糖蛋白的分离鉴定方法

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脑脊液糖蛋白分离推荐度：
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A sub-proteome of Arabidopsis thaliana mature stems trapped on Concanavalin A is enriched in cell wall glycoside hydrolases

Michel Zivy3, and

90% of plant cell walls and constitute three different kinds of polymers: cellulose,

hemicelluloses and pectins. Plant cell wall polysaccharide composition and structure change

during plant development and are different from one plant species to another (Cosgrove,

1997; Popper and Fry, 2003). Cell wall proteins (CWPs) contribute to wall architecture or are

involved in the regulation of growth and development, or defence against biotic or abiotic

stresses (Lee et al., 2004; Jamet et al., 2006). Cell wall modifying proteins such as glycoside

hydrolases (GHs), esterases, transglycosylases, lyases and peroxidases are involved in the

construction, remodelling or turnover of cell wall components (Heredia et al., 1995; Cosgrove,

1997; Fry, 2004; Stolle-Smits et al., 1999; Obel et al., 2002; Reiter, 2006).

The enzymes of GH and transglycosylase superfamilies are particularly important for the

reorganization of cell wall polysaccharides after their deposition (Fry, 2004; Minic and

Jouanin, 2006). They fall into several families whose distinction is based on amino acid

sequence similarities (Henrissat, 1991; 1998). Exo-glycanases attack polysaccharides

progressively from the non-reducing end or substituted side groups, thus releasing

monosaccharides. Endo-glycanases attack polysaccharide backbones in an endo-fashion. They

have a large impact on the molecular mass of polysaccharides. A third group of hydrolases can

break some substituted non-carbohydrate groups linked to wall polysaccharides such as O -

acetyl, O -methyl and O -feruloyl groups (Fry, 2004). Xyloglucan transglycosylase hydrolases

(XTHs) can exhibit both endo-glycanase and transglycosylase activities (Fry, 2004).

Significant progress has been made in proteomic analysis of plant cell walls (Jamet et al.,

2006). Interesting results were obtained using cell cultures, culture medium of seedlings,

leaves, etiolated hypocotyls, protoplasts and roots (Chivasa et al., 2002; Borderies et al.,

2003; Boudart et al., 2005; Charmont et al., 2005; Kwon et al., 2005; Jamet et al., 2006; Zhu

et al., 2006). One cell wall proteome of mature stems was described in Medicago sativa L.

(Watson et al., 2004). All these studies were based either on elution of cell wall proteins from

living cells or on extraction of proteins from purified cell walls with salt solutions. However,

since all CWPs are secreted proteins, they can be N -glycosylated with sugars such as D-glucose

and D-mannose during their passage through endoplasmic reticulum and Golgi (Lerouge et al.,

1998). It should be possible to trap them on Concanavalin A (Con A) which is a lectin extracted

from Canavalia ensiformis L. able to bind molecules containing α-D-mannopyranosyl, β-D-

glucopyranosyl or sterically-related residues (Carlsson et al., 1998). Recently, the N-

glycoproteomes of human urine and human bile were analysed using Con A Sepharose affinity

chromatography followed by 2D-electrophoresis and mass spectrometry (Kristiansen et al.,

2004; Wang et al., 2006). The majority of the proteins identified were predicted to be

extracellular or membrane components. Con A affinity chromatography was also used for the

characterisation of N -linked glycoproteins of Ceanorhabditis elegans (Kaji et al., 2003) and

of GHs from various plant organs (Sheldon et al., 1998; Wilson and Altmann, 1998; Minic et

al., 2004; Li and Kushad, 2005; Minic et al., 2006; Van Riet et al., 2006). In this work, we

have developed a new proteomic approach starting from a crude protein extract and using Con

A Sepharose affinity chromatography to identify soluble cell wall N -linked glycoproteins. This

glycoproteome is significantly enriched for putative cell wall GHs compared to previous cell

wall proteomes.Materials and methods

Plant material

Wild-type Arabidopsis thaliana , Wassilewskija ecotype, was grown in the greenhouse at 20°

C to 22°C with a 16 h-photoperiod at 150 μE.m ?2.s ?1. Inflorescence stems of plants at mature

stage (18–22 cm) were used for analysis.

Minic et al.Page 2

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Preparation of protein extracts from stems of A. thaliana

Mature stems of A. thaliana measuring 18–22 cm in length at the late flowering stage were

used for analysis. Approximately 10 g of stem tissues were suspended in 12 mL of ice-cold

extraction buffer and grinded in a mortar with a pestle for 5 min. The extraction buffer consisted

in 25 mM BisTris pH 7.0 (HCl), 200 mM CaCl 2, 10% (v/v) glycerol, 4 μM Na-cacodylate,

1/200 (v/v) protease inhibitor cocktail (P-9599, Sigma Chemical, St Louis, MO, USA). The

ground material was centrifuged twice at 4°C for 3 min at 10,000 g , and the supernatant was

further centrifuged for 15 min at 17,000 g . The resulting supernatant was used for

chromatographic analyses.

Con A Sepharose affinity chromatography

A 1 x 6-cm column was filled with 3 mL of Con A Sepharose (Sigma Chemical, St Louis, MO,

USA) and washed with 6 mL of 20 mM Tris pH 7.4 (HCl), 1 mM CaCl 2/MgCl 2/MnCl 2 and

0.5 M NaCl buffer. The soluble protein extract (10 mL) was added and then washed with 15

mL of this buffer at a flow rate of 5 mL.h ?1. Proteins were eluted with 0.2 M methyl-α-

glucopyranoside in the same buffer. The eluate was collected, concentrated by “Ultrafree-

CL” (10 kDa) (Sigma Chemical, St Louis, MO, USA) to 300 μL and dialysed against 7 M urea,

5 mM K 2CO 3, 0.125% SDS, 0.6% Triton X-100, 1 mM DTT, 2% carrier ampholytes 3–10

(GE Healthcare Europe GmbH, Orsay, France).

