Hydroxyproline: its asymmetric distribution in a cell wall glycoprotein

The main structural glycoprotein of the cell wall of Chlamydomonas reinhardii has been cleaved by thermolysin into glycopeptides which have been separated into three fractions, T1, T2 and T3. These correspond to three distinct domains within the glycoprotein, characterized by the asymmetric distribution of both sugars and amino acids, in particular hydroxyproline. T2 is very rich in hydroxyproline (43 mol %) and is highly glycosylated, while T3 is poor in hydroxyproline and contains very little carbohydrate. The results are discussed in terms of cell wall glycoproteins and their function.Abbreviations PAGE polyacrylamide gel electrophoresis - Tris Tris(hydroxymethyl)-methylamine - SDS Sodium dodecyl sulphate - PAS periodic acid-Schiff This is the seventh paper in a series entitled Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. The last paper in this series was Catt et al. (1978)     

Reinforced Polyproline II Conformation in a Hydroxyproline-Rich Cell Wall Glycoprotein from Carrot Root

The salt-extractable hydroxyproline-rich cell wall glycoprotein from carrot (Daucus carota L.) roots is composed of 35% (w/w) protein, 3% (w/w) galactose, and 62% (w/w) arabinose. The arabinose is attached to hydroxyproline as tetra- and trisaccharides. The circular dichroism of the glycoprotein shows that it is completely in the polyproline II conformation. After deglycosylation of the glycoprotein, the polyproline II conformation of the peptide backbone was lost. This indicates that the carbohydrate reinforces the polyproline II conformation.     

Assembly of cell-wall glycoproteins of Chlamydomonas reinhardii: Oligosaccharides are added in medial and trans Golgi compartments

A series of monoclonal antibodies and a polyclonal antiserum have been used to investigate the localisation and pathway of biosynthesis of the cell-wall hydroxyproline-rich glycoprotein 2BII in the alga Chlamydomonas reinhardii. Glyco-protein precursors were detected within the endoplasmic reticulum using a polyclonal antiserum raised to the deglycosylated 2BII. Monoclonal antibodies which are known to recognise different carbohydrate epitopes of 2BII were found to label two distinct regions of the Golgi stack. The immunolabelling results demonstrate that there is compartmentation of protein synthesis and glycosylation steps for these O-glycosidically linked glycoproteins. Newly synthesised glycoproteins are transported from the Golgi apparatus to the cell surface via two distinct routes. They then undergo assembly into a cell wall, the inner wall layer being formed first and probably functionaing as a template within which the outer crystalline wall layers are assembled.Abbreviations DGP deglycosylated glycoprotein - ER endoplasmic reticulum - MAC monoclonal antibody centre - M _r relative molecular mass     

Cell wall glycoproteins from Chlamydomonas reinhardii,and their self-assembly

We have investigated the in vitro reassembly of the salt soluble, hydroxyproline rich, glycoproteins from the cell wall of Chlamydomonas reinhardii, into structured cell wall fragments. We have devised an assay which has been used to follow the reassembly of the unfractionated and fractionated (2BI and 2BII) cell wall glycoproteins. Reassembly has a pH optimum of 5, a temperature optimum of 20°C, and the final size of the reassembled fragments appears to be promoted by the minor component 2BI. Periodate oxidation experiments show that sugar residues, in particular mannose, are important for accurate reassembly. Using electron microscopy, the structure of the reassembled products has been elucidated, as have intermediate stages in the reassembly process.Abbreviations TRIS Tris(hydroxymethyl)-methylamine - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis This is the fifth paper in a series entitled Structure composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. The last paper in this series was Catt et al. (1976)     

Cell wall glycoproteins from Chlamydomonas reinhardii are sulphated

Abstract Both the two major structural cell wall glycoproteins and the soluble excreted glycoproteins of Chlamydomonas reinhardii Levine WT II/32 contain low levels (approx. 1–4%) of sugar O-sulphate esters, asymmetrically distributed within the molecules. Preliminary characterization of their structure is described through [³⁵S] sulphate labelling experiments. The function of the sulphated glycoproteins is discussed in terms of their structural role and their water retaining properties.     

