Determination of subunit composition of Clostridium cellulovorans cellulosomes that degrade plant cell walls

Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of C. cellulovorans and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.     

Determination of Subunit Composition of Clostridium cellulovorans Cellulosomes That Degrade Plant Cell Walls

Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of C. cellulovorans and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.     

Isolation of β-galactosidase fractions from Japanese pear: Activity against native cell wall polysaccharides

β-Galactosidase (EC 3.2.1.23) was purified from the cell wall of the fruit of Japanese pear ( Pyrus serotina Rehder var. culta Rehder cv. Hosui) and characterized. Five peaks of β-galactosidase activity, designated as Gal I to V, were separated by hydrophobic chromatography on butyl toyopearl and ion exchange chromatography on Mono S. These isolated β-galactosidases were investigated with regard to their abilities to release monomeric galactose from the fractionated polymers of native cell wall (cyclo-hexane-trans-1,2-diamine tetraacetic acid-, Na₂CO₃-, guanidine thiocyanate- and KOH-soluble fractions) and arabinogalactan (from larch wood). All the β-galactosidase fractions were active against native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall and arabinogalactan. Gal II released much galactose only from KOH-soluble polymers and arabinogalactan. Gal III released the most galactose. from cyclohexane- trans -1,2-diamine tetraacetic acid-, Na₂CO₃- and guanidine thiocyanate-soluble cell wall polymers which probably contained galactosyl side chains of pectic polymers, although it did not react much to arabinogalactan. In addition, the activity of Gal Ill dramatically increased as ripening proceeded. Furthermore, Gal III was purified to homogeneity by gel filtration on TSKgel 3000SW and the size of a polypeptide was 80 kDa on SDS-PAGE.     

Study of the composition of cell wall of group A Streptococcus after hydrolysis using muramidase from Streptomyces levoris

The aim of the experiment was to study the lysis products of cell walls of group A streptococci resulting from exposure to N-acetylmuramidase. It was shown that for isolating surface proteins free of polysaccharide and peptidoglycan fragments it was necessary to treat the streptococcal cell walls with endo-beta-N-acetylmuramidase for no more than 30 minutes. Prolonged hydrolysis with muramidase led to the presence of polysaccharide and the peptidoglycan fragments in the protein fractions, intracellular wall proteins covalently bound to the peptidoglycan fragments and polysaccharide being also released.     

Identification of envelope proteins of Candida albicans by vectorial iodination

Intact Candida albicans yeast cells were radiolabelled with 125I, and cell wall, mixed membrane and soluble fractions prepared. The majority (67%) of the 125I was detected in the protein of the cell wall fraction at a specific activity 70-fold higher than that in the membrane or soluble fractions. SDS treatment of the cell wall fraction released 52% of the total protein but only 3% of the wall-bound 125I, and the extract was shown to be severely contaminated with cytosolic and membrane proteins. Zymolyase digestion of SDS treated walls liberated material which contained 93% of the 125I and on electrophoresis migrated as a single diffuse zone (average Mr 260 kD) typical of heterogenous mannoproteins. Protein (1.5%), GlcN (0.08%) and hexose (98.4%) content was measured and amino acid analysis showed enrichment in Ser (15.9%) and Thr (20.2%). These results indicate that the major iodinated protein(s) in the cell envelope is a 260 kD mannoprotein fraction containing both O-linked and Asn-linked oligosaccharides.     

Carrot arabinogalactan proteins are interlinked with pectins

Cell wall extracts from a carrot cell culture and tap roots were obtained by sequential extraction with water, EDTA buffer solution and cold sodium hydroxide solution. Arabinogalactan proteins (AGPs) were isolated from the extracts and from the medium of the cell culture and analysed for their molecular weight distribution and carbohydrate composition. Copper ions were used to separate the Yariv positive fractions into AGP fractions with a high and a low level of galacturonic acid (GalA). The GalA rich AGP fractions were incubated with pectin methylesterase and polygalacturonase. This enzyme incubation released GalA fragments from the AGP fractions as monitored by HPAEC and MALDI-TOF MS. At least part of carrot AGPs from the medium and cell walls may be covalently linked to pectin containing a homogalacturonan structural element.     

