Molecular Cloning of a b-Galactosidase from Radish That Specifically Hydrolyzes b-(1!3)- and b-(1!6)-Galactosyl Residues of Arabinogalactan Protein 1 Toshihisa Kotake*, Soraya Dina, Tomoyuki Konishi, Satoshi Kaneko, Kiyohiko Igarashi, Masahiro Samejima, Yoko Watanabe, Kazumasa Kimura, and Yoichi Tsumuraya Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, Sakura-ku, Saitama 338–8570, Japan (T.K., S.D., T.K., Y.T.); Biological Function Division, National Food Research Institute, Tsukuba, Ibaraki 305–8642, Japan (S.K.); Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan (K.I., M.S.); and Yakult Central Institute for Microbiological Research, Tokyo 186–8650, Japan (Y.W., K.K.) A basic b-galactosidase with high specificity toward b-(1/3)- and b-(1/6)-galactosyl residues was cloned from radish (Raphanus sativus) plants by reverse transcription-PCR. The gene, designated RsBGAL1, contained an open reading frame consisting of 2,532 bp (851 amino acids). It is expressed in hypocotyls and young leaves. RsBGAL1 was highly similar to b-galactosidases having exo-b-(1/4)-galactanase activity found in higher plants and belongs to family 35 of the glycosyl hydrolases. Recombinant RsBGAL1 was expressed in Pichia pastoris and purified to homogeneity. The recombinant enzyme specifically hydrolyzed b-(1/3)- and b-(1/6)-galactooligosaccharides, the same substrates as the native enzyme isolated from radish seeds (Sekimata et al., 1989). It split off about 90% of the carbohydrate moieties of an arabinogalactan protein extracted from radish roots in concerted action with microbial a-L-arabinofuranosidase and b-glucuronidase. These results suggest that RsBGAL1 is a new kind of b-galactosidase with different substrate specificity than other b-galactosidases that exhibit exo-b-(1/4)-galactanase activity. The C-terminal region (9.6 kD) of RsBGAL1 is significantly similar to the Gal lectin- like domain, but this region is not retained in the native enzyme. Assuming posttranslational processing of RsBGAL1 with elimination of the Gal lectin-like domain results in a protein consisting of two subunits with molecular masses of 46 and 34 kD (calculated from the RsBGAL1 gene sequence). This is in good agreement with the SDS-PAGE and matrix-assisted laser desorption/ionization-time-of flight mass spectrometry measurements for subunits of the native enzyme (45 and 34 kD) and may thus partially explain the formation process of the native enzyme. The enzyme group of b-galactosidases (EC 3.2.1.23) is widely distributed in higher plants. Plant b-galac- tosidases can be divided into at least two classes according to substrate specificity: one class that com- prises exo-b-(1/4)-galactanases that specifically act on pectic b-(1/4)-galactan (sugars in this study are D series unless designated otherwise), and a second class that prefers p-nitrophenyl-b-galactoside (PNP- b-Gal) and lacks hydrolytic activity toward b-(1/4)- galactan (in this study, we call the former enzymes b-galactosidase/exo-b-(1/4)-galactanases). In the tomato (Lycopersicon esculentum), b-galactosidase/exo- b-(1/4)-galactanase activity significantly increases due to specific expression of the enzyme proteins during fruit ripening. This indicates their role in the degradation of b-(1/4)-galactan side chains of pec- tins as part of the ripening process. On the other hand, the activity level of the second class of b-galactosidase does not markedly change during ripening (Carey et al., 1995; Smith et al., 1998; Smith and Gross, 2000). Since the in vivo substrates for the second class of b-galactosidase is not yet identified, their functions in plant growth and development remain elusive. Arabinogalactan proteins (AGPs), a family of pro- teoglycans found in higher plants, consist of a Hyp- rich core protein and carbohydrate moieties attached to the Hyp, Ser, and/or Thr residues. The carbohy- drate moieties of AGPs have a common structure of b-(1/3)-galactosyl backbones to which side chains of b-(1/6)-linked galactosyl residues are attached through O-6. The b-(1/6)-linked galactosyl chains are further substituted with L-arabinofuranose (L-Ara) and lesser amounts of other auxiliary sugars, such as GlcUA, 4-O-methyl-GlcUA (4-Me-GlcUA), L-rhamnose, and L-Fuc. AGPs are implicated in many physiological processes, such as cell-to-cell signaling, cell adhesion, cell elongation, cell death, and stress responses (Fincher et al., 1983; Nothnagel, 1997; Majewska-Sawka and Nothnagel, 2000; Shi et al., 2003). In tobacco (Nicotiana tabacum), transmitting tissue-specific (TTS) protein that belongs to the AGP family is incorporated into pollen tube walls and undergoes degradation of the carbohydrate moieties, leading to stimulation of elongation growth (Cheung et al., 1995; Wu et al., 1995). Although the enzymes 1 This work was supported in part by a Grant for Ground Research for Space Utilization (to T.K.) from the Japan Space Forum. * Corresponding author; e-mail [email protected]; fax 81–48–858–3384. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.062562. Plant Physiology, July 2005, Vol. 138, pp. 1563–1576, www.plantphysiol.org Ó 2005 American Society of Plant Biologists 1563 Downloaded from https://academic.oup.com/plphys/article/138/3/1563/6103112 by guest on 05 January 2022
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Molecular Cloning of a b-Galactosidase from Radish ThatSpecifically Hydrolyzes b-(1!3)- and b-(1!6)-GalactosylResidues of Arabinogalactan Protein1
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, Sakura-ku,Saitama 338–8570, Japan (T.K., S.D., T.K., Y.T.); Biological Function Division, National Food ResearchInstitute, Tsukuba, Ibaraki 305–8642, Japan (S.K.); Graduate School of Agricultural and Life Sciences,University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan (K.I., M.S.); and Yakult Central Institute forMicrobiological Research, Tokyo 186–8650, Japan (Y.W., K.K.)
