Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa

doi:10.1016/j.carres.2010.09.006

Carbohydrate Research

Volume 345, Issue 17, 22 November 2010, Pages 2516-2522

https://doi.org/10.1016/j.carres.2010.09.006 Get rights and content

Abstract

Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-l-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-l-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.

Graphical abstract

β-(1→6)-Galactan side chains of arabinogalactan-proteins are reduced to one or two galactan residues by a combination of an α-l-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, of Neurospora crassa.

Introduction

Arabinogalactan-proteins (AGPs) are a family of proteoglycans involved in various physiological phenomena including cell adhesion, stress resistance, signal transduction, and growth of pollen tubes in higher plants.1, 2, 3, 4, 5, 6 AGPs generally have large (more than 90% of total weight) and complex carbohydrate components rich in Gal and l-Ara.1, 2, 3 The carbohydrate moieties of AGPs have a common structure consisting of β-(1→3)-galactan main chains to which β-(1→6)-galactan side chains are attached through O-6. The β-(1→6)-linked galactosyl chains are further substituted with l-Ara and lesser amounts of other auxiliary sugars such as GlcA, 4-O-methyl-GlcA (4-Me-GlcA), l-Rha, and l-Fuc.1, 2, 3, 7 AGPs undergo in vivo hydrolysis by endogenous glycoside hydrolases in plants. In sycamore cells, the turnover of AGPs is quite rapid,⁸ indicating active metabolism of AGPs by glycoside hydrolases in plants. These enzymes are categorized into glycoside hydrolase families (GHFs) based on amino acid sequence and structural similarity.9, 10 In higher plants, GHF 3 α-l-arabinofuranosidase (EC 3.2.1.55), GHF 35 β-galactosidase (EC 3.2.1.23), and GHF 79 β-glucuronidase (EC 3.2.1.31) are presumed to participate in the hydrolysis of the carbohydrate moieties of AGPs.11, 12, 13, 14, 15

The carbohydrate moieties of AGPs are also degraded by glycoside hydrolases secreted from bacteria and fungi in nature. exo-β-(1→3)-Galactanase (EC 3.2.1.145), belonging to GHF 43, has been isolated from fungi such as Irpex lacteus and Phanerochaete chrysosporium and shown to hydrolyze β-(1→3)-galactan main chains of the carbohydrate moieties of AGPs.16, 17, 18 endo-β-(1→6)-Galactanase (EC 3.2.1.164) categorized into GHF 5 based on the amino acid sequence, specifically acts on the β-(1→6)-galactosyl side chains of AGPs, releasing Gal and β-(1→6)-galactooligosaccharides.19, 20 GHF 79 β-glucuronidases from Aspergillus niger and Neurospora crassa release GlcA and/or 4-Me-GlcA from AGPs.21, 22 exo-β-(1→3)-Galactanase and endo-β-(1→6)-galactanase have also been found in bacteria.23, 24, 25 In addition to these enzymes, α-l-arabinofuranosidases secreted from fungi and bacteria probably participate in the hydrolysis of carbohydrate moieties of AGPs. Indeed, treatment with α-l-arabinofuranosidase makes AGPs susceptible to exo-β-(1→3)-galactanase,16, 18 endo-β-(1→6)-galactanase,19, 20 and β-glucuronidase.²² Additionally, the removal of β-(1→6)-galactan side chains makes the β-(1→3)-galactan main chain a better substrate for exo-β-(1→3)-galactanase, as evidenced by the increasing hydrolysis of β-(1→3)-galactans prepared by successive Smith degradation of gum arabic.16, 18 These facts strongly suggest that the carbohydrate moieties of AGPs are degraded by the collaborative actions of several glycoside hydrolases. However, the degradation process of AGPs by the glycoside hydrolases secreted from a single fungus or bacterium remains elusive.

