Isolation and Characterization of a Sex-Specific Lectin in a MarineRed Alga, Aglaothamnion oosumiense Itono

Jong Won Han,a Tatyana A. Klochkova,a Jun Bo Shim,a Kangsup Yoon,b and Gwang Hoon Kima

Department of Biology, Kongju National University, Kongju, South Korea,a and Laboratory for Algae Research and Biotechnology, Arizona State University, Mesa, Arizona,USAb

In red algae, spermatial binding to female trichogynes is mediated by a lectin-carbohydrate complementary system. Aglaotham-nion oosumiense is a microscopic filamentous red alga. The gamete recognition and binding occur at the surface of the hairliketrichogyne on the female carpogonium. Male spermatia are nonmotile. Previous studies suggested the presence of a lectin re-sponsible for gamete recognition on the surface of female trychogynes. A novel N-acetyl-D-galactosamine-specific protein wasisolated from female plants of A. oosumiense by affinity chromatography and named AOL1. The lectin was monomeric and didnot agglutinate horse blood or human erythrocytes. The N-terminal amino acid sequence of the protein was analyzed, and de-generate primers were designed. A full-length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends-PCR (RACE-PCR). The cDNA was 1,095 bp in length and coded for a protein of 259 amino acids with a deduced molecular massof 21.4 kDa, which agreed well with the protein data. PCR analysis using genomic DNA showed that both male and female plantshave this gene. However, Northern blotting and two-dimensional electrophoresis showed that this protein was expressed 12 to15 times more in female plants. The lectin inhibited spermatial binding to the trichogynes when preincubated with spermatia,suggesting its involvement in gamete binding.

The precise point of gamete recognition varies along the con-tinuum of reproduction and development, from directional

movements that bring the compatible gametes together throughmany steps of fertilization to the formation of embryonic off-spring. Fertilization in red algae, however, begins with direct con-tact between a male spermatium and a female trichogyne becauseboth male and female gametes are nonmotile (7, 32). As sperma-tial binding to trichogynes is highly selective, some recognitionfactors are expected to be present along their surfaces (11, 16, 17,25). Cell surface glycoconjugates have been reported as importantfactors for cell-cell recognition in many organisms (24). Such rec-ognition systems depend on complementary binding betweencarbohydrate moieties on one cell with specific sugar-binding lec-tins on another cell.

Lectin-carbohydrate complementary systems have been re-ported in gamete recognition of marine algae for a long time (1,8–10, 17, 22, 31), but most studies used indirect evidence frominhibition experiments using carbohydrates or foreign lectins(mostly from land plants) as blocking agents of gamete binding.Although several studies have reported on the isolation of marinealgal lectins, the number of these proteins that have been purifiedand characterized is still small (4, 33).

Our previous cytochemical study on the fertilization of Aglaoth-amnion oosumiense Itono suggested the presence of N-acetyl-D-galactosamine (GalNAc) and/or D-methyl mannose-specific lec-tin(s) on the surface of female trichogynes (11). Here we reportthe purification and molecular characterization of a novelGalNAc-binding lectin from this species. The sex-specific expressionof the lectin was analyzed, and its role in gamete binding wasexamined.

MATERIALS AND METHODSOrganism and laboratory culture. Tetrasporic plants of A. oosumiensewere collected from Wando, on the southern coast of Korea, and main-tained in unialgal cultures in IMR medium (13). Plants were grown at

15°C in 16:8-h light and dark cycles with �20 �mol photons m�2 s�1

provided by cool-white fluorescent bulbs.Preparation of algal extract and purification of AOL1. For prepara-

tion of algal extract and purification of AOL1 (Aglaothamnion oosumienselectin 1), materials were frozen with liquid nitrogen and stored at �80°Cbefore homogenization. Samples were homogenized using a mortar andpestle to fine powder, and then 10 volumes of Tris-buffered saline (TBS)(25 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 1 mM phenylmethylsul-fonyl fluoride [PMSF], pH 7.4) was added. Homogenates were centri-fuged at 5,000 � g for 20 min to sediment out cell organelles and debris.Then, the supernatant was recentrifuged at 30,000 � g for 30 min andconcentrated using an ultrafiltration unit (model 8200, MWCO, 10K;Amicon) at 75 lb/in2 (5.3 kg/cm2). All procedures were performed at 4°C.

