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Characterization of the Arabinogalactan Protein 31 (AGP31) of Arabidopsis thaliana

NEW ADVANCES ON THE HYP-O-GLYCOSYLATION OF THE PRO-RICH DOMAIN*
  • May Hijazi
    Affiliations
    Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France
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  • Jessica Durand
    Affiliations
    Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France
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  • Carole Pichereaux
    Affiliations
    FR3450, Plateforme de Protéomique Toulouse Midi-Pyrénées, Institut de Pharmacologie et Biologie Structurale, Université de Toulouse, F-31077 Toulouse, France
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  • Frédéric Pont
    Affiliations
    INSERM, Plateau Technique Interactions et Profils d'Expression des Protéines, Plateforme de Protéomique Toulouse Midi-Pyrénées, F-31300 Toulouse, France
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  • Elisabeth Jamet
    Affiliations
    Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France
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  • Cécile Albenne
    Correspondence
    To whom correspondence should be addressed: UPS, CNRS, UMR 5546, Laboratoire de Recherche en Sciences Végétales, BP 42617, F-31326 Castanet-Tolosan, France. Tel.: 33-534-32-38-59; Fax: 33-534-32-38-02
    Affiliations
    Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France
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  • Author Footnotes
    * This work was supported by the Université Paul Sabatier (Toulouse, France), the CNRS (France), and a grant from the Lebanon ecological association (to M. H.). This work has been done in the LRSV, part of the “Laboratoire d'Excellence” (LABEX) entitled TULIP (ANR-10-LABX-41).
    This article contains supplemental Figs. S1–S3.
Open AccessPublished:January 23, 2012DOI:https://doi.org/10.1074/jbc.M111.247874
      Proteins are important actors in plant cell walls because they contribute to their architecture and their dynamics. Among them, hydroxyproline (Hyp)-rich glycoproteins constitute a complex family of O-glycoproteins with various structures and functions. In this study, we characterized an atypical Hyp-rich glycoprotein, AGP31 (arabinogalactan protein 31), which displays a multidomain organization unique in Arabidopsis thaliana, consisting of a short arabinogalactan protein (AGP) motif, a His stretch, a Pro-rich domain, and a C-terminal PAC (PRP-AGP containing Cys) domain. The use of various mass spectrometry strategies was innovative and powerful: it permitted us to locate Hyp residues, to demonstrate the presence of carbohydrates, and to refine their distribution over the Pro-rich domain. Most Hyp were isolated within repeated motifs such as KAOV, KSOV, K(PO/OP)T, K(PO/OP)V, T(PO/OP)V, and Y(PO/OP)T. A few extensin-like motifs with contiguous Hyp (SOOA and SOOT) were also found. The Pro-rich domain was shown to carry Gal residues on isolated Hyp but also Ara residues. The existence of new type Hyp-O-Gal/Ara-rich motifs not recognized by the β-glucosyl Yariv reagent but interacting with the peanut agglutinin lectin was proposed. In addition, the N-terminal short AGP motif was assumed to be substituted by arabinogalactans. Altogether, AGP31 was found to be highly heterogeneous in cell walls because arabinogalactans could be absent, Hyp-O-Gal/Ara-rich motifs of different sizes were observed, and truncated forms missing the C-terminal PAC domain were found, suggesting degradation in muro and/or partial glycosylation prior to secretion.

      Introduction

      Hydroxyproline (Hyp)-rich
      The abbreviations used are: Hyp
      hydroxyproline(s)
      AG
      arabinogalactan
      AGP
      arabinogalactan protein
      CEC
      cation exchange chromatography
      CWP
      cell wall protein
      ETD
      electron transfer dissociation
      EXT
      extensin
      GalT
      galactosyl transferase
      HRGP
      hydroxyproline-rich glycoprotein
      NAC
      nickel affinity chromatography
      PAC
      PRP-AGP containing Cys
      PMF
      peptide mass fingerprinting
      PNA
      peanut agglutinin lectin
      PRP
      Pro-rich protein
      Ara
      arabinose
      Gal
      galactose
      DIG
      digoxigenin.
      glycoproteins (HRGPs) are a superfamily of plant cell wall proteins (CWPs) identified several decades ago (
      • Lamport D.
