlcNAc, a degradation product of plant complex-type N -glycans, by sequential actions of vacuolar α-mannosidase; β1,2-xylosidase; and endo-β-mannosidase. The AtFUC1-deficient mutant showed no distinct phenotypic plant growth features; however, it accumulated GlcNAcβ1-4(Fucα1-3)GlcNAc, a substrate of AtFUC1. These results showed that AtFUC1 is an α1,3-fucosidase acting on plant complex-type N -glycans and elucidated the degradation pathway of plant complex-type N -glycans.
... As described above, insects also exhibit core-α-1,3-fucosylated glycans (Fabini et al., 2001;Kajiura et al., 2015;Kubeka et al., 1993, Kubelka et al., 1995Liu et al., 2019;Mabashi-Asazuma et al., 2015;Minagawa et al., 2015;Soya et al., 2016;Stanton et al., 2017;Walski et al., 2016). Tomato and Arabidopsis α-fucosidases classified as GH29-B are involved in N-glycan degradation and hydrolyze α-1,3-fucosyl linkages of 3-FGn 2 but not 3fucosyl-N-acetylglucosamine and longer core-α-1,3-fucosyl N-glycan substrates (Kato et al., 2018;Rahman et al., 2017, Rahman et al., 2018Wilson et al., 2001). In addition, almond α-L-fucosidase can hydrolyze 3-FL and lacto-N-fucopentanose Ⅱ but not showed activity core-α-1,3fucosylated N-glycans (Zeleny et al., 2006). ...
N-glycans play a role in physiological functions, including glycoprotein conformation, signal transduction, and antigenicity. Insects display both α-1,6- and α-1,3-linked fucose residues bound to the innermost N-acetylglucosamine of N-glycans whereas core α-1,3-fucosylated N-glycans are not found in mammals. Functions of insect core-fucosylated glycans are not clear, and no α-L-fucosidase related to the N-glycan degradation has been identified. In the genome of the domestic silkworm, Bombyx mori, a gene for a protein, BmFucA, belonging to the glycoside hydrolase family 29 is a candidate for an α-L-fucosidase gene. In this study, BmFucA was cloned and recombinantly expressed as a glutathione-S-transferase tagged protein (GST-BmFucA). Recombinant GST-BmFucA exhibited broad substrate specificity and hydrolyzed p-nitrophenyl α-L-fucopyranoside, 2′-fucosyllactose, 3-fucosyllactose, 3-fucosyl-N,N′-diacetylchitobiose, and 6-fucosyl-N,N′-diacetylchitobiose. Further, GST-BmFucA released fucose from both pyridylaminated complex-type and paucimannose-type glycans that were core-α-1,6-fucosylated. GST-BmFucA also shows hydrolysis activity for core-fucosylated glycans attached to phospholipase A2 from bee venom. BmFucA may be involved in the catabolism of core-fucosylated N-glycans in B. mori.
... As for alternative enzymes that could digest xyloglucan in species without apoplastic members of family 95, the only other known plant fucosidases are in family 29 of glycosyl hydrolases. There are two divergent clades in this family but members of both branches have been characterized as α(1 → 3), α(1 → 4) fucosidases with no activity on α(1 → 2) linkages (Zeleny et al. 2006;Rahman et al. 2016;Ziaur Rahman et al. 2017). ...
Key message
Brachypodium distachyon has a full set of exoglycosidases active on xyloglucan, including α-xylosidase, β-galactosidase, soluble and membrane-bound β-glucosidases and two α-fucosidases. However, unlike in Arabidopsis, both fucosidases are likely cytosolic.
Abstract
Xyloglucan is present in primary walls of all angiosperms. While in most groups it regulates cell wall extension, in Poaceae its role is still unclear. Five exoglycosidases participate in xyloglucan hydrolysis in Arabidopsis: α-xylosidase, β-galactosidase, α-fucosidase, soluble β-glucosidase and GPI-anchored β-glucosidase. Mutants in the corresponding genes show alterations in xyloglucan composition. In this work putative orthologs in the model grass Brachypodium distachyon were tested for their ability to complement Arabidopsis mutants. Xylosidase and galactosidase mutants were complemented, respectively, by BdXYL1 (Bd2g02070) and BdBGAL1 (Bd2g56607). BdBGAL1, unlike other xyloglucan β-galactosidases, is able to remove both galactoses from XLLG oligosaccharides. In addition, soluble β-glucosidase BdBGLC1 (Bd1g08550) complemented a glucosidase mutant. Closely related BdBGLC2 (Bd2g51280), which has a putative GPI-anchor sequence, was found associated with the plasma membrane and only a truncated version without GPI-anchor complemented the mutant, proving that Brachypodium also has soluble and membrane-bound xyloglucan glucosidases. Both BdXFUC1 (Bd3g25226) and BdXFUC2 (Bd1g28366) can hydrolyze fucose from xyloglucan oligosaccharides but were unable to complement a fucosidase mutant. Fluorescent protein fusions of BdXFUC1 localized to the cytosol and both proteins lack a signal peptide. Signal peptides appear to have evolved only in some eudicot lineages of this family, like the one leading to Arabidopsis. These results could be explained if cytosolic xyloglucan α-fucosidases are the ancestral state in angiosperms, with fucosylated oligosaccharides transported across the plasma membrane.
We recently demonstrated the presence of a new asparagine-linked complex glycan on plant glycoproteins that harbors the Lewis a (Le a), or Gal(1-3)[Fuc(1-4)]GlcNAc, epitope, which in mam-malian cells plays an important role in cell-to-cell recognition. Here we show that the monoclonal antibody JIM 84, which is widely used as a Golgi marker in light and electron microscopy of plant cells, is specific for the Le a antigen. This antigen is present on glycoproteins of a number of flowering and non-flowering plants, but is less apparent in the Cruciferae, the family that includes Arabidopsis. Le a-containing oligosaccharides are found in the Golgi apparatus, and our immunocytochemical experiments suggest that it is synthesized in the trans-most part of the Golgi apparatus. Le a epitopes are abundantly present on extracellular glycoproteins, either soluble or membrane bound, but are never observed on vacuolar glycopro-teins. Double-labeling experiments suggest that vacuolar glycopro-teins do not bypass the late Golgi compartments where Le a is built, and that the absence of the Le a epitope from vacuolar glycoproteins is probably the result of its degradation by glycosidases en route to or after arrival in the vacuole.
Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades. Here we show that Notch1 endogenously expressed in Chinese hamster ovary cells is modified with O-linked fucose andO-linked glucose saccharides, two unusual forms ofO-linked glycosylation found on epidermal growth factor-like (EGF) modules. Interestingly, both modifications occur as monosaccharide and oligosaccharide species. Through exoglycosidase digestions we determined that the O-linked fucose oligosaccharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-α2,3-Gal-β1,4-GlcNAc-β1,3-Fuc-α1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a trisaccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose orO-linked glucose modifications. It also represents the second protein discovered with an elongated form ofO-linked fucose. The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs. Since Notch is known to interact with its ligands through subsets of EGF modules, these results suggest that the O-linked carbohydrate modifications of these modules may influence receptor-ligand interactions.
Human seminal plasma α-L-fucosidase (EC 3.2.1.51) has been purified 7100-fold to very high purity and specific activity (83 000 nmol/min/mg protein) by affinity chromatography on agarose-ε-aminocaproyl-fucopyranosylamine. The purified α-L-fucosidase appeared to contain a single subunit of 56–57 kDa (as determined by SDS–PAGE and Western analysis). Lectin blotting and N-glycanase treatment studies indicated that this subunit is N-glycosylated and contains sialic acid residues. Human seminal plasma α-L-fucosidase was shown to contain three multimeric forms of 110, 236 and 314 kDa respectively (as determined by Sephadex ® G-200 chromatography) and therefore probably exists in dimeric, tetrameric and hexameric forms. Kinetic analysis with the 4-methylumbelliferyl-α-L-fucopyranoside (4MU-Fuc) substrate indicated a broad acidic optimum (pH 4.0–4.5) with a second neutral optimum (pH 6.4–7.4) with 60–80% of maximal activity. Apparent K M and V max values for the 4MU-Fuc substrate were determined to be 0.06 mmol/l and 92 µmol/min/mg protein respectively, using Lineweaver–Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment studies provided further evidence that the purified seminal plasma α-L-fucosidase is a sialoglycoprotein with several isoforms between pI values 5–7. The acidic isoforms between pI values 5–6 appear to be related chemically to the more neutral isoforms by sialic acid residues since neuraminidase treatment converted the former into the latter isoforms.
Asparagine (N)-linked protein glycosylation is a ubiquitous co- and post-translational modification which can alter the biological function of proteins and consequently affects the development, growth, and physiology of organisms. Despite an increasing knowledge of N-glycan biosynthesis and processing, we still understand very little about the biological function of individual N-glycan structures in plants. In particular, the N-glycan-processing steps mediated by Golgi-resident enzymes create a structurally diverse set of protein-linked carbohydrate structures. Some of these complex N-glycan modifications like the presence of β1,2-xylose, core α1,3-fucose or the Lewis a-epitope are characteristic for plants and are evolutionary highly conserved. In mammals, complex N-glycans are involved in different cellular processes including molecular recognition and signaling events. In contrast, the complex N-glycan function is still largely unknown in plants. Here, in this short review, I focus on important recent developments and discuss their implications for future research in plant glycobiology and plant biotechnology.
beta-D-𝑁-Acetylhexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of 𝛽-1,4-linked 𝑁-acetylhexosamine
residues from oligosaccharides and their conjugates. We constructed phylogenetic tree of 𝛽-hexosaminidases to analyze the
evolutionary history and predicted functions of plant hexosaminidases. Phylogenetic analysis reveals the complex history
of evolution of plant 𝛽-hexosaminidase that can be described by gene duplication events. The 3D structure of tomato 𝛽-
hexosaminidase (𝛽-Hex-Sl) was predicted by homology modeling using 1now as a template. Structural conformity studies of the
best fit model showed that more than 98% of the residues lie inside the favoured and allowed regions where only 0.9% lie in the
unfavourable region. Predicted 3D structure contains 531 amino acids residues with glycosyl hydrolase20b domain-I and glycosyl
hydrolase20 superfamily domain-II including the (𝛽/𝛼)8 barrel in the central part.The 𝛼 and 𝛽 contents of the modeled structure
were found to be 33.3% and 12.2%, respectively. Eleven amino acids were found to be involved in ligand-binding site; Asp(330) and
Glu(331) could play important roles in enzyme-catalyzed reactions.The predicted model provides a structural framework that can
act as a guide to develop a hypothesis for 𝛽-Hex-Sl mutagenesis experiments for exploring the functions of this class of enzymes in
plant kingdom.
Plant pathogenic fungi deploy secreted effectors to suppress plant immunity responses. These effectors operate either in the apoplast or within host cells, so they are putatively glycosylated, but the posttranslational regulation of their activities has not been explored. In this study, the ASPARAGINE-LINKED GLYCOSYLATION3 (ALG3)-mediated N-glycosylation of the effector, Secreted LysM Protein1 (Slp1), was found to be essential for its activity in the rice blast fungus Magnaporthe oryzae. ALG3 encodes an α-1,3-mannosyltransferase for protein N-glycosylation. Deletion of ALG3 resulted in the arrest of secondary infection hyphae and a significant reduction in virulence. We observed that Δalg3 mutants induced massive production of reactive oxygen species in host cells, in a similar manner to Δslp1 mutants, which is a key factor responsible for arresting infection hyphae of the mutants. Slp1 sequesters chitin oligosaccharides to avoid their recognition by the rice (Oryza sativa) chitin elicitor binding protein CEBiP and the induction of innate immune responses, including reactive oxygen species production. We demonstrate that Slp1 has three N-glycosylation sites and that simultaneous Alg3-mediated N-glycosylation of each site is required to maintain protein stability and the chitin binding activity of Slp1, which are essential for its effector function. These results indicate that Alg3-mediated N-glycosylation of Slp1 is required to evade host innate immunity.