Glycoside hydrolase activities

The reaction mixture contained 2 mM p NP-glycosides (Sigma Chemical, St Louis, MO, USA),

0.1 M acetate buffer (pH 5.0), 2 mM sodium azide, and 50 μL of protein extract in a total

volume of 0.5 mL. The reaction was carried out at 37°C for 5 to 60 min (depending on activity)

and stopped by the addition of 0.5 mL 0.4 M sodium chloride. Controls were stopped at time

0. Concentration of the resulting p NP was determined spectrophotometrically at 405 nm by

comparison to a calibration curve. Standard deviations values for 3 replicate assays were less

than 5%.

2D-electrophoresis

Isoelectric focusing (IEF) was performed using 24 cm-immobilized pH gradient (IPG) strips

(GE Healthcare Europe GmbH, Orsay, France) with a linear pH gradient from 4 to 7 and 250

μg of protein were applied on an IPG strip for in-gel rehydration in 7 M urea, 2 M thiourea,

2% CHAPS, 10 mM DTT, 2% IPG buffer pH 4–7 (Méchin et al., 2004). Focusing was achieved

using a Protean IEF Cell (Bio-Rad, Hercules, CA, USA). An active rehydration was performed

at 22°C during 12 h at 50 V prior to focusing. To improve sample entry, the voltage was

increased step by step from 50 to 10,000 V (0.5 h at 200 V, 0.5 h at 500 V, 1 h at 1000 V then

10,000 V for a total of 94,000 V h). After IEF, IPG strips were successively incubated in 50

mM Tris pH 8.8 (HCl), 6 M urea, 30% glycerol, 2 % SDS, 1% DTT for 15 min, and in 50 mM

Tris pH 8.8 (HCl), 6 M urea, 30% glycerol, 2 % SDS, 2.5% iodoacetamide for 15 min (G?rg

et al., 1987). Strips were further sealed on top of the 1 mm-thick second dimensional gel (24

x 24 cm) with the help of 1% low-melting agarose in SDS–electrophoresis buffer (25 mM Tris,

0.2 M glycine, 0.1% SDS). Continuous gels (11% T, 2.67% C gels with PDA as cross-linking

agent) were used. Separation was carried out at 20 V for 1 h and subsequently at a maximum

of 30 mA/gel, 120 V overnight, until the bromophenol blue front had reached the end of the

gel.

Protein staining

Following 2D-electrophoresis, gels were stained with colloidal Coomassie blue G250

according to Mechin et al. (2004).Minic et al.Page 3J Exp Bot . Author manuscript; available in PMC 2008 June 16.HAL-AO Author Manuscript HAL-AO Author Manuscript HAL-AO Author Manuscript

Identification of proteins by mass spectrometry

The inpidual protein spots obtained after 2D-electrophoresis were excised and in-gel digested

with trypsin according to a standard protocol (Santoni et al., 2003). Tryptic peptides from each

protein were analyzed by nanoHPLC-MS/MS or MALDI-TOF MS as previously described

(Minic et al., 2004; Mechin et al., 2004). Proteins analysed by MALDI-TOF MS were identified

via automated NCBI non redundant protein database (2539bf120b4e767f5acfce7c/) searching

using the MASCOT programme (2539bf120b4e767f5acfce7c/search_form_select).

Only mowse scores exceeding threshold (p<0.5) were considered as positive results.

Identification of proteins with nanoHPLC-MS/MS (ion trap) was performed with Biowoks ?

(Thermo scientific, San Jose, USA). The main search parameters were methionine oxidation

as differential modification and trypsin as enzyme. One miss cleavage was allowed. The A.

thaliana protein database was downloaded from the mips website

(http://mips.gsf.de/projects/plants). Identification was considered significant when the proteins

were identified with at least 2 different tryptic peptides as first candidate, Xcorr > 1.7, 2.2 and

3.3 for respectively mono-, di- and tri-charged peptides and delta Cn >0.1.

Bioinformatics analyses

Sub-cellular localization and length of signal peptides were predicted using PSORT

(http://psort.nibb.ac.jp/) and TargetP (http://www.cbs.dtu.dk/services/TargetP/) (Nielsen et al.,

1997; Emanuelsson et al., 2000). Prediction of transmembrane domains was done with

Aramemnon (http://aramemnon.botanik.uni-koeln.de/) (Schwacke et al, 2003). Molecular

masses and pI values were calculated using the aBi program

(http://www.up.univ-mrs.fr/~wabim/d_abim/compo-p). Homologies to other proteins

were searched for using BLAST programs (2539bf120b4e767f5acfce7c/BLAST/) (Altschul

et al., 1990). Identification of protein families and functional domains was performed using

MyHits (http://myhits.isb-sib.ch/cgi-bin/motif_scan) and InterProScan

(2539bf120b4e767f5acfce7c/InterProScan/) (Quevillon et al., 2005). GHs and CEs were classified

according to the CAZy database (2539bf120b4e767f5acfce7c/CAZY/) (Coutinho et al., 1999).

Peroxidases were named as in the PeroxiBase (http://peroxidase.isb-sib.ch/index.php)

(Bakalovic et al., 2006).

Protein measurements

Protein concentration was determined by the method of Bradford (1996) using bovine serum

albumin dissolved in extraction buffer as the standard.Results and discussion

Extraction of glycoside hydrolases from stem tissues of A. thaliana

In a first attempt to study GHs from stem tissues of A. thaliana by using a proteomic approach

it was necessary to establish a protocol for the extraction of these enzymes. Based on published

experimental data on the purification of GHs from various plant organs (Sheldon et al., 1998;

Wilson and Altmann, 1998; Minic et al., 2004; Li and Kushad, 2005; Van Riet et al., 2006),

we developed a 2-step extraction procedure. Stems were ground in a buffer containing 200

mM CaCl 2, followed by Con A Sepharose affinity chromatography. CaCl 2 was chosen as the

most efficient salt for CWP extraction (Boudart et al., 2005). This protocol is different from

those used in previous cell wall proteomic studies (Feiz et al., 2006): (i) the initial step is a

grinding in a buffer containing 200 mM CaCl 2 to release CWPs instead of a low ionic strength

buffer usually used to prevent CWP elution; (ii) there is no step of cell wall isolation to avoid

loosing CWPs weakly-bound to cell walls during the centrifugation steps required for cell wall

isolation; (iii) the last step is an affinity chromatography to trap N -glycosylated proteins.

