Plant Cell Wall Glycoproteins Research Articles

Engineering hydroxyproline-O-glycosylated biopolymers to reconstruct the plant cell wall for improved biomass processability.

Reconstructing the chemical and structural characteristics of the plant cell wall represents a promising solution to overcoming lignocellulosic biomass recalcitrance to biochemical deconstruction. This study aims to leverage hydroxyproline (Hyp)-O-glycosylation, a process unique to plant cell wall glycoproteins, as an innovative technology for de novo design and engineering in planta of Hyp-O-glycosylated biopolymers (HypGP) that facilitate plant cell wall reconstruction. HypGP consisting of 18 tandem repeats of "Ser-Hyp-Hyp-Hyp-Hyp" motif or (SP4)18 was designed and engineered into tobacco plants as a fusion peptide with either a reporter protein enhanced green fluorescence protein or the catalytic domain of a thermophilic E1 endoglucanase (E1cd) from Acidothermus cellulolyticus. The engineered (SP4)18 module was extensively Hyp-O-glycosylated with arabino-oligosaccharides, which facilitated the deposition of the fused protein/enzyme in the cell wall matrix and improved the accumulation of the protein/enzyme in planta by 1.5-11-fold. The enzyme activity of the recombinant E1cd was not affected by the fused (SP4)18 module, showing an optimal temperature of 80°C and optimal pH between 5 and 8. The plant biomass engineered with the (SP4)18 -tagged protein/enzyme increased the biomass saccharification efficiency by up to 3.5-fold without having adverse impact on the plant growth.

Anaerobic 4‐Hydroxyproline Metabolism by a Widespread Microbial Glycyl Radical Enzyme

Microbiome research has rapidly advanced through the use of high‐through put sequencing technologies, but the functions of most genes in sequence databases remain unknown. Glycyl radical enzymes represent an ancient protein super family, which is one of the most abundant protein families in the human gut microbiome. These enzymes use radical chemistry to catalyze a diverse set of chemically difficult transformations in metabolic pathways essential for anaerobic growth.Here, we describe the biochemical characterization of a new member of the glycyl radical enzyme superfamily, 4‐hydroxyproline dehydratase (HypD). This enzyme is responsible for the first step of anaerobic 4‐hydroxyproline (Hyp) metabolism. Hyp is the most abundant post‐translationally modified amino acid in the human proteome, with collagen as its principle source. It is also abundant in plant cell wall glycoproteins and functions as a site for O‐glycosylation. Hyp is present at significant levels in the human gut, deriving from both dietary sources and endogenous collagen turnover. Anaerobic Hyp metabolism had only been observed in Clostridiales as a part of Stickland fermentation in energy metabolism, but the enzymes and intermediates involved had not been identified until our work.We predicted the catalytic activity of HypD based on its sequence similarity to characterized glycyl radical enzymes and its genomic context in Clostridial species. Using purified enzymes, we demonstrated that HypD catalyzes the removal of water from Hyp to form pyrroline‐5‐carboxylate, a central intermediate in amino acid metabolism. As with all other glycyl radical enzymes, HypD activity requires post‐translational modification by a partner radical SAM enzyme to install a radical on a conserved glycine in the active site. We next examined the substrate scope and kinetics of HypD and demonstrated that it is stereospecific for trans‐4‐hydroxy‐L‐proline. Additional biochemical work has yielded insights into the radical‐based mechanism of this intriguing transformation. Our discovery of the biochemical basis for anaerobic Hyp metabolism has enabled us to uncover hundreds of strains with the potential for metabolizing Hyp. This under appreciated metabolism is encoded in species not known to ferment amino acids, where Hyp is likely used as a precursor for non‐Stickland pathways. In addition, HypD is widespread in common gut commensals as well as pathogens like Clostridiodes difficile, suggesting that it may represent a core function in the human gut microbiota. Furthermore, our work on the characterization of HypD has led us to new research directions to investigate related pathways in the gut microbiome.Support or Funding InformationFunded by Harvard University, a Packard Fellowship for Science and Engineering, and NSERC Postgraduate Scholarship‐Doctoral Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intermolecular interactions between glycomodules of plant cell wall arabinogalactan-proteins and extensins