BioLiP (http://zhanglab.ccmb.med.umich.edu/BioLiP/) is a semi-manually curated database for biologically relevant ligand-protein interactions. Establishing interactions between protein and biologically relevant ligands is an important step toward understanding the protein functions. Most ligand-binding sites prediction methods use the protein structures from the Protein Data Bank (PDB) as templates. However, not all ligands present in the PDB are biologically relevant, as small molecules are often used as additives for solving the protein structures. To facilitate template-based ligand-protein docking, virtual ligand screening and protein function annotations, we develop a hierarchical procedure for assessing the biological relevance of ligands present in the PDB structures, which involves a four-step biological feature filtering followed by careful manual verifications. This procedure is used for BioLiP construction. Each entry in BioLiP contains annotations on: ligand-binding residues, ligand-binding affinity, catalytic sites, Enzyme Commission numbers, Gene Ontology terms and cross-links to the other databases. In addition, to facilitate the use of BioLiP for function annotation of uncharacterized proteins, a new consensus-based algorithm COACH is developed to predict ligand-binding sites from protein sequence or using 3D structure. The BioLiP database is updated weekly and the current release contains 204 223 entries.
Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.
We have developed a new COFACTOR webserver for automated structure-based protein function annotation. Starting from a structural
model, given by either experimental determination or computational modeling, COFACTOR first identifies template proteins of
similar folds and functional sites by threading the target structure through three representative template libraries that
have known protein–ligand binding interactions, Enzyme Commission number or Gene Ontology terms. The biological function insights
in these three aspects are then deduced from the functional templates, the confidence of which is evaluated by a scoring function
that combines both global and local structural similarities. The algorithm has been extensively benchmarked by large-scale
benchmarking tests and demonstrated significant advantages compared to traditional sequence-based methods. In the recent community-wide
CASP9 experiment, COFACTOR was ranked as the best method for protein–ligand binding site predictions. The COFACTOR sever and
the template libraries are freely available at http://zhanglab.ccmb.med.umich.edu/COFACTOR.
α-L-fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially α-L-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an α-L-fucosynthase that specifically introduces Le(a) and Le(x) antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Galβ1-3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an α-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-α-L-fucosidase (EC 3.2.1.111) and α-L-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Le(a/x) epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds.
The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure. Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures.
An Arabidopsis thaliana mutant with an altered structure of its hemicellulose xyloglucan (XyG; axy-8) identified by a forward genetic screen facilitating oligosaccharide mass profiling was characterized. axy8 exhibits increased XyG fucosylation and the occurrence of XyG fragments not present in the wild-type plant. AXY8 was identified to encode an α-fucosidase acting on XyG that was previously designated FUC95A. Green fluorescent protein fusion localization studies and analysis of nascent XyG in microsomal preparations demonstrated that this glycosylhydrolase acts mainly on XyG in the apoplast. Detailed structural analysis of XyG in axy8 gave unique insights into the role of the fucosidase in XyG metabolism in vivo. The genetic evidence indicates that the activity of glycosylhydrolases in the apoplast plays a major role in generating the heterogeneity of XyG side chains in the wall. Furthermore, without the dominant apoplastic glycosylhydrolases, the XyG structure in the wall is mainly composed of XXXG and XXFG subunits.
Motivation:
Quality assessment of protein structures is an important part of experimental structure validation and plays a crucial role in protein structure prediction, where the predicted models may contain substantial errors. Most current scoring functions are primarily designed to rank alternative models of the same sequence supporting model selection, whereas the prediction of the absolute quality of an individual protein model has received little attention in the field. However, reliable absolute quality estimates are crucial to assess the suitability of a model for specific biomedical applications.
Results:
In this work, we present a new absolute measure for the quality of protein models, which provides an estimate of the 'degree of nativeness' of the structural features observed in a model and describes the likelihood that a given model is of comparable quality to experimental structures. Model quality estimates based on the QMEAN scoring function were normalized with respect to the number of interactions. The resulting scoring function is independent of the size of the protein and may therefore be used to assess both monomers and entire oligomeric assemblies. Model quality scores for individual models are then expressed as 'Z-scores' in comparison to scores obtained for high-resolution crystal structures. We demonstrate the ability of the newly introduced QMEAN Z-score to detect experimentally solved protein structures containing significant errors, as well as to evaluate theoretical protein models. In a comprehensive QMEAN Z-score analysis of all experimental structures in the PDB, membrane proteins accumulate on one side of the score spectrum and thermostable proteins on the other. Proteins from the thermophilic organism Thermatoga maritima received significantly higher QMEAN Z-scores in a pairwise comparison with their homologous mesophilic counterparts, underlining the significance of the QMEAN Z-score as an estimate of protein stability.
Availability:
The Z-score calculation has been integrated in the QMEAN server available at: http://swissmodel.expasy.org/qmean.
It has been reported that acidic α-mannosidase activity increases during tomato fruit ripening, suggesting the turnover of N-glycoproteins is deeply associated with fruit ripening. As part of a study to reveal the relationship between the plant α-mannosidase activity and fruit maturation at the molecular level, we have already purified and characterized an α-mannosidase from tomato fruit (Hossain et al., Biosci. Biotechnol. Biochem. 2009;73:140-146). In this article, we describe the identification and expression of the tomato acidic α-mannosidase gene using the yeast-expression system. The α-mannosidase-gene located at chomosome 6 is a 10 kb spanned containing 30 exons. The gene-encoded-protein is single polypeptide chain of 1,028 amino acids containing glycosyl hydrolase domain-38 with predicted molecular mass of 116 kDa. The recombinant enzyme showed maximum activity at pH 5.5, and was almost completely inhibited by both of 1-deoxymannojirimycin and swainsonine. The recombinant α-mannosidase, like the native enzyme, could cleave α1-2, 1-3 and 1-6 mannosidic linkage from both high-mannose and truncated complex-type N-glycans. A molecular 3D modelling shows that catalytically important residues of animal lysosomal α-mannosidase could be superimposed on those of tomato α-mannosidase, suggesting that active site conformation is highly conserved between plant acidic α-mannosidase and animal lysosomal α-mannosidase.
Plant acidic peptide:N-glycanase (PNGase) is one of the deglycosylation enzymes and has been considered to be involved in the catabolism of glycoproteins
in plant cells. However, the tangible physiological significance involved in plant differentiation or growth is yet unclear.