Results show that the affinity chromatography step resulted in a significant increase in specific

Minic et al.Page 4J Exp Bot . Author manuscript; available in PMC 2008 June 16.HAL-AO Author Manuscript HAL-AO Author Manuscript

HAL-AO Author Manuscript

activities of several exo-GHs using artificial substrates compared to what was measured in the

dialysed crude protein extract. These increases varied from 2.0 for β-D-xylosidase to 6.1 for

β-D glucosidase (Table 1). On the basis of this observation, this protocol was adapted to analyze

the N -glycoproteome of mature stems.

Proteomic analysis after enrichment of the soluble protein extract in glycoside hydrolases by lectin affinity chromatography

The proteomic analysis was performed using prot ein extracts from 18–22 cm mature stems at

the late flowering stage. About 10 g of stem tissues were used for the extraction of proteins.

After grinding and centrifugation, the crude protein extract contained 2.5 mg of protein as

determined by the Bradford method (1996). A fraction of this protein extract was subjected to

Con A Sepharose affinity chromatography. Eluted proteins were concentrated and dialysed,

resulting in a fraction of 400 μL containing about 300 μg of protein. A sample containing 250

μg of protein was subjected to 2D-electrophoresis. Proteins were detected by colloidal

Coomassie blue staining (Fig. 1). The number of resolved spots was about 200. Fifty-seven

spots resolved by 2D-electrophoresis were analyzed using MALDI-TOF. Spots corresponding

to proteins having molecular mass smaller than 20 kDa were not analyzed since they are not

expected to contain GHs on the basis of calculations made from genes predicted to encode such

proteins (Minic and Jouanin, 2006). The other proteins visible on the gel were also analyzed,

but due to small quantity or mixture with other proteins their scores were not significant. Fifteen

spots localized at the basic side of the gel were subjected to tryptic digestion and proteins were

identified using nanoHPLC-MS/MS. Each of them was expected to contain more than one

protein since previous studies showed that most CWP are basic (Jamet et al., 2006). A total

number of 102 different proteins was identified (Table 2; Tables S1 and S2 at JXB online).

Many of these proteins were present in several of the spots resolved by 2D-electrophoresis

suggesting post-translational modifications such as glycosylations. On the basis on these

results, those spots were collected into thirty-five groups as shown in Fig 1. Conversely, as

expected, most of the 15 spots at the basic side of the gel contained more than one protein.

Bioinformatic prediction of sub-cellular localization and N-glycosylation of identified

proteins

PSORT, TargetP and Aramemnon programmes were used to predict the sub-cellular

localization of the identified proteins. Seventy-seven out of the 102 identified proteins (77%)

were predicted to be localized in the cell wall matrix, 13 at the plasma membrane, 6 into the

endoplasmic reticulum, 2 in the cytoplasm and 3 in the chloroplast. Six proteins were predicted

to be either targeted to vacuoles or to the cell wall. However, vacuolar targeting is not well-

established in plants and the predictions are not yet very reliable (Hadlington and Denecke,

2000). Altogether, about 90% of the proteins have a predicted N-terminal signal peptide, which

means that all of these proteins are targeted to the secretory pathway. Two proteins could not

be assigned to any sub-cellular compartment due to discrepancies between predictions with

PSORT and TargetP. Seven proteins were predicted to harbour a glycosyl phosphatidyl inositol

(GPI) anchor. As expected, all of the identified proteins contained N -glycosylation sites as

predicted by the MyHits programme (see Supplementary Table S1 at JXB).

These results show that the proposed protocol allowed the isolation of a protein fraction

essentially composed of N -linked glycoproteins targeted to either the cell wall or to the plasma

membrane. Despite the absence of a cell wall purification step, it should be noted that the

proportion of proteins with a predicted intracellular localization was very low (12%). This

protocol thus appears as an efficient alternative to previously described protocols used for A.

thaliana cell wall proteomic analyses. Previous protocols include: (i) non-destructive methods

such as analysis of culture media (Borderies et al., 2003; Charmont et al. 2005), washing of

cells cultured in liquid medium with salt solutions (Borderies et al., 2003; Kwon et al. 2005)

Minic et al.Page 5

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and vacuum infiltration of leaves (Boudart et al., 2005); (ii) destructive methods, i.e. cell wall

purification, prior to CWP extraction with various buffers (Chivasa et al. 2002; Bayer et al.,

2006; Feiz et al., 2006). The choice of a particular protocol will depend on the aim of the study

and on the plant organ of interest.

Identification and functional classification of proteins

Identification of protein families and functional domains were performed using several

bioinformatic programmes. Proteins were classified according to the predicted functional

classes of CWPs proposed by Jamet et al. (2006) (Tables 2, 3). Proteins belonging to seven

functional classes were found according to the presence of functional domains predicted as

described in the Material and methods: (i) proteins acting on polysaccharides include GH and

esterases; (ii) oxido-reductases mainly include peroxidases and multicopper oxidases; (iii)

proteins with interacting domains include proteins with lectin or LRR (leucine rich repeat)

domains as well as enzyme inhibitors; (iv) proteins involved in signaling processes include

fasciclin AGPs (arabinogalactan proteins); (v) proteases; (vi) proteins of yet unknown function;

(vii) miscellaneous. However, this classification is provisional since the biological role of many

of these proteins remains to be determined (Tatosov et al., 1997). For example, an enzyme of

the GH 3 family (XYL3) shows amino acid homology with β-D-xylosidase. However, it was

identified as an enzyme that efficiently hydrolyzed arabinosyl residues from arabinans,

suggesting that it works as an α-L-arabinofuranosidase (Minic et al., 2006).

Thirty-three proteins were expected to act on polysaccharides (Table 2). Furthermore, 30

proteins (29%) belong to the superfamily of GHs, 29 of which were predicted to be extracellular

or plasma membrane-associated. The second largest group comprises 16 proteases, 14 of which

were predicted to be localized in the extracellular matrix. Together GHs and proteases represent

47% of identified proteins. Among other proteins, oxido-reductases, proteins with interacting

domains, miscellaneous proteins, proteins of unknown function, signalling and intracellular

proteins were identified.