Monoclonal antibodies to the major structural glycoprotein of the Chlamydomonas cell wall

A family of monoclonal antibodies (McAb) has been raised to the major cell-wall structural glycoprotein of the green alga Chlamydomonas reinhardii. The work represents an approach using McAb to the structure and function of plant cell wall-components. On the basis of cross-competition assays, the McAb have been subdivided into six groups. Various lines of evidence indicate that some of these group's e.g. group III, contain McAb which recognise specific oligosaccharide side chains of the glycoprotein. Heterogeneity of the oligosaccharide side chains is demonstrated, by probing Western blots of separated glycoproteins and glycopeptides with the McAb. The results are discussed in relation to the possibility of raising McAb as probes for the structure and function of higher-plant cell-wall oligosaccharides, which are increasingly being shown to be important in cell-cell recognition phenomena and host-pathogen interactions.Abbreviations McAb monoclonal antibody (bodies) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Protein conformation of potato (Solanum tuberosum) lectin determined by circular dichroism.

The structure of potato (Solanum tuberosum) lectin, which is a hydroxyproline-rich glycoprotein, has been investigated by circular dichroism. The spectra of the native lectin, and of the oxidized, reduced and carboxymethylated and deglycosylated derivatives were examined, as was a hydroxyproline-rich glycopeptide and its deglycosylated derivative. It is concluded that the lectin contains about 35% polyproline II conformation, 34% type II beta-turn and 31% irregular conformation. No indications were found for the presence of alpha-helix or beta-sheet conformations. The polyproline II conformation is heat-stable, but is markedly destabilized by deglycosylation. The type II beta-turn is destabilized by cleavage of disulphide bonds.     

Methylation analysis of cell wall glycoproteins and glycopeptides from Chlamydomonas reinhardii

Cell wall glycoproteins from Chlamydomonas reinhardii and the glycopeptides produced by the action of thermolysin were subjected to standard methylation analysis. GC-MS of the methylated alditol acetates revealed short oligosaccharides some of which show branching. O-glycosidically linked galactofuranosyl residues are present. The asymmetric distribution of the major O-glycosidic linkages is also reported.     

The lithium-chloride-soluble cell-wall layers of Chlamydomonas reinhardii contain several immunologically related glycoproteins

Cell-wall glycoproteins of the unicellular green alga Chlamydomonas reinhardii have been purified from LiCl extracts of intact cells by gel exclusion chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised against several polypeptide components isolated from the LiCl extracts. All these antibodies specifically reacted with the cell surface of formaldehyde-fixed cells. They showed cross-reactivity with the different antigens and were also reactive against some other polypeptides present in the LiCl extracts of intact wild-type cells as shown by double-diffusion assays and immunoblot analyses. These antigens were largely missing in LiCl extracts from the cell-wall-deficient mutant CW-15. The pattern of immunologically related cell-wall polypeptides of C. reinhardii varied during the vegetative cell cycle and was found to be also dependent on the growth conditions. Dot-immunobinding assays on chemically modified cell-wall glycoproteins demonstrated differences between the various antibodies with respect to their specificities. Differences were observed especially with respect to their reactivities against chemically deglycosylated cell-wall polypeptides. Chemical deglycosylation generally reduced the binding of the different antibodies indicating that all these antibodies recognize carbohydrate side chains. Only two of these antibody preparations, raised against cell-wall glycoproteins of relative molecular mass 35 and 150 kilodaltons, were found to be strongly reactive against deglycosylated cell-wall polypeptides. When these antibodies were saturated with cell-wall-derived glycopeptides in order to abolish the binding to carbohydrate side chains, they still recognized the same cell-wall polypeptides as did the untreated antibodies. These findings indicate that the cross-reactivity of the different cell-wall polypeptides with the antibodies is not exclusively the consequence of similar glycosylation patterns but is also the result of the presence of similar structures within the non-glycosylated stretches of the polypeptide backbones. Cell walls isolated from growing tobacco pollen tubes contained a single polypeptide component which showed crossreactivity with the antibodies to the cell-wall glycoproteins of C. reinhardii.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - kDa kilodalton - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

A structural glycoprotein,containing hydroxyproline,isolated from the cell wall of Chlamydomonas reinhardii

Summary A soluble extract from purified cell walls of C. reinhardii has been separated by gel filtration into three fractions which together account for 94% of the cell wall. The major fraction (accounting for 70% of the extract) is a glycoprotein, with a molecular wt. in sodium perchlorate of 298,000, which can be split into 4 electrophoretically distinct species. It contains 35% protein with high levels of hydroxyproline, arabinose and galactose, and is capable of self assembly into crystalline structures identical to those found within the cell wall. The second fraction (25% of the extract) is a similar glycoprotein, but contains 24% protein, a higher proportion of mannose, and is incapable of self assembly. The third fraction (3–6% of the extract) is shown to be an adsorbed impurity from the growth medium used.Abbreviations CB or CBB coomassie brilliant blue - PAS periodic acid Schiff's reagent - PPO 2,5 diphenyloxazole - PO-POP 1,4-di[2-(5phenyloxazolyl]benzene - SDS sodium dodecyl sulphate This is the fourth paper in a series entitled Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. The last paper in this series was Hills et al. (1975)     