In this study, as a first step to elucidate the physiological role of free N-glycans and the de-N-glycosylation machinery working in developing plant cells, we have succeeded in expressing a cDNA from tomato fruits in Pichia pastoris and identified an acidic peptide:N-glycanase in the culture supernatant. The PNGase-gene-encoded protein is a single polypeptide chain of 588 amino acids with
a predicted molecular mass of 65.8 kDa. The deduced amino acid sequence showed 57.9% similarity with almond PNGase A. The
recombinant tomato PNGase showed optimum activity at pH 4.5 and 40°C. It did not require any metal ions for full enzymatic
activity and could release the complex-type N-glycan from glycopeptides. Our phylogenetic analysis reveals that the plant acidic PNGase is completely different from the
ubiquitous cytosolic PNGase and is involved in a different de-N-glycosylation mechanism associated with plant growth and development.
The Carbohydrate-Active Enzyme (CAZy) database is a knowledge-based resource specialized in the enzymes that build and breakdown
complex carbohydrates and glycoconjugates. As of September 2008, the database describes the present knowledge on 113 glycoside
hydrolase, 91 glycosyltransferase, 19 polysaccharide lyase, 15 carbohydrate esterase and 52 carbohydrate-binding module families.
These families are created based on experimentally characterized proteins and are populated by sequences from public databases
with significant similarity. Protein biochemical information is continuously curated based on the available literature and
structural information. Over 6400 proteins have assigned EC numbers and 700 proteins have a PDB structure. The classification
(i) reflects the structural features of these enzymes better than their sole substrate specificity, (ii) helps to reveal the
evolutionary relationships between these enzymes and (iii) provides a convenient framework to understand mechanistic properties.
This resource has been available for over 10 years to the scientific community, contributing to information dissemination
and providing a transversal nomenclature to glycobiologists. More recently, this resource has been used to improve the quality
of functional predictions of a number genome projects by providing expert annotation. The CAZy resource resides at URL: http://www.cazy.org/.
Fucosylated oligosaccharides and glycoconjugates have been implicated in several biological events, including the cell-cell adhesion processes that mediate inflammation. Alpha-L-fucosidase (ALF) is an exoglycosidase that is involved in the hydrolytic degradation of alpha-L-fucose from glycoconjugates. In this study, we investigated the potential role of ALF in regulation of leukocyte migration. Measurement of transendothelial migration in response to CCL5 demonstrated that pretreatment of monocytic cells with ALF reduced migration (p = 0.0004) to a greater extent than treatment of the endothelial monolayer (p = 0.0374). Treatment with ALF significantly reduced the adhesion of monocytic cells to immobilized P-selectin.Fc. A murine model of experimental autoimmune uveitis was then used to show that treatment of splenic cells with ALF produced an 8.6-fold decrease in rolling and a 3.2-fold decrease in cell migration across the retinal vasculature. Further in vitro studies demonstrated that treatment of monocytes with the chemokines CCL3 or CCL5 increased the level of mRNA encoding ALF; this was accompanied by the detection of significant increases in both the 51- and 56-kDa components of ALF by Western blotting. Treatment of monocytic cells with ALF for 2 h significantly reduced the cell surface expression of CD31, with a further decrease in expression observed after 5 h (p = 0.002). Thus, CD31 and fucosylated ligands of P-selectin seem to be the candidates through which ALF mediates its effect in vitro. These data identify a previously unrecognized immunoregulatory role for ALF in late stages of inflammation.
This paper reports for the first time the presence of the human Lewis(a) type determinant in glycoproteins secreted by plant cells. A single glycopeptide was identified in the tryptic hydrolysis of the peroxidase VMPxC1 from Vaccinium myrtillus L. by HPLC/ESI-MS. The oligosaccharide structures were elucidated by ESI-MS-MS and by methylation analysis before and after removal of fucose by mild acid hydrolysis. The major structure determined is of the biantennary plant complex type containing the outer chain motif Lewis(a) [structure in text]. A corresponding fucosyltransferase activity catalyzing the formation of Lewis(a) type structures in vitro was identified in cellular extracts of the suspension cultures.
We recently demonstrated the presence of a new asparagine-linked complex glycan on plant glycoproteins that harbors the Lewis a (Lea), or Galbeta(1-3)[Fucalpha(1-4)]GlcNAc, epitope, which in mammalian cells plays an important role in cell-to-cell recognition. Here we show that the monoclonal antibody JIM 84, which is widely used as a Golgi marker in light and electron microscopy of plant cells, is specific for the Lea antigen. This antigen is present on glycoproteins of a number of flowering and non-flowering plants, but is less apparent in the Cruciferae, the family that includes Arabidopsis. Lea-containing oligosaccharides are found in the Golgi apparatus, and our immunocytochemical experiments suggest that it is synthesized in the trans-most part of the Golgi apparatus. Lea epitopes are abundantly present on extracellular glycoproteins, either soluble or membrane bound, but are never observed on vacuolar glycoproteins. Double-labeling experiments suggest that vacuolar glycoproteins do not bypass the late Golgi compartments where Lea is built, and that the absence of the Lea epitope from vacuolar glycoproteins is probably the result of its degradation by glycosidases en route to or after arrival in the vacuole.
The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures. Though the common mushroom exclusively contained N-glycans of the oligomannosidic type, all plant foods contained mixtures of the above-mentioned types. Apple, asparagus, avocado, banana, carrot, celery, hazelnut, kiwi, onion, orange, pear, pignoli, strawberry, and walnut were particularly rich in Le(a)-carrying N-glycans. Although traces of Le(a)-containing structures were also present in almond, pistachio, potato, and tomato, no such glycans could be found in cauliflower. Coconut exhibited almost exclusively N-glycans containing only xylose but no fucose. Oligomannosidic N-glycans dominated in buckwheat and especially in the legume seeds mung bean, pea, peanut, and soybean. Papaya presented a unique set of hybrid type structures partially containing the Le(a) determinant. These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Le(a)-type structure is very widespread among plants.
A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed.
The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition.
Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF(3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF(3)/Gn(GF)XF(3) (with a Lewisa epitope on one arm) in the molar ratio 67:8:23.
The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.
An alpha-L-fucosidase (EC 3.2.1.51) able to release the t-fucosyl residue from the side chain of xyloglucan oligosaccharides has been detected in the leaves of Arabidopsis plants. Moreover, an alpha-L-fucosidase with similar substrate specificity was purified from cabbage (Brassica oleracea) leaves to render a single band on SDS-PAGE. Two peptide sequences were obtained from this protein band, and they were used to identify an Arabidopsis gene coding for an alpha-fucosidase that we propose to call AtFXG1. In addition, an Arabidopsis gene with homology with known alpha-L-fucosidases has been also found, and we proposed to name it as AtFUC1. Both AtFXG1 and ATFUC1 were heterologously expressed in Pichia pastoris cells and the alpha-L-fucosidase activities secreted to the culture medium. The alpha-L-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2'-fucosyl-lactitol but not against p-nitrophenyl-alpha-L-fucopyranoside. However, the AtFUC1 heterologously expressed was active only against 2'-fucosyl-lactitol. Thus, the former must be related to xyloglucan metabolism.
Human seminal plasma alpha-L-fucosidase (EC 3.2.1.51) has been purified 7100-fold to very high purity and specific activity (83,000 nmol/min/mg protein) by affinity chromatography on agarose-epsilon-aminocaproyl-fucopyranosylamine. The purified alpha-L-fucosidase appeared to contain a single subunit of 56-57 kDa (as determined by SDS-PAGE and Western analysis). Lectin blotting and N-glycanase treatment studies indicated that this subunit is N-glycosylated and contains sialic acid residues. Human seminal plasma alpha-L-fucosidase was shown to contain three multimeric forms of 110, 236 and 314 kDa respectively (as determined by Sephadex G-200 chromatography) and therefore probably exists in dimeric, tetrameric and hexameric forms. Kinetic analysis with the 4-methylumbelliferyl-alpha-L-fucopyranoside (4MU-Fuc) substrate indicated a broad acidic optimum (pH 4.0-4.5) with a second neutral optimum (pH 6.4-7.4) with 60-80% of maximal activity. Apparent K(M) and V(max) values for the 4MU-Fuc substrate were determined to be 0.06 mmol/l and 92 micromol/min/mg protein respectively, using Lineweaver-Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment studies provided further evidence that the purified seminal plasma alpha-L-fucosidase is a sialoglycoprotein with several isoforms between pI values 5-7. The acidic isoforms between pI values 5-6 appear to be related chemically to the more neutral isoforms by sialic acid residues since neuraminidase treatment converted the former into the latter isoforms.
An α-L-fucosidase (EC 3.2.1.51) able to release the t-fucosyl residue from the side chain of xyloglucan oligosaccharides has been detected in the leaves of Arabidopsis plants. Moreover, an α-L-fucosidase with similar substrate specificity was purified from cabbage (Brassica oleracea) leaves to render a single band on SDS-PAGE. Two peptide sequences were obtained from this protein band, and they were used to identify an Arabidopsis gene coding for an α-fucosidase that we propose to call AtFXG1. In addition, an Arabidopsis gene with homology with known α-L-fucosidases has been also found, and we proposed to name it as AtFUC1. Both AtFXG1 and ATFUC1 were heterologously expressed in Pichia pastoris cells and the α-L-fucosidase activities secreted to the culture medium. The α-L-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2′-fucosy-lactitol but not against p-nitrophenyl-α-Lfucopyranoside. However, the AtFUC1 heterologously expressed was active only against 2′-fucosyl-lactitol. Thus, the former must be related to xyloglucan metabolism.
The Japanese cedar pollen allergen (Cry j1) and the mountain cedar pollen allergen (Jun a1) are glycosylated with plant complex type N-glycans bearing Lewis a epitope(s) (Galβ1-3[Fucα1-4]GlcNAc-). The biological significance of Lewis a type plant N-glycans and their effects on the human immune system remain to be elucidated. Since a substantial amount of such plant specific N-glycans are required to evaluate immunological activity, we have searched for good plant-glycan sources to characterize Lewis a epitope-containing plant N-glycans. In this study, we have found that three water plants, Elodea nuttallii, Egeria densa, and Ceratophyllum demersum, produce glycoproteins bearing Lewis a units. Structural analysis of the N-glycans revealed that almost all glycoproteins expressed in these three water plants predominantly carry plant complex type N-glycans including the Lewis a type, suggesting that these water plants are good sources for preparation of Lewis a type plant N-glycans in substantial amounts.
Rice α-fucosidase (α-fucosidase Os, 58 kDa) that is active for α1-4 fucosyl linkage in Lewis a unit of plant N-glycans was purified to homogeneity. α-fucosidase Os showed activity against α1-3 fucosyl linkage in Lacto-N-fucopentaose III but not α1-3 fucosyl linkage in the core of plant N-glycans. The N-terminal sequence of α-fucosidase Os was identified as A-A-PT- P-P-P-L-, and this sequence was found in the amino acid sequence of the putative rice α-fucosidase 1 (Os04g0560400). © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry.
Arabinogalactan proteins (AGPs) are among the most
intriguing sets of macromolecules, specific to plants,
structurally complex, and found abundantly in all plant
organs including roots, as well as in root exudates.
AGPs have been implicated in several fundamental
plant processes such as development and reproduction.
Recently, they have emerged as interesting actors of
root–microbe interactions in the rhizosphere. Indeed,
recent findings indicate that AGPs play key roles at
various levels of interaction between roots and soilborne
microbes, either beneficial or pathogenic. Therefore,
the focus of this review is the role of AGPs in the
interactions between root cells and microbes. Under-
standing this facet of AGP function will undoubtedly
improve plant health and crop protection.