Proteins from the same functional classes as in previous cell wall proteomic studies were found,

but 37 proteins were not identified before (Chivasa et al. 2002; Borderies et al. 2003; Borner

et al., 2003; Schultz et al., 2004; Boudart et al., 2005; Charmont et al. 2005; Kwon et al.

2005; Bayer et al., 2006; Feiz et al. 2006). This stem N-glycoproteome thus appears to be very

specific. Among the 90 proteins predicted to be at the plasma membrane or in the cell wall,

only 5 also have been found in previously described proteomes: cell suspension cultures, rosette

leaves and etiolated hypocotyls (Jamet et al., 2006). They encode a β-xylosidase that belongs

to the GH 31 family (At1g68560), a multicopper oxidase (At1g76160), two lectins

(At1g78850, At1g78860) and a protein homologous to the carrot extracellular dermal

glycoprotein (EDGP) and to the tomato xyloglucan specific endoglucanase inhibitor protein

(XEGIP) (At1g03220) (Qin et al., 2003). However, some protein families are missing. Since

proteins with molecular masses lower than 20 kDa were not analyzed, it was not possible to

identify homologs to protease or pectin methylesterase inhibitors, non-specific lipid transfer

proteins, and blue copper binding proteins. Only one protein homolog to germins was

identified. Although several expansins, which molecular masses are between 25 and 30 kDa,

were previously identified in cell wall proteomes (Jamet et al., 2006), none was found in this

study. This might be explained either by their low abundance or their low level of N -

glycosylation. Finally, no structural protein could be identified either because of their strong

binding to the extracellular matrix, or the absence of N-glycans.

Possible roles of proteins identified in stem tissues of A. thaliana

Proteins acting on polysaccharides constitute the major functional class. According to Coutinho

et al. (1999), they belong to 12 GH families and to 2 carbohydrate esterase (CE) families (Fig.Minic et al.Page 6

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2, Table 2). These enzyme families have perse biological functions in defence, signalling,hydrolysis of starch, and cell wall modifications (Minic and Jouanin, 2006). A total of 18 GHs belonging to 7 different GH families were found. Three of them, αL-arabinofuranosidase (At3g10740), α-L-arabinofuranosidase/β-D-xylosidase (At5g49360) and β-glucosidase AtGLU1 (At5g11720), were recently purified and characterized (Minic et al., 2004; Monroe et al., 1999). The XYL1 β-xylosidase and two XTHs (Meri5/At4g30270, EXGT-A1/At2g06850) have been studied previously using both biochemical and genetic approaches (Akamatsu et al., 1999; Sampedro et al., 2001; Rose et al., 2002). A pectin methyl- and a pectin acyl-esterase were also identified. Previous studies have shown that pectin methylesterase activity is inversely correlated to the growth rate of expanding tissues, suggesting its possible involvement in wall rigidification (McQueen-Mason and Cosgrove, 1995).Possible substrates in muro of the majority of these enzymes are xyloglucans and pectins. Most of them act as exo-enzymes whereas only enzymes belonging to GH families 16 and 28 act as endo-GHs on xyloglucans and homogalacturonans, respectively. Other cell wall GHs can hydrolyse β1,4 glucan, arabinoxylan and xylan. These results suggest that xyloglucan and pectins, that are composed of homogalacturonans (HG), arabinans and galactans (RG-I),undergo structural changes in mature stem. However, some GH families can act on several natural polysaccharides showing broad substrate specificity. This low specificity has been reported in the case of several purified cell wall GHs (Leach et al, 1995; Kim and al., 2000;Sampedro et al., 2001; Steele et al., 2001; Rose et al., 2002; Lee et al., 2003; Minic et al,2004; Minic et al., 2006). It has been hypothesized that it allows efficient modification of complex cell wall polysaccharides without requiring an extremely high number of enzymes (Minic et al, 2004; 2006).Many GH families described here could have other functions than cell wall modifications.Predicted extracellular GHs such as β-D-glucuronidase (GH 79), α-D-mannosidase (GH 38)and acetyl-N-hexasaminase (chitinase-like enzymes, GH 19) could be involved in post-

translational modifications of glycoproteins. Recently, an A. thaliana β-D-glucuronidase

(AtGUS) was shown to hydrolyze glucuronic acids from carbohydrate chains of AGPs (Eudes,personal communication). Kinetic and structural analyses of Ginkgo α-D-mannosidase acting on a pyridylamino derivative of oligo mannosides strongly suggested its involvement in the catabolism and turnover of N -linked glycoproteins (Woo et al., 2004). The pumpkin endo-β-N-acetylglucosaminidase, partially purified from cotyledons, was highly active towards high-mannose type glycans (Kimura et al., 2002).

Among other GHs, one thioglucosyl hydrolase (GH 1), 3 β-1,3-D-glucanase (GH 17) and 3chitinase-like enzymes (GH 19 and 20) were identified. Thioglucosidases, also known as

myrosinases, play perse roles in cruciferous plant during growth, development and defence against microorganisms and insects (Rodman, 1991). Chitinase-like enzymes are able to

degrade chitin in cell walls of fungal pathogens. However, the substrates and functions of most chitinase-like enzymes are not completely known. For example, a mutation in the chitinase-like gene classified in GH 19 family (AtCTL1/At1g05850) caused a cellulose deficiency as well as aberrant patterns of lignification with incomplete cell walls in the stem pith (Zhong et al., 2002; Rogers et al., 2005).

The second largest class of identified proteins comprises proteases. Seventeen putative

proteinases including aspartyl and serine type proteases were found (Table 2). Fifteen of them were predicted to be secreted (Supplementary Table S1 at JXB online). The abundance of proteases in mature stem suggests that these enzymes may be actively involved in secondary wall formation. Proteases may play various roles in plant development and during plant pathogen interactions through maturation of CWPs and generation of active peptides. It has been shown that the extracellular subtilisin-like serine protease SDD1 (STOMATAL

Minic et al.