      • Northcote D.
      Hydroxyproline in primary cell walls of higher plants.
      ). Their study remains challenging because HRGPs constitute a very complex protein group with various structures and functions whose molecular bases are not fully elucidated (
      • Kieliszewski M.
      • Lamport D.
      Extensin: repetitive motifs, functional sites, post-translational codes, and phylogeny.
      ,
      • Kieliszewski M.
      The latest hype on Hyp-O-glycosylation codes.
      ,
      • Showalter A.
      Structure and function of plant cell wall proteins.
      ,
      • Seifert G.J.
      • Roberts K.
      The biology of arabinogalactan proteins.
      ,
      • Nothnagel E.
      Proteoglycans and related components in plant cells.
      ). HRGPs fall into three families: (i) hyperglycosylated arabinogalactan proteins (AGPs) having diverse functions (
      • Seifert G.J.
      • Roberts K.
      The biology of arabinogalactan proteins.
      ); (ii) moderately glycosylated extensins (EXTs) that can form cell wall scaffolds essential for cytokinesis (
      • Cannon M.C.
      • Terneus K.
      • Hall Q.
      • Tan L.
      • Wang Y.
      • Wegenhart B.L.
      • Chen L.
      • Lamport D.T.
      • Chen Y.
      • Kieliszewski M.J.
      Self-assembly of the plant cell wall requires an extensin scaffold.
      ); and (iii) Pro-rich proteins (PRPs) that may be nonglycosylated, weakly glycosylated, or highly glycosylated and possibly involved in various aspects of development (
      • Showalter A.
      Structure and function of plant cell wall proteins.
      ,
      • Sommer-Knudsen J.
      • Clarke A.E.
      • Bacic A.
      A galactose-rich, cell-wall glycoprotein from styles of Nicotiana alata.
      ). Hybrid HRGPs are composed of different types of HRGP domains, and chimeric HRGPs contain at least one HRGP domain within a non-HRGP protein. A recent bioinformatics analysis achieved on the Arabidopsis thaliana genome identified 166 HRGPs classified in 85 AGPs, 59 EXTs, 18 PRPs, and 4 AGP/EXT hybrid proteins, highlighting the question why plants need so many HRGPs (
      • Showalter A.M.
      • Keppler B.
      • Lichtenberg J.
      • Gu D.
      • Welch L.R.
      A bioinformatics approach to the identification, classification, and analysis of hydroxyproline-rich glycoproteins.
      ).
      HRGPs are characterized by the presence of Hyp-rich repetitive motifs, called glycomodules, containing from 3 to 16 amino acids. Glycomodule sequences determine Pro hydroxylation and the subsequent Hyp O-glycosylation. These two consecutive sequence-driven post-translational modifications define the so-called Hyp-O-glycosylation code (
      • Kieliszewski M.
      The latest hype on Hyp-O-glycosylation codes.
      ). AGPs have repetitive variants of (Xaa1–3-Hyp)n motifs (where Xaa is commonly Ser, Ala, or Thr) with O-linked arabinogalactan (AG) polysaccharides. EXTs display clusters of Ser-(Hyp)2,4 containing short O-Hyp arabinose (Ara)-oligosaccharides. Finally, PRPs are probably the most heterogeneous group, regarding their repetitive sequences and their carbohydrate patterns. They are characterized by the repeating occurrence of (Pro)2 or (Pro)3 motifs within a variety of larger repeated units. It was shown that PRPs contain approximately equimolar quantities of Pro and Hyp (
      • Sommer-Knudsen J.
      • Clarke A.E.
      • Bacic A.
      A galactose-rich, cell-wall glycoprotein from styles of Nicotiana alata.
      ,
      • Averyhart-Fullard V.
      • Datta K.
      • Marcus A.
      A hydroxyproline-rich protein in the soybean cell wall.
      ,
      • Datta K.
      • Schmidt A.
      • Marcus A.
      Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes.
      ,
      • Liu C.