In this study, we purified and characterized the β-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified β-xylosidase (β-Xyl'ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that β-Xyl'ase Le-1 has a monomeric structure in plant cells. The N-terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N-glycans were used as substrates, β-Xyl'ase Le-1 showed optimum activity at about pH 5 at 40 °C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. β-Xyl'ase Le-1 hydrolyzed the β1-2 xylosyl residue from Man(1)Xyl(1)GlcNAc(2)-PA, Man(1)Xyl(1)Fuc(1)GlcNAc(2)-PA, and Man(2)Xyl(1)Fuc(1)GlcNAc(2)-PA, but not that from Man(3)Xyl(1)GlcNAc(2)-PA or Man(3)Xyl(1)Fuc(1)GlcNAc(2)-PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of β-Xyl'ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the β1-2 xylosyl residue by β-Xyl'ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.
Recent studies suggest that α-L-fucosidases of glycoside hydrolase family 29 can be divided into two subfamilies based on substrate specificity and phylogenetic clustering. To explore the validity of this classification, we enzymatically characterized two structure-solved α-L-fucosidases representing the respective subfamilies. Differences in substrate specificities are discussed in relation to differences in active-site structures between the two enzymes.
The expression of single rol genes of the TL-DNA of Agrobacterium rhizogenes strain A4 in transgenic tobacco (Nicotiana tabacum L.) and potato (Solanum tuberosum L.) plants alters the internal concentrations of, and the sensitivity to, several plant hormones. The levels of immunoreactive cytokinins, abscisic acid, gibberellins and indole-3-acetic acid were analysed in tissues of the apical shoots, stems, leaves, roots and undifferentiated callus tissue. The addition of the dominant and morphogenetically active rolA, rolB, or rolC genes resulted in alterations in the content of several hormones. rolC overexpression in particular led to an up to fourfold increase in the content of isopentenyladenosine, dihydrozeatin riboside and trans-zeatin riboside-type cytokinins in potato plants. This increase correlated well with different levels of expression of the rolC gene in different transgenic plants. Furthermore it was shown that the dwarfism of P35s-rolC transgenic tobacco and potato plants is correlated with a 28–60% reduction of gibberellic acid A1 concentration in apical shoots. Exogenous addition of gibberellic acid completely restored stem elongation in P35s-rolC transgenic plants. Apical shoots of dwarf rolA transgenic tobacco plants also contained 22% less gibberellic acid A1 than control plants, but growth cannot be restored completely by exogenously added gibberellic acid. Similarly, the sensitivity of transgenic tobacco seedlings or callus tissues towards different phytohormone concentrations can be altered by the expression of single rol genes. The overexpression of the rolC gene in seedlings led to an altered response to auxins, cytokinins, abscisic acid, gibberellic acid and the ethylene precursor 1-aminocyclopropane-carboxylic acid. The overexpression of the rolB gene in tobacco calli led to necrosis at lower auxin concentrations than in the wild-type, while other parameters of auxin action, like the induction of cell growth, remained unchanged.
Proteins perform functions through interacting with other molecules. However, structural details for most of the protein-ligand interactions are unknown. We present a comparative approach (COFACTOR) to recognize functional sites of protein-ligand interactions using low-resolution protein structural models, based on a global-to-local sequence and structural comparison algorithm. COFACTOR was tested on 501 proteins, which harbor 582 natural and drug-like ligand molecules. Starting from I-TASSER structure predictions, the method successfully identifies ligand-binding pocket locations for 65% of apo receptors with an average distance error 2 Å. The average precision of binding-residue assignments is 46% and 137% higher than that by FINDSITE and ConCavity. In CASP9, COFACTOR achieved a binding-site prediction precision 72% and Matthews correlation coefficient 0.69 for 31 blind test proteins, which was significantly higher than all other participating methods. These data demonstrate the power of structure-based approaches to protein-ligand interaction predictions applicable for genome-wide structural and functional annotations.
This chapter provides an overview of the biological roles of glycans and attempts to synthesize some general principles for understanding and exploring these roles. For details, see the reviews cited and the other chapters in this book.
Asparagine-linked glycosylation involves the sequential assembly of an oligosaccharide onto a polyisoprenyl donor, followed by the en bloc transfer of the glycan to particular asparagine residues within acceptor proteins. These N-linked glycans play a critical role in a wide variety of biological processes, such as protein folding, cellular targeting and motility, and the immune response. In the past decade, research in the field of N-linked glycosylation has achieved major advances, including the discovery of new carbohydrate modifications, the biochemical characterization of the enzymes involved in glycan assembly, and the determination of the biological impact of these glycans on target proteins. It is now firmly established that this enzyme-catalyzed modification occurs in all three domains of life. However, despite similarities in the overall logic of N-linked glycoprotein biosynthesis among the three kingdoms, the structures of the appended glycans are markedly different and thus influence the functions of elaborated proteins in various ways. Though nearly all eukaryotes produce the same nascent tetradecasaccharide (Glc(3)Man(9)GlcNAc(2)), heterogeneity is introduced into this glycan structure after it is transferred to the protein through a complex series of glycosyl trimming and addition steps. In contrast, bacteria and archaea display diversity within their N-linked glycan structures through the use of unique monosaccharide building blocks during the assembly process. In this review, recent progress toward gaining a deeper biochemical understanding of this modification across all three kingdoms will be summarized. In addition, a brief overview of the role of N-linked glycosylation in viruses will also be presented.
Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-alpha-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885-4893). Here, we identified a gene encoding a novel 1,3-1,4-alpha-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 alpha-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released alpha1,3- and alpha1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing alpha1,2-fucosyl residue or on synthetic alpha-fucoside (p-nitrophenyl-alpha-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic alpha-l-fucosidase. The transformant carrying afcA could utilize 2'-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading alpha1,2- and alpha1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.
In this study, we identified a gene encoding tomato ENGase (Endo-LE) using the gene information of rice ENGase, and expressed the Endo-LE protein in Escherichia coli. The substrate specificity of the recombinant Endo-LE was the same as that of the native enzyme, showing strong activity towards the high-mannose type N-glycans with the Manalpha1-2Manalpha1-3Manbeta1-4GlcNAcbeta1-4GlcNAc unit. Real-time PCR analysis revealed that the gene expression of Endo-LE did not vary significantly with the tomato ripening process, indicating that Endo-LE activity is ubiquitously expressed.