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DENSITY AND DISTRIBUTION 1) is involved in the regulation of stomatal density and

distribution in A. thaliana (Berger and Altmann, 2000). ALE1 (ABNORMAL LEAF

EPIDERMIS1) is also predicted to encode a subtilisin-like serine protease and is assumed to

produce a peptide required for proper differentiation of epidermis (Tanaka et al., 2001). CDR1

(CONSTITUTIVE DISEASE RESISTANT 1) encodes a putative aspartic protease (Xia et al.,

2004). Overexpression of CDR1 causes dwarfing and resistance to virulent Pseudomonas

syringae . It was shown that CDR1 generates a small mobile signal (3–10 kDa) sensitive to

heating and to protease. The substrates of these three proteases are yet unknown. On the

contrary, it was shown that the CLE (CLV3/ESR-related) basic secreted proteins are processed

at their C-terminus to generate 14 amino-acid peptides that carry a biological activity (Ito et

al., 2006;Kondo et al. 2006). In those cases, the proteases have not yet been identified. Finally,

several plant cell wall proteomic analyses show a large discrepancy between the observed and

the expected molecular masses of proteins (Boudart et al., 2005;Kwon et al., 2005;Zhu et al.,

2006). Proteases could be involved in processing and/or turnover of cell wall proteins.

Several oxido-reductases such as multicopper oxidase-like (6 proteins), peroxidases (4

proteins), germin-like protein (1 protein) and a homolog to berberine bridge enzyme were

identified. In contrast to previously characterized cell wall proteomes, this study allowed the

identification of numerous multicopper oxidase-like proteins. They catalyse full, four-electron

reduction of dioxygen (O 2) to water (H 2O) using a variety of substrates (Solomon et al.,

1997). They belong to a large gene family of 19 members in A. thaliana (Jacob and Roe, 2005).

Only two members of the family have been previously studied, SKU5 (At4g12420) and SKS6

(At1g41830). It was shown that SKU5 is involved in the control of root growth (Sedbrook et

al., 2002) and that SKS6 contributes to cotyledon vascular patterning during development

(Jacob and Roe, 2005). Peroxidases are involved in many physiological and developmental

processes that have been reviewed recently (Passardi et al., 2004). They can be involved in

both cell elongation processes and in their arrest. In the latter case, they catalyze the formation

of bridges across phenolic residues of lignins and between lignins and adjacent cell wall

proteins or polysaccharides.

Three extracellular acid phosphatases were identified in this work. The presence of

phosphorylated proteins and phosphatases in plant cell wall has been reported in several

proteomes (Chivasa et al, 2002; Kwon et al., 2005; Jamet et al., 2006). However, no

extracellular kinase has yet been found (Chivasa et al., 2005). Acid phosphatases may

participate in extracellular signalling events or in regulation of cell wall proteins.

Some proteins contained interacting domains, such as LRRs. The polygalacturonase-inhibiting

protein (PGIP2/At5g06870) plays a role in plant defence (Di Matteo et al., 2006). Two proteins

identified as fasciclin-like AGPs (AtFLA8/At2g45470, AtFLA13/At5g45130) can participate

in cell-to-cell adhesion in plant (Johnson et al., 2003; Groover and Robischon, 2006). Finally,

10 proteins of unknown function were found.Concluding remarks

This study demonstrates the effectiveness of the purification procedure to isolate cell wall

glycoproteins. This includes novel GHs, multicopper oxidases and proteases. In contrast to

these analyses, we did not identify homologs to protease or pectin methylesterase inhibitors,

non-specific lipid transfer proteins, blue copper binding proteins, expansins and structural

proteins. The abundance of GHs suggests a great plasticity of polysaccharides in cell walls,

even in well-differentiated tissues such as mature stems. Finally, the presence of phosphatases,

proteases and GHs suggests a complex regulation of cell wall proteins involving various types

of post-translational modifications such as de-phosphorylation and hydrolytic processing by

proteases or glycosidases.

Minic et al.Page 8

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Supplementary data

Refer to Web version on PubMed Central for supplementary material.

Acknowledgements

This work was partly funded by the Génoplante program Af2001-009. We thank Bruno Letarnec for growing A.

thaliana plants, Dr Christain Malosse for mass spectrometry analyses, Drs Jorun Johansen and Herman H?fte for

improving this manuscript.References

Akamatsu T, Hanzawa Y, Ohtake Y, Takahashi T, Nishitani K, Komeda Y. Expression of endoxyloglucan transferase genes in acaulis mutants of Arabidopsis. Plant Physiology 1999;121:715–722. [PubMed:10557219]Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of Molecular Biology 1990;215:403–410. [PubMed: 2231712]Bakalovic N, Passardi F, Ioannidis V, Cosio C, Penel C, Falquet L, Dunand C. PeroxiBase: A class III plant peroxidase database. Phytochemistry 2006;67:534–539. [PubMed: 16442574]Bayer EM, Bottrill AR, Walshaw J, Vigouroux M, Naldrett MJ, Thomas CL, Maule AJ. Arabidopsis cell wall proteome defined using multidimensional protein identification technology. Proteomics 2006;6:301–311. [PubMed: 16287169]Berger D, Altmann T. A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana . Genes and Development 2000;14:1119–1131. [PubMed:10809670]Bhattacharyya L, Brewer CF. Formation of homogeneous carbohydrate-lectin cross-linked precipitates from mixtures of D-galactose/N-acetyl-D-galactosamine-specific lectins and multiantennary galactosyl carbohydrates. European Journal of Biochemistry 1992;208:179–185. [PubMed: 1511686]Borderies G, Jamet E, Lafitte C, Rossignol M, Jauneau A, Boudart G, Monsarrat B, Esquerre-Tugaye MT, Boudet A, Pont-Lezica R. Proteomics of loosely bound cell wall proteins of Arabidopsis

thaliana cell suspension cultures: a critical analysis. Electrophoresis 2003;24:3421–3432. [PubMed:

14595688]

Borner GH, Lilley KS, Stevens TJ, Dupree P. Identification of glycosylphosphatidylinositol-anchored

proteins in Arabidopsis. A proteomic and genomic analysis. Plant Physiology 2003;132:568–577.

