Structure of the Threonine-Rich Extensin from Zea mays

Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete post-translational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the `P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains alternate with high regularity. The THRGP is not in the polyproline-II conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HF-solvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot.     

An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins

A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex.Abbreviations AGP arabinogalactan protein - ELISA enzyme-linked immunosorbent assay - HRGP hydroxyproline-rich glycoprotein - THRGP threonine- and hydroxyproline-rich glycoprotein This work was supported by The Leverhulme Trust. We also acknowledge support from The Royal Society and thank Prof. L.A. Staehelin for the carrot extensin, N. Stacey for the rice cell culture and Dr. J. Keen for protein sequencing.     

Extensin: repetitive motifs, functional sites, post-translational codes, and phylogeny

Homologous hydroxyproline-rich glycoproteins (HRGPs) of the plant extracellular matrix include extensins, repetitive proline-rich proteins (RPRPs), some nodulins, gum arabic glycoprotein (GAGP), arabinogalactan-proteins (AGPs), and chimeric proteins such as potato lectin which contain an extensin module fused to a lectin. The key to the role of HRGPs in cell wall self-assembly and cell extension lies in their chemistry, which is dependent on extensive post-translational modifications (PTMs): hydroxylation, glycosylation, and cross-linking. Repetitive peptide motifs characterize HRGPs. One or more repetitive peptide motifs and their variants, singly or in combination, may constitute functional sites involved in various aspects of cell wall assembly, as follows:

Rules for the post-translational modifications are emerging:

Their protistan origin obscures the phylogenetic affinities of a single extensin-HRGP family due to their sequence divergence. We propose a phylogenetic series ranging from the minimally glycosylated basic RPRPs to the highly glycosylated acidic AGPs. Furthermore, based on similarities between dicots and gymnosperm extensins, and their marked difference from graminaceous monocot extensins, graminaceous monocot and dicot lines may have diverged as early as the progymnosperms. The origin of the embryophytes (land plants) is an even more open question, the nearest extant algal relatives being unknown. The Charophyta, frequently suggested as ancestral to the embryophytes, seem unlikely as the Charalean cell wall of Chara and Nitella lacks hydroxyproline.     

Extensin from suspension-cultured potato cells: a hydroxyproline-rich glycoprotein, devoid of agglutinin activity

Enhanced deposition and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in the plant cell wall is acknowledged to contribute to the formation of a resistant barrier against pathogen infection. We have isolated, from suspension-cultured potato (Solanum tuberosum L. cv. Desiree) cells, two forms of soluble HRGP, a cross-linked and a monomeric form; the latter can be converted to the cross-linked form by incubation with tomato extensin peroxidase and H₂O₂. The monomeric form was purified by Sephacryl S-200 gel-filtration, reverse-phase high-performance liquid chromatography and Mono-S cation-exchange chromatography into two isoforms (A, a minor form; B, a major form). The properties of the B isoform were further investigated. A quantitative enzyme-linked immuno-sorbent assay of the B isoform, using tomato extensin antiserum, showed a titration curve at a high antibody-dilution range comparable to that of purified tomato extensin monomer (M.D. Brownleader and P.M. Dey, 1993, Planta 191: 457–469). The amino acid and carbohydrate compositions were similar to those of tomato extensin, but did not match well with the other two HRGPs from potato, potato lectin and potato bacterial agglutinin. These observations demonstrate the similarities of the B isoform to extensin. The homogeneity of the B isoform was demonstrated by its ability to be fully cross-linked in vitro, leaving no residual protein, into a high-molecular-weight form by the action of extensin peroxidase. The trifluoroacetic acid-deglycosylated sample migrated as a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, SDS-PAGE and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry indicated a molecular weight of approximately 67 kDa. Circular-dichroism spectroscopy demonstrated that the molecule possesses an extended polyproline II helix conformation with no evidence of α- helix or β- sheet secondary structure. In conclusion, we refer to this HRGP as potato extensin. As proposed for other extensins, potato extensin is likely to play a role in cell wall architecture and plant disease resistance. Received: 25 November 1996 / Accepted 13 January 1997     