Hydroxyproline-rich glycoproteins (HRGPs) are a unique component of plant cell walls, undergoing extensive posttranslational modification such as proline hydroxylation and hydroxyproline-O-glycosylation. Arabinogalactan proteins (AGPs) and extensins are major members of the HRGP superfamily. AGPs have repetitive AlaHyp, SerHyp, and ThrHyp peptides, the Hyp residues being glycosylated with large type II arabinogalactan polysaccharides, while extensins contain characteristic SerHyp4 and SerHyp2 motifs with arabinosylated (1–4 residues) Hyp. Although they are less than ten percent in all wall materials, AGPs and extensins play important roles in all aspects of plant growth and development. The detailed mechanisms of their functions are still under investigation. However, many of the functions may be attributed to their adhesive properties. Here, we used a forced unbinding technique to measure relative adhesive potential of the well characterized (AlaHyp)51 and (SerHyp4)18 glycomodules representing AGPs and extensins, respectively. In the presence of different wall ions such as protons, Ca2+, and boron, the glycomodules exhibited different adhesive patterns, suggesting that the wall ion-regulated intermolecular interactions/adhesions between AGPs and/or extensins may be involved in maintaining wall-plasma membrane integrity during wall loosening processes such as wall elongation or expansion. This research applies a biophysical approach to understand the biological function of plant cell wall glycoproteins.

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1–5 β- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-β1,4Araf-β1,2Araf-β1,2Araf) side chains in an α-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its’ product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.

The pgip family in soybean and three other legume species: evidence for a birth-and-death model of evolution.

BackgroundPolygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) plant cell wall glycoproteins involved in plant immunity. They are typically encoded by gene families with a small number of gene copies whose evolutionary origin has been poorly investigated. Here we report the complete characterization of the full complement of the pgip family in soybean (Glycine max [L.] Merr.) and the characterization of the genomic region surrounding the pgip family in four legume species.ResultsBAC clone and genome sequence analyses showed that the soybean genome contains two pgip loci. Each locus is composed of three clustered genes that are induced following infection with the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary, and remnant sequences of pgip genes. The analyzed homeologous soybean genomic regions (about 126 Kb) that include the pgip loci are strongly conserved and this conservation extends also to the genomes of the legume species Phaseolus vulgaris L., Medicago truncatula Gaertn. and Cicer arietinum L., each containing a single pgip locus. Maximum likelihood-based gene trees suggest that the genes within the pgip clusters have independently undergone tandem duplication in each species.ConclusionsThe paleopolyploid soybean genome contains two pgip loci comprised in large and highly conserved duplicated regions, which are also conserved in bean, M. truncatula and C. arietinum. The genomic features of these legume pgip families suggest that the forces driving the evolution of pgip genes follow the birth-and-death model, similar to that proposed for the evolution of resistance (R) genes of NBS-LRR-type.

Cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum and transgenic CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). The PGIPs directly reduce the aggressive potential of PGs. Here, we isolated and functionally characterized three members of the pepper (Capsicum annuum) PGIP gene family. Each was up-regulated at a different time following stimulation of the pepper leaves by Phytophthora capcisi and abiotic stresses including salicylic acid, methyl jasmonate, abscisic acid, wounding and cold treatment. Purified recombinant proteins individually inhibited activity of PGs produced by Alternaria alternata and Colletotrichum nicotianae, respectively, and virus-induced gene silencing in pepper conferred enhanced susceptibility to P. capsici. Because three PGIP genes acted similarily in conferring resistance to infection by P. capsici, and because individually purified proteins showed consistent inhibition against PG activity of both pathogens, CaPGIP1 was selected for manipulating transgenic tobacco. The crude proteins from transgenic tobacco exhibited distinct enhanced resistance to PG activity of both fungi. Moreover, the transgenic tobacco showed effective resistance to infection and a significant reduction in the number of infection sites, number of lesions and average size of lesions in the leaves. All results suggest that CaPGIPs may be involved in plant defense response and play an important role in a plant's resistance to disease.