[PubMed: 12805588]

Boudart G, Jamet E, Rossignol M, Lafitte C, Borderies G, Jauneau A, Esquerré-Tugayé MT, Pont-Lezica

R. Cell wall proteins in apoplastic fluids of Arabidopsis thaliana rosettes: identification by mass

spectrometry and bioinformatics. Proteomics 2005;5:212–221. [PubMed: 15593128]

Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein

utilizing the principle of protein-dye binding. Analytical Biochemistry 1976;72:248–254. [PubMed:

942051]

Carlsson, J.; Janson, J-C.; Sparrman, M. Affinity chromatography. In: Janson, J-C.; Ryden, L., editors.

Protein Purification, Principles, High Resolution and Applications. VCH Publishers Inc; New York:

1998. p. 275-329.

Carpita NC, Gibeaut DM. Structural models of primary cell walls in flowering plants: consistency of

molecular structure with the physical properties of the walls during growth. The Plant Journal

1993;3:1–30. [PubMed: 8401598]

Charmont S, Jamet E, Pont-Lezica R, Canut H. Proteomic analysis of secreted proteins from Arabidopsis

thaliana seedlings: improved recovery following removal of phenolic compounds. Phytochemistry

2005;66:453–461. [PubMed: 15694452]

Chivasa S, Ndimba BK, Simon WJ, Lindsey K, Sladas AR. Extracellular ATP functions as an endogenous

external metabolite regulating plant cell viability. The Plant Cell 2005;17:3019–3034. [PubMed:

16199612]

Minic et al.Page 9J Exp Bot . Author manuscript; available in PMC 2008 June 16.HAL-AO Author Manuscript

HAL-AO Author Manuscript

Chivasa S, Ndimba BK, Simon WJ, Robertson D, Yu XL, Knox JP, Bolwell P, Slabas AR. Proteomic analysis of the Arabidopsis thaliana cell wall. Electrophoresis 2002;23:1754–1765. [PubMed:12179997]Cosgrove DJ. Assembly and enlargement of the primary cell wall in plants. Annual Review of Cell Developmental Biology 1997;13:171–201.Coutinho, PM.; Henrissat, B. Carbohydrate-active enzymes: an integrated database approach. In: Gilbert,HJ.; Davies, G.; Henrissat, B.; Svensson, B., editors. Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry; Cambridge: 1999. p. 3-12.Di Matteo A, Bonivento D, Tsernoglou D, Federici L, Cervone F. Polygalacturonase-inhibiting protein (PGIP) in plant defence: a structural view. Phytochemistry 2006;67:528–533. [PubMed: 16458942]Emanuelsson O, Nielsen H, Brunak S, von Heijne G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Journal of Molecular Biology 2000;300:1005–1016.[PubMed: 10891285]Feiz L, Irshad M, Pont-Lezica RF, Canut H, Jamet E. Evaluation of cell wall preparations for proteomics:a new procedure for purifying cell walls from Arabidopsis hypocotyls. Plant Methods 2006;2:10.[PubMed: 16729891]Fry SC. Primary cell wall metabolism: tracking the careers of wall polymers in living plant cells. New Phytologist 2004;161:641–675.G?rg A, Postel W, Weser J, Günther S, Strahler JR, Hanash S, Somerlot L. Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer.Electrophoresis 1987;8:122–124.Hadlington JL, Denecke J. Sorting of soluble proteins in the secretory pathway of plants. Current Opinion in Plant Biology 2000;3:461–468. [PubMed: 11074376]Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities.Biochemistry Journal 1991;280:309–316.Henrissat B. Glycosidase families. Biochemical Society Transactions 1998;26:153–156. [PubMed:9649738]Ito Y, Nakanomyo I, Motose H, Iwamoto K, Sawa S, Dohmae N, Fukuda H. Dodeca-CLE peptides as

suppressors of plant stem differentiation. Science 2006;313:842–845. [PubMed: 16902140]

Jacobs J, Roe JL. SKS6, a multicopper oxidase-like gene, participates in cotyledon vascular patterning

during Arabidopsis thaliana development. Planta 2005;222:652–666. [PubMed: 15986216]

Jamet E, Canut H, Boudart G, Pont-Lezica RF. Cell wall proteins: a new insight through proteomics.

Trends in Plant Sciences 2006;11:33–39.

Johnson KL, Jones BJ, Bacic A, Schultz CJ. The fasciclin-like arabinogalactan proteins of Arabidopsis.

A multigene family of putative cell adhesion molecules. Plant Physiology 2003;133:1911–1925.

[PubMed: 14645732]

Kaji H, Saito H, Yamauchi Y, Shinkawa T, Taoka M, Hirabayashi J, Kasai K, Takahashi N, Isobe T.

Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked

glycoproteins. Nature Biotechnology 2003;21:667–672.

Kim JB, Olek AT, Carpita NC. Cell wall and membrane-associated exo-beta-D-glucanases from

developing maize seedlings. Plant Physiology 2000;123:471–486. [PubMed: 10859178]

Kimura Y, Matsuo S, Tsurusaki S, Kimura M, Hara-Nishimura I, Nishimura M. Subcellular localization

of endo-β-N -acetylglucosaminidase and high-mannose type free N -glycans in plant cell. Biochimica and Biophysica Acta 2002;1570:38–46.

Kondo T, Sawa S, Kinoshita A, Mizuno S, Kakimoto T, Fukuda H, Sakagami Y. A plant peptide encoded

by CLV3 identified by in situ MALDI-TOF MS analysis. Science 2006;313:845–848. [PubMed:16902141]

Kristiansen TZ, Bunkenborg J, Gronborg M, Molina H, Thuluvath PJ, Argani P, Goggins MG, Maitra

A, Pandey A. A proteomic analysis of human bile. Molecular Cell Proteomics 2004;3:715–728.Kwon HK, Yokoyama R, Nishitani K. A proteomic approach to apoplastic proteins involved in cell wall

regeneration in protoplasts of Arabidopsis suspension-cultured cells. Plant Cell Physiology

2005;46:843–857. [PubMed: 15769804]

Leah R, Kigel J, Svendsen I, Mundy J. Biochemical and molecular characterization of a barley seed beta-

glucosidase. Journal of Biological Chemistry 1995;270:15789–15797. [PubMed: 7797581]

Minic et al.