A basic b-galactosidase with high specificity toward b-(1/3)- and b-(1/6)-galactosyl residues was cloned from radish(Raphanus sativus) plants by reverse transcription-PCR. The gene, designated RsBGAL1, contained an open reading frameconsisting of 2,532 bp (851 amino acids). It is expressed in hypocotyls and young leaves. RsBGAL1 was highly similar tob-galactosidases having exo-b-(1/4)-galactanase activity found in higher plants and belongs to family 35 of the glycosylhydrolases. Recombinant RsBGAL1 was expressed in Pichia pastoris and purified to homogeneity. The recombinant enzymespecifically hydrolyzed b-(1/3)- and b-(1/6)-galactooligosaccharides, the same substrates as the native enzyme isolatedfrom radish seeds (Sekimata et al., 1989). It split off about 90% of the carbohydrate moieties of an arabinogalactan proteinextracted from radish roots in concerted action with microbial a-L-arabinofuranosidase and b-glucuronidase. These resultssuggest that RsBGAL1 is a new kind of b-galactosidase with different substrate specificity than other b-galactosidases thatexhibit exo-b-(1/4)-galactanase activity. The C-terminal region (9.6 kD) of RsBGAL1 is significantly similar to the Gal lectin-like domain, but this region is not retained in the native enzyme. Assuming posttranslational processing of RsBGAL1 withelimination of the Gal lectin-like domain results in a protein consisting of two subunits with molecular masses of 46 and 34 kD(calculated from the RsBGAL1 gene sequence). This is in good agreement with the SDS-PAGE and matrix-assisted laserdesorption/ionization-time-of flight mass spectrometry measurements for subunits of the native enzyme (45 and 34 kD) andmay thus partially explain the formation process of the native enzyme.
The enzyme group of b-galactosidases (EC 3.2.1.23)is widely distributed in higher plants. Plant b-galac-tosidases can be divided into at least two classesaccording to substrate specificity: one class that com-prises exo-b-(1/4)-galactanases that specifically acton pectic b-(1/4)-galactan (sugars in this study areD series unless designated otherwise), and a secondclass that prefers p-nitrophenyl-b-galactoside (PNP-b-Gal) and lacks hydrolytic activity toward b-(1/4)-galactan (in this study, we call the former enzymesb-galactosidase/exo-b-(1/4)-galactanases). In thetomato (Lycopersicon esculentum), b-galactosidase/exo-b-(1/4)-galactanase activity significantly increasesdue to specific expression of the enzyme proteinsduring fruit ripening. This indicates their role in thedegradation of b-(1/4)-galactan side chains of pec-tins as part of the ripening process. On the other hand,the activity level of the second class of b-galactosidasedoes not markedly change during ripening (Carey
et al., 1995; Smith et al., 1998; Smith and Gross, 2000).Since the in vivo substrates for the second class ofb-galactosidase is not yet identified, their functionsin plant growth and development remain elusive.
Arabinogalactan proteins (AGPs), a family of pro-teoglycans found in higher plants, consist of a Hyp-rich core protein and carbohydrate moieties attachedto the Hyp, Ser, and/or Thr residues. The carbohy-drate moieties of AGPs have a common structure ofb-(1/3)-galactosyl backbones to which side chainsof b-(1/6)-linked galactosyl residues are attachedthrough O-6. The b-(1/6)-linked galactosyl chainsare further substituted with L-arabinofuranose(L-Ara) and lesser amounts of other auxiliary sugars,such as GlcUA, 4-O-methyl-GlcUA (4-Me-GlcUA),L-rhamnose, and L-Fuc. AGPs are implicated in manyphysiological processes, such as cell-to-cell signaling,cell adhesion, cell elongation, cell death, and stressresponses (Fincher et al., 1983; Nothnagel, 1997;Majewska-Sawka and Nothnagel, 2000; Shi et al.,2003). In tobacco (Nicotiana tabacum), transmittingtissue-specific (TTS) protein that belongs to the AGPfamily is incorporated into pollen tube walls andundergoes degradation of the carbohydrate moieties,leading to stimulation of elongation growth (Cheunget al., 1995; Wu et al., 1995). Although the enzymes
1 This work was supported in part by a Grant for GroundResearch for Space Utilization (to T.K.) from the Japan Space Forum.
Article, publication date, and citation information can be found atwww.plantphysiol.org/cgi/doi/10.1104/pp.105.062562.
Plant Physiology, July 2005, Vol. 138, pp. 1563–1576, www.plantphysiol.org � 2005 American Society of Plant Biologists 1563
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involved in the degradation of TTS protein have notyet been identified, the incorporated carbohydratemoieties of TTS protein are thought to serve asnutrients for the pollen tubes. It is also known thatthe sugar composition and structure of the carbohy-drate moieties of AGPs are organ specific and regu-lated depending on the stage of development,implying that the carbohydrate moieties undergorapid metabolism (Tsumuraya et al., 1988). Indeed,the rate of turnover of the carbohydrate moieties ofAGPs in proso millet cells is extremely high (Gibeautand Carpita, 1991). This means that a substantialportion of liberated sugars must be reutilized for thesynthesis of new polymers.
It seems likely that hydrolysis of the carbohydratemoieties of AGPs is the result of concerted actionof several glycosidases, such as b-galactosidase, a-L-arabinofuranosidase (a-L-arafase), and b-glucuroni-dase (b-GlcUAase). We have previously foundb-galactosidases in radish (Raphanus sativus) seedsand spinach (Spinacia oleracea) leaves, which werecapable of hydrolyzing b-(1/3)- and b-(1/6)-galac-tosyl sequences of AGPs but not pectic b-(1/4)-galactan (Sekimata et al., 1989; Hirano et al., 1994).These enzymes belong to the second class of b-galac-tosidase. The monomeric sugars released by b-galac-tosidase and other glycosidases may be utilized asnutrients during the development of plant tissues, asstated above, and/or salvaged via incorporation intothe cytoplasm followed by conversion to nucleotidesugars by the successive actions of respective mono-saccharide kinases and pyrophosphorylases (Reiterand Vanzin, 2001). This last metabolic process hasbeen partially verified in Pisum sativum by our recentfinding of a novel pyrophosphorylase that catalyzesconversion of various monosaccharide-1 phosphates(including Gal, Glc, GlcUA, L-arabinopyranose, andXyl-1 phosphates) in the presence of UTP to the re-spective UDP sugars (Kotake et al., 2004).