A gymnosperm extensin contains the serine-tetrahydroxyproline motif

The extensin family is a diverse group of hydroxyproline-rich glycoproteins located in the cell wall and characterized by repetitive peptide motifs glycosylated to various degrees. The origin of this diversity and its relationship to function led us earlier to compare extensins of the two major groups of angiosperms from which we concluded that the highly glycosylated Ser-Hyp₄ motif was characteristic of advanced herbaceous dicots, occurring rarely or not at all in a representative graminaceous monocot (Zea mays) and a chenopod (Beta vulgaris) representative of primitive dicots. Because these results could arise either from loss or acquisition of a characteristic feature, we chose a typical gymnosperm representing seed-bearing plants more primitive than the angiosperms. Thus, salt eluates of Douglas fir (Pseudotsuga menziesii) cell suspension cultures yielded two monomeric extensins differing in size and composition. The larger extensin reported earlier lacked the Ser-Hyp₄ motif, was rich in proline and hydroxyproline, and contained peptide motifs similar to the dicot repetitive proline-rich proteins. The smaller extensin monomer reported here (Superose-6 peak 2 [SP2]) was compositionally similar to typical dicot extensins such as tomato P1, mainly consisting of Hyp, Thr, Ser, Pro, Val, Tyr, Lys, His, abundant arabinose, and a small but significant galactose content. A chymotryptic peptide map (on Hamilton PRP-1) of anhydrous hydrogen fluoride-deglycosylated SP2 yielded eight peptides sequenced after further purification on a high-resolution fast-sizing column (polyhydroxyethyl aspartamide; Poly LC). Significantly, two of the eight peptides contained the Ser-Hyp₄ motif, consistent both with the SP2 amino acid composition as well as the presence of hydroxyproline tetraarabinoside as a small (4% of total Hyp) component of the hydroxyproline arabinoside profile; thus, hydroxyproline tetraarabinoside corroborates the presence of Ser-Hyp₄, in agreement with our earlier observation that Hyp contiguity and Hyp glycosylation are positively correlated. Interestingly, other peptide sequences indicate that SP2 contains motifs such as Ser-Hyp₃-Thr-Hyp-Tyr, Ser-Hyp₄-Lys, and (Ala-Hyp)_n repeats that are related to and typify dicot extensins P1, P3, and arabinogalactan proteins, respectively. Overall, these peptide sequences confirm our previous prediction that Ser-Hyp₄ is indeed an ancient motif and also strongly support our suggestion that the extensins comprise an extraordinarily diverse, but nevertheless phylogenetically related, family of cell wall hydroxyproline-rich glycoproteins.     

Isolation of extensin precursors by direct elution of intact tomato cell suspension cultures

Dilute salt solutions eluted peroxidase and hydroxyproline-rich glycoproteins (HRGP's) very rapidly (60 % within 10s) from the surface of intact tomato cells grown in suspension culture. Further purification of the HRGPs based on (a) their solubility in 10% trichloroacetic acid and (b) chromatography on carboxymethyl cellulose, gave two components (P1 and P2) rich in serine, tyrosine, lysine and arabinosylated hydroxyproline. The sum of the hydroxyproline arabinoside profiles of P1 and P2 approximated that of the wall. P1, unlike P2, was histidine-rich and also contained proline. Significantly, isodityrosine (IDT) was absent from P1 and P2 but present in cell wall hydrolysates where, the Hyp:IDT molar ratio was ca 15: 1. In cells 4 days after subculture, ³H-proline pulse-chase data indicated turnover of P1 and P2 presumably resulting from covalent attachment to the wall as neither P1 nor P2 appeared in the growth medium. At day four the cell mean generation time (MGT) was 4.6 days, the cell hydr oxyproline content was 0.7 % (w/w), the half lives of P1 and P2 were both ca 12 hr, and the combined CaCl₂ elutable P1 and P2 precursor pools contained ca 400 μg Hyp/g cells (dry weight). Calculated from the MGT and Hyp content, the cell demand was 44.μg Hyp/g cells (dry weight)/hr. The precursor pool size was therefore sufficient for 9 hours growth. However the pool turnover calculated from half life and pool size was 5.6 %/hr or 22.4μg Hyp/g cells (dry weight)/hr. Thus the supply of P1 and P2 precursors met > 50 % of the cell wall demand. Corroborative experiments showed that after depletion of the P1 and P2 pools by salt elution, washed cells resuspended in growth medium repleted the precursor pools at a rate corresponding to a synthesis of 43μg Hyp/g cells (dry weight)/hr, or 98 % of the demand. These data allow us to make the following suggestions: P1 and P2 represent monomeric extensin precursor subunits. Salt elution of P1 and P2 indicates their ionic binding by pectic carboxyl groups. The rapidity of elution indicates a high diffusivity of these extended rodlike macromolecules through the cell wall. This may imply a preferred orientation for P1 and P2 perpendicular rather than parallel to the plane of the wall. The lack of IDT in P1 and P2 implies that IDT forms in muro, possibly via peroxidase. We speculate that some of these IDT residues may crosslink an extensin precursor ‘tweft’ around a cellulose microfibrillar ‘twarp’. Such formation of heteromultimeric extensin interpenetrated by microfibrils would create a mechanically coupled extensin-cellulose network.     

A repetitive proline-rich protein from the gymnosperm douglas fir is a hydroxyproline-rich glycoprotein