Membrane glycoproteins of Chlamydomonas eugametos flagella

The flagellar glycoproteins exposed on Chlamydomonas eugametos gametes were labeled by means of lactoperoxidase, diiodosulfanilic acid and chloramine T, and characterised in SDS-electrophoresis gels. The medium from gamete cultures contains particles (isoagglutinins) that agglutinate gametes of the opposite mating type. When crude preparations of these particles were subjected to isopycnic centrifugation in a caesium chloride gradient, two bands of particles were found. The lighter, active band consisted of membrane vesicles. The denser, inactive band consisted of cell wall material. The active band had the same glycoprotein composition as membrane vesicles artificially made from isolated flagella. Preparations of glagella were also separated on a caesium chloride cushion into pure flagella and cell wall material. The flagella, but not the cell wall material, isoagglutinated opposite gametes. Again the glycoprotein composition of pure flagella was similar to that of pure isoagglutinin vesicles. No difference was detected between the protein and glycoprotein compositions of flagella and isoagglutinins from both mating types.Abbreviations LPO lactoperoxidase - PB phosphate buffer - DISA diazotized ¹²⁵I-iodo-sulfanilic acid - SDS sodium dodecyl sulphate - CBD coomassie Brilliant Blue - PAS periodic acid Schiff     

Evidence for a gamma-turn motif in antifreeze glycopeptides.

Knowledge of the secondary structure of antifreeze peptides (AFPs) and glycopeptides (AFGPs) is crucial to understanding the mechanism by which these molecules inhibit ice crystal growth. A polyproline type II helix is perhaps the most widely accepted conformation for active AFGPs; however, random coil and alpha-helix conformations have also been proposed. In this report we present vibrational spectroscopic evidence that the conformation of AFGPs in solution is not random, not alpha-helical, and not polyproline type II. Comparison of AFGP amide vibrational frequencies with those observed and calculated for beta and gamma-turns in other peptides strongly suggests that AFGPs contain substantial turn structure. Computer-generated molecular models were utilized to compare gamma-turn, beta-turn, and polyproline II structures. The gamma-turn motif is consistent with observed amide frequencies and results in a molecule with planar symmetry with respect to the disaccharides. This intriguing conformation may provide new insight into the unusual properties of AFGPs.     

14-3-3 proteins are constituents of the insoluble glycoprotein framework of the chlamydomonas cell wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists predominantly of Hyp-rich glycoproteins, which also occur in the extracellular matrix of multicellular green algae and higher plants. In addition to the Hyp-rich polypeptides, the insoluble glycoprotein framework of the Chlamydomonas cell wall contains minor amounts of 14-3-3 proteins, as revealed by immunochemical studies and mass spectroscopic analysis of tryptic peptides. Polypeptides immunologically related to the 14-3-3 proteins also were found in the culture medium of Chlamydomonas. The levels of two of these 14-3-3-related polypeptides were decreased in the culture medium of the wall-deficient mutant cw-15. These findings indicate that 14-3-3 proteins are involved in the cross-linking of Hyp-rich glycoproteins in the Chlamydomonas cell wall.     

Extensin—a major cell wall glycoprotein

Abstract The structure of extensin is described in detail. It has a hydroxyproline-rich backbone, which contains repeating peptides glycosylated by short side chains and it adopts a polyproline II helical conformation. The glycoprotein is synthesized intracellularly and soluble precursors are secreted to the wall, where they are bound, perhaps, by the formation of isodityrosine cross-links. The various hypotheses, including the most recent ‘warp and weft’ model, which have been suggested to explain the attachment of extensin to the other wall polymers are discussed. The possible functions of extensin in defence and in the control of extension growth are described in addition to its probable structural role. Other glycoproteins which resemble extensin are also mentioned.     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

The zygote cell wall of Chlamydomonas reinhardii: a structural,chemical and immunological approach

