Fractionation and Structural Characterization of Arabinogalactan-Proteins from the Cell Wall of Rose Cells

Arabinogalactan-proteins (AGPs) have been purified from Paul's Scarlet rose (Rosa sp.) cell walls. As estimated by gel permeation chromatography, the apparent molecular masses of the two major cell-wall AGP fractions were 130 and 242 kD. Since the 130-kD AGP had a ratio of arabinose/glucuronic acid that was 12 times higher than that of the 242-kD AGP, the fractions were named cell-wall AGP1 (CW-AGP1) and glucuronogalactan-protein (GGP), respectively. CW-AGP1 and GGP contained predominantly t-arabinofuranosyl residues; 3-linked, 6-linked, and 3,6-branched galactopyranosyl residues; and 4-linked and t-glucuronopyranosyl residues. The 1H-nuclear magnetic resonance spectra of CW-AGP1 and GGP showed that the arabinofuranosyl and galactopyranosyl residues were predominantly in [alpha]- and [beta]-anomeric configuration, respectively, and that GGP contained a few O-acetyl residues. The protein moieties of CW-AGP1 and GGP were both rich in hydroxyproline and alanine but differed in the percentage of various amino acids, including hydroxyproline, alanine, serine, and glycine. Cell-wall AGPs bound to ([beta]-D-glucosyl)3 Yariv phenylglycoside, but the stoichiometry of binding was about 6 times greater in GGP than in other Rosa AGPs. GGP seems to be peculiar to the cell wall, since no similar molecule was found in the culture medium.     

Arabinogalactan-proteins from the suspension culture medium and plasma membrane of rose cells

Arabinogalactan-proteins (AGPs) that bind to beta-glycosyl Yariv antigens have been purified from the culture medium and plasma membrane of "Paul's Scarlet" rose cells. Starting from culture medium or from plasma membrane vesicles prepared by aqueous two-phase partitioning, the purification procedure involved Yariv antigen-induced precipitation and subsequent chromatographic steps. Two fractions, AGP-(a) and AGP-(b), were obtained from the culture medium, and one AGP fraction was obtained from the plasma membrane. The glycosyl compositions of all three fractions were dominated by arabinosyl and galactosyl residues and included glucuronosyl and other minor residues. Methylation analysis showed that AGP-(a) and AGP-(b) were both highly branched 3,6-galactans with terminal arabinofuranosyl substituents. The amino acid compositions of all three AGPs were high in alanine, hydroxyproline, and serine and/or threonine. The amino-terminal sequence of AGP-(b) contained an alanine-hydroxyproline repeat. While sharing general structural similarity, the AGPs from the plasma membrane and the culture medium were distinguishable by composition and by size and charge, with the plasma membrane AGPs being larger and more negatively charged than the culture medium AGPs.     

Localization of arabinogalactan proteins in the membrane system of etiolated hypocotyls of Phaseolus vulgaris L.

The subcellular distribution of arabinogalactan protein (AGP) in etiolated bean hypocotyls was studied by isopycnic density centrifugation on sucrose gradients at different Mg²⁺ concentrations. The distribution of hydroxyproline (a major amino acid in AGP) in the membrane-containing fractions indicated that hydroxyproline-containing proteins were associated with rough endoplasmic reticulum, possibly with the Golgi apparatus, and with the plasma membrane. Non-specific binding of hydroxyproline-containing molecules to membranes could be excluded. To detect AGPs, fractions obtained after isopycnic density centrifugation were isoelectrofocused on polyacrylamide gels, and the gels were stained with β-Gal-Yariv reagent. Bands appeared only at low pH values, where also most hydroxyproline was found. In the fractions at low densities (presumably membranefree), several bands were visible supporting the idea of the heterogeneous character of soluble AGP. The distribution of AGP in the membranous fractions strongly indicated that AGP was associated with the plasma membrane. Specific agglutination of protoplasts in the presence of β-Gal-Yariv reagent indicated that AGP was exposed at the outside of the cell membrane.     