Intact cell elution of suspension cultures derived from Douglas fir, Pseudotsuga menziesii (Mirbel) Franco, yielded two extensin monomers, the first hydroxyproline-rich glycoproteins (HRGPs) to be isolated from a gymnosperm. These HRGPs resolved on Superose-6 gel filtration. The smaller monomer was compositionally similar to angiosperm extensins like tomato P1. The larger monomer had a simple composition reminiscent of repetitive proline-rich proteins (RPRPs) from soybean cell walls and contained proline, hydroxyproline, and sugar; hence designated a proline-hydroxyproline-rich glycoprotein (PHRGP). The simple composition of the PHRGP implied a periodic structure which was confirmed by the simple chymotryptic map and 45-residue partial sequence of the major proline-hydroxyproline-rich glycoprotein chymotryptide 5: Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp-Val- Tyr-Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp- Val-Tyr-Lys-Ile-Pro-Pro(Hyp)-Val-Ile-Lys-Pro. Proline-hydroxyproline-rich glycoprotein chymotryptide 5 contained an 18-residue tandem repeat devoid of tetra(hydroxy)-proline or serine; it also contained two instances of the five-residue motif Hyp-Hyp-Val-Tyr-Lys and five of the general Pro-Pro-X-X-Lys motif, thereby establishing its homology with typical angiosperm RPRPs and extensins from tomato, petunia, carrot, tobacco, sugar beet, and Phaseolus. Unlike the nonglycosylated soybean RPRP, the highly purified Douglas fir PHRGP was lightly glycosylated, confirmed by a quantitative hydroxyproline glycoside profile, indicating that extensins can range from highly glycosylated hydroxyproline to little or no glycosylated hydroxyproline. Comparison of extensin sequence data strongly indicates that a major determinant of hydroxyproline glycosylation specificity is hydroxyproline contiguity: extensins with tetrahydroxyproline blocks are very highly arabinosylated (>90% hydroxyproline glycosylated), tri- and dihydroxyproline are less so, and single hydroxyproline residues perhaps not at all. Despite high yields of extensins eluted from intact cells, the Douglas fir cell wall itself was hydroxyproline poor yet remarkably rich in protein (>20%), again emphasizing the existence of other structural cell wall proteins that are neither HRGPs nor glycine-rich proteins.     

Antibody localization of extensin in cell walls of carrot storage roots

The accumulation and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants has been correlated with a number of wall-strengthening phenomena. Polyclonal antibodies raised against glycosylated extensin-1, the most abundant HRGP in carrot (Daucus carota L.) cell walls, recognize this antigen on gel and dot blots and on thin sections of epoxy-embedded carrot-root cell walls. Since wall labeling can be largely reduced by preincubating the antibodies with purified extensin-1, most labeling can be attributed to recognition of this antigen. The remaining label may be the result of recognition of extensin-2, a second carrot HRGP, or other wall components (cellulose, hemicellulose and pectin are not recognized). Extensin-1 label was distributed quite uniformly across the cell wall but was absent from the expanded middle lamella at the intersection of three or more cells and was reduced in the narrow middle lamella between two cells. This distribution is essentially the same as that of cellulose. Because of limitations of this labeling technique, it is not possible to construct a complete model of the structure of the cross-linked extensin matrix. Nonetheless, short, linear arrays of gold particles may represent small portions of the extensin matrix or of individual extensin molecules as they are exposed on the surface of sections. These and other results presented here indicate that: a) newly synthesized extensin is added to the wall by intussusception; b) extensin cannot cross the middle lamella separating the walls of adjacent cells; and c) incorporation of extensin is a late event in the development of phloem-parenchyma cell walls in carrot.Abbreviations dE-1 antibodies antibodies raised against deglycosylated extensin 1 - ELISA enzyme-linked immunosorbant assay - gE-1 antibodies antibodies raised against glycosylated extensin 1 - HRGP hydroxyproline-rich glycoprotein - PAGE polyacrylamide gel electrophoresis - RG-1 rhamnogalacturonan I - SDS sodium dodecyl sulfate     

Characterization of a hydroxyproline-rich glycoprotein in pearl millet and its differential expression in response to the downy mildew pathogen <Emphasis Type="Italic">Sclerospora graminicola</Emphasis>

A monoclonal antibody, JIM 20, derived against an extensin type of hydroxyproline-rich glycoprotein (HRGP) from pea, showed high affinity for HRGP in pearl millet [Pennisetum glaucum (L.) R. Br.]. Electrophoretic separation of Tris–SDS extracted proteins from suspension cells of pearl millet revealed a range of PM-HRGP polypeptides having a glycan epitope, which reacted with JIM 20. A high molecular mass band, probably an HRGP aggregate or polymer, and a few low molecular mass polypeptides were recognized by JIM 20 during Western blot analysis. Treatment of pearl millet suspension cells with hydrogen peroxide in the presence of an endogenous peroxidase resulted in insolubilization of HRGP polypeptides with molecular weights between 45 and 33 kDa. To investigate the gene coding for an extensin type of HRGP, a fosmid-based genomic library of pearl millet having a fourfold genome coverage was constructed. A partial sequence of 378 bp of an HRGP gene was obtained by PCR amplification of pearl millet DNA with a primer pair designed from the conserved regions of monocotyledon extensin type of HRGPs. Screening the genomic library using the homologous probe developed from the 378-bp PCR product resulted in the isolation of five fosmid clones. Restriction mapping of these fosmids resulted in an 11.8-kb region around an HRGP gene in pearl millet. The newly characterized gene, PM-HRGP, had all the characteristic features of a monocotyledon extensin type of HRGP. An intron at the 3′ untranslated region of the gene was identified by cDNA cloning. Differential expression of the PM-HRGP gene was observed during compatible and incompatible interactions of pearl millet with the downy mildew pathogen Sclerospora graminicola (Sacc) Schroet. Induced expression of the gene was observed only in case of an incompatible interaction.     