In this article, we report the isolation of a cDNAclone encoding a b-galactosidase from radish and theproperties of the recombinant protein expressed in
Pichia pastoris. Based on the substrate specificity ofthe recombinant protein, we suggest that the b-galac-tosidase has specificity toward b-(1/3)- andb-(1/6)-galactosyl residues and plays a key role inthe degradation of the carbohydrate moieties of AGPs.
RESULTS
Nature of the Native b-Galactosidase Protein
A native b-galactosidase was purified from radishseeds by the methods employed in our previous study(Sekimata et al., 1989) and analyzed on SDS-PAGE(Fig. 1A). The purified enzyme was resolved into twosubunits with relative molecular masses of 45 and34 kD on SDS-PAGE. Determination of the molecularmass of the fragments by matrix-assisted laser de-sorption/ionization-time-of flight mass spectrometry(MALDI-TOF/MS) gave 44,981 and 33,654 D, respec-tively (Fig. 1B). The small signals corresponding tomolecular masses of 7,973 and 17,621 D seem to becaused by contaminations and could not be detectedon SDS-PAGE. The faint band at molecular mass 29 kDon SDS-PAGE seems to be a contaminant that ap-pears only on SDS-PAGE; it was not detectable byMALDI-TOF/MS. The dimeric nature and the molec-ular masses of the two subunits of the radish enzymeare similar to those observed for a persimmonb-galactosidase (44 and 34 kD; Kang et al., 1994) andan apple (Malus domestica) b-galactosidase (44 and32 kD; Ross et al., 1994). The N-terminal aminoacid sequences of the radish b-galactosidase wereASVTYDHRAL VIDGKRKILI SGSIHY for the 45-kDsubunit and AELGSQWSYP KEPVGADAFD VKP forthe 34-kD subunit. The peptide sequences of both sub-units are highly similar to those of the correspondingsubunits of b-galactosidase/exo-b-(1/4)-galactanaseand the second class of b-galactosidases found inhigher plants.
Based on the sequence determined for the purifiedb-galactosidase, reverse transcription-PCR followedby 3# and 5# RACE procedures were performed. The
Figure 1. Molecular masses of the sub-units of a native b-galactosidase fromradish seeds. A, Enzyme protein (0.5 mg)purified from imbibed radish seeds byseveral chromatographic procedures wasseparated on SDS-PAGE. Lane S, Molecu-lar mass markers; lane 1, purified enzyme.Protein in the gel was stained with Coo-massie Brilliant Blue R-250. The proteinbands subjected to peptide sequenceanalysis are indicated by arrows. B, Mo-lecular masses of the subunits weredetermined byMALDI-TOF/MS. The num-bers at the top of the peaks indicate theobserved molecular mass.
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Figure 2. Amino acid sequence of RsBGAL1. A, Amino acid sequence of RsBGAL1 was aligned with plant b-galactosidase andb-galactosidase/exo-b-(1/4)-galactanase sequences by the pairwise method using the ClustalW program. The amino acidresidues are numbered from the first Met. Gaps (-) were introduced to achieve maximum similarity. Residues identical toRsBGAL1 are highlighted in black. The solid lines indicate amino acid sequences corresponding to those determined for thenative enzyme purified from radish seeds and the dotted line indicates a domain with similarity to Gal lectin. B, Phylogenetic
b-Galactosidase from Radish
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cloned cDNA, designated RsBGAL1 (Raphanus sativusbeta-galactosidase 1), appeared to encode a polypeptideof 851 amino acids (molecular mass 92,534 D; Fig. 2A).The calculated pI value for RsBGAL1 (from Ala-31to Ala-851) was 9.72. The N-terminal sequence of the45-kD subunit of the native enzyme coincided with theregion from Ala-31 to Tyr-56 of RsBGAL1, but thatfor the 34-kD subunit differed considerably from thecorresponding sequence from Ala-445 to Pro-469. Thereason for this discrepancy is not clear, but it may bea consequence of low purity of the native enzymeand/or a difference between the cultivars used forthe purification of the native enzyme and for thecDNA cloning. The cDNA contained a putative signalsequence (30 amino acid residues) preceding theN-terminal sequence of the native enzyme, and thissignal sequence agreed well with the prediction bythe SignalP program (Bendtsen et al., 2004). Twoputative N-glycosylation sites were found in the de-duced protein sequence. The molecular mass (45 kD)determined for the larger fragment by MALDI-TOF/MS was almost identical to the calculated molecularmass (46.2 kD) of the region from Ala-31 to Ser-444; themass of the smaller (34 kD) fragment, however, did notagree with that expected for the region from Ala-445to Ala-851 (43.2 kD). This discrepancy between therelative molecular mass observed and the calculatedmolecular mass from the cDNA sequence is likely aresult of posttranslational processing occurringaround the Ser-759 residue and at Ser-444. Assumingthat the posttranslational processing occurred betweenSer-444 and Ala-445 and between Ser-759 and Asp-760, the polypeptide (from Ala-31 to Ala-851) wouldbe divided into three fragments with molecularmasses of 46.2 (from Ala-31 to Ser-444), 33.6 (fromAla-445 to Ser-759), and 9.6 kD (from Asp-760 toAla-851). Since we could not find the small C-terminalfragment (9.6 kD) in the native enzyme, we surmisethat the missing C-terminal fragment has likely beenremoved in the posttranslational processing, but wecannot rule out the possibility that it has been lostduring the purification procedures.