Partial purification and characterization of polygalacturonase-inhibitor proteins from pearl millet

Polygalacturonase-inhibitor proteins (PGIPs) are plant cell wall glycoproteins, involved in the inhibition of microbial endo-polygalacturonases (EPGs). The present study involved activity guided partial purification of pearl millet [Pennisetum glaucum (L.) R.Br.] protein extract by cation exchange chromatography, which resulted in two pooled protein peaks – Peak-A and Peak-B, both of which showed inhibitory activity against the Aspergillus niger EPG. Protein separation of the two peaks by gel electrophoresis showed prominent bands between 29 and 43 kDa, consistent with the molecular weights of the known plant PGIPs. The two PGIP peaks were further studied for their inhibitory activities with respect to three parameters viz., inhibitor concentration, pH and temperature effects. Enzyme inhibition was partial and increased with inhibitor concentration. The Peak-B was found to be the more active inhibitor of the two. The results indicate the presence of at least two isoforms of PGIP in pearl millet. This is the first such study to be undertaken in understanding the presence of the PGIPs in millets.

Overexpression of polygalacturonase-inhibiting protein 2 (PGIP2) of Chinese cabbage (Brassica rapa ssp. pekinensis) increased resistance to the bacterial pathogen Pectobacterium carotovorum ssp. carotovorum

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit microbial polygalacturonase (PG) activity. In this study, we cloned five PGIP genes from Chinese cabbage (Brassica rapa ssp. pekinensis). Reverse transcription PCR expression analysis showed that the accumulation of BrPGIP transcripts differed among various tissues and in response to biotic (bacterial innoculation) and abiotic stresses (i.e., wounding, jasmonic acid, cold, NaCl, and dehydration treatment). Transcripts of BrPGIP1, BrPGIP3, and BrPGIP5 were detected in all tissues tested except the stamen, while BrPGIP2 transcripts were expressed in all tissues tested. Transcripts of BrPGIP4 were not expressed in cabbage. Innoculation with a bacterium that causes soft rot, Pectobacterium carotovorum ssp. carotovorum (Pcc), strongly induced transcripts of BrPGIP2; and expression occurred more rapidly in the resistant compared to the susceptible line. BrPGIP2 showed 50–99% similarity in amino acid sequences to extracellular PGIPs from other plants. In order to assess the role of BrPGIP2 protein in protecting plants from Pcc, we generated a number of transgenic tobacco and Chinese cabbage lines over-expressing BrPGIP2. PGIP from transgenic tobacco plants inhibited Pcc PG activity by 74%, while PGIP from wild-type tobacco plants gave only 43% Pcc PG inhibition. Transgenic Chinese cabbage plants also exhibited improved resistance to bacterial soft rot (up to 54%). PGIP from transgenic Chinese cabbage plants inhibited Pcc PG activity by 77%, while PGIP from wild-type plants showed only 8% Pcc PG inhibition. This is the first report showing the role of BrPGIPs in resistance to bacterial soft rot caused by Pcc.

A mixture of peptides and sugars derived from plant cell walls increases plant defense responses to stress and attenuates ageing-associated molecular changes in cultured skin cells

Small peptides and aminoacid derivatives have been extensively studied for their effect of inducing plant defense responses, and thus increasing plant tolerance to a wide range of abiotic stresses. Similarly to plants, these compounds can activate different signaling pathways in mammalian skin cells as well, leading to the up-regulation of anti-aging specific genes. This suggests the existence of analogous defense response mechanisms, well conserved both in plants and animal cells. In this article, we describe the preparation of a new mixture of peptides and sugars derived from the chemical and enzymatic digestion of plant cell wall glycoproteins. We investigate the multiple roles of this product as potential “biostimulator” to protect plants from abiotic stresses, and also as potential cosmeceutical. In particular, the molecular effects of the peptide/sugar mixture of inducing plant defense responsive genes and protecting cultured skin cells from oxidative burst damages were deeply evaluated.