Fresh crude extract was applied to a GalNAc-agarose affinity column(Sigma-Aldrich, Seoul, South Korea) equilibrated with TBS buffer. Thecolumn was washed with the same solution until the absorbency (at 280nm) of the washed fraction was lowered to 0.001. The bound proteinswere eluted with the same solution containing 0.5 M GalNAc (12). Twentysequential fractions of 2 ml each were collected and analyzed with sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (14).The fractions showing a single protein band were pooled, concentrated,and dialyzed against TBS buffer. The protein content was determined bythe method of Lowry et al. (15) or Bradford (2).

SDS-PAGE. SDS-PAGE was performed according to the method de-scribed by Laemmli (14) with a 4% stacking gel and a 12% separating gel.Samples were treated with 4% 2-mercaptoethanol to reduce disulfidebonds. Control samples without 4% 2-mercaptoethanol were preparedseparately. Proteins were stained with Coomassie brilliant blue R-250 andtime-limited silver staining.

Received 10 February 2012 Accepted 1 August 2012

Published ahead of print 3 August 2012

Address correspondence to Gwang Hoon Kim, ghkim@kongju.ac.kr.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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MS determination. The purified lectin was dialyzed extensivelyagainst double-distilled water and lyophilized. The exact molecular massof the protein was determined by matrix-assisted laser desorption ioniza-tion–time of flight mass spectrometry (MALDI-TOF MS) (Applied Bio-systems, CA) at the Korean Basic Science Institute (KBSI) (Daejeon,South Korea).

Analysis of N-terminal and internal amino acid sequence. The puri-fied protein was electrophoresed on a 12% SDS-polyacrylamide gel andelectroblotted onto a polyvinylidene difluoride (PVDF) membrane. Thefirst 20 amino acids of the N-terminal sequence and seven internal se-quences were determined with an Applied Biosystems Precise Sequencer(Applied Biosystems) at KBSI.

Construction of cDNA libraries. Male and female cDNA librarieswere constructed. Double-stranded cDNA was synthesized using 5 �g ofpoly(A) RNA as a template, directionally cloned into a UniZAP-XR vectorphage (ZAP-cDNA synthesis kit; Stratagene, La Jolla, CA), and packagedusing the ZAP-cDNA Gigapack III Gold packaging extract. Approxi-mately 1.8 million and 1.5 million recombinants were represented in themale and female cDNA libraries, respectively.

Cloning of cDNA encoding AOL1. Based on the amino acid se-quences derived from the purified protein, degenerate primers of bothsense and antisense strands were designed (Table 1). The first round ofPCR was performed with a set of primers (AOL1-NDF and T7) withcDNA library as a template using an Ex Taq polymerase (TaKaRa, Tokyo,Japan). For the second round of nested PCR, primer set AOL1-IDF/T7and AOL1-NDF/AOL1-IDR was used. PCR was performed under thefollowing conditions: cDNA was denatured at 95°C for 3 min followed by40 cycles of amplification (95°C for 30 s, 53°C for 30 s, 72°C for 1 min) andby 10 min at 72°C. The PCR products were cloned into pDrive CloningVector (Qiagen), and their DNA sequences were determined.

Based on the sequences of the 3= end PCR products, the specific prim-ers were designed. The initial PCR was performed with a gene-specificprimer (AOL1-ISR1) and vector primer (M13 reverse). To increase thespecificity and identify the desired amplification product, an aliquot ofreaction product from the initial PCR was reamplified using the nestedgene-specific primer (AOL1-ISR2) and vector primer (SK). The resultingPCR product was subcloned and sequenced as described above, and thesequence was deposited in GenBank.

Screening of the cDNA library. To isolate a full-length cDNA, thecDNA library was screened by plaque hybridization. A probe for plaquehybridization was generated by PCR using primers AOL1-NSF andAOL1-CSR, which amplified the coding sequence of AOL1. The probewas labeled with digoxigenin (DIG)-dUTP using the PCR DIG probesynthesis kit (Roche, Mannheim, Germany) and purified using theQIAquick gel extraction kit (Qiagen). For cDNA library screening, 3 �105 plaques were transferred onto Hybond-N� membranes (AmershamPharmacia Bioscience). After UV cross-linking, the membranes were pre-hybridized for 3 h at 42°C in DIG EasyHyb Solution (Roche, USA), fol-lowed by hybridization with DIG-labeled DNA probe at 42°C for 16 h.After washing twice with 0.5� SSC (1�SSC is 0.15 M NaCl plus 0.015 Msodium citrate) containing 0.1% SDS at 68°C for 15 min, colorimetric

detection was performed using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate in the DIG-DNA detection kit as describedin the manufacturer’s instructions. Five positive plaques were convertedto plasmids using the VCSM13 helper phage (Stratagene, La Jolla, CA)and sequenced.