      • Mehdy M.
      A nonclassical arabinogalactan protein gene highly expressed in vascular tissues, AGP31, is transcriptionally repressed by methyl jasmonic acid in Arabidopsis.
      ,
      • Kleis-San Francisco S.
      • Tierney M.
      Isolation and characterization of a proline-rich cell wall protein from soybean seedlings.
      ). Many PRPs contain the repeated pentapeptide Pro-Hyp-Xaa-Yaa-Lys, where Xaa and Yaa can be Val, Tyr, His, or Glu (
      • Showalter A.
      Structure and function of plant cell wall proteins.
      ,
      • Fowler T.
      • Bernhardt C.
      • Tierney M.
      Characterization and expression of four proline-rich cell wall protein genes in Arabidopsis encoding two distinct subsets of multiple domain proteins.
      ). PRPs were initially assumed to be non- or weakly glycosylated (
      • Datta K.
      • Schmidt A.
      • Marcus A.
      Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes.
      ,
      • Kieliszewski M.
      • de Zacks R.
      • Leykam J.F.
      • Lamport D.T.
      A repetitive proline-rich protein from the gymnosperm Douglas fir is a hydroxyproline-rich glycoprotein.
      ). However, the characterization of a Gal-rich glycoprotein (GaRSGP) from Nicotiana alata styles revealed a new class of PRPs with repetitive motifs never described before and a carbohydrate content of 75%, unusual for PRPs (
      • Sommer-Knudsen J.
      • Clarke A.E.
      • Bacic A.
      A galactose-rich, cell-wall glycoprotein from styles of Nicotiana alata.
      ). Recently, an A. thaliana homologue of GaRSGP has been described (
      • Liu C.
      • Mehdy M.
      A nonclassical arabinogalactan protein gene highly expressed in vascular tissues, AGP31, is transcriptionally repressed by methyl jasmonic acid in Arabidopsis.
      ). This glycoprotein mostly contains Gal residues and was called AGP31 because of its positive interaction with the β-glucosyl Yariv reagent. This protein, encoded by At1g28290, was shown to be a multidomain protein and was classified as a chimeric AGP (
      • Showalter A.M.
      • Keppler B.
      • Lichtenberg J.
      • Gu D.
      • Welch L.R.
      A bioinformatics approach to the identification, classification, and analysis of hydroxyproline-rich glycoproteins.
      ).
      Here, we provide a detailed characterization of AGP31 extracted from cell walls of A. thaliana etiolated hypocotyls. Combining two isolation processes and three detection methods, we demonstrated that the native AGP31 displays a huge heterogeneity with various O-glycans and truncated forms. After enrichment, MS analyses were permitted to determine the location of Hyp residues within the Pro-rich domain, to demonstrate the presence of carbohydrates, and to refine their distribution.

      DISCUSSION

      AGP31 is a remarkable cell wall protein with a multidomain organization unique in A. thaliana. Although it was already reported that AGP31 was a glycoprotein, no data about the distribution of Hyp and O-glycans on the protein were available so far (
      • Liu C.
      • Mehdy M.
      A nonclassical arabinogalactan protein gene highly expressed in vascular tissues, AGP31, is transcriptionally repressed by methyl jasmonic acid in Arabidopsis.
      ). The use of diverse MS technologies was innovative and provided an in depth characterization of the AGP31 Pro-rich domain. Combining various experimental approaches, we demonstrated that native AGP31 displays a huge heterogeneity in cell walls, with various O-glycans and truncated forms of its protein core.
      Our results provided for the first time a full MS coverage of the Pro-rich region of a native HRGP, whereas most available data were obtained by Edman sequencing of small peptides (reviewed in Refs.
      • Kieliszewski M.
      • Lamport D.
      Extensin: repetitive motifs, functional sites, post-translational codes, and phylogeny.
      ,
      • Showalter A.
      Structure and function of plant cell wall proteins.
      , and
      • Sommer-Knudsen J.
      • Bacic A.
      • Clarke A.
      Hydroxyproline-rich plant glycoproteins.
      ) and on recombinant HRGPs (
      • Tan L.