Tandem Mass Spectrometry and Structural Elucidation of Glycopeptides from a Hydroxyproline-rich Plant Cell Wall Glycoprotein Indicate That Contiguous Hydroxyproline Residues Are the Major Sites of Hydroxyproline O-Arabinosylation

Hydroxyproline-rich glycoproteins (HRGPs) occur in the extracellular matrix of land plants and green algae. HRGPs contain from 2 to 95% of their dry weight as carbohydrate, predominantly as oligoarabinosides and/or as heteropolysaccharides which are O-linked to the hydroxyproline residues. A glycosylation code that determines the presence or absence and extent of arabinosylation at each hydroxyproline residue is likely, as each HRGP has a unique arabinosylation profile. Previously we noted a positive correlation between the contiguity of hydroxyproline residues and the extent of HRGP O-arabinosylation (Kieliszewski, M., deZacks, R., Leykam, J.F., and Lamport, D.T.A. (1992) Plant Physiol. 98, 919-926); most arabinosylated hydroxyproline residues and the longer arabinofuranoside chains occur in HRGPs where Hyp residues occur as blocks of tetrahydroxyproline, while those with little or no contiguous Hyp exhibit very little Hyp arabinosylation. In order to test this Hyp contiguity hypothesis, we have for the first time determined the arabinosylation site specifics of an HRGP, namely the proline and hydroxyproline-rich glycoprotein (PHRGP) isolated from Douglas fir (Pseudotsuga menziesii). Pronase digests of PHRGP yielded a major peptide and three glycopeptides whose structures were determined directly from the unfractionated underivatized Pronase digest by tandem mass spectrometry using collisionally induced dissociation. We corroborated the peptide and glycopeptide structures by Edman degradation, neutral sugar analyses, hydroxyproline arabinoside profiles, and further mass spectrometric analyses after purification of the major peptide and glycopeptides by a combination of hydrophilic interaction and reverse phase column chromatography. Consistent with the Hyp contiguity hypothesis, the structural analyses indicate that while the sequence Ile-Pro-Pro-Hyp is never arabinosylated and Lys-Pro-Hyp-Val-Hyp is only occasionally monoarabinosylated at Hyp-5, the peptide containing contiguous Hyp, Lys-Pro-Hyp-Hyp-Val, is always arabinosylated at Hyp-3, mainly by a triarabinoside. We also obtained precise molecular masses for both intact and anhydrous hydrogen fluoride-deglycosylated PHRGPs (73.113 and 53.834 kDa) via matrix-assisted laser desorption/ionization time of flight mass spectrometry, representing the first HRGP to be analyzed by this method.

Polyphenols, astringency and proline-rich proteins

Recent, NMR and precipitation, studies of molecular recognition of proline-rich proteins and peptides by plant polyphenols are described and rationalized. The action of polysaccharides and caseins in the moderation of the astringent response, which is engendered by polyphenols present in foodstuffs and beverages, is described. The possible influence of plant cell wall glycoproteins on the process of lignification is discussed in the light of the observed affinity of phenolic substrates for prolyl residues in protein structures.