Amino Acid Sequence of RsBGAL1
Based on amino acid sequence and structuralsimilarities, glycoside hydrolases are classified intomore than 90 families (Henrissat, 1991; Henrissat andBairoch, 1993). RsBGAL1 is quite similar to other plantb-galactosidases and b-galactosidase/exo-b-(1/4)-galactanases, such as TBG5 (AF154423; 67% identical)
and TBG4 (AF023847; 54% identical) from tomato(Smith et al., 1998), indicating that RsBGAL1 is a mem-ber of family 35 of the glycoside hydrolases (Fig. 2A).Phylogenetic analysis of plant b-galactosidases re-vealed that RsBGAL1 forms, together with Arabidop-sis (Arabidopsis thaliana) BGAL8 (AtBGAL8, At2g28470)and tomato TBG5, a small subgroup apart from plantb-galactosidase/exo-b-(1/4)-galactanases, such as to-mato TBG4 and apple ABG1 (Ross et al., 1994; Fig. 2B).These results suggest that RsBGAL1, AtBGAL8, andTBG5 have properties and functions distinct fromthose of b-galactosidase/exo-b-(1/4)-galactanases inthe cell wall metabolism. As in a previous study onb-galactosidase genes from strawberry (Trainotti et al.,2001), RsBGAL1 was found to possess a Gal-bindinglectin-like domain at the C terminus (Leu-773-Ala-851), which shows significant similarities with aGal-specific lectin from Anthocidaris crassispina (32%identical; A37961) and an L-rhamnose-binding lectinfrom Silurus asotus (31% identical; Q9PVW8). Interest-ingly, the lectin-like domain is a structural character-istic of the RsBGAL1 subfamily, but not common inmembers of the b-galactosidases/exo-b-(1/4)-galac-tanase family, such as TGB4 (Fig. 2, A and B).
Organization and Expression Pattern of theRsBGAL1 Gene
The expression pattern of RsBGAL1 in young radishseedlings was analyzed. The transcript of RsBGAL1was detected in hypocotyls and leaves, but not inroots. Relatively strong expression of RsBGAL1 wasobserved in the uppermost part of the hypocotyls(Fig. 3).
The number of RsBGAL1-related genes in the radishgenome was determined by Southern-blot analysis. Alabeled cDNA probe hybridized to several restrictionfragments of the genomic DNA digested with DraI,EcoRI, or HindIII. A few faint bands were also detectedin the genomic DNA digested with BamHI or XbaI (Fig.4). These results suggest that several related genesexist in the radish genome.
Heterologous Expression of RsBGALI in P. pastoris
The RsBGAL1 open reading frame, except for thesignal peptides, was fused to a yeast secretion signalsequence (a-factor) and introduced into the methylo-trophic yeast P. pastoris. Recombinant RsBGAL1(rRsBGAL1) was induced under the control of the al-cohol oxidase promotor and purified from the culture
Figure 2. (Continued.)relationships of RsBGAL1, b-galactosidases, and b-galactosidases/exo-b-(1/4)-galactanases were analyzed using ClustalW.Clones containing a Gal lectin-like domain are underlined. Accession numbers for the clones are as follows: ABG1, AAA62324;TBG1, CAA58734; TBG2, AAF70821; TGB3, CAA10173; TBG4, AAC25984; TBG5, AAF70824; TBG6, AAF70825; TBG7,AAF70823; AtBGAL1, At3g13750; AtBGAL3, At4g36360; AtBGAL4, At5g56870; AtBGAL6, At5G63800; AtBGAL8,At2g28470; AtBGAL9, At2g32810; AtBGAL10, At5g63810; AtBGAL11, At4g35010; AtBGAL12, At4g26140; AtBGAL13,At2g16730; AtBGAL14, At4g38590; RsBGAL1, AB180725.
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medium by conventional chromatography (Table I).The specific activity (64.9 units mg protein21) ofrRsBGAL1 was more than 10 times (5.6 units mgprotein21) that of the native enzyme (Sekimata et al.,1989). The purified rRsBGAL1 appeared as a singleband, with a relative molecular mass of approximately100 kD on SDS-PAGE, indicating that the recombinantenzyme had not undergone posttranslational proteol-ysis in the yeast (Fig. 5). The molecular mass forrRsBGAL1 was determined as 106,449 D by MALDI-TOF/MS analysis (data not shown). The higher mo-lecular mass of the recombinant enzyme compared tothe value (89.4 kD) expected from the cDNA sequenceis likely the result of a difference in the N-glycosylationof the protein between plants and yeast. It is knownthat yeast attaches large high-Man-type glycans tosecreted proteins (Gemmill and Trimble, 1999).
Properties of rRsBGAL1
The properties of rRsBGAL1 were examined usingPNP-b-Gal as the substrate. The recombinant enzymeshowed maximum activity between pH 3.5 and pH 4.0and became almost inactive on PNP-b-Gal below pH2.0 and above pH 6.0. The optimum temperature forenzyme action was 50�C, and the enzyme lost about90% of its activity at 65�C. The native b-galactosidasefrom radish seeds shows maximal activity at 40�C andcompletely loses activity when exposed to 55�C for10 min (Sekimata et al., 1989). The greater stabilityof rRsBGAL1 under high temperature may be attrib-
uted to the N-glycosylation performed by P. pastoris.The effects of heavy metal ions were examined ata final concentration of 1 mM. The metal ions Fe31,Mn21, Cu21, Mg21, Ba21, Ca21, Co21, Zn21, and Cd21
did not significantly affect enzyme activity, whereasHg21 almost completely inactivated the enzyme. Iodo-acetic acid and SDS also inhibited more than 80% ofthe enzyme activity when applied at a concentrationof 1 mM.
Substrate Specificity of rRsBGAL1toward Oligosaccharides
The activity of rRsBGAL1 toward oligosaccharideswas examined using various b-(1/3)-, b-(1/4)-, andb-(1/6)-galactooligosaccharides. Whereas rRsBGAL1extensively hydrolyzed b-(1/3)- and b-(1/6)-galac-tooligosaccharides, it failed to act on b-(1/4)-galac-tooligosaccharides. The action of this enzyme seemsthus specific to b-(1/3)- and b-(1/6)-linked galac-tosyl residues (Table II). The hydrolysis rates of theseb-(1/3)- and b-(1/6)-galactooligomers tended toincrease with increasing degree of polymerization.The weak action on methyl-b-galactoside and lactose[b-Gal-(1/4)-Glc], and the failure of hydrolysis ofb-Gal-(1/3)-GalNAc and b-Gal-(1/3)-GlcNAc, sug-gest that adjacent residues linked to the galactosyl re-sidues also affect the enzymatic action of rRsBGAL1.On heterooligosaccharides substituted with a-L-arabi-nofuranosyl, b-glucuronosyl, or 4-O-methyl-b-glucur-onosyl, residues at the nonreducing terminals ofgalactooligomers, rRsBGAL1 did not act (Table II).