The zygote cell wall of Chlamydomonas reinhardii has been studied using structural, chemical and immunological methods. Monoclonal antibodies and polyclonal antisera that were originally raised to the major hydroxyproline-rich glycoproteins of the vegetative cell wall were used to probe the zygote wall for common antigenic components. These antibodies cross-reacted strongly and specifically with components of the zygote cell wall, and were used to show the origin, route of transport, and the location of these antigens within the zygote cell wall. The zygote cell wall contained about 10% protein, with hydroxyproline accounting for 22.5 mol % of the total amino acids present. Glucose was the most abundant sugar residue, and accounted for 56% of the total sugar present. Gas liquid chromatography-mass spectrometry showed the presence of a (1-3)-d-glucan as the major structural polysaccharide within the zygote cell wall. The (1-3)-d-glucan was detected and localised within the zygote cell wall by immunogold labelling of thin sections. Using an antiserum directed against (1-3)-d-linked glucose units, this polysaccharide was found to be consistently present within the non-staining layer of both young and mature zygote cell walls. (1-3)-d-Glucan was also detected in other wall layer using higher concentrations of antiserum. No intracellular labelling was found, indicating that the plasmamembrane is the site for the synthesis of this polysaccharide within the Chlamydomonas zygote.Abbreviations DGP antiserum to deglycosylated 2-BII glycoprotein - GLC-MS gas liquid chromatography-mass spectrometry - MAC monoclonal antibody centre     

Spectroscopic rationalization of the separation abilities of decaproline chiral selector in dichloromethane-isopropanol solvent mixture

A chiral column, with decaproline as the chiral selector, has broad chiral selectivity. To understand the separation mechanism of this chiral column, multiple spectroscopic techniques, including optical rotation, electronic circular dichroism, infrared absorption and vibrational circular dichroism, have been used here to study the conformation of the decaproline oligomer in isopropanol(IPA)/dichloromethane(DCM) mixtures. These studies indicate that decaproline oligomer adopts polyproline II conformation in IPA/DCM solvent system (0% IPA approximately 100% IPA). Hydrogen bonding interactions between C=O groups of decaproline and IPA molecules increase as the content of IPA in the solvent mixture increases up to 60% and become less significant from then onwards. These spectroscopic observations are found to have a good correlation with the enantiomeric separation of racemic 2,2,2-trifluoro-1-[10-(2,2,2-trifluoro-1-hydroxy-ethyl-anthracen-9-yl]-ethanol by the decaproline column.     

Variability of mitochondrial population in Chlamydomonas reinhardii

Several details have been published cocerning the mitochondrial number and shapes at various stages of the synchronized vegetative and generative cell cycle in Chlamydomonas reinhardii. The present study, based on ultrathin serial sections and threedimensional reconstructions, completes these data. Quantitative analysis of serial micrographs makes it possible to give specific details of mitochondrial volumes in cells at early intermediate stages of the vegetative life cycle. Our investigations clearly show that mitochondria have a relatively wide range of sizes, within certain limits, and vary like the mitochondrial shapes; in fact, they vary in various cells at various stages as well as in several cells at the same stage and even in one and the same cell. Thus, we present a plastic insight into the dynamically changing micromorphology of the mitochondrial population in Chlamydomonas reinhardii.     

Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergenCry j I: relationship between the structures and antigenic epitopes of plantN-linked complex-type glycans

The oligosaccharide structures ofCry j I, a major allergenic glycoprotein ofCryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz¹H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides asCry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing 1–6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin andClerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides onCry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.Abbreviations Cry j I a major allergenic glycoprotein ofCryptomeria japonica - B-SJA-II Sophora japonica bark lectin II - CTA Clerodendron trichotomum lectin - TFMS trifluoromethanesulfonic acid - HRP horseradish peroxidase     

In Vitro Localization of Hydroxyproline O-Glycosyl Transferases in Chlamydomonas reinhardii

The intracellular location of the two major O-glycosylatingenzymes (hydroxyproline-arabinosyl and -galactosyl transferases)involved in the synthesis of the cell wall glycoproteins ofChlamydomonas reinhardii was determined by isopycnic sucrosedensity gradient centrifugation. A comparison of gradients preparedunder low and high Mg²⁺-conditions has enabled us to clearlyallocate the galactosyl transferase to membranes of the Golgiapparatus. In contrast, the membranes which bear the arabinosyltransferase respond to a change in Mg²⁺-concentration in justthe same way as the endoplasmic reticulum does. Analysis ofthe product formed in vitro from UDP-[¹⁴C]arabinose and microsomalmembranes has confirmed the synthesis of an arabinose-containinghydroxyproline-rich glycoprotein. Our results indicate thatwhilst the Golgi apparatus is responsible for some of the glycosylationreactions in hydroxyproline-rich glycoprotein biosynthesis anappreciable portion of the arabinosylation is accomplished whilethe polypeptide is still in the lumen of the endoplasmic reticulum. ³This paper is dedicated to Professor Lothar Jaenicke on theoccasion of his 65th birthday. (Received July 2, 1988; Accepted March 8, 1989)