Page 10J Exp Bot . Author manuscript; available in PMC 2008 June 16.HAL-AO Author Manuscript

HAL-AO Author Manuscript

Lee RC, Hrmova M, Burton RA, Lahnstein J, Fincher GB. Bifunctional family 3 glycoside hydrolases from barley with α-L-arabinofuranosidase and β-D-xylosidase activity. Journal of Biological Chemistry 2003;278:5377–5387. [PubMed: 12464603]Lee SJ, Saravanan RS, Damasceno CM, Yamane H, Kim BD, Rose JK. Digging deeper into the plant cell wall proteome. Plant Physiology and Biochemistry 2004;42:979–988. [PubMed: 15707835]Lerouge P, Cabanes-Macheteau M, Rayon C, Fischette-Lainé AC, Gomord V, Faye L. N -glycoprotein biosynthesis in plants: recent developments and future trends. Plant Molecular Biology 1998;38:31–48. [PubMed: 9738959]Li X, Kushad MM. Purification and characterization of myrosinase from horseradish (Armoracia rusticana ) roots. Plant Physiolology and Biochemistry 2005;43:503–511.McQueen-Mason SJ, Cosgrove DJ. Expansin mode of action on cell walls. Analysis of wall hydrolysis,stress relaxation, and binding. Plant Physiology 1995;107:87–100. [PubMed: 11536663]Méchin V, Balliau T, Chateau-Joubert S, Davanture M, Langella O, Negroni L, Prioul JL, Thevenot C,Zivy M, Damerval C. A two-dimensional proteome map of maize endosperm. Phytochemistry 2004;65:1609–1618. [PubMed: 15276456]Minic Z, Jouanin L. Plant glycosyl hydrolases involved in cell wall polysaccharide degradation. Plant Physiology and Biochemistry 2006;44:435–449. [PubMed: 17023165]Minic Z, Do C-T, Rihouey C, Morin H, Lerouge P, Jouanin L. Purification, functional characterization,cloning and identification of mutants of a seed specific arabinan hydrolase in Arabidopsis. Journal of Experimental Botany 2006;57:2339–2351. [PubMed: 16798843]Minic Z, Rihouey C, Do CT, Lerouge P, Jouanin L. Purification and characterization of enzymes exhibiting beta-D-xylosidase activities in stem tissues of Arabidopsis. Plant Physiology 2004;135:867–878. [PubMed: 15181203]Monroe JD, Gough CM, Chandler LE, Loch CM, Ferrante JE, Wright PW. Structure, properties, and tissue localization of apoplastic alpha-glucosidase in crucifers. Plant Physiology 1999;119:385–397.[PubMed: 9952433]Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 1997;10:1–6. [PubMed: 9051728]

Obel N, Porchia AC, Scheller HV. Dynamic changes in cell wall polysaccharides during wheat seedling

development. Phytochemistry 2002;60:603–610. [PubMed: 12126707]

Passardi F, Penel C, Dunand C. ; Performing the paradoxica: how plant peroxidases modify the cell wall.

Trends in Plant Sciences 2004;9:534–540.

Qin O, Bergmann CW, Rose JK, Saladie M, Kolli VS, Albersheim P, Darvill AG, York WS.

Characterization of a tomato protein that inhibits a xyloglucan-specific endoglucanase. The Plant Journal 2003;34:327–38. [PubMed: 12713539]

Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R. InterProScan: protein

domains identifier. Nucleic Acids Research 2005;33:W116–W120. [PubMed: 15980438]

Reiter WD. Biosynthesis and properties of the plant cell wall. Current Opinion in Plant Biology

2002;5:536–542. [PubMed: 12393017]

Roberts K. The plant extracellular matrix: in a new expansive mood. Current Opinion in Cell Biology

1994;6:688–694. [PubMed: 7833049]

Rodman JE. A taxonomic analysis of glucosinolate- producing plants. Systematic Botany 1991;16:598–

618.

Rogers LA, Dubos C, Surman C, Willment J, Cullis IF, Mansfield SD, Campbell MM. Comparison of

lignin deposition in three ectopic lignification mutants. New Phytologist 2005;168:123–140.

[PubMed: 16159327]

Rose JK, Braam J, Fry SC, Nishitani K. The XTH family of enzymes involved in xyloglucan

endotransglucosylation and endohydrolysis: current perspectives and a new unifying nomenclature.Plant Cell Physiology 2002;43:1421–1435. [PubMed: 12514239]

Sampedro J, Sieiro C, Revilla G, Gonzalez-Villa T, Zarra I. Cloning and expression pattern of a gene

encoding an alpha-xylosidase active against xyloglucan oligosaccharides from Arabidopsis . Plant Physiology 2001;126:910–920. [PubMed: 11402218]

Minic et al.

Page 11J Exp Bot . Author manuscript; available in PMC 2008 June 16.HAL-AO Author Manuscript