Genes possibly encoding the glycoside hydrolases active on AGPs can be seen in the genome of several fungi. N. crassa possesses genes for GHF 5 and 54 enzymes, together with one for a GHF 79 β-glucuronidase, NcGlcAase, which has been characterized in our previous study.²² For the present study, a GHF 54 α-l-arabinofuranosidase and a GHF 5 endo-β-(1→6)-galactanase of N. crassa were characterized using their recombinant enzymes expressed in Pichia pastoris. In addition, the synergistic action of these enzymes in the hydrolysis of the carbohydrate moieties of AGPs was investigated.

Section snippets

Identification of genes for GHF 54 α-l-arabinofuranosidase and GHF 5 endo-β-(1→6)-galactanase in N. crassa

The α-l-arabinofuranosidases can be categorized into GHF 3, 43, 51, 54, and 62 based on sequence and structural similarities.9, 10 On the basis of the genome, N. crassa is expected to have open reading frames (ORFs) for two GHF 3, seven GHF 43, one GHF 51, and one GHF 54 enzyme.²⁶ It is known that α-l-arabinofuranosidases belonging to GHF 54 substantially hydrolyze polysaccharides containing α-l-arabinofuranosyl residues.27, 28 Hence, we designated as NcAraf1 a GHF 54 protein (accession number,

Materials

Arabino-(1→4)-xylan from wheat flour, α-(1→5)-arabinooligosaccharides, carboxymethyl (CM)-cellulose 4 M, β-(1→4)-galactan from lupin, β-glucan from barley, and native and debranched arabinans from sugar beet were purchased from Megazyme (Wicklow, Ireland). Galactomannans from guar and locust bean, gum arabic from acacia tree, laminarin from Laminaria digitata, β-(1→4)-xylan from birch wood, PNP-α-L-arabinofuranoside, PNP-α-l-fucoside, PNP-β-galactoside, PNP-β-glucoside, PNP-β-glucuronide, and