      • Varnai P.
      • Lamport D.T.
      • Yuan C.
      • Xu J.
      • Qiu F.
      • Kieliszewski M.J.
      Plant O-hydroxyproline arabinogalactans are composed of repeating trigalactosyl subunits with short bifurcated side chains.
      ). Combining literature data with our fragmentation results on tryptic peptides from the Pro-rich domain, we could locate all of the Hyp residues in the Pro-rich domain of AGP31. Indeed, the so-called Hyp-O-glycosylation code based on protein sequence data assumes that Lys, Phe, and Tyr prevent hydroxylation of the following Pro (
      • Kieliszewski M.
      The latest hype on Hyp-O-glycosylation codes.
      ). In addition, TPK motifs were found in the maize THRGP (
      • Kieliszewski M.J.
      • Leykam J.F.
      • Lamport D.T.
      Structure of the threonine-rich extensin from Zea mays.
      ). Then we propose that KPOT, KPOV, TPOV, and YPOT motifs are present in AGP31 (Fig. 5). The sequences of other repetitive motifs determined experimentally, i.e. SOOA, SOOT, KSOV, and KAOV, are consistent with the Hyp-O-glycosylation code. It should be noted that SOOA/T motifs are similar to those found in EXTs (
      • Kieliszewski M.
      The latest hype on Hyp-O-glycosylation codes.
      ,
      • Shpak E.
      • Leykam J.F.
      • Kieliszewski M.J.
      Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes.
      ). The only exception to the Hyp-O-glycosylation code is the YPPK motif in which none of the Pro is hydroxylated (Fig. 5). The amino acids preceding Pro are assumed to provide a specific local conformation permitting or preventing the action of prolyl 4-hydroxylases involved in Pro hydroxylation. Enzymatic characterization of A. thaliana prolyl 4-hydroxylases provided insight into their substrate specificity (
      • Hieta R.
      • Myllyharju J.
      Cloning and characterization of a low molecular weight prolyl 4-hydroxylase from Arabidopsis thaliana. Effective hydroxylation of proline-rich, collagen-like, and hypoxia-inducible transcription factor α-like peptides.
      ,
      • Tiainen P.
      • Myllyharju J.
      • Koivunen P.
      Characterization of a second Arabidopsis thaliana prolyl 4-hydroxylase with distinct substrate specificity.
      ). Recently, the crystal structure of an algal prolyl 4-hydroxylase complexed with a Ser-Pro5 substrate highlighted the molecular bases of Pro hydroxylation (
      • Koski M.K.
      • Hieta R.
      • Hirsilä M.
      • Rönkä A.
      • Myllyharju J.
      • Wierenga R.K.
      The crystal structure of an algal prolyl 4-hydroxylase complexed with a proline-rich peptide reveals a novel buried tripeptide binding motif.
      ). The mechanisms of Pro hydroxylation within Pro-rich domains of PRPs will require further investigation to check whether it follows the same rules as in EXTs and AGPs. As suggested, this code may also depend on the organ and/or plant (
      • Shpak E.
      • Barbar E.
      • Leykam J.F.
      • Kieliszewski M.J.
      Contiguous hydroxyproline residues direct hydroxyproline arabinosylation in Nicotiana tabacum.
      ,
      • Estevez J.
      • Kieliszewski M.
      • Khitrov N.
      • Somerville C.
      Characterization of synthetic hydroxyproline-rich proteoglycans with arabinogalactan protein and extensin motifs in Arabidopsis.
      ).
      Figure thumbnail gr5
      FIGURE 5Location of Hyp residues within the Pro-rich domain. Hyp residues were located combining MALDI-TOF/TOF MS/MS fragmentation results on deglycosylated tryptic peptides of the AGP31 Pro-rich domain (see ) and literature data (
      • Kieliszewski M.
      The latest hype on Hyp-O-glycosylation codes.
      ,
      • Sommer-Knudsen J.
      • Bacic A.
      • Clarke A.
      Hydroxyproline-rich plant glycoproteins.