A Novel Hydroxyproline-Deficient Arabinogalactan Protein Secreted by Suspension-Cultured Cells of Daucus carota (Purification and Partial Characterization)

Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to [beta]-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, [beta]-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.     

Molecular interactions of arabinogalactan proteins with cortical microtubules and F-actin in Bright Yellow-2 tobacco cultured cells

Arabinogalactan proteins (AGPs), a superfamily of plant hydroxyproline-rich glycoproteins, are present at cell surfaces. Although precise functions of AGPs remain elusive, they are widely implicated in plant growth and development. A well-characterized classical tomato (Lycopersicon esculentum) AGP containing a glycosylphosphatidylinositol plasma membrane anchor sequence was used here to elucidate functional roles of AGPs. Transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells stably expressing green fluorescent protein (GFP)-LeAGP-1 were plasmolysed and used to localize LeAGP-1 on the plasma membrane and in Hechtian strands. Cytoskeleton disruptors and beta-Yariv reagent (which binds and perturbs AGPs) were used to examine the role of LeAGP-1 as a candidate linker protein between the plasma membrane and cytoskeleton. This study used a two-pronged approach. First, BY-2 cells, either wild type or expressing GFP-microtubule (MT)-binding domain, were treated with beta-Yariv reagent, and effects on MTs and F-actin were observed. Second, BY-2 cells expressing GFP-LeAGP-1 were treated with amiprophosmethyl and cytochalasin-D to disrupt MTs and F-actin, and effects on LeAGP-1 localization were observed. beta-Yariv treatment resulted in terminal cell bulging, puncta formation, and depolymerization/disorganization of MTs, indicating a likely role for AGPs in cortical MT organization. beta-Yariv treatment also resulted in the formation of thicker actin filaments, indicating a role for AGPs in actin polymerization. Similarly, amiprophosmethyl and cytochalasin-D treatments resulted in relocalization of LeAGP-1 on Hechtian strands and indicate roles for MTs and F-actin in AGP organization at the cell surface and in Hechtian strands. Collectively, these studies indicate that glycosylphosphatidylinositol-anchored AGPs function to link the plasma membrane to the cytoskeleton.     

Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the beta-glucosyl Yariv reagent

An arabinogalactan-protein (AGP) from pressed juice of Echinacea purpurea herb was isolated from a high molecular weight fraction by precipitation with the beta-glucosyl Yariv reagent, followed by gel-permeation chromatography. It revealed characteristic features of other AGPs: i.e., a high amount of polysaccharide (83%) with a ratio of galactose to arabinose of 1.8:1, some uronic acids (4-5%), and a low protein content (7%) with high levels of serine, alanine and hydroxyproline. The molecular weight was estimated to be 1.2 x 10(6) Da. Linkage and 13C NMR analyses showed that the AGP is composed of a highly branched core polysaccharide of 3-, 6-, and 3,6-linked Galp residues with terminal Araf, GlcAp and terminal units of Araf-(1-->5)-Araf-(1-->. Partial acid hydrolysis resulted in loss of Araf residues at the periphery of the molecule. Complete loss of reactivity toward the beta-glucosyl Yariv antigen was then noticed.     

Functional Identification of a Hydroxyproline-O-galactosyltransferase Specific for Arabinogalactan Protein Biosynthesis in Arabidopsis

Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [¹⁴C]Gal from UDP-[¹⁴C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)₇ and anhydrous hydrogen fluoride-deglycosylated d(AO)₅₁. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [¹⁴C]Gal to (AO)₇, and the resulting product co-eluted with (AO)₇ by reverse-phase HPLC. Acid hydrolysis of the [¹⁴C]Gal-(AO)₇ product released ¹⁴C-radiolabel as Gal only. Base hydrolysis of the [¹⁴C]Gal-(AO)₇ product released a ¹⁴C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg²⁺ or Mn²⁺ for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> arabinogalactan proteins contain abundant terminal 3-<Emphasis Type="Italic">O</Emphasis>-methyl-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosyl residues not found in angiosperms