HAL-AO Author Manuscript

Santoni V, Vinh J, Pflieger D, Sommerer N, Maurel C. A proteomic study reveals novel insights into the persity of aquaporin forms expressed in the plasma membrane of plant roots. Biochemistry Journal 2003;373:289–296.Schultz CJ, Ferguson KL, Lahnstein J, Bacic A. Post-translational modifications of arabinogalactan-peptides of Arabidopsis thaliana . Endoplasmic reticulum and glycosylphosphatidylinositol-anchor signal cleavage sites and hydroxylation of proline. Journal of Biological Chemistry 2004;279:45503–45511. [PubMed: 15322080]Schwacke R, Schneider A, Van Der Graaff E, Fischer K, Catoni E, Desimone M, Frommer WB, Flugge UI, Kunze R. ARAMEMNON, a Novel Database for Arabidopsis Integral Membrane Proteins. Plant Physiology 2003;131:16–26. [PubMed: 12529511]Sedbrook JC, Carroll KL, Hung KF, Masson PH, Somerville CR. The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth. The Plant Cell 2002;14:1635–1648. [PubMed: 12119380]Sheldon PS, Keen JN, Bowles DJ. Purification and characterization of N-glycanase, a concanavalin A binding protein from jackbean (Canavalia ensiformis ). Biochemical Journal 1998;330:13–20.[PubMed: 9461484]Solomon, EI.; Machonkin, TE.; Sundaram, UM. Spectroscopy of multi-copper oxidases. In:Messerschmidt, A., editor. Multicopper oxidases. World Scientific Publishing Co; Singapore: 1997.p. 103-127.Steele NM, Sulova Z, Campbell P, Braam J, Farkas V, Fry SC. Ten isoenzymes of xyloglucan endotransglycosylase from plant cell walls select and cleave the donor substrate stochastically.Biochemical Journal 2001;355:671–679. [PubMed: 11311129]Stolle-Smits T, Beekhuizen JG, Kok MT, Pijnenburg M, Recourt K, Derksen J, Voragen AG. Changes in cell wall polysaccharides of green bean pods during development. Plant Physiology 1999;121:363–372. [PubMed: 10517827]Tanaka H, Onouchi H, Kondo M, Hara-Nishimura I, Nishimura M, Machida C, Machida Y. A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants. Development 2001;128:4681–4689. [PubMed: 11731449]Tatusov RL, Koonin EV, Lipman DJ. A genomic perspective on protein families. Science 1997;278:631–

637. [PubMed: 9381173]

Van Riet L, Nagaraj V, Van den Ende W, Clerens S, Wiemken A, Van Laere A. Purification, cloning

and functional characterization of a fructan 6-exohydrolase from wheat (Triticum aestivum L.).Journal of Experimental Botany 2006;57:213–223. [PubMed: 16330524]

Wang L, Li F, Sun W, Wu S, Wang X, Zhang L, Zheng D, Wang J, Gao Y. Concanavalin A captured

glycoproteins in healthy human urine. Molecular and Cell Proteomics 2006;5:560–562.

Watson BS, Lei Z, Dixon RA, Sumner LW. Proteomics of Medicago sativa cell walls. Phytochemistry

2004;65:1709–1720. [PubMed: 15276432]

Wilson IB, Altmann F. Concanavalin A binding and endoglycosidase D resistance of beta1,2-xylosylated

and alpha1,3-fucosylated plant and insect oligosaccharides. Glycoconjugate Journal 1998;15:203–206. [PubMed: 9557883]

Woo KK, Miyazaki M, Hara S, Kimura M, Kimura Y. Purification and characterization of a co(II)-

sensitive alpha-mannosidase from Ginkgo biloba seeds. Bioscience Biotechnology and Biochemistry 2004;68:2547–2556.

Xia Y, Suzuki H, Borevitz J, Blount J, Guo Z, Patel K, Dixon RA, Lamb C. An extracellular aspartic

protease functions in Arabidopsis disease resistance signaling. EMBO Journal 2004;23:980–988.

[PubMed: 14765119]

Zhong R, Kays SJ, Schroeder BP, Ye ZH. Mutation of a chitinase-like gene causes ectopic deposition of

lignin, aberrant cell shapes, and overproduction of ethylene. The Plant Cell 2002;14:165–179.

[PubMed: 11826306]

Zhu J, Chen S, Alvarez S, Asirvatham VS, Schachtman DP, Wu Y, Sharp R. Cell wall proteome in the

maize primary root elongation zone. I. Extraction and identification of water-soluble and lightly ionically bound proteins. Plant Physiology 2006;140:311–325. [PubMed: 16377746]

Minic et al.

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Fig 1.Analysis of A. thaliana proteins by 2D-electrophoresis. The 2D-gel was loaded with 250 μg of the fraction obtained after Con A Sepharose affinity chromatography from stem tissues of A. thaliana. The gel was stained with colloidal Coomassie blue. Fifteen spots were picked for nanoHPLC-MS/MS (1 to 15) and fifty-seven for MALDI-TOF analyses. In the latter case,

numbering refers to groups of spots containing the same protein. Arrows in the circles represent same identified proteins. Molecular mass markers are indicated on the right.

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Fig. 2.Distribution of families of glycoside hydrolases (GH) and carbohydrate esterases (CE) in the A. thaliana stem sub-proteome. Proteins are listed in Table 2. Proteins have been classified according to the CAZy nomenclature (Henrissat et al., 1998; 2539bf120b4e767f5acfce7c/). Glycoside

hydrolase (GH) families from 1 to 79 are on the left whereas carbohydrate esterase (CE)

families 8 and 13 are on the right. White bars correspond to families that might participate in cell wall modification and reorganization.

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Table 1

Specific activities of several glycoside hydrolases after Con A Sepharose affinity chromatography

All enzyme activities were measured in vitro at 37 °C, using 50 μL of protein and pNP-glycosides as substrates.

Enzyme Specific activities (nmol/min/mg protein)Recovery (%)Ratio of specific activities

Crude protein extract After Con A Sepharose Con A Sepharose/Crude protein extract β-D-xylosidase328943 2.8

α-L-arabinofuranosidase10344266 4.3

β-D-glucuronidase102234 2.0

β-D-mannosidase2610864 4.2

α-D-mannosidase4717256 3.6

β-D-glucosidase8249793 6.1

α-D-galactosidase8546884 5.5

β-D-galactosidase39382532 2.1

α-D-glucosidase83159 3.9

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Table 3

Predicted functional classes of proteins in the A. thaliana stem sub-proteome

Functional classes have been defined according to Jamet et al. (2006). A list of all proteins identified in this study is provided in Table 2. The detailed bioinformatics functional analysis is given in Supplementary Table S1 at JXB online.

Functional classes Number of proteins Proteins acting on polysaccharides31

Glycoside hydrolases29

Esterases2

Oxido-reductases13

Peroxidases4

Multicopper oxidases6

Others3

Proteins with interacting domains12

Lectin domains6

LRR domains3

Others3

Signalling2