Since the carbohydrate moieties of AGPs commonlyhave branched b-(1/3)(1/6)-galactan structures astheir backbones, the action of rRsBGAL1 on mixed-linkage galactotrioses, b-Gal-(1/6)-b-Gal-(1/3)-Galand b-Gal-(1/3)[b-Gal-(1/6)]-Gal, was examined inorder to explore the mechanism for the completedigestion of the galactan backbones by plant b-galac-tosidases. These galactotrioses were good substratesfor rRsBGAL1, almost to the same extent as b-(1/3)-and b-(1/6)-galactotrioses (Table II). In the case ofb-Gal-(1/3)[b-Gal-(1/6)]-Gal, the hydrolysate con-tained more b-(1/3)-galactobiose than b-(1/6)-gal-actobiose as intermediate products (Fig. 6), whichindicates that the enzyme preferred the b-(1/6)-linked galactosyl residues to the b-(1/3)-linkedgalactosyl residues of b-Gal-(1/3)[b-Gal-(1/6)]-Gal.Since the intermediate products from mixed-linkagegalactotrioses, b-(1/3)- and b-(1/6)-galactobioses
Table I. Purification of rRsBGAL1 expressed in P. pastoris
Total Protein Total Activitya Specific Activity Recoveryb Purification Factorb
aActivity determined with PNP-b-Gal as the substrate. bRecoveries are expressed as a percentage of initial activity, andpurification factors are calculated on the basis of specific activities.
Figure 3. Northern-blot analysis of RsBGAL1. Total RNAwas extractedfrom young leaves, hypocotyls, and roots and then subjected tonorthern hybridization using the labeled RsBGAL1 fragment excisedfrom RsBGAL1 cDNA as the probe. The position of the segment excisedfrom the hypocotyls for RNA preparation is indicated in parentheses(distance from the cotyledons). The methylene blue-stained 18S rRNAused as a loading control is shown at the bottom.
b-Galactosidase from Radish
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are, in turn, good substrates for rRsBGAL1, it seemsthat not only the linear b-(1/3)- and b-(1/6)-galac-tosyl stretches but also the branch points ofb-(1/3)(1/6)-galactans of AGPs can be completelydegraded by this enzyme.
The effect of substrate concentration on the activityof rRsBGAL1 was examined using PNP-b-Gal,b-(1/3)-galactobiose, and b-(1/6)-galactobiose. Theresulting Km, kcat, and catalytic efficiency (kcat/Km)values are listed in Table III. Although the Km value(0.30 mM) of rRsBGAL1 for PNP-b-Gal was compara-ble to that (0.46 mM) of the native enzyme (Sekimataet al., 1989), the catalytic efficiency (4.10 3 105) ofrRsBGAL1 was much higher than that (0.16 3 105,calculated from the Km value, 0.46 mM, and the Vmaxvalue, 5.36 mmol min21 mg protein21) of the nativeenzyme (Sekimata et al., 1989). The lower Km value (i.e.higher affinity) of rRsBGAL1 toward b-(1/6)-galactobiose than toward b-(1/3)-galactobiose isconsistent with the preference of the enzyme forthe b-(1/6)-galactosyl residues over the b-(1/3)-galactosyl residues of b-Gal-(1/3)[b-Gal-(1/6)]-Gal(Fig. 6).
Substrate Specificity of rRsBGAL1toward Polysaccharides
Compared to the activity against PNP-b-Gal, which istaken as 100 in Table IV, b-(1/3)-galactan degradationproceeded slowly. Under exhaustive digestion,rRsBGAL1 released about 30% of Gal from b-(1/3)-galactan, whereas b-(1/4)-galactan and Protothecazopfii b-(1/3)(1/6)-galactan were essentially resis-tant to the enzyme. Native root and leaf AGPs wereresistant to enzymatic hydrolysis even after prolongedincubation. However, partial removal of L-arabinosylresidues from the carbohydrate moieties made the
modified AGPs more accessible to rRsBGAL1, result-ing in the release of 16% and 25% of Gal based on totalsugars in the modified AGPs, respectively. The loweractivity of rRsBGAL1 toward a-L-arafase-treated rootAGP is likely attributable to obstruction by L-arabino-syl residues remaining even after treatment. Themicrobial a-L-arafase used releases almost all L-arabi-nosyl residues from the leaf AGP, whereas 10% to 30%of total L-arabinosyl residues remain in root AGP evenafter exhaustive digestion (see Table V; Tsumurayaet al., 1984, 1988). Overall, the action pattern ofrRsBGAL1 on AGPs and galactans was quite similarto that of the native b-galactosidase (Sekimata et al.,1989). Other polysaccharides, such as b-(1/3)(1/4)-glucan from barley, b-(1/3)(1/6)-glucan fromLaminaria digitata, b-(1/6)-glucan from Umbilicariapapullosa, CM-curdlan [b-(1/3)-glucan], CM-cellulose[b-(1/4)-glucan], galactomannans from guar andlocust bean, b-(1/4)-xylan from birchwood (Betulaspp.), debranched arabinan, and chitosan from crabshells were not hydrolyzed by rRsBGAL1 at all.
Degradation of AGP by rRsBGAL1 withOther Glycosidases
To gain an insight into the mechanism of turnover ofAGPs in vivo, the synergistic action of rRsBGAL1 withother glycosidases on an AGP was investigated. Asshown in Figure 7, the action of rRsBGAL1 aloneliberated only a limited amount (approximately 14% ofthe total sugars) of Gal as the sole hydrolysis productfrom a-L-arafase-treated radish root AGP under ex-haustive digestion (Table IV). This relatively limitedhydrolysis of the carbohydrate moieties of the modi-fied AGP can be attributed to the presence of uronic
Figure 5. SDS-PAGE of rRsBGAL1 at different purification steps. TherRsBGAL1 proteins (0.5 mg) obtained after different purification steps ofthe recombinant protein were analyzed by SDS-PAGE. Lane S, Molec-ular mass markers; lane 1, supernatant from P. pastoris culture medium;lane 2, rRsBGAL1 purified on CM-cellulose column; lane 3, rRsBGAL1purified on hydroxyapatite column. Protein in the gel was stained withCoomassie Brilliant Blue R-250. The arrow indicates the purifiedrRsBGAL1.