Glycosylation motifs that direct arabinogalactan addition to arabinogalactan-proteins

Hydroxyproline (Hyp)-rich glycoproteins (HRGPs) participate in all aspects of plant growth and development. HRGPs are generally highly O-glycosylated through the Hyp residues, which means carbohydrates help define the interactive molecular surface and, hence, HRGP function. The Hyp contiguity hypothesis predicts that contiguous Hyp residues are sites of HRGP arabinosylation, whereas clustered noncontiguous Hyp residues are sites of galactosylation, giving rise to the arabinogalactan heteropolysaccharides that characterize the arabinogalactan-proteins. Early tests of the hypothesis using synthetic genes encoding only clustered noncontiguous Hyp in the sequence (serine [Ser]-Hyp-Ser-Hyp)(n) or contiguous Hyp in the series (Ser-Hyp-Hyp)(n) and (Ser-Hyp-Hyp-Hyp-Hyp)(n) confirmed that arabinogalactan polysaccharide was added only to noncontiguous Hyp, whereas arabinosylation occurred on contiguous Hyp. Here, we extended our tests of the codes that direct arabinogalactan polysaccharide addition to Hyp by building genes encoding the repetitive sequences (alanine [Ala]-proline [Pro]-Ala-Pro)(n), (threonine [Thr]-Pro-Thr-Pro)(n), and (valine [Val]-Pro-Val-Pro)(n), and expressing them in tobacco (Nicotiana tabacum) Bright-Yellow 2 cells as fusion proteins with green fluorescent protein. All of the Pro residues in the (Ala-Pro-Ala-Pro)(n) fusion protein were hydroxylated and consistent with the hypothesis that every Hyp residue was glycosylated with arabinogalactan polysaccharide. In contrast, 20% to 30% of Pro residues remained non-hydroxylated in the (Thr-Pro-Thr-Pro)(n), and (Val-Pro-Val-Pro)(n) fusion proteins. Furthermore, although 50% to 60% of the Hyp residues were glycosylated with arabinogalactan polysaccharide, some remained non-glycosylated or were arabinosylated. These results suggest that the amino acid side chains of flanking residues influence the extent of Pro hydroxylation and Hyp glycosylation and may explain why isolated noncontiguous Hyp in extensins do not acquire an arabinogalactan polysaccharide but are arabinosylated or remain non-glycosylated.     

Peptides isolated from cell walls of Medicago truncatula nodules and uninfected root

The hydroxyproline-rich root nodules of legumes provide a microaerobic niche for symbiotic nitrogen-fixing Rhizobacteria. The contributions of the cell wall and associated structural proteins, particularly the hydroxyproline-rich glycoproteins (HRGPs), are therefore of interest. Our approach involved identification of the protein components by direct chemical analysis of the insoluble wall. Chymotryptic peptide mapping showed a "P3-type" extensin containing the highly arabinosylated Ser-Hyp4-Ser-Hyp-Ser-Hyp4-Tyr3-Lys motif as a major component. Cell wall amino acid analyses and quantitative hydroxyproline arabinoside profiles, predominantly of tri- and tetraarabinosides, confirmed this extensin as the major structural protein in the cell walls of both root nodules and uninfected roots. On the other hand, judging from the Pro, Glu and non-glycosylated Hyp content, the nodule-specific proline-rich glycoproteins, such as the early nodulins (ENOD-PRPs), are present in much lesser amounts. Although we isolated no PRP peptides from nodule cell walls, a single PRP peptide from root cell walls confirmed the presence of a PRP in roots and represented the first direct evidence for a crosslinked PRP in muro. Compared with root cell walls (approximately 7% protein dry weight) nodule cell walls contained significantly more protein (approximately 13% dry weight) with an overall amino acid and peptide composition indicating the presence of structural protein unrelated to the HRGPs.     

Tomato LeAGP-1 arabinogalactan-protein purified from transgenic tobacco corroborates the Hyp contiguity hypothesis

Functional analysis of the hyperglycosylated arabinogalactan-proteins (AGPs) attempts to relate biological roles to the molecular properties that result largely from O-Hyp glycosylation putatively coded by the primary sequence. The Hyp contiguity hypothesis predicts contiguous Hyp residues as attachment sites for arabino-oligosaccharides (arabinosides) and clustered, non-contiguous Hyp residues as arabinogalactan polysaccharide sites. Although earlier tests of naturally occurring hydroxyproline-rich glycoproteins (HRGPs) and HRGPs designed by synthetic genes were consistent with a sequence-driven code, the predictive value of the hypothesis starting from the DNA sequences of known AGPs remained untested due to difficulties in purifying a single AGP for analysis. However, expression in tobacco (Nicotiana tabacum) of the major tomato (Lycopersicon esculentum) AGP, LeAGP-1, as an enhanced green fluorescent protein fusion glycoprotein (EGFP)-LeAGP-1, increased its hydrophobicity sufficiently for chromatographic purification from other closely related endogenous AGPs. We also designed and purified two variants of LeAGP-1 for future functional analysis: one lacking the putative glycosylphosphatidylinositol (GPI)-anchor signal sequence; the other lacking a 12-residue internal lysine-rich region. Fluorescence microscopy of plasmolysed cells confirmed the location of LeAGP-1 at the plasma membrane outer surface and in Hechtian threads. Hyp glycoside profiles of the fusion glycoproteins gave ratios of Hyp-polysaccharides to Hyp-arabinosides plus non-glycosylated Hyp consistent with those predicted from DNA sequences by the Hyp contiguity hypothesis. These results demonstrate a route to the purification of AGPs and the use of the Hyp contiguity hypothesis for predicting the Hyp O-glycosylation profile of an HRGP from its DNA sequence.     