A biochemical investigation of arabinogalactan proteins (AGPs) in Physcomitrella patens was undertaken with particular emphasis on the glycan chains. Following homogenization and differential centrifugation of moss gametophytes, AGPs were obtained by Yariv phenylglycoside-induced precipitation from the soluble, microsomal membrane, and cell wall fractions. Crossed-electrophoresis indicated that each of these three AGP fractions was a mixture of several AGPs. The soluble AGP fraction was selected for further separation by anion-exchange and gel-permeation chromatography. The latter indicated molecular masses of ∼100 and 224 kDa for the two major soluble AGP subfractions. The AGPs in both of these subfractions contained the abundant (1,3,6)-linked galactopyranosyl residues, terminal arabinofuranosyl residues, and (1,4)-linked glucuronopyranosyl residues that are typical of many angiosperm AGPs. Unexpectedly, however, the moss AGP glycan chains contained about 15 mol% terminal 3-O-methyl-l-rhamnosyl residues, which have not been found in angiosperm AGPs. This unusual and relatively nonpolar sugar, also called l-acofriose, is likely to have considerable effects on the overall polarity of Physcomitrella AGPs. A review of the literature indicates that the capacity to synthesize polymers containing 3-O-methyl-l-rhamnosyl residues is present in a variety of bacteria, algae and lower land plants but became less common through evolution to the extent that this sugar has been found in only a few species of angiosperms where it occurs as a single residue on steroidal glycosides.     

Isolation of the protein backbone of an arabinogalactan-protein from the styles of Nicotiana alata and characterization of a corresponding cDNA.

Arabinogalactan-proteins (AGPs) from the styles of Nicotiana alata were isolated by ion exchange and gel filtration chromatography. After deglycosylation by anhydrous hydrogen fluoride, the protein backbones were fractionated by reversed-phase HPLC. One of the protein backbones, containing mainly hydroxyproline, alanine, and serine residues (53% of total residues), was digested with proteases, and the peptides were isolated and sequenced. This sequence information allowed the cloning of a 712-bp cDNA, AGPNa1. AGPNa1 encodes a 132-amino acid protein with three domains: an N-terminal secretion signal sequence, which is cleaved from the mature protein; a central sequence, which contains most of the hydroxyproline/proline residues; and a C-terminal hydrophobic region. AGPNa1 is expressed in many tissues of N. alata and related species. The arrangement of domains and amino acid composition of the AGP encoded by AGPNa1 are similar to that of an AGP from pear cell suspension culture filtrate, although the only sequence identity is at the N termini of the mature proteins.     

Immunolocalization of LM2 arabinogalactan protein epitope associated with endomembranes of plant cells

Summary Arabinogalactan proteins (AGPs) are proteoglycans detected in high amounts at plant cell surfaces; however, details of their subcellular localization are largely unknown. Immunolocalization studies with the anti-AGP monoclonal antibody LM2 have indicated that this AGP epitope is associated with secretory compartments such as endoplasmic reticulum and Golgi apparatus within plant cells actively producing and secreting AGPs. The LM2 epitope contains a -linked glucuronic acid residue and occurs in the polysaccharide moiety of AGPs. We have localized this AGP epitope also to the tonoplast and to cytoplasmic strands. Endomembrane association of AGPs was confirmed with two other monoclonal antibodies, JIM13 and MAC207, both reacting with carbohydrate AGP epitopes containing GlcpA-(13)-D-GalpA-(12)-L-Rha residues. Immunocytochemistry is supported by biochemical analysis which shows that LM2 reacts with the microsomal fraction and also with low-molecular-weight material of the detergent phase after Triton X-114 phase separation prepared from maize roots. Our results indicate that some AGP epitopes are closely associated with endomembranes.Abbreviations AGP arabinogalactan protein - ER endoplasmic reticulum - GlcA glucuronic acid Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of Pyrus communis and Nicotiana alata

This paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGP Pc 2) and the other from suspension cultures of Nicotiana alata (AGP Na 2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGP Pc 2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGP Na 2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Ser, Ala, Thr and the other rich in Asn, Tyr and Ser. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the 'classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well as differential glycosylation, can contribute to the tissue specificity of AGPs.     