The Role of Carbohydrate in Maintaining Extensin in an Extended Conformation

Monomers of the plant cell wall glycoprotein extensin are secreted into the wall where they become cross-linked to each other to form a rigid matrix. Expression of the extensin matrix is correlated with the inhibition of further cell elongation during normal development, with increased resistance to virulent pathogens and with other physiological responses characterized by wall strengthening. Carbohydrates make up about two-thirds of the mass of extensin. Arabinose oligomers linked to hydroxyproline residues represent 95% of the total carbohydrate with the remainder occurring as single residues of galactose linked to some serine residues. Electron microscopy of shadowed extensin shows the glycosylated form to be an easily visualized and highly elongated molecule. In contrast, extensin that has been deglycosylated with anhydrous hydrogen fluoride is difficult to resolve in the EM. Glycosylated extensin elutes from a gel filtration column much more rapidly than does the deglycosylated form, and from this analysis we have calculated respective Stokes' radii of 89 and 11 Angstroms for these molecules. Others have shown that inhibition of extensin glycosylation has no effect on its secretion or insolubilization in the cell wall, but that this extensin cannot inhibit cell elongation. It is likely that carbohydrate moieties keep extensin in an extended conformation and that extensin must be in this conformation to form a cross-linked matrix that can function properly in vivo.

The three-dimensional structure of the cell wall glycoprotein of Chlorogonium elongatum.

The green alga Chlorogonium elongatum, a member of the Volvocales, possesses a crystalline cell wall composed of hydroxyproline-rich glycoprotein similar to the primary cell wall glycoproteins of higher plants. Electron microscopy and computer image processing have been used to determine the crystal structure of the Chlorogonium cell wall in three dimensions to a resolution of 2.0 nm. The structure is composed of heterologous dimers. Each subunit of the dimer comprises a long, thin spacer domain and a large globular domain, which is the site of the intra- and inter-dimer interactions. There are also sites of intersubunit interactions at the opposite ends of the rod domains. We suggest that the rods are composed predominantly of glycosylated polyproline helix, as has been suggested for higher plant cell wall glycoproteins and has been shown for the cell wall glycoprotein of Chlamydomonas reinhardtii, which is closely related to Chlorogonium.

Glycoprotein conformation in plant cell walls

The major structural glycoprotein of the cell wall of Chlamydomonas reinhardii has a protein core, at least 50% of which is in the unusual polyproline II conformation. This has been demonstrated by examining the circular dichroism of the cell wall, its constituent glycoproteins, and thermolysin released wall glycopeptides. One of these glycopeptides, T2, has a high hydroxyproline and sugar content, and possesses upward of 85% polyproline II structure. The main extracellular matrix glycoprotein therefore has a rigid, rod-like structure and the significance of this and its relation to higher plant cell wall glycoproteins is discussed. The unusual conformation appears to confer great stability on the glycoprotein as it is unchanged either by certain denaturing agents or during the transition from protomer to assembled cell wall.

Effect of a Fungal Disease on Extensin, the Plant Cell Wall Glycoprotein

Effect of a Fungal Disease on Extensin, the Plant Cell Wall Glycoprotein

Hydroxyproline-O-glycosidic Linkage of the Plant Cell Wall Glycoprotein Extensin

PRIMARY cell walls contain a protein, extensin, which is rich in trans 4-L-hydroxyproline1–3. I suggested earlier that this protein could cross link wall polysaccharides4; if some of these cross linkages were labile, it would provide a chemical basis for changes in cell wall plasticity which are necessary for extension of plant cells. The recent isolation of several glycopeptides, rich in hydroxyproline from enzymatic digests of tomato cell walls5, confirmed the inference of a covalent carbohydrate–protein linkage in extensin. The chemical composition and properties of these glycopeptides also led me to the preliminary conclusion that attachment of the carbohydrate is by a glycosidic link through the hydroxyl group of hydroxyproline5. This rather unexpected possibility receives additional support from results presented in this communication, describing the isolation of hydroxyproline-O-glycosides from partial alkaline hydrolysates of tomato cell walls. These cell wall preparations account for more than 95 per cent of the hydroxyproline of the cells3. Most of this hydroxyproline (about 70 per cent) can be released as hydroxyproline glycosides. The method is based on the fact that glycosidic linkages are usually stable to those alkaline conditions which lead to rapid hydrolysis of peptide bonds6.