Gum arabic glycoprotein is a twisted hairy rope : a new model based on o-galactosylhydroxyproline as the polysaccharide attachment site

Separation of the wound exudate from Acacia senegal (L.) Willd., “gum arabic,” on a preparative Superose-6 column gave two major fractions: a high molecular weight gum arabic glycoprotein (GAGP) containing about 90% carbohydrate and a lower molecular weight heterogenous gum arabic polysaccharide fraction. Hydrogen fluoride-deglycosylation of GAGP gave a large (~400 residue) hydroxyproline-rich polypeptide backbone (dGAGP). Alkaline hydrolysis of GAGP showed that most of the carbohydrate was attached to the polypeptide backbone as small (~30 residue) hydroxyproline (Hyp)-polysaccharide substituents. After partial acid hydrolysis of the Hyp-polysaccharide fraction we identified O-galactosylhydroxyproline as the glycopeptide linkage, identical with that of hydroxyproline-rich arabinogalactan-proteins (AGPs). However, unlike the acidic alanine-rich AGPs, GAGP is basic and notably deficient in alanine. Thus, while the GAGP polypeptide backbone more closely resembles that of the Hyp-rich cell wall protein extensin, the GAGP polysaccharide sidechains resemble AGPs. Possibly all three proteins comprise a phylogenetically related extensin superfamily of extended rod-like macromolecules. The “wattle-blossom” model for AGP and gum arabic predicts a few large polysaccharide substituents along the polypeptide backbone of a spheroidal macromolecule. On the contrary, our data imply a rodlike molecule with numerous small polysaccharide substituents (attached to 24% of the Hyp residues), regularly arranged along a highly periodic polypeptide backbone based, hypothetically, on a 10 to 12 residue repetitive peptide motif. Thus, a simple statistical model of the gum arabic glycoprotein predicts a repeating polysaccharide-peptide subunit of about 7 kilodaltons. The small polysaccharide substituents will maximize intramolecular hydrogen bonding if aligned along the long axis of the molecule, forming in effect a twisted hairy rope. Electron micrographs of rotary shadowed GAGP molecules support that prediction and may also explain how such apparently large molecules can exit the cell by endwise reptation through the small pores of the primary cell wall.     

Cross-linking patterns in salt-extractable extensin from carrot cell walls

Extensins are hydroxyproline-rich glycoproteins (HRGPs) found in the primary cell walls of dicots. Extensin monomers are secreted into the wall and covalently bound to each other, presumably by isodityrosine (IDT) cross-links, to form a rigid matrix. Expression of the extensin matrix is correlated with inhibition of cell elongation during normal development and with increased resistance to virulent pathogens. We have isolated extensin from carrot root tissue (Daucus carota L.) by published techniques and have used gel filtration chromatography to purify fractions enriched in monomers and oligomers. We refer to this protein as “extensin-1” to distinguish it from “extensin-2,” a second extensin-like HRGP from carrot which we will describe later. We prepared extensin-1 for electron microscopy by shadowing it with platinum. Monomers are highly elongated (84 nanometers) and kinked at several sites. Kinks occur at all sites on molecules with nearly equal probability, but do not appear to occur at their ends. The distribution of kinks is similar to that of tyrosine-lysine-tyrosine sequences, which have been shown to be capable of forming intramolecular IDT cross-links, so we suggest that kinks are visible manifestations of intramolecular IDTs. Oligomers likely result from IDT cross-links between monomers, and may be regarded as transient precursors of the fully cross-linked matrix. Nearly 60% of cross-links involve the ends of molecules while the rest are scattered among internal sites. We discuss how the relative positions and proportions of intra- and intermolecular cross-links in extensin-1 may affect the structure, and in turn the function, of the extensin matrix.     

Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture

Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of -l-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid) of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240–300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif — Ser (Hyp)₄ — within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H₂O₂ was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.Abbreviations CM-cellulose carboxymethyl-cellulose - FPLC fast protein liquid chromatography - HF hydrogen fluoride - HRGP hydroxyproline-rich glycoprotein - Hyp hydroxyproline - V_c retention volume - TCA trichloroacetic acid - TFA tri-fluoroacetic acid This work was supported by a A.F.R.C. postdoctoral assistantship to Michael D. Brownleader. We thank Dr. Anthony K. Allen (Department of Biochemistry, Charing Cross and Westminster Hospital, London, UK) for performing the amino-acid analysis and Mrs. Margaret Pickering (Department of Biochemistry, Royal Holloway) for performing the peptide-sequence analysis of extensin. We also express our gratitide to Dr. A. Mort (Oklahoma State University) for performing the HF-deglycosylation of extensin.     