Characterization of degradation products of the cell wall released during growth and sporulation ofBacillus megaterium

Asporogenic and sporogenic strains ofBacillus megaterium KM release during growth heterogeneous fragments of the cell wall into the medium the non-dialyzable fraction representing 50–90% by the total. During lysis of sporangia the non-dialyzable fraction represents only 30% of the soluble fraction of autolyzed walls. Gel filtration on Sephadex permits to separate the non-dialyzable fragments of the cell wall released during growth into two fractions contaning simultaneously peptidoglycan and phosphorus. The two fractions contain peptidoglycan components in the same ratio as in the cell wall. Only one peptidoglycan macromolecular fraction, smaller than the fractions released during growth, was detected by gel filtration in the material released during lysis of sporangia.     

Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae

Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows. The sequence encoding the 40 C-terminal amino acids of each was fused with the structural gene for a reporter protein consisting of a secretion signal, α-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall. On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of α-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence. Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them.     

The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls

It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher at pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.     

Analysis of human T cell responses to group A streptococci using fractionated Streptococcus pyogenes proteins

Cell extract and spent culture supernatant proteins from Streptococcus pyogenes Manfredo strain (type M5) were each separated to give 22 narrow range molecular weight fractions by blot-elution from SDS-polyacrylamide gels. Eluted samples and unfractionated proteins were screened for T cell stimulatory activity using human peripheral blood mononuclear cells (PBMC) from healthy adults in proliferation assays. Responses were measured in 4- and 7d cultures. Responses to a wide range of cell extract proteins were revealed by fractionation, the degree of response to each fraction varying between donors. Unfractionated culture supernatant proteins elicited proliferative responses by PBMC from all individuals examined. Responses to culture supernatant fractions containing 25–33 kDa proteins could be attributed to known superantigens. Furthermore, samples from culture supernatants containing higher molecular weight fractions (>45 kDa) elicited responses in 50% of donors in 7d cultures, suggesting that these fractions contained common recall antigens. The efficacy of using electroeluted samples to identify T lymphocyte stimulatory proteins was confirmed by demonstrating that a known superantigen of S. pyogenes Manfredo strain, streptococcal pyrogenic exotoxin C (SPEC), could be fractionated successfully using this method and its activity recovered. Our results show that human T cell responses to group A streptococci involve a remarkably wide range of both cell-associated and released streptococcal proteins.     

Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae

Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows. The sequence encoding the 40 C-terminal amino acids of each was fused with the structural gene for a reporter protein consisting of a secretion signal, α-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall. On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of α-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence. Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them. Received: 1 September 1997 / Accepted: 20 November 1997     

The acropetal wave of developmental cell death of tobacco corolla is preceded by activation of transglutaminase in different cell compartments

The activity of transglutaminase (TGase), an enzyme responsible for polyamine conjugation to proteins, was analyzed in relationship to developmental cell death (DCD) during the flower life span stages of the tobacco (Nicotiana tabacum) corolla. As the DCD exhibits an acropetal gradient, TGase was studied in corolla proximal, medial, and distal parts. TGase was immunorecognized by three TGase antibodies; the main 58-kD band decreased during corolla life, whereas a 38-kD band localized progressively from basal to distal parts. The former was present in the soluble, microsomal, plastidial (together with the 38-kD band), and cell wall fractions. The endogenous TGase activity increased during DCD reaching a maximum soon after the corolla opening. The activity maximum shifted from proximal to distal part, preceding the DCD acropetal pattern. A similar activity increase was observed by the exogenous TGase substrate (histidine(6)-Xpr-green fluorescent protein). Subcellular activities were detected in (1) the microsomes, where TGase activity is in general higher in the proximal part, peaking at the corolla opening; (2) the soluble fraction, where it is present only in the proximal part at senescence; (3) the plastids, where it shows an increasing trend; and (4) cell walls, prevailing in the distal part and progressively increasing. These data suggest a relationship between DCD and TGase; the latter, possibly released in the cell wall through the Golgi vesicles, could cooperate to cell wall strengthening, especially at the abscission zone and possibly during corolla shape change. The plastid TGase, stabilizing the photosystems, could sustain the energy requirements for the senescence progression.     