Reverse transcriptase PCR (RT-PCR) and genomic DNA-PCR. TotalRNA and genomic DNA (gDNA) were isolated simultaneously from thesame tissue sample. Contaminated DNA in the RNA preparation wasremoved by RNase-free DNase I treatment, while contaminated RNA inDNA preparations was removed by DNase-free RNase A treatment (Pro-mega, Madison, WI). The total RNA underwent oligo(dT)-primed re-verse transcription using StrataScriptase according to the manufacturer’sinstructions. The cDNA and gDNA (at 0.1 �g/�l) were used as the tem-plate for PCRs. PCRs were done with Ex Taq Polymerase (TaKaRa, Tokyo,Japan) using the same primers (AOL1-NSF/AOL1-CSR) and annealingconditions.

2-DE. For two-dimensional polyacrylamide gel electrophoresis (2-DE), male and female plants were frozen with liquid nitrogen and storedat �80°C before use. Materials were homogenized using a mortar-drivenhomogenizer (PowerGen125) in a lysis solution consisting of 7 M urea, 2M thiourea, 4% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfate (CHAPS), 1% (wt/vol) dithiothreitol (DTT), 2% (vol/vol)pharmalyte, and 1 mM benzamidine. Freezing and thawing steps for thesamples were repeated 5 times in 1 day. After centrifugation at 15,000 � gfor 60 min at 18°C, insoluble material was discarded and the soluble frac-tion was used for 2-DE. Protein loading was normalized using a modifiedBradford assay (2, 21).

Isoelectric focusing (IEF) was performed with 200 �g of sample at20°C using a Multiphor II electrophoresis unit and EPS 3500 XL powersupply (Amersham Biosciences) following the manufacturer’s instruc-tion, and then SDS-PAGE (20 by 24 cm, 10 to 16% polyacrylamide) wasperformed using Hoefer DALT 2-DE system (Amersham Biosciences)following the manufacturer’s instruction. The gels were stained with CBBG250.

Northern blot analysis. The DNA probe was directly amplified andlabeled with DIG-dUTP by PCR from the cDNA library using a DIG probesynthesis kit (Roche, USA). The PCR product was separated on a 1.2%(wt/vol) agarose gel. Total RNA was extracted from male and femaleplants developing sexual reproductive structures. Equal amounts of totalRNA (5 �g) were loaded and separated on 1.2% (wt/vol) formaldehyde-agarose gels and photographed to confirm RNA quality and to verify equalsample loading. RNA was transferred onto Biodyne Nylon B membranes(Pall Life Science) by capillary transfer with 20� SSC (3 M NaCl, 0.3 Msodium citrate [pH 7.0]), and immobilized to the membranes by UVcross-linking. Membrane was incubated with prehybridization solution(DIG Easy Hyb; Roche) for 2 h and then hybridization solution (DIG EasyHyb with 25 ng/ml DIG probe) overnight at 50°C. The blots were washedin 2� SSC– 0.1% SDS and then 0.5� SSC– 0.1% SDS at room tempera-ture. The mRNA was immunologically detected by antidigoxigeninprobes. The blots were then exposed to X-ray films (CP-BU; Agfa) for 2 h.

Computer-aided sequence analyses and secondary structure predic-tions. Nucleotide and amino acid sequence homology searches and com-parisons were carried out using BLAST in GenBank (http://www.ncbi.nlm.nih.gov/GenBank/), EMBL (http://www.ebi.ac.uk/embl/), PDB(http://www.rcsb.org/), and Uniprot (http://www.uniprot.org/). Post-translational modifications of protein were identified using the CBS pre-diction service (http://www.cbs.dtu.dk/services/).

Hemagglutinating activity assay. Fresh crude extract was preparedbefore the experiment. Horse blood and rabbit blood used in the assaywere purchased from Hanil Comed (Seongnam, South Korea), and hu-man blood types O, A, and B were obtained from the Chungnam NationalUniversity hospital (Daejeon, South Korea). Erythrocytes were firstwashed from the blood plasma with saline solution (0.9% NaCl in double-distilled water). For the investigation of hemagglutinating activity, wefollowed protocols described by Hori et al. (5). A serial 2-fold dilution of

TABLE 1 Primers used in isolation of AOL1 cDNA

Primername Sequence

Correspondingamino acidsequence

AOL1-NDF TAY GTN AAY GTN GAY GA YVNVDDAOL1-IDF GCN TTY GGN GGN TTY CC AFGGFPAOL1-IDR GGR AAN CCN CCR AA AFGGFPAOL1-ISR1 CGG ACG TAC ACG TCA GTT TTC GAOL1-ISR2 CAT TGG CAC GCA TCG TAT GCCAOL1-NSF GAT ATG TCC GGC TCG TAC ACG CTCAOL1-CSR CTA GTT TCC GCA CCA ATG ATA ATC

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the crude extract was made in a final volume of 25 �l saline in microtiterplate wells, and 25-�l erythrocyte suspensions were added sequentially toeach well. The reciprocal of the highest dilution of the lectin showingcomplete agglutination was taken as the agglutination titer. The mini-mum amount of lectin required for complete agglutination was defined as1 agglutination unit (AU).