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Type II arabinogalactans (AGs) are a highly diverse class of plant polysaccharides generally encountered as the carbohydrate moieties of certain extracellular proteoglycans, the so-called arabinogalactan-proteins (AGPs), which are found on plasma membranes and in cell walls. The basic structure of type II AG is a 1,3-β-D-galactan main chain with 1,6-β-D-galactan side chains. The side chains are further decorated with other sugars such as α-l-arabinose and β-d-glucuronic acid. In addition, AGs with 1,6-β-D-galactan as the main chain, which are designated as ‘type II related AG’ in this review, can also be found in several plants. Due to their diverse and heterogenous features, the determination of carbohydrate structures of type II and type II related AGs is not easy. On the other hand, these complex AGs are scientifically and commercially attractive materials whose structures can be modified by chemical and biochemical approaches for specific purposes. In the current review, what is known about the chemical structures of type II and type II related AGs from different plant sources is outlined. After that, structural analysis techniques are considered and compared. Finally, structural modifications that enhance or alter functionality are highlighted.
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However, the fungal clade of GH30_5 only contained sequences from ascomycetes (Fig. 1d). The fungal and bacterial GH30_5 showing similar activity (β-(1→6)-galactanase) shared more than 50 % amino acid sequence identity [12,13,33]. GH30_7 contained sequences from both ascomycetes and basidiomycetes (Fig. 1e) and the characterized enzymes showed several xylanolytic activities (Table 1).
Efficient bioconversion of agro-industrial side streams requires a wide range of enzyme activities. Glycoside Hydrolase family 30 (GH30) is a diverse family that contains various catalytic functions and has so far been divided into ten subfamilies (GH30_1-10). In this study, a GH30 phylogenetic tree using over 150 amino acid sequences was contructed. The members of GH30 cluster into four subfamilies and eleven candidates from these subfamilies were selected for biochemical characterization. Novel enzyme activities were identified in GH30. GH30_3 enzymes possess β-(1→6)-glucanase activity. GH30_5 targets β-(1→6)-galactan with mainly β-(1→6)-galactobiohydrolase catalytic behavior. β-(1→4)-Xylanolytic enzymes belong to GH30_7 targeting β-(1→4)-xylan with several activities (e.g. xylobiohydrolase, endoxylanase). Additionally, a new fungal subfamily in GH30 was proposed, i.e. GH30_11, which displays β-(1→6)-galactobiohydrolase. This study confirmed that GH30 fungal subfamilies harbor distinct polysaccharide specificity and have high potential for the production of short (non-digestible) di- and oligosaccharides.
Expression of recombinant fungal proteins in Pichia Pastoris
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Production of recombinant proteins, including enzymes, in a heterologous host is often necessary to obtain sufficient amounts for functional studies. In particular, yeast expression systems are cost-effective and convenient for producing recombinant proteins having post-translational modifications characteristic of eukaryotes. The yeast Pichia pastoris is a unicellular eukaryote that can be handled in the same environment as Escherichia coli. Here, we present an overview of the construction of expression systems in P. pastoris and their application for the production of recombinant proteins.
Properties of arabinogalactan-proteins in European pear (Pyrus communis L.) fruits
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It releases Gal from unsubstituted β-(1 → 3)-galactan chains, and can bypass branching points at which β-(1 → 6)-galactosyl side chains are attached, resulting in the release of the side chains as oligosaccharides. Recombinant endo-β-(1 → 6)-galactanase (rTv6GAL) (EC 3.2.1.164) from Trichoderma viride and endo-β-(1 → 3)-galactanase (rNcEn3GAL) (EC 3.2.1.181) from Neurospora crassa expressed in Pichia pastoris were prepared as reported previously [8,9]. The endo-β-(1 → 6)-galactanase has the specific capacity to release Gal, β-Gal-(1 → 6)-Gal, and 4-Me-β-GlcA-(1 → 6)-Gal as the main final reaction products from radish root AGP pretreated with α-L-Arafase [10].
Arabinogalactans (AGs) and arabinogalactan-proteins (AGPs) were partially purified from an extract of fruits of the European pear (Pyrus communis L.) by DEAE-cellulose ion-exchange and Sepharose 6B gel-filtration chromatography. Among 7 AG(P)-containing fractions, a neutral AGP (SE-1) was confirmed to be highly purified (M_r 67,000) and rich in L-Ara and Gal; this fraction included a small amount (2.6%, w/w) of protein and showed the highest reactivity forming precipitate with β-Glc Yariv reagent among the 7 fractions, the intensity of which was comparable to that of gum arabic, a standard AGP. Another accompanying minor low-M_r neutral AGP (SE-2; M_r approx. 7200) still contained other polysaccharide (starch fragments) and did not show Yariv reactivity. The carbohydrate moieties of SE-1 consisted of consecutive (1 → 3)-linked β-galactosyl backbone chains substituted with side chains of (1 → 6)-linked β-galactosyl residues at O-6, to which mainly single α-l-arabinofuranosyl residues were attached through O-3. This structural feature was also observed for SE-2. Successive digestion of SE-1 with α-l-arabinofuranosidase and exo-β-(1 → 3)-galactanase with the aid of endo-β-(1 → 3)-galactanase released most (more than 98%, w/w) of the carbohydrate moieties as low-M_r fragments. These consisted of free L-Ara and Gal, and a series of β-(1 → 6)-galactooligosaccharides with degree of polymerization (dp) up to at least 17, indicative of attachment of (1 → 6)-linked β-galactosyl side chains of varying length along the (1 → 3)-linked β-galactosyl backbone chains.