      ). The sequence should be read from left to right and from top to bottom. The Hyp residues of the AGP31 Pro-rich domain are represented by O. The repeated motifs (repeats) are indicated in bold type at the bottom of the figure.
      Our experimental data gave new insight into the O-glycosylation of the Pro-rich domain of AGP31. MALDI-TOF MS analyses showed the presence of hexoses and pentoses, assumed to be Gal and Ara according to the PNA-lectin blot detection and the monosaccharide composition of AGP31. The ETD fragmentation technology was employed to describe the distribution of carbohydrates onto the Pro-rich domain, providing the first successful study of a plant protein O-glycosylation using this method. ETD MS/MS experiments performed onto P2 O-glycopeptides containing hexoses showed that Gal are uniformly distributed on isolated Hyp within the KAOV, KPOT, KPOV, and YPOT motifs. Because such experiments were not possible on P1 and P4 O-glycopeptides containing pentoses, we could not locate Ara on these peptides. However, we suggest that Ara or short Ara-oligosaccharides may be carried by contiguous Hyp in SOOA/T motifs, like in EXTs (
      • Kieliszewski M.
      The latest hype on Hyp-O-glycosylation codes.
      ,
      • Shpak E.
      • Leykam J.F.
      • Kieliszewski M.J.
      Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes.
      ). In addition, we cannot exclude that Ser residues within these EXT-like motifs could also be O-galactosylated (
      • Kieliszewski M.
      The latest hype on Hyp-O-glycosylation codes.
      ,
      • Shpak E.
      • Leykam J.F.
      • Kieliszewski M.J.
      Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes.
      ).
      An important feature highlighted in this study is the huge heterogeneity of AGP31. After separation by SDS-PAGE and various detection methods, AGP31, whose predicted molecular mass is 38 kDa, was found as a smear from 30 to ∼250 kDa. Combining all of our results, we could propose structural models schematized on Fig. 6. The Pro-rich domain on which carbohydrates were detected by MALDI-TOF MS is probably also substituted by larger O-glycans that escaped MS analyses because of the large size of the corresponding tryptic glycopeptides. According to the monosaccharide composition of AGP31-enriched fraction obtained using CEC and NAC (53.2% Gal and 39.5% Ara), we propose to call them Hyp-O-Gal/Ara-rich motifs. Note that our monosaccharide analysis showed a higher proportion of Ara than previously reported, probably because our expression and purification procedures were different (
      • Liu C.
      • Mehdy M.
      A nonclassical arabinogalactan protein gene highly expressed in vascular tissues, AGP31, is transcriptionally repressed by methyl jasmonic acid in Arabidopsis.
      ). These Pro-rich domain O-glycans were specifically recognized by PNA as a smear from 34 to 170 kDa, but not by the β-glucosyl Yariv reagent, suggesting a structure different from that of type II arabino-3,6-galactans predominantly found in AGPs (
      • Gaspar Y.
      • Johnson K.
      • McKenna J.
      • Bacic A.
      • Schultz C.
      The complex structures of arabinogalactan-proteins and the journey towards understanding function.
      ). The higher molecular mass glycoforms of AGP31 (Fig. 6, smear above 170 kDa) are also assumed to carry AG motifs on the short N-terminal AGP sequence (APAPAP) as suggested by the positive signal with the β-glucosyl Yariv reagent. The absence of a signal above 170 kDa by PNA-lectin blot analysis was probably due to electrophoretic transfer limitations. Finally, truncated forms of AGP31 missing the C-terminal PAC domain also exist (Fig. 6, smear of 30–40 kDa after SDS-PAGE). These latter forms were found to be weakly O-glycosylated on their Pro-rich domain.
      Figure thumbnail gr6
      FIGURE 6AGP31 structural models. The different domains of the mature AGP31 are represented from left to right: N-terminal AGP motif (AGP), His stretch (His), Pro-rich domain (Pro-rich), and C-terminal PAC domain (PAC). The various glycoforms and truncated forms of AGP31 are schematized, as well as the results obtained for isolating, detecting, and analyzing each of them using the different approaches carried out in this study. The limits encountered at each step are indicated. AG motifs assumed to substitute the short N-terminal AGP motif are indicated. The size-heterogeneous Hyp-O-Gal/Ara-rich motifs expected on the Pro-rich domain are represented with green lines, and putative short Ara-oligosaccharides are represented with purple lines.