Immunogold localization of THRGP-like epitopes in the haustorial interface of obligate,biotrophic fungi on monocots

Summary Immunoelectron microscopy was used to determine the subcellular distribution of threonine-hydroxyproline-rich glycoprotein (THRGP) epitopes in host-parasite interactions between obligate, biotrophic fungi and cereals. Infection sites of stem rust (Puccinia graminis f. sp.tritici) and leaf rust (Puccinia recondita) on primary leaves of wheat (Triticum aestivum), as well as of powdery mildew (Erysiphe graminis f. sp.hordei) on coleoptiles of barley (Hordeum vulgare), wete probed with a polyclonal antiserum to maize THRGP. A few immunogold particles were found over the cell walls of wheat mesophyll tissue and barley coleoptile epidermis. Unlike previous examples in dicot plants, no enhanced accumulation of THRGP was observed in cereal cell walls adjacent to sites of pathogen ingress. Instead, the most pronounced accumulation of THRGP-like molecules occurred over the extrahaustorial matrix in both incompatible and compatible plant-pathogen interactions. For powdery mildew of barley, immunogold staining was distinctly increased over the center of the penetration sites; however, no labeling was found over papillae that formed during incompatible and compatible interactions. In addition, no cross-reactivity of the anti-THRGP antiserum with intercellularly growing rust pathogens was observed. The highly localized deposition of THRGP-like molecules in the extrahaustorial matrix suggests that the host plant establishes a modified barrier between itself and the pathogen.Abbreviations C chloroplast - EC plant epidermal cell - EM extrahaustorial membrane - EMA extrahaustorial matrix - GO Golgi body - GRP glycine-rich protein - HP high pressure - HRGP hydroxyprolinerich glycoprotein - Hyp hydroxyproline - LT low temperature - PBS phosphate-buffered saline - PBST PBS with Tween-20 - THRGP threonine-hydroxyproline-rich glycoprotein - VA vesicular arbuscular     

Differential accumulation of hydroxyproline-rich glycoproteins in bean root nodule cells infected with a wild-type strain or a C4-dicarboxylic acid mutant of Rhizobium leguminosarum bv. phaseoli

An antiserum raised against deglycosylated hydroxyproline-rich glycoproteins (HPGPs) from melon (Cucumis melo L.) was used to study the relationship between Rhizobium infection and induction of HRGPs in bean (Phaseolus vulgaris L.) root nodule cells infected with either the wild-type or a C₄-dicarboxylic acid mutant strain of Rhizobium leguminosarum bv. phaseoli. In effective nodules, where fixation of atmospheric dinitrogen is taking place, HRGPs were found to accumulate mainly in the walls of infected cells and in peribacteroid membranes surrounding groups of bacteroids. Internal ramifications of the peribacteroid membrane were also enriched in HRGPs whereas the peribacteroid space as well as the bacteroids themselves were free of these glycoproteins. In mutant-induced root nodules, HRGPs were specifically associated with the electron-dense, laminated structures formed in plastids as a reaction to infection by this mutant. The presence of HRGPs was also detected in the host cytoplasm. The aberrant distribution of HRGPs in infected cells of mutant-induced nodules likely reflects one aspect of the altered host metabolism in relation to peribacteroid-membrane breakdown. The possibility that the antiserum used for HRGP localization may have cross-reacted with ENOD 2 gene products is discussed in relation to amino-acid sequences and sites of accumulation.     

The O-Hyp glycosylation code in tobacco and Arabidopsis and a proposed role of Hyp-glycans in secretion

Most aspects of plant growth involve cell surface hydroxyproline (Hyp)-rich glycoproteins (HRGPs) whose properties depend on arabinogalactan polysaccharides and arabinosides that define the molecular surface. Potential glycosylation sites are defined by an O-Hyp glycosylation code: contiguous Hyp directs arabinosylation. Clustered non-contiguous Hyp directs arabinogalactosylation. Elucidation of this code involved a single species, tobacco (Nicotiana tabacum) BY-2 cells. However, recent work suggests species variation, perhaps tissue specific Hyp glycosylation. Thus, the extent to which the Hyp glycosylation code is 'global' needs testing. We compared the ability of distantly related Arabidopsis cell cultures to process putative HRGP glycosylation motifs encoded by synthetic genes. The genes included: repetitive Ser-Pro, Ser-Pro2, Ser-Pro4 and an analog of the tomato arabinogalactan-protein, LeAGP-1DeltaGPI. All were expressed as enhanced green fluorescent protein (EGFP) fusion glycoproteins, designated: AtSO-EGFP (O=Hyp), AtSO2-EGFP, AtSO4-EGFP and AtEGFP-LeAGP-1DeltaGPI, respectively. The Arabidopsis glycosylation patterns were essentially similar to those observed in Nicotiana: non-contiguous Hyp residues in AtSO-EGFP were glycosylated exclusively with arabinogalactan polysaccharides while contiguous Hyp in AtSO2-EGFP and AtSO4-EGFP was exclusively arabinosylated. Mixed contiguous and non-contiguous Hyp residues in AtEGFP-LeAGP-1DeltaGPI were also arabinosylated and arabinogalactosylated consistent with the code. However, slightly more arabinogalactosylated Hyp and less non-glycosylated Hyp in AtEGFP-LeAGP-1DeltaGPI than tobacco NtEGFP-LeAGP-1DeltaGPI suggested Arabidopsis prolyl hydroxylases have a slightly broader specificity. Arabidopsis Hyp-arabinogalactans differed from tobacco in decreased glucuronic acid content and lack of rhamnose. Yields of the EGFP fusion glycoproteins were dramatically higher than targeted EGFP lacking Hyp-glycomodules. This validates earlier suggestions that the glycosylation of proteins facilitates their secretion.     