Lignification related enzymes in Picea abies suspension cultures

Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient medium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cellular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is suggested to be responsible for hydrogen peroxide (H2O2) production. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for peroxidase-catalysed H2O2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly basic and highly basic isoenzyme groups by isoelectric focusing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoenzymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellular protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H2O2. The total activity of this oxidase towards coniferyl alcohol was, however, several orders of magnitude smaller than that of peroxidases in vitro. According to 2D 1H-13C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension culture lignin are present in the oxidase produced dehydrogenation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coniferin beta-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium.     

Mechanism of pneumococcal cell wall degradation in vitro and in vivo.

We compared the products of autolytic amidase-catalyzed wall degradation in vivo (in penicillin-induced lysis) and in vitro. Pneumococci labeled in their cell wall stem peptides by radioactive lysine were treated with penicillin, and the nature of wall degradation products released to the medium during lysis of the bacteria was determined. At early times of lysis (20% loss of wall label), virtually all the radioactive peptides released (greater than 94%) were of high molecular size and were still attached to glycan and teichoic acid. At times of more extensive bacterial lysis (56%), progressively larger and larger fractions of the released peptides became free, i.e., detached from glycan and teichoic acid. Analysis of the nondegraded residual wall material by high-resolution high-pressure liquid chromatography revealed that this in vivo-triggered autolysis did not involve selective hydrolysis of some of the chemically distinct stem peptides. Parallel in vitro experiments yielded completely different results. Purified pneumococcal cell walls labeled with radioactive lysine were treated in vitro with low concentrations of pure amidase, and the nature of wall degradation products released during limited hydrolysis and after more extensive degradation was determined. In sharp contrast to the in vivo experiments, the main products of in vitro hydrolysis were free peptides. After a short treatment with amidase (resulting in a 20% loss of label), the material released was enriched for the monomeric stem peptides. At all times of hydrolysis (including the time of extensive degradation), only a relatively small fraction of the released wall peptides was covalently attached to glycan and teichoic acid components (17% as compared with 40% in the intact cell wall). We propose that the in vivo-triggered amidase activity first attacks the amide bonds in some strategically located (or unprotected) stem peptides that hold large segments of cell wall material together. The observations indicate that the in vivo activity of the pneumococcal autolysin is under topographic constraints.     

EFFECT OF AUXIN ON β-1, 3-GLUCANASE ACTIVITY IN AVENA COLEOPTILE

When the homogenate of Avena coleoptile segments was fractionated, the specific activity of β-1, 3-glucanase was remarkably associated with the cell wall, partly to be released from it by a detergent. The cell wall-bound glucanase activity was increased by the treatment of coleoptile segments with auxin. Only in 10 min of the treatment the glucanase activity and the incorporation of labeled leucine into the proteins were found to be increased in the fraction to be liberated by detergent from the cell wall fraction. These effects of auxin were inhibited by 10 μg/ml cycloheximide.     

Properties and biosynthesis of cell wall-bound acid phosphatase in Aspergillus nigerx

Acid phosphatase (EC 3.1.3.2 [EC] ) of Aspergillus niger myceliumwas distributed exclusively in the cell wall and soluble fractions,whereas alkaline phosphatase was distributed in the solubleand particulate fractions but only slightly in the cell wallfraction. Cell wall-bound acid phosphatase was released by fungal-walllytic enzymes such as snail gut juice. Cell wall-bound, released,and soluble acid phosphatases showed very similar enzymaticproperties except that the bound enzyme was more stable to heatand detergents. By DEAE-cellulose chromatography, the releasedacid phosphatase was found to correspond to acid phosphatasesI A, IB and II in the soluble fraction. When phosphate in the medium was consumed, the acid phosphataseactivity of the soluble fraction increased more rapidly thanthat of the cell wall fraction. When phosphate was added tothe derepressed culture, the acid phosphatase activity of thesoluble fraction decreased after a short lag period, while thatof the cell wall fraction continued to increase. When labeledamino acid was added to the derepressed culture, it was incorporatedinto the soluble acid phosphatase without a lag period, whileit was incorporated into the cell wall phosphatase after a lagperiod. From these observations, acid phosphatase was consideredto be synthesized first as the soluble form and then integratedinto the cell wall. ¹ The present experiments were carried out, for the most part,at the Institute of Applied Microbiology of the University ofTokyo. (Received January 19, 1976; )     