The structures of the N-linked oligosaccharides of momordin-a, which is a ribosome-inactivating protein from the seeds of Momordica charantia, were analyzed. First, the N-linked oligosaccharides of this glycoprotein were liberated by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides were coupled with 2-aminopyridine and the pyridylamino (PA-) derivatives were purified by gel filtration and high performance liquid chromatography (HPLC) on an ODS-silica column. Three kinds of oligosaccharide fractions were separated by HPLC. The structure of each oligosaccharide isolated was analyzed by a combination of sugar component analysis, exoglycosidase digestion, another kind of HPLC using an amide-silica column, and 500-MHz 1H NMR spectroscopy. The structures of two main oligosaccharides were established to be: [Formula; see text] and [Formula; see text]. These two oligosaccharides were the first examples having xylose (or fucose) but no alpha-mannosyl linkage among the N-linked oligosaccharides of glycoproteins from both animal and plant origins.
Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P23∗ (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P23∗, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells.
An alpha-fucosidase that releases fucosyl residues from oligosaccharide fragments of xyloglucan, a plant cell wall hemicellulosic polysaccharide, was purified to homogeneity from pea (Pisum sativum) epicotyls using a combination of cation exchange chromatography and isoelectric focusing. The alpha-fucosidase has a molecular mass of 20 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The alpha-fucosidase has an isoelectric point of 5.5. The substrate specificity of the alpha-fucosidase was determined by high performance anion exchange chromatographic analysis of oligosaccharide substrates and products. The enzyme hydrolyzes the terminal alpha-1,2-fucosidic linkage of oligosaccharides and does not cleave p-nitrophenyl-alpha-L-fucoside. The enzyme does not release measurable amounts of fucosyl residues from large polysaccharides. The subcellular localization of alpha-fucosidase in pea stems and leaves has been studied by immunogold cytochemistry. The alpha-fucosidase accumulates in primary cell walls and is not detectable in the middle lamella or in the cytoplasm of 8-day-old stem tissue and 14-day-old leaf tissue. alpha-Fucosidase activity was readily detected in extracts of 8-day-old stem tissue. No significant alpha-fucosidase activity or immunogold labeling of the alpha-fucosidase was detected in 2- and 4-day-old stem tissue indicating that production of alpha-fucosidase is developmentally regulated.
In plants, N-linked glycans are processed in the Golgi apparatus to complex-type N-glycans of limited size containing a beta(1,2)-xylose and/or an alpha(1,3)-fucose residue. Larger mono- and bi-antennary N-linked complex glycans have not often been described. This study has re-examined the structure of such plant N-linked glycans, and, through both immunological and structural data, it is shown that the antennae are composed of Lewis a (Le(a)) antigens, comprising the carbohydrate sequence Gal beta 1-3[Fuc alpha 1-4]GlcNAc. Furthermore, a fucosyltransferase activity involved in the biosynthesis of this antigen was detected in sycamore cells. This is the first characterization in plants of a Lewis antigen that is usually found on cell-surface glycoconjugates in mammals and involved in recognition and adhesion processes. Le(a)-containing N-linked glycans are widely distributed in plants and highly expressed at the cell surface, which may suggest a putative function in cell/cell communication.
Apoptosis is a well defined physiological process characterised by many specific features including DNA fragmentation and protease activation. Cell membrane-associated changes such as the altered exposure of phosphatidylserine and glycosylation patterns have also been described. We investigate here the change in exposure of surface alpha-L-fucose residues during thymocyte and P815 cell apoptosis. We show that apoptosis in these cells induced by dexamethasone, gliotoxin or thapsigargin was associated with an increase in the exposure of terminal fucose residues. Furthermore, this increase in fucose exposure occurred late in the apoptotic process. The observation of increased fucose exposure in two different cell types by three different apoptosis-inducing agents suggests it may be part of the normal apoptotic process.
Cross-reacting carbohydrate determinants (CCDs) are antigenic structures shared by allergenic components from taxonomically distant sources. The case history of a patient with a great discrepancy between skin test and specific IgE results led us to investigate the role of these determinants in his specific case and in an allergic population.
We sought to determine the role of CCDs in causing false-positive and clinically irrelevant results in in vitro tests.
The involvement of CCDs was studied by specific IgE inhibition by using glycoproteins with a known carbohydrate structure. Direct and inhibition assays were performed by commercially available systems, in-house ELISA, and the immunoblotting technique. The binding to the periodate-oxidated carbohydrate structure of glycoproteins and allergenic extracts was also evaluated. A comparative study between skin test and specific IgE responses to the antigens studied was carried out in 428 consecutive allergic subjects.
All the tests performed suggested that cross-reacting carbohydrate epitopes were the cause of false-positive specific IgE results in one of the commercial systems and the high reactivity in all the solid-phase in vitro tests. None of the cross-reacting carbohydrate allergens yielded a positive skin test response. Periodate treatment caused variable degrees of reduction of IgE binding to the different antigens studied, indicating that CCDs played a different role in each of them. About 41% of patients allergic to pollen had specific IgE for a glycoprotein, without a positive skin test response to the same molecule.
CCDs must be taken into account when evaluating the clinical relevance of positive results in in vitro specific IgE assays, at least in the diagnosis of patients with pollen allergy. Commercial systems should be carefully assessed for the ability to detect specific IgE for carbohydrate determinants to avoid false-positive or clinically irrelevant results.
Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades. Here we show that Notch1 endogenously expressed in Chinese hamster ovary cells is modified with O-linked fucose and O-linked glucose saccharides, two unusual forms of O-linked glycosylation found on epidermal growth factor-like (EGF) modules. Interestingly, both modifications occur as monosaccharide and oligosaccharide species. Through exoglycosidase digestions we determined that the O-linked fucose oligosaccharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-alpha2,3-Gal-beta1, 4-GlcNAc-beta1,3-Fuc-alpha1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a trisaccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose or O-linked glucose modifications. It also represents the second protein discovered with an elongated form of O-linked fucose. The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs. Since Notch is known to interact with its ligands through subsets of EGF modules, these results suggest that the O-linked carbohydrate modifications of these modules may influence receptor-ligand interactions.