Properties of two fungal endo-β-1,3-galactanases and their synergistic action with an exo-β-1,3-galactanase in degrading arabinogalactan-proteins
2017, Carbohydrate Research
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Interestingly, the mycelia of F. velutipes can utilize gum arabic as a carbon source [25]. The genome of N. crassa has genes for exo-β-1,3-galactanase and endo-β-1,6-galactanase in addition to the gene for NcEn3GAL [18,23]. These observations suggest that endo-β-1,3-galactanases contribute to the degradation of AGPs by acting on different structure than the other galactanases.
Arabinogalactan-proteins (AGPs) are plant proteoglycans, which are widely encountered in the plant kingdom, usually localized on the cell surface. The carbohydrate moieties of AGPs consist of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. To date, FvEn3GAL isolated from Flammulina velutipes is the sole β-1,3-galactanase acting on β-1,3-galactan in an endo-manner. Here we cloned two homologous genes, designated Af3G and NcEn3GAL, possibly encoding endo-β-1,3-galactanase from Aspergillus flavus and Neurospora crassa, respectively. The recombinant Af3G (rAf3G) and rNcEn3GAL expressed in Pichia pastoris specifically hydrolyzed β-1,3-galactan in an endo-manner, as did the rFvEn3GAL. Among galactooligosaccharides, β-1,3-galactotriose was identified as the smallest substrate for these enzymes. These results suggest that enzymatic characteristics are conserved in many endo-β-1,3-galactanases belonging to the glycoside hydrolase 16 family. On the other hand, rAf3G and rNcEn3GAL generated more β-1,3-galactobiose from β-1,3-galactotetraose than did rFvEn3GAL, suggesting that rAf3G and rNcEn3GAL prefer hydrolyzing the central β-1,3-glycosidic linkage of three in β-1,3-galactotetraose. Although rAf3G and rNcEn3GAL alone hardly hydrolyze native AGP, they acted synergistically with a fungal exo-β-1,3-galactanase on the AGP. These endo-β-1,3-galactanases presumably aid hydrolysis by internally breaking up AGPs, which creates more sites of attack for exo-β-1,3-galactanase.
Differences in the expression profile of endo-β-(1,6)-D-galactanase in pathogenic and non-pathogenic races of Colletotrichum lindemuthianum grown in the presence of arabinogalactan, xylan or Phaseolus vulgaris cell walls
2017, Physiological and Molecular Plant Pathology
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Despite the critical role of endo-β-(1,6)-D-galactanases in CW degradation, there have been few studies of these enzymes in fungi and bacteria, and so far their research has focused on the application in biotechnology. Accordingly, native endo-β-(1,6)-D-galactanases from Aspergillus niger and Trichoderma viride have been characterized [12,13] and cDNAs from T. viride and Neurospora crassa have been sequenced, characterized and expressed in Escherichia coli and Pichia pastoris, respectively [14,15]. There is only one report of an endo-β-(1,6)-d-galactanase in the actinomycete Streptomyces avermitilis [16].
The scope of this study was the isolation and molecular characterization of endo-β-1,6-galactanase (ebg) from pathogenic (1472) and non-pathogenic (0) races of Colletotrichum lindemuthianum cultivated with arabinogalactan, xylan or Phaseolus vulgaris cell walls as carbon sources. Characterization of the ebg cDNA and 3D protein modeling revealed typical elements of the GH30 family of galactanases. The growth of both races with glucose showed basal transcription levels of ebg. When glucose was replaced with arabinogalactan, xylan or plant cell walls, ebg transcription markedly increased in race 1472 but not in race 0. Putative DNA-binding sites for Cre, Xlnr, ACEI, PacC and Gal4 transcriptional factors were predicted in ebg genes from Colletotrichum species.

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Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa☆

Abstract

Graphical abstract

Introduction

Section snippets

Identification of genes for GHF 54 α-l-arabinofuranosidase and GHF 5 endo-β-(1→6)-galactanase in N. crassa

Materials

Int. Rev. Cytol.

Cell

J. Biol. Chem.

J. Biol. Chem.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

J. Biosci. Bioeng.

Biochim. Biophys. Acta

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Carbohydr. Res.

Anal. Biochem.

Anal. Biochem.

Annu. Rev. Plant Physiol.

Ann. Rev. Plant Biol.

Plant Cell

Nature

Plant Physiol.

Plant Physiol.