NaAGP4 is an arabinogalactan protein whose expression is suppressed by wounding and fungal infection inNicotiana alata

Arabinogalactan proteins (AGPs) are proteoglycans secreted by plant cells that have been implicated in plant growth and development. Most AGPs cloned to date possess highly labile glycosylphosphatidylinositol (GPI) lipid anchors. These anchors transiently attach AGPs to the plasma membrane before they are released into the cell wall following GPI anchor hydrolysis. We have isolated and partially sequenced the protein core of an AGP purified from styles of Nicotiana alata. The protein sequence data were utilised to clone the AGP's gene, NaAGP4. This AGP shares about 78% sequence identity with the tomato AGP LeAGP-1. RNA gel blot analyses of different plant organs indicate that NaAGP4 is expressed in the same tissues and at similar levels as LeAGP-1. Furthermore, NaAGP4 like LeAGP-1 is rapidly suppressed by tissue wounding and by pathogen infection. We believe NaAGP4 and LeAGP-1 are the first described examples of orthologous AGPs from different plant species. In contrast, another AGP from N. alata, NaAGP1, is comparatively unaffected by wounding and pathogen infection, although this AGP is expressed in similar tissues and at similar levels as NaAGP4.     

An agarose gel electrophoresis method for the separation of arabinogalactan proteins

A method for resolving plasma membrane associated arabinogalactan proteins (AGPs) has been developed. Plasma membranes purified by aqueous polymer two-phase partitioning were first subjected to Triton X-114 fractionation. The resulting water phase contained all detectable plasma membrane-bound AGPs. Plasma membrane AGPs were then resolved in an SDS-agarose gel electrophoresis system (SDS-AGE). For separating plasma membrane AGP species of the same apparent molecular weight but with different net charge, a two-dimensional electrophoresis system was used, utilizing isoelectric focusing in an immobilized pH gradient in the first dimension and SDS-AGE in the second dimension. These methods enabled the separation of individual plasma membrane AGPs. In comparison, SDS-PAGE methods left AGPs as unresolved high molecular-weight smears. The methods described here may help to establish some basic features of AGPs, such as the number, organization, and protein and carbohydrate characteristics of plasma membrane AGPs, as well as the relationship between plasma membrane and extracellular AGPs.     

Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.     

The lysine-rich arabinogalactan-protein subfamily in Arabidopsis: gene expression, glycoprotein purification and biochemical characterization

AtAGP17, AtAGP18 and AtAGP19 are homologous genes encoding three putative glycosylphosphatidylinositol (GPI)-anchored classical arabinogalactan-proteins (AGPs) in Arabidopsis. They are distinguished from other AGPs by a short, C-terminal lysine-rich region. Organ-specific expression of these genes was revealed by Northern blot analysis. AtAGP17 was strongly expressed in leaves and stems, and weakly expressed in flowers and roots; AtAGP18 was strongly expressed in flowers, and moderately expressed in roots, stems and young leaves; and AtAGP19 was strongly expressed in stems, moderately expressed in flowers and roots, and weakly expressed in young leaves. One of these genes, AtAGP17, was expressed and purified as a green fluorescent protein (GFP) fusion protein in transgenic tobacco cells using hydrophobic interaction chromatography, size exclusion chromatography and reverse phase high-performance liquid chromatography. The fusion (glyco)protein produced a characteristic AGP 'smear' with a molecular mass of 80-150 kDa when detected by Western blot analysis. Glycosyl composition and linkage analyses of purified GFP-AtAGP17 showed that carbohydrate accounted for approximately 86% of the molecule, with arabinose and galactose as major, and rhamnose and glucuronic acid as minor glycosyl residues and with 1,3,6-galactose, 1,4-glucuronic acid, 1,3-galactose and terminal arabinose as major linkages. GFP-AtAGP17 was also precipitated by beta-Yariv reagent, further confirming that AtAGP17 is a bona fide AGP. Confocal fluorescence microscopy of plasmolysed, transformed cells indicated that AtAGP17 is localized on the plasma membrane and in Hechtian strands. Hydroxyproline (Hyp) glycoside profiles of GFP-AtAGP17 in conjunction with the deduced protein sequence also served to corroborate the Hyp contiguity hypothesis, which predicts contiguous Hyp residues as attachment sites for arabinosides and clustered, non-contiguous Hyp residues as attachment sites for arabinogalactan polysaccharides.     