      The heterogeneity of AGP31 in cell walls raises the question of its origin. O-Glycans might be processed by glycoside hydrolases. Galactosidases and arabinosidases were found in A. thaliana cell wall proteomes (
      • Minic Z.
      • Jouanin L.
      Plant glycoside hydrolases involved in cell wall polysaccharide degradation.
      ,
      • Jamet E.
      • Canut H.
      • Boudart G.
      • Pont-Lezica R.
      Cell wall proteins. A new insight through proteomics.
      ), and several studies provided evidence for enzymatic digestion of AGP O-glycans (
      • Kotake T.
      • Tsuchiya K.
      • Aohara T.
      • Konishi T.
      • Kaneko S.
      • Igarashi K.
      • Samejima M.
      • Tsumuraya Y.
      An α-l-arabinofuranosidase/β-d-xylosidase from immature seeds of radish (Raphanus sativus L.).
      ,
      • Kotake T.
      • Dina S.
      • Konishi T.
      • Kaneko S.
      • Igarashi K.
      • Samejima M.
      • Watanabe Y.
      • Kimura K.
      • Tsumuraya Y.
      Molecular cloning of a β-galactosidase from radish that specifically hydrolyzes β-(1–3)- and β-(1–6)-galactosyl residues of arabinogalactan protein.
      ,
      • Hata K.
      • Tanaka M.
      • Tsumuraya Y.
      • Hashimoto Y.
      α-l-Arabinofuranosidase from radish (Raphanus sativus L.) seeds.
      ,
      • Eudes A.
      • Mouille G.
      • Thévenin J.
      • Goyallon A.
      • Minic Z.
      • Jouanin L.
      Purification, cloning and functional characterization of an endogenous beta-glucuronidase in Arabidopsis thaliana.
      ). Their degradation has been assumed to produce signal molecules involved in plant development (
      • Nothnagel E.
      Proteoglycans and related components in plant cells.
      ,
      • Gaspar Y.
      • Johnson K.
      • McKenna J.
      • Bacic A.
      • Schultz C.
      The complex structures of arabinogalactan-proteins and the journey towards understanding function.
      ). The turnover of AGP O-glycans was also described as part of a salvage pathway allowing the recycling of sugars for the synthesis of new polymers (
      • Gibeaut D.
      • Carpita N.
      Tracing cell wall biogenesis in intact cells and plants: selective turnover and alteration of soluble and cell wall polysaccharides in grasses.
      ). This turnover of O-glycans could explain the observed low molecular mass glycoforms of AGP31. It should be noted that truncated forms of AGP31 missing the C-terminal PAC domain are (i) not glycosylated on the AGP motif and (ii) weakly O-glycosylated on their Pro-rich domain. We suggest that the PAC domain may be degraded by proteases present in A. thaliana cell wall proteomes (
      • Jamet E.
      • Albenne C.
      • Boudart G.
      • Irshad M.
      • Canut H.
      • Pont-Lezica R.
      Recent advances in plant cell wall proteomics.
      ) and that O-glycosylation protects AGP31 from proteolysis, as assumed for AGPs (
      • Showalter A.
      Structure and function of plant cell wall proteins.
      ). Alternatively, the heterogeneity of the Pro-rich domain O-glycosylation may reflect a partial O-glycosylation of AGP31 along the secretory pathway. Then our ETD MS/MS data suggest that isolated Hyp of the Pro-rich domain may be first galactosylated before the subsequent elongation step. Galactosylation would require two different galactosyltransferases (GalTs) as recently reported for AGPs in A. thaliana and Nicotiana tabacum (
      • Oka T.
      • Saito F.
      • Shimma Y.
      • Yoko-o T.
      • Nomura Y.
      • Matsuoka K.
      • Jigami Y.
      Characterization of endoplasmic reticulum-localized UDP-d-galactose. Hydroxyproline O-galactosyltransferase using synthetic peptide substrates in Arabidopsis.