Focus Issue on Plant Cell Walls: A Bioinformatics Approach to the Identification,Classification, and Analysis of Hydroxyproline-Rich Glycoproteins

Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall proteins that function in diverse aspects of plant growth and development. This superfamily consists of three members: hyperglycosylated arabinogalactan proteins (AGPs), moderately glycosylated extensins (EXTs), and lightly glycosylated proline-rich proteins (PRPs). Hybrid and chimeric versions of HRGP molecules also exist. In order to “mine” genomic databases for HRGPs and to facilitate and guide research in the field, the BIO OHIO software program was developed that identifies and classifies AGPs, EXTs, PRPs, hybrid HRGPs, and chimeric HRGPs from proteins predicted from DNA sequence data. This bioinformatics program is based on searching for biased amino acid compositions and for particular protein motifs associated with known HRGPs. HRGPs identified by the program are subsequently analyzed to elucidate the following: (1) repeating amino acid sequences, (2) signal peptide and glycosylphosphatidylinositol lipid anchor addition sequences, (3) similar HRGPs via Basic Local Alignment Search Tool, (4) expression patterns of their genes, (5) other HRGPs, glycosyl transferase, prolyl 4-hydroxylase, and peroxidase genes coexpressed with their genes, and (6) gene structure and whether genetic mutants exist in their genes. The program was used to identify and classify 166 HRGPs from Arabidopsis (Arabidopsis thaliana) as follows: 85 AGPs (including classical AGPs, lysine-rich AGPs, arabinogalactan peptides, fasciclin-like AGPs, plastocyanin AGPs, and other chimeric AGPs), 59 EXTs (including SP₅ EXTs, SP₅/SP₄ EXTs, SP₄ EXTs, SP₄/SP₃ EXTs, a SP₃ EXT, “short” EXTs, leucine-rich repeat-EXTs, proline-rich extensin-like receptor kinases, and other chimeric EXTs), 18 PRPs (including PRPs and chimeric PRPs), and AGP/EXT hybrid HRGPs.The genomics era has produced vast amounts of biological data that await examination. In order to “mine” such data effectively, a bioinformatics approach can be utilized to identify genes of interest, subject them to various in silico analyses, and extract relevant biological information on them from various public databases. Examination of such data produces novel insights with respect to the genes in question and can be used to facilitate and guide further research in the field. Such is the case here, where bioinformatics tools were developed to identify, classify, and analyze members of the Hyp-rich glycoprotein (HRGP) superfamily encoded by the Arabidopsis (Arabidopsis thaliana) genome.HRGPs are a superfamily of plant cell wall proteins that are subdivided into three families, arabinogalactan proteins (AGPs), extensins (EXTs), and Pro-rich proteins (PRPs), and extensively reviewed (Showalter, 1993; Kieliszewski and Lamport, 1994; Nothnagel, 1997; Cassab, 1998; José-Estanyol and Puigdomènech, 2000; Seifert and Roberts, 2007). However, it has become increasingly clear that the HRGP superfamily is perhaps better represented as a spectrum of molecules ranging from the highly glycosylated AGPs to the moderately glycosylated EXTs and finally to the lightly glycosylated PRPs. Moreover, hybrid HRGPs, composed of HRGP modules from different families, and chimeric HRGPs, composed of one or more HRGP modules within a non-HRGP protein, also can be considered part of the HRGP superfamily. Given that many HRGPs are composed of repetitive protein sequences, particularly the EXTs and PRPs, and many have low sequence similarity to one another, particularly the AGPs, BLAST searches typically identify only a few closely related family members and do not represent a particularly effective means to identify members of the HRGP superfamily in a comprehensive manner.Building upon the work of Schultz et al. (2002) that focused on the AGP family, a new bioinformatics software program, BIO OHIO, developed at Ohio University, makes it possible to search all 28,952 proteins encoded by the Arabidopsis genome and identify putative HRGP genes. Two distinct types of searches are possible with this program. First, the program can search for biased amino acid compositions in the genome-encoded protein sequences. For example, classical AGPs can be identified by their biased amino acid compositions of greater then 50% Pro (P), Ala (A), Ser (S), and Thr (T), as indicated by greater than 50% PAST. Similarly, arabinogalactan peptides (AG peptides) are identified by biased amino acid compositions of greater then 35% PAST, but the protein (i.e. peptide) must also be between 50 and 90 amino acids in length. Likewise, PRPs can be identified by a biased amino acid composition of greater then 45% PVKCYT. Second, the program can search for specific amino acid motifs that are commonly found in known HRGPs. For example, SP₄ pentapeptide and SP₃ tetrapeptide motifs are associated with EXTs, a fasciclin H1 motif is found in fasciclin-like AGPs (FLAs), and PPVX(K/T) (where X is any amino acid) and KKPCPP motifs are found in several known PRPs (Fowler et al., 1999). In addition to searching for HRGPs, the program can analyze proteins identified by a search. For example, the program checks for potential signal peptide sequences and glycosylphosphatidylinositol (GPI) plasma member anchor addition sequences, both of which are associated with HRGPs (Showalter, 1993, 2001; Youl et al., 1998; Sherrier et al., 1999; Svetek et al., 1999). Moreover, the program can identify repeated amino acid sequences within the sequence and has the ability to search for bias amino acid compositions within a sliding window of user-defined size, making it possible to identify HRGP domains within a protein sequence.Here, we report on the use of this bioinformatics program in identifying, classifying, and analyzing members of the HRGP superfamily (i.e. AGPs, EXTs, PRPs, hybrid HRGPs, and chimeric HRGPs) in the genetic model plant Arabidopsis. An overview of this bioinformatics approach is presented in Figure 1. In addition, public databases and programs were accessed and utilized to extract relevant biological information on these HRGPs in terms of their expression patterns, most similar sequences via BLAST analysis, available genetic mutants, and coexpressed HRGP, glycosyl transferase (GT), prolyl 4-hydroxylase (P4H), and peroxidase genes in Arabidopsis. This information provides new insight to the HRGP superfamily and can be used by researchers to facilitate and guide further research in the field. Moreover, the bioinformatics tools developed here can be readily applied to protein sequences from other species to analyze their HRGPs or, for that matter, any given protein family by altering the input parameters.Open in a separate windowFigure 1.Bioinformatics workflow diagram summarizing the identification, classification, and analysis of HRGPs (AGPs, EXTs, and PRPs) in Arabidopsis. Classical AGPs were defined as containing greater than 50% PAST coupled with the presence of AP, PA, SP, and TP repeats distributed throughout the protein, Lys-rich AGPs were a subgroup of classical AGPs that included a Lys-rich domain, and chimeric AGPs were defined as containing greater than 50% PAST coupled with the localized distribution of AP, PA, SP, and TP repeats. AG peptides were defined to be 50 to 90 amino acids in length and containing greater than 35% PAST coupled with the presence of AP, PA, SP, and TP repeats distributed throughout the peptide. FLAs were defined as having a fasciclin domain coupled with the localized distribution of AP, PA, SP, and TP repeats. Extensins were defined as containing two or more SP₃ or SP₄ repeats coupled with the distribution of such repeats throughout the protein; chimeric extensins were similarly identified but were distinguished from the extensins by the localized distribution of such repeats in the protein; and short extensins were defined to be less than 200 amino acids in length coupled with the extensin definition. PRPs were identified as containing greater than 45% PVKCYT or two or more KKPCPP or PVX(K/T) repeats coupled with the distribution of such repeats and/or PPV throughout the protein. Chimeric PRPs were similarly identified but were distinguished from PRPs by the localized distribution of such repeats in the protein. Hybrid HRGPs (i.e. AGP/EXT hybrids) were defined as containing two or more repeat units used to identify AGPs, extensins, or PRPs. The presence of a signal peptide was used to provide added support for the identification of an HRGP but was not used in an absolute fashion. Similarly, the presence of a GPI anchor addition sequence was used to provide added support for the identification of classical AGPs and AG peptides, which are known to contain such sequences. BLAST searches were also used to provide some support to our classification if the query sequence showed similarity to other members of an HRGP subfamily. Note that some AGPs, particularly chimeric AGPs, and PRPs were identified from an Arabidopsis database annotation search and that two chimeric extensins were identified from the primary literature as noted in the text.     

Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlamydomonas reinhardtii and two other Volvocales

The unicellular alga Chlamydomonas reinhardtii Dang, has a cell wall made entirely from hydroxyproline-rich glycoproteins (HRGPs). We recently employed a quantiative in vitro reconstitution system (Adair et al. 1987, J. Cell Biol. 105, 2373–2382) to assign outer-wall HRGPs of C. reinhardtii to specific sublayers, and describe the major interactions responsible for their assembly. Some of these interactions appear to involve relatively conserved HRGP domains, as evidenced by interspecific cell-wall reconstitution between C. reinhardtii and two multicellular Volvocales (Volvoxcarteri lyengar and Gonium pectorale Müller). In the present report we provide biochemical and immunological evidence that the outer cell-walls of V. carteri and G. pectorale both contain prominent HRGPs closely related to C. reinhardtii GP2. Identification of conserved GP2 homologues indicates a molecular basis for interspecific reconstitution and provides a useful avenue for characterization of HRGP domains mediating cell-wall formation in these algae.Abbreviations GP1, 2, 3 outer-cell wall glycoproteins 1, 2, and 3 - GP2_dg deglycosylated GP2 - HRGP hydroxyprolinerich glycoprotein - SDS-PAGE sodium docecyl sulfate polyacrylamide gel electrophoresis