Effect of anti-wall protein antibodies on auxin-induced elongation, cell wall loosening, and β-D-glucan degradation in maize coleoptile segments

Antiserum raised against the LiCl extract of maize shoot cell walls suppresses auxin-induced elongation of maize coleoptile segments. A series of polyclonal antibodies were raised against protein fractions separated from the LiCl extract of maize ( Zea mays L. cv. B73 x Mo17) coleoptiles by SP-Sephadex and Bio-Gel P-150 chromatography. To understand the role of cell wall proteins in growth regulation, the effect of these antibodies on auxin-induced elongation and changes in the cell walls of maize coleoptiles was examined. Four of the fractions prepared reacted with the antiserum raised against the total LiCl extract and effectively suppressed its growth-inhibiting activity. Only these fractions contained the proteins responsible for eliciting growthinhibiting antibodies. The antibodies capable of growth inhibition of auxin-induced elongation of segments also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. The antibodies raised against one of the protein fractions separated by SP-Sephadex inhibited the autolytic reactions of isolated cell walls and the auxin-induced decrease in (1→3), (1→4)-β-D-glucans in the cell walls. Thus, the degradation of β-D-glucans by cell wall enzymes may be associated with the cell wall loosening that is responsible for cell elongation. Because the other antibodies did not influence the auxin-induced degradation of (1→3), (1→4)-β-D-glucanses, β-D-glucanases and other cell wall enzymes may cooperate in regulation of cell elongation in maize coleoptiles.     

Invertase in cultured Daucus carota cells

Invertase activity of cultured carrot cells was distributed between cell wall and supernatant fractions of the cell homogenate. The enzyme associated with the cell wall fraction was solubilized by alkaline NaCl solution and the proportions found in the cell wall and soluble fractions depended on the concentration of NaCl. Formation of protoplasts by the action of cellulase and pectinase was accompanied by release of 50–60% of the invertase activity from the cells.     

A transglutaminase immunologically related to tissue transglutaminase catalyzes cross-linking of cell wall proteins in Chlamydomonas reinhardtii

The addition of primary amines to the growth medium of the unicellular green alga Chlamydomonas reinhardtii disrupts cell wall assembly in both vegetative and zygotic cells. Primary amines are competitive inhibitors of the protein-cross-linking activity of transglutaminases. Two independent assays for transglutaminase confirmed a burst of extracellular activity during the early stages of cell wall formation in both vegetative cells and zygotes. When non-inhibiting levels of a radioactive primary amine ((14)C-putrescine) were added to the growth medium, both cell types were labeled in a reaction catalyzed by extracellular transglutaminase. The radioactive label was found specifically in the cell wall proteins of both cell types, and acid hydrolysis of the labeled material released unmodified (14)C-putrescine. Western blots of the proteins secreted at the times of maximal transglutaminase activity in both cell types revealed a single highly cross-reactive 72-kD band when screened with antibodies to guinea pig tissue transglutaminase. Furthermore, the proteins immunoprecipitated by this antiserum in vivo exhibited transglutaminase activity. We propose that this transglutaminase is responsible for an early cell wall protein cross-linking event that temporally precedes the oxidative cross-linking mediated by extracellular peroxidases.     

Partial purification of cell wall α-galactosidases and α-arabinosidases from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes

Two protein fractions with activity as α-galactosidase (EC 3.2.1.22) and α-arabinosidase (EC 3.2.1.55), respectively, were identified in the proteins of cell wall of Cicer arietinum L. cv. Castellana extracted with 3 M LiCl. These fractions were partially purified by gel filtration chromatography (Bio Gel P-150), increasing the specific arabinosidase activity 57-fold and the α-galactosidase activity 6-fold. Other protein fractions with glucosidase (EC 3.2.1.21) and glucanase (EC 3.2.1.6) activity also appeared. According to earlier authors, α-arabinosidases and α-galactosidases are related to alterations in linkages occurring in cell walls, since the enzymes are able to hydrolyze isolated wall polymers. However, our preparations hydrolyze intact cell walls only to a very limited extent, such that their participation in the autolytic processes of cell walls can be ruled out.