Figure 4. Southern-blot analyses of RsBGAL1 in the radish genome.Genomic DNA (10 mg) of radish was digested with ApaI (lane 1),BamHI (lane 2), DraI (lane 3), EcoRI (lane 4), HindIII (lane 5), and XbaI(lane 6) and then subjected to Southern hybridization using the labeledRsBGAL1 fragment excised from RsBGAL1 cDNA as the probe.Locations of lDNA markers digested with HindIII are shown on theleft side.
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acids at the nonreducing ends of b-(1/6)-linkedgalactosyl side chains of most of the AGPs (Fincheret al., 1983; Nothnagel, 1997), which renders galactosylresidues inaccessible to the enzyme. Indeed, more than70% of the total side chains of radish root AGP areknown to carry 4-Me-GlcUA groups at their nonre-ducing ends (Tsumuraya et al., 1990). As anticipated,the simultaneous action of rRsBGAL1 and a microbialb-GlcUAase on the modified AGP liberated muchmore (approximately 80%) reducing sugar by expos-ing nonreducing galactosyl residues, making the sidechains susceptible to rRsBGAL1. Further addition ofa microbial a-L-arafase to the above reaction mixtureliberated nearly 90% of total sugar from the modifiedAGP. Finally, a large portion (91%) of Gal, togetherwith 4-Me-GlcUA (8%) and L-Ara (1%), was liberatedas free monosaccharides. The control experimentusing b-GlcUAase alone liberated only very little4-Me-GlcUA, and a-L-arafase alone did not act onthe modified AGP. Since a-L-arafase-treated radishroot AGP still retains a small portion (approximately5%) of L-arabinosyl residues in the carbohydrate
moiety, possibly at the inner parts of the side chains(Tsumuraya et al., 1988), our results imply that theL-arabinosyl residues become accessible to a-L-arafaseduring stepwise elimination of galactosyl and 4-O-methyl-glucuronosyl residues by the action of bothrRsBGAL1 and b-GlcUAase, leading to liberation ofmost of the sugar residues. These conclusions areconsistent with the substrate specificity of rRsBGAL1toward oligosaccharides (Table II) in that the substi-tution with L-Ara or uronic acids at nonreducing endsof b-(1/6)-galactooligosaccharides renders those gal-actooligomers impervious to the enzyme.
Digestion of a-L-arafase-treated AGP with rRsBGAL1in the presence of b-GlcUAase and a-L-arafase ina large-scale reaction yielded a small portion (15%)of a high-Mr component possibly representing a corepart of the AGP consisting of a polypeptide with shortoligosaccharide remnants that have an increased pro-portion (25% of total sugars) of L-arabinosyl residuesresistant to a-L-arafase attack (Table V). However, thechain lengths and numbers of the remnants along thesingle polypeptide backbone are unknown. Structuralanalysis of the high-Mr component indicated an in-crease in the proportion of nonreducing terminal andO-2-linked L-arabinosyl residues, as well as O-3-linkedgalactosyl residues and fewer O-6-linked galactosylresidues, when compared with the data obtained forthe initial AGP. These observations suggest that theb-(1/3)- and b-(1/6)-linked galactosyl sequences in
Figure 6. Hydrolysis of mixed-linkage galactooligosaccharides. Hy-drolysis products of b-Gal-(1/6)-b-Gal-(1/3)-Gal and b-Gal-(1/3)[b-Gal-(1/6)]-Gal by action of rRsBGAL1 were analyzed onTLC. Lane S, Standard Gal, b-(1/3)-galactobiose, and b-(1/6)-galactobiose; lane 1, b-Gal-(1/6)-b-Gal-(1/3)-Gal before hydroly-sis; lane 2, hydrolysis products of b-Gal-(1/6)-b-Gal-(1/3)-Gal after1 h; lane 3, those after 12 h; lane 4, b-Gal-(1/3)[b-Gal-(1/6)]-Galbefore hydrolysis; lane 5, hydrolysis products of b-Gal-(1/3)[b-Gal-(1/6)]-Gal after 1 h; lane 6, those after 12 h. Localization of thestandard sugars is indicated on the left side.
Table II. Substrate specificity of rRsBGAL1 toward oligosaccharides
aThe enzyme was incubated with substrates at a concentration of5 mM, and 2 mM was employed for PNP-b-Gal. bActivity isexpressed as percent of that (64.9 units mg protein21) of PNP-b-Gal.
b-Galactosidase from Radish
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the a-L-arafase-treated AGP are removed in a con-certed stepwise enzymatic degradation process withrRsBGAL1 in the presence of b-GlcUAase and a-L-arafase, leaving only the core protein of the root AGP.On the other hand, a clearly different high-Mr compo-nent obtained after digestion of the modified AGPwith rRsBGAL1 alone showed sugar composition andstructure similar to those of the initial AGP. Onlya small amount (15% of total sugar) of galactosylresidues was eliminated by the action of rRsBGAL1 inthis case.