It has been reported that N-linked glycan moieties of glycoproteins function as IgE-reactive determinants. Gly m Bd 28K, a soybean allergen, was a glycoprotein with glycan moieties, which are supposed to be the Man(3)GlcNAc(2) backbone with the beta1-->2 xylose and alpha1-->3 fucose branches. The purpose of the present study was to examine the IgE-binding ability of the glycan moiety of Gly m Bd 28K in the binding reaction with patients' sera.
A peptide containing the glycan moiety was prepared from Gly m Bd 28K by digestion with lysyl endopeptidase. The binding site of the glycan moiety was determined by amino acid sequence analyses. The glycan moiety of the allergen was characterized using anti-horseradish peroxidase antibody (anti-HRP) recognizing the N-linked glycan moieties of glycoproteins. The binding of patients' IgE antibodies with their glycan moiety was examined by an immunostaining technique using the glycopeptide and its deglycosylated peptide derived from Gly m Bd 28K.
The binding site of the glycan moiety in Gly m Bd 28K was shown to be its Asn20 residue. Gly m Bd 28K did react with anti-HRP and the sera of soybean-sensitive patients, but the binding of IgE antibodies was inhibited by the preincubation with anti-HRP. Moreover, the glycopeptide also reacted with the sera of soybean-sensitive patients, but its deglycosylated peptide did not react with any IgE antibodies of patients' sera.
The specific IgE antibodies recognizing the N-linked glycan moieties of Gly m Bd 28K and other glycoproteins with homologous glycan moieties occur in the sera of soybean-sensitive patients. It was indicated that the N-linked glycan moieties such as that of Gly m Bd 28K may be one of the common IgE-reactive determinants distributed in various plant food proteins.
The number of microbes associated with our gut likely exceeds our total number of somatic and germ cells. Despite their numbers, almost nothing is known about the molecular mechanisms that determine whether the interaction between a microbial species and its host will be beneficial. Recent results obtained from in vivo models have revealed critical roles for glycoconjugates in helping define the outcome of two such host-microbial relationships. In one case, attachment of Helicobacter pylori to fucosylated or sialylated glycans produced by various gastric epithelial lineages and their progenitors skews the destiny of colonization toward pathogenicity. In the second case, a molecular dissection of how Bacteroides thetaiotaomicron, a normal inhabitant of the distal small intestine, is able to communicate with intestinal epithelial cells has revealed a novel role for host fucosylated glycans in forging a mutually beneficial relationship. These observations lend support to the hypothesis that the capacity to synthesize diverse carbohydrate structures may have arisen in part from our need to both evade pathogenic relationships and to coevolve symbiotic relationships with our nonpathogenic resident microbes.
A protein structure model generally needs to be evaluated to assess whether or not it has the correct fold. To improve fold assessment, four types of a residue-level statistical potential were optimized, including distance-dependent, contact, �/ � dihedral angle, and accessible surface statistical potentials. Approximately 10,000 test models with the correct and incorrect folds were built by automated comparative modeling of protein sequences of known structure. The criterion used to discriminate between the correct and incorrect models was the Z-score of the model energy. The performance of a Z-score was determined as a function of many variables in the derivation and use of the corresponding statistical potential. The performance was measured by the fractions of the correctly and incorrectly assessed test models. The most discriminating combination of any one of the four tested potentials is the sum of the normalized distancedependent and accessible surface potentials. The distance-dependent potential that is optimal for assessing models of all sizes uses both C � and C � atoms as interaction centers, distinguishes between all 20 standard residue types, has the distance range of 30 Å, and is derived and used by taking into account the sequence separation of the interacting atom pairs. The terms for the sequentially local interactions are significantly less informative than those for the sequentially nonlocal interactions. The accessible surface potential that
The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to approximately 2.2 A resolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.
The distance-dependent structure-derived potentials developed so far all employed a reference state that can be characterized as a residue (atom)-averaged state. Here, we establish a new reference state called the distance-scaled, finite ideal-gas reference (DFIRE) state. The reference state is used to construct a residue-specific all-atom potential of mean force from a database of 1011 nonhomologous (less than 30% homology) protein structures with resolution less than 2 A. The new all-atom potential recognizes more native proteins from 32 multiple decoy sets, and raises an average Z-score by 1.4 units more than two previously developed, residue-specific, all-atom knowledge-based potentials. When only backbone and C(beta) atoms are used in scoring, the performance of the DFIRE-based potential, although is worse than that of the all-atom version, is comparable to those of the previously developed potentials on the all-atom level. In addition, the DFIRE-based all-atom potential provides the most accurate prediction of the stabilities of 895 mutants among three knowledge-based all-atom potentials. Comparison with several physical-based potentials is made.
Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group. It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract.
To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart. To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one.
Recombinant Cup a1 was expressed in E. coli. IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils.
Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity-after periodate deglycosylation of the allergen. Moreover, only native molecule was capable of inducing histamine release by this group of sera. Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation.
A large number of sera reactive with the major allergen recognize carbohydrate epitopes only. IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy.
An alpha-L-fucosidase purified from pea (Pisum sativum L. cv Alaska) epicotyl was previously described as a cell wall enzyme of 20 kDa that hydrolyses terminal alpha-L-fucosidic linkages from oligosaccharide fragments of xyloglucan. cDNA and genomic copies were further isolated and sequenced. The predicted product of the cDNA and the genomic clone (fuc1), was a 20 kDa protein containing a signal peptide and five cysteines. This was the first alpha-L-fucosidase gene to be cloned in plants but its fucosidase activity has not been demonstrated. Here, our biochemical and immuno analyses suggest that fuc1 does not encode an alpha-L-fucosidase. Pea fuc1 expressed in Escherichia coli, insect cells and Arabidopsis thaliana produced recombinant proteins without alpha-L-fucosidase activity. Pea plants had endogenous alpha-L-fucosidase activity, but the enzyme was not recognised by an antibody produced against recombinant FUC1 protein expressed in E. coli. In contrast, the antibody immunoprecipitated a 20 kDa protein which was inactive. By chromatographic analysis of pea protein extracts, we separated alpha-L-fucosidase-active fractions from the 20 kDa protein fractions. We conclude that the alpha-L-fucosidase activity is not attributable to the 20 kDa FUC1 protein. A new function for fuc1 gene product, now named PIP20 (for protease inhibitor from pea) is proposed.