      ,
      • Liang Y.
      • Faik A.
      • Kieliszewski M.
      • Tan L.
      • Xu W.L.
      • Showalter A.
      Identification and characterization of in vitro galactosyltransferase activities involved in arabinogalactan-protein glycosylation in tobacco and Arabidopsis.
      ). Two distinct GalT activities were identified from in vitro assays: a Hyp:GalT activity catalyzing the addition of Gal onto peptidyl-Hyp residues and a Gal:GalT activity extending the sugar chain. Twenty putative β-(1,3)-GalTs were predicted by bioinformatics in A. thaliana (
      • Qu Y.
      • Egelund J.
      • Gilson P.R.
      • Houghton F.
      • Gleeson P.A.
      • Schultz C.J.
      • Bacic A.
      Identification of a novel group of putative Arabidopsis thaliana β-(1,3)-galactosyltransferases.
      ). It will be necessary to characterize the substrate specificity of each of them to elucidate the mechanisms of O-galactosylation of AGPs and PRPs.
      Altogether, AGP31 is a glycoprotein far more complex than previously assumed. The next challenge will be to perform the structural characterization of the new Hyp-O-Gal/Ara-rich motifs of its Pro-rich domain. Such work is mostly performed using biochemical approaches like methylation analysis and NMR spectroscopy and requires large amounts of pure protein. In this way, several models were proposed for type II AGs isolated from recombinant AGPs (
      • Tan L.
      • Varnai P.
      • Lamport D.T.
      • Yuan C.
      • Xu J.
      • Qiu F.
      • Kieliszewski M.J.
      Plant O-hydroxyproline arabinogalactans are composed of repeating trigalactosyl subunits with short bifurcated side chains.
      ). Interestingly, Tryfona et al. (
      • Tryfona T.
      • Liang H.C.
      • Kotake T.
      • Kaneko S.
      • Marsh J.
      • Ichinose H.
      • Lovegrove A.
      • Tsumuraya Y.
      • Shewry P.R.
      • Stephens E.
      • Dupree P.
      Carbohydrate structural analysis of wheat flour arabinogalactan protein.
      ) carried out an alternative approach to elucidate the structure of native wheat flour AGP, combining AG enzymatic digestion and product analysis using polysaccharide analysis by carbohydrate gel electrophoresis and MS. Note that type III AGs have also been described in Art v 1 and Amb a allergens, which have Pro-rich domains (
      • Léonard R.
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      A new allergen from ragweed (Ambrosia artemisiifolia) with homology to art v 1 from mugwort.
      ,
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      • Altmann F.
      Two novel types of O-glycans on the mugwort pollen allergen Art v 1 and their role in antibody binding.
      ).
      Finally, it will be important to elucidate the function of each of the AGP31 domains and the role of the various glycans. As a first clue, this work highlighted the remarkable affinity between AGP31 and PNA. Hyp-O-Gal/Ara-rich motifs of the AGP31 Pro-rich domain may interact with cell wall lectins in muro. Legume lectins, which are the closest homologues of PNA in A. thaliana, were found in A. thaliana cell wall proteomes (
      • Irshad M.
      • Canut H.
      • Borderies G.
      • Pont-Lezica R.
      • Jamet E.
      A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana. Confirmed actors and newcomers.
      ,
      • Jamet E.
      • Canut H.
      • Boudart G.
      • Pont-Lezica R.
      Cell wall proteins. A new insight through proteomics.
      ). This trail may be interesting to explore the structure/function relationships of AGP31 and other proteins with O-glycosylated Pro-rich domains.

      Acknowledgments

      We thank Dr. S. Fournier and Dr. A. Lemassu for advice on trimethylsilylated derivative analyses, Dr. G. Boudart for HF deglycosylation experiments, the late Prof. R. Pont-Lezica for critical reading of the manuscript, and Dr. H. Canut for stimulating discussions. We also thank Prof. M. Kieliszewski (University of Ohio) for welcoming Dr. G. Boudart into her laboratory.

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