DISCUSSION
To date, hundreds of cDNA sequences for b-galacto-sidase/exo-b-(1/4)-galactanases and b-galactosidasesfrom higher plants have been cloned. Although theseenzymes are widely distributed in higher plants, untilnow their functions have been discussed primarilywith respect to the degradation of pectic b-(1/4)-galactan. The reason for this is that the structure ofpectic b-(1/4)-galactan is regulated spatially duringthe development of plant tissues, and pectin thusplays an important role in the architecture of the cellwall and intercellular attachment (McCartney et al.,2000; Sørensen et al., 2000). On the other hand, wehave previously shown that b-galactosidase speci-mens isolated from radish seeds and spinach leaves(spinach b-Gal I) hydrolyze specifically b-(1/3)- andb-(1/6)-galactooligosaccharides besides PNP-b-Gal,and are thereby able to degrade the b-(1/3)(1/6)-galactan backbones of AGPs, but not pectic b-(1/4)-galactan (Sekimata et al., 1989; Hirano et al., 1994).These enzymes can be classified into the second classof b-galactosidases, the b-galactosidase/exo-b-(1/3)(1/6)-galactanases. However, we cannot ruleout the possibility that this second group of b-galac-tosidases participates in the degradation of both pecticb-(1/4)-galactan and the carbohydrate moieties ofAGPs, because a b-galactosidase specimen (b-Gal II)purified from spinach leaves shows a broad substratespecificity, acting not only on b-(1/3)- and b-(1/6)-galactooligosaccharides but also weakly on b-(1/4)-galactooligosaccharides (Hirano et al., 1994). Furtherstudies are required to clarify the functional differ-
ences of the second class of b-galactosidases fromb-galactosidase/exo-b-(1/4)-galactanases. For thisstudy, we have cloned a gene (RsBGAL1) encodingradish b-galactosidase and expressed the recombinantenzyme (rRsBGAL1) in P. pastoris. We then investi-gated the nature of the RsBGAL1 protein and com-pared the enzymatic properties of rRsBGAL1 withthose of the native enzyme.
Substrate specificity was quite similar to that of thenative enzyme (Sekimata et al., 1989); that is,rRsBGAL1 preferred b-(1/3)- and b-(1/6)-galacto-oligosaccharides, and hydrolyzed b-(1/3)-galactanand b-(1/3)(1/6)-galactan backbones of AGPs. Al-though the amino acid sequence of the smaller subunitdeduced from RsBGAL1 cDNA was not completelyidentical to that determined for the corresponding34-kD subunit of the native enzyme, RsBGAL1 likelyencodes the native radish enzyme or, at least, ab-galactosidase with properties quite similar to thenative enzyme. Purification of rRsBGAL1 yieldeda single polypeptide with a molecular mass of106 kD, including a Gal lectin-like domain at the Cterminus, whereas the native RsBGAL1 consisted oftwo subunits with molecular masses of 45 and 34 kDand lacked the Gal lectin-like domain. These observa-tions clearly indicate that the RsBGAL1 polypeptideundergoes posttranslational processing in radishplants. A similar case of posttranslational process-ing in the C-terminal region of the native enzymehas been observed for a-L-arabinofuranosidase andb-xylosidase from barley (Lee et al., 2003). Elucidating
Table IV. Substrate specificity of rRsBGAL1 toward polysaccharides
Substratea Relative Activityb Limit of Hydrolysisc
b-(1/3)-Galactan 8 29b-(1/3)(1/6)-Galactan
from P. zopfii2 3
b-(1/4)-Galactan fromlupin
0.8 1
Native AGP fromradish roots
3 3
a-L-Arafase-treated AGPfrom radish roots
4 16
Native AGP fromradish leaves
4 1
a-L-Arafase-treated AGPfrom radish leaves
8 25
PNP-b-Gal 100 –d
aThe enzyme was incubated with polymers at a concentration of5 mg mL21 and 2 mM was employed for PNP-b-Gal. bActivity isexpressed as percent of that (64.9 units mg protein21) of PNP-b-Gal. cThe reaction was carried out under the standard condi-tions, except for the concentration of the substrate (1 mg mL21) and theamount (20 mU) of the enzyme for 16 h, followed by furtherincubation with the addition of an equal amount of the enzyme foranother 10 h. The limit of hydrolysis was determined after theliberation of reducing sugars reached a plateau, and expressed asGal equivalent against the total sugars in each substrate. dNotdetermined.
Table III. Kinetic values of rRsBGAL1
Substratea Km kcat kcat/Km
mM s21M21 s21
PNP-b-Gal 0.30 123.0 4.10 3 105
b-(1/3)-Galactobiose 1.77 28.6 0.16 3 105
b-(1/6)-Galactobiose 0.77 32.5 0.42 3 105
aTo examine the effects of galactooligosaccharides on the activity,reactions were carried out with varying concentrations of PNP-b-Gal(0.1–5 mM), b-(1/3)-galactobiose (0.5–10 mM), and b-(1/6)-galac-tobiose (0.5–10 mM). The Km and kcat values were calculated froma Hanes-Woolf plot using the obtained activities.
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the precise mechanism of posttranslational processingand its significance to kinetic properties and functionsof plant glycosidases is an important problem, butwe are not yet able to give a satisfactory explana-tion concerning rRsBGAL1. In our experiments, eventhough the substrate specificities of recombinant andnative enzymes were quite similar, several kineticparameters differed considerably. The kcat value(123.0 s21) of rRsBGAL1 for PNP-b-Gal was muchhigher than that (7.48 s21) of the native enzyme(Sekimata et al., 1989). This difference affected otherkinetic data obtained in this study. For example, therelatively lower hydrolysis rates (corresponding to8% of that for PNP-b-Gal) of b-(1/3)-galactan anda-L-arafase-treated radish leaf AGP by rRsBGAL1(Table IV) when compared with those (17 and 49%,respectively) by the native enzyme seem to reflect thehigh preference of rRsBGAL1 for PNP-b-Gal. Never-theless, the limits of hydrolysis are almost the same forboth enzymes. In addition, the recombinant enzymeshowed a lower Km value (1.77 mM) for b-(1/3)-galactobiose compared with that (7.79 mM) of thenative enzyme (Sekimata et al., 1989). It seems likelythat these catalytic differences result from the post-translational processing of the enzyme protein in theradish plant. However, the processing does not affectthe essential recognition of b-(1/3)- and b-(1/6)-linked galactosyl sequences.