Arabinogalactan proteins in root and pollen-tube cells: distribution and functional aspects

BACKGROUND: Arabinogalactan proteins (AGPs) are complex proteoglycans of the cell wall found in the entire plant kingdom and in almost all plant organs. AGPs encompass a large group of heavily glycosylated cell-wall proteins which share common features, including the presence of glycan chains especially enriched in arabinose and galactose and a protein backbone particularly rich in hydroxyproline residues. However, AGPs also exhibit strong heterogeneities among their members in various plant species. AGP ubiquity in plants suggests these proteoglycans are fundamental players for plant survival and development. SCOPE: In this review, we first present an overview of current knowledge and specific features of AGPs. A section devoted to major tools used to study AGPs is also presented. We then discuss the distribution of AGPs as well as various aspects of their functional properties in root tissues and pollen tubes. This review also suggests novel directions of research on the role of AGPs in the biology of roots and pollen tubes.     

Arabinogalactan proteins during the development of soybean root nodules

In soybean (Glycine max (L.) Merr.) root nodules the level of hydroxyproline-containing molecules is developmentally regulated. Hydroxyproline accumulates in both nodule cortex and medulla. In the cortex, the hydroxyproline is mainly localized in the cell wall, presumably as extensin, but in the medulla it is mainly in the soluble fraction as an arabinogalactan protein (AGP). Nodule-specific AGPs are present at early nodulation. The highest concentration of AGP is in the nodule medulla, followed by nodule cortex, uninfected roots, leaves, flowers, pods and seeds. Root nodules and all organs of the soybean plant that were tested were found to express a tissue-specific set of arabinogalactan proteins.Abbreviation AGP Arabinogalactan protein     

Arabinogalactan proteins are required for apical cell extension in the moss Physcomitrella patens

Cell biological, structural, and genetic approaches have demonstrated the presence of arabinogalactan proteins (AGPs) in the moss Physcomitrella patens and provided evidence for their function in cell expansion and specifically in the extension of apical tip-growing cells. Inhibitor studies indicated that apical cell expansion in P. patens is blocked by synthetic AGP binding beta-glucosyl Yariv reagent (betaGlcYR). The anti-(1-->5)-alpha-L-arabinan monoclonal antibody LM6 binds to some AGPs in P. patens, to all plasma membranes, and to the cell wall surface at the most apical region of growing protonemal filaments. Moreover, LM6 labeling of cell walls at the tips of apical cells of P. patens was abolished in the presence of betaGlcYR, suggesting that the localized movement of AGPs from the plasma membrane to the cell wall is a component of the mechanism of tip growth. Biochemical and bioinformatic analyses were used to identify seven P. patens ESTs encoding putative AGP core proteins from homology with Arabidopsis thaliana, Brassica napus, and Oryza sativa sequences and from peptide fragments isolated from betaGlcYR-precipitated AGPs. Gene knockout by homologous recombination of one of these genes, P. patens AGP1, encoding a classical AGP core protein, resulted in reduced cell lengths in protonemal filaments, indicating a role for AGP1 in apical cell expansion in P. patens.     

Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation

Two polymorphic forms of an extracellular arabinogalactan protein (AGP1 and AGP2), obtained from the conditioned media of two carrot suspension-cultured cell lines, have been identified in terms of binding of the anti-plasma membrane antibodies JIM4 and MAC207. AGP1 and AGP2 have been used as immunogens to generate further anti-AGP monoclonal antibodies. JIM14 identified an epitope carried by AGP2 and also by glycoproteins of low molecular weight localized to the plant cell wall. In addition, further antibodies (JIM13 and JIM15) identified carbohydrate epitopes of the AGPs that also occur on plasma membrane glycoproteins and are expressed by patterns of cells that reflect cell position at the carrot root apex. Indirect immunofluorescence microscopy indicated that JIM13 recognized the surface of cells forming the epidermis and cells marking the region and axis of the future xylem. JIM15 recognized a pattern of cells directly complementary to the JIM13 pattern. The panel of anti-AGP monoclonal antibodies now available indicates groups of cells within the root meristem that may reflect an early pre-pattern of the tissues of the mature root structure and suggests extensive modulation of cell surface AGPs during cell development and the positioning of cells within the apex.     

Displacement currents associated with the insertion of Alzheimer disease amyloid beta-peptide into planar bilayer membranes

The role of endogenous amyloid beta-peptides as causal factors of neurodegenerative diseases is largely unknown. We have previously reported that interactions between Alzheimer's disease A beta P[1-40] peptide in solution and planar bilayer membranes made from anionic phospholipids lead to the formation of cation-selective channels. We now find and report here that the spontaneous insertion of free A beta P[1-40] across the bilayer can be detected as an increase in bilayer capacity. To this end we recorded the displacement currents across planar bilayers (50 mM KCl on both sides) in response to sudden displacements of the membrane potential, from -300 to 300 mV in 20-mV increments. To monitor the A beta P[1-40]-specific displacement currents, we added A beta P[1-40] (1-5 microM) to the solution on either side of the membrane and noted that the direction of the displacement current depended on the side with A beta P[1-40]. The size of the A beta P[1-40]-specific charge displaced during a pulse was always equal to the charge returning to the original configuration after the pulse, suggesting that the dipole molecules are confined to the membrane. As a rule, the steady-state distribution of the A beta P[1-40]-specific charges within the bilayer could be fit by a Boltzmann distribution. The potential at which the charges were found to be equally distributed (V(o)) were approximately -135 mV (peptide added to the solution in the compartment electrically connected to earth) and 135 mV (peptide added to the solution connected to the input of the amplifier). The A beta P[1-40]-specific transfer of charge reached a maximum value (Q(max)) when the electrical potential of the side containing the amyloid beta-protein was taken to either -300 or 300 mV. For a circular membrane of 25-microm radius ( approximately 2000 microm(2)), the total A beta P[1-40]-specific charge Q(max) was estimated as 55 fC, corresponding to some 170 e.c./microm(2). Regardless of the side selected for the addition of A beta P[1-40], at V(o) the charge displaced underwent an e-fold change for a approximately 27-mV change in potential. The effective valence (a) of the A beta P[1-40] dipole (i.e., the actual valence Z multiplied by the fraction of the electric field chi acting on the dipole) varied from 1 to 2 electronic charges. We also tested, with negative results, the amyloid peptide with the reverse sequence (A beta P[40-1]). These data demonstrate that A beta P[1-40] molecules can span the low dielectric domain of the bilayer, exposing charged residues (D(1), E(3), R(5), H(6), D(7), E(11), H(13), and H(14)) to the electric field. Thus the A beta P[1-40] molecules in solution must spontaneously acquire suitable conformations (beta-pleated sheet) allowing specific interactions with charged phospholipids. Interestingly, the domain from residues 676 to 704 in the APP(751) is homologous with the consensus sequence for lipid binding found in other membrane proteins regulated by anionic phospholipids.