Previous studies have explored the physiologicalroles of AGPs by application of b-glycosyl-Yariv re-agent, chemical name 1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene, that specifically binds to thecarbohydrate moieties of AGPs and perturbs theirfunctions (Komalavilas et al., 1991; Majewska-Sawka
and Nothnagel, 2000). In cultured cells of Arabidopsis,b-glycosyl-Yariv reagent induces programmed celldeath, possibly by disrupting the plasma membrane-cell wall connections (Gao and Showalter, 1999). Re-cently, Motose et al. (2004) have reported that xylogen,a nonclassical AGP, induces differentiation of zinnia
Figure 7. Hydrolysis of a-L-arafase-treated radish root AGP byrRsBGAL1. a-L-Arafase-treated radish root AGP was digested withrRsBGAL1 (black square), b-GlcUAase (white triangle), or a-L-arafase(white square), each acting alone or by simultaneous action ofrRsBGAL1 and b-GlcUAase (black circle), or simultaneously byrRsBGAL1, b-GlcUAase, and a-L-arafase (white circle). The amountsof sugars released were determined reductometrically and the extent(percentage) of hydrolysis was calculated based on the total sugarcontent of the modified AGP. Arrows indicate the addition of additionalrRsBGAL1 into three reaction mixtures as described in ‘‘Materials andMethods.’’
Table V. Characterization of a-L-arafase-treated AGP and high-Mr products obtained by digestionwith rRsBGAL1 with or without the presence of b-GlcUAase and a-L-arafase
aMethylation analysis was done without carboxyl reduction of uronosyl residues. The amounts ofnonreducing terminal 4-Me-GlcUA groups are thus not accounted for. bCalculated from data inTsumuraya et al. (1988). cNot detectable. dLess than 1%.
b-Galactosidase from Radish
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(Zinnia elegans) mesophyll cells. The inductive func-tion of xylogen, however, was suppressed when thezinnia cells were treated with b-glycosyl-Yariv re-agent. It was also lost when the carbohydrate moietieswere removed from the xylogen by chemical treat-ment. We think it likely that the carbohydrate moietiesof AGPs are required for intercellular communicationand that various glycosidases, such as b-galactosidase,are involved in the structural modification of thecarbohydrate moieties of AGPs in response to thedevelopmental stage. In this study, rRsBGAL1 was notvery active toward native AGP from radish roots, but
removed nearly 90% of the carbohydrate moieties ofthe AGP when aided by other microbial glycosidases(Fig. 7). It is highly probable that the carbohydratemoieties of AGPs are degraded by the concerted actionof b-galactosidase, a-L-arafase, and b-GlcUAase, to-gether with other auxiliary glycosidases, dependingon the sugar compositions of the various AGPs in vivo.This has been postulated previously in a study on bothradish b-galactosidase and a-L-arafase (Hata et al.,1992). Here, we have shown thatb-galactosidase plays akey role in the degradation of the carbohydrate moietiesof AGPs, leading to their structural modification.
Figure 8. 13C-NMR spectra of galactotriose 1 (A) and 2 (B).
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There have been only a small number of studies ofplant b-GlcUAases, such as that found in suspensioncells of skullcap (Scutellaria baicalensis), which partic-ipates in the metabolism of b-GlcUA groups in fla-vones (Sasaki et al., 2000), and no plant b-GlcUAasescapable of hydrolyzing plant cell wall polysaccharideshave been found so far. However, a preliminary ex-periment we performed showed that radish plantscontain low, but detectable, levels of enzyme activityhydrolyzing both PNP-b-GlcUA and b-GlcUA-(1/6)-Gal. It is possible that the radish b-GlcUAase(s) are notunimportant as degrading enzymes for the recyclingof AGPs in vivo.
We found that rRsBGAL1 hydrolyzed a-L-arafase-treated radish leaf AGP in preference to a-L-arafase-treated radish root AGP (Table IV). Except for theoccurrence of L-Fuc residues as a minor (5%–6% oftotal sugar) constituent in leaf AGP, the structuresof the carbohydrate moieties of both AGPs are quitesimilar. Both native AGPs possess a commonb-(1/3)(1/6)-galactan backbone, towhichaconsider-able amount of a-L-arabinofuranosyl and other minorsugar residues are attached, and have comparable rel-ative molecular masses (88 kD for the root AGP, 75 kDfor the leaf AGP; Nakamura et al., 1984; Tsumurayaet al., 1984, 1988). We are not able, at the moment, togive a detailed explanation of the particular structuralfeatures that influence the susceptibility to b-galacto-sidase.
The physiological functions of most b-galactosidaseand b-galactosidase-like genes cloned so far fromhigher plants have not yet been established. Arab-idopsis BGAL8 and tomato TBG5 are close homologsof RsBGAL1 and form a putative subfamily ofb-galactosidases distinct from that of the b-galactosi-dase/exo-b-(1/4)-galactanases, which includes TBG4(Fig. 2B). The substrate specificity of these two homo-logs has not yet been examined, but they seem tobehave similarly to RsBGAL1; that is, they recognizespecifically b-(1/3)- and b-(1/6)-linked galactosylsequences. We are planning to further investigate thephysiological functions of plant b-galactosidases us-ing T-DNA knockout lines of Arabidopsis.
MATERIALS AND METHODS
Plant Material
Seeds of radish (Raphanus sativus L. var hortensis cv aokubi-miyashige-
nagajiri) were purchased from Tokita Seed and Plant (Saitama, Japan). For
DNA and RNA preparations, the radish seeds were sown on moist plastic
mesh and grown at 25�C for 6 d.
Oligo- and Polysaccharides
The b-(1/3)- and b-(1/6)-linked galactobioses and -trioses used were
prepared from larch wood (Larix decidua) arabinogalactan (Aspinall et al.,
1958b), b-(1/6)-galactotetraose was prepared from gum ghatti (Aspinall
et al., 1958a), and b-(1/4)-galactooligosaccharides with degree of polymer-
ization 2 to 4 were prepared from soybean arabinan galactan (Sekimata et al.,
1989). The a-L-Ara-(1/3)-Gal-b-(1/6)-Gal was prepared from enzymatic
hydrolysate of the Smith degradation product of acacia (Acacia senegal) gum by
incubation with exo-b-(1/3)-galactanase (Tsumuraya et al., 1990), b-GlcUA-