Binding assay. Dense suspensions of spermatia were obtained frommale plants with actively developing spermatial clusters as described pre-viously (11). Male plants were incubated in 10 ml of fresh culture mediumwith mild shaking for 12 h. After male plants were removed, the solutionwas diluted to various concentrations (102 to 104 spermatia/ml) by addingculture medium. Released spermatia were preincubated for 60 min at20°C in a solution containing various concentrations of purified proteinsor foreign lectins (1 to 25 �g/ml) as well as carbohydrates (10 mM).Spermatial binding to trichogynes was assayed with ca. 100 trichogynesplaced in various concentrations of spermatia (10 to 400 spermatia pertrichogyne) for 2 h at 20°C with mild rotation. The total number of tricho-gynes and the percentage of trichogynes with one or more attached sper-matia were recorded. The average number of spermatia attached to a

trichogyne was also determined. Assays were conducted in triplicate,counting a minimum of 100 trichogynes from each replicate. Variationbetween replicates was within 5%.

Nucleotide sequence accession number. The sequence determined inthis study has been deposited in GenBank under accession numberJX164251.

RESULTSIsolation and purification of the lectin. We chose a GalNAc-aga-rose affinity column to purify the lectin from A. oosumiense be-cause previous cytochemical studies suggested the presence of acomplementary lectin for GalNAc on the surface of female tricho-gynes. Crude extract of female plants was loaded onto the affinitycolumn, and the eluate fractions showed only one protein band(Fig. 1A). SDS-PAGE analyses of the protein contents showed thatthis one-step column purification was good enough to yield ahomogeneous protein (Fig. 1B). The molecular mass of the intactlectin was determined as 21.4 kDa by MALDI-TOF MS (Fig. 2).

FIG 1 Purification of female-specific lectin AOL1 from Aglaothamnion oosumiense by the use of agarose-bound GalNAc affinity chromatography. (A) SDS-PAGE of eluted proteins collected from GalNAc-agarose column. Lanes 1 to 13, gel electrophoresis according to the order of eluted fractions 1 through 13. Thearrow points to the protein bands with a molecular mass of ca. 22 kDa. (B) SDS-PAGE of samples according to purification step. Lane 1, crude extract; lane 2,washing fraction; lane 3, purified AOL1. M, molecular mass markers.

FIG 2 Molecular mass determination of AOL1 by MALDI-TOF MS.

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The lectin showed a single protein band pattern on SDS-PAGEwith or without the reducing agent, 2-mercaptoethanol, and themobility of the protein band was not changed (Fig. 3). The puri-fied lectin did not show agglutinating activity to rabbit and horseblood cells or to human blood groups (data not shown).

Molecular characterization and cloning. The N-terminal 20amino acids of the lectin were Asp-Met-Ser-Gly-Ser-Tyr-Thr-

Leu-Tyr-Val-Asn-Val-Asp-Asp-Ala-Ala-Asp-Val-Tyr-Leu, whichshows no sequence homology with any proteins in public data-bases (NCBI, PDB, EMBL, etc.). To develop effective degenerateprimers, we analyzed an internal sequence (Table 1; Fig. 4). Threedegenerate primers were also designed based on the internalamino acid sequences of the protein. A fragment of about 850 bpwas amplified using primers AOL1-NDF and T7, and a 3= untrans-lated region (UTR) of 240 bp was found downstream from thestop codon in the amplified sequence. The deduced amino acidsequence of the 850-bp fragment contained those of the twoknown internal tryptic peptide sequences obtained from the pro-tein. Based on the sequence of the 3= rapid amplification of cDNAends (RACE) fragment, two reverse specific primers were de-signed and used for the amplification of the 5= end sequence of thecDNA. A 550-bp fragment was obtained, in which a 5= UTR of 80bp was found upstream of the first ATG codon. Based on thesequences of the 3= and 5= RACE products, a full-length cDNAfragment was obtained (Fig. 4).

cDNA cloning of the lectin was performed using an A. oosumi-ense cDNA library by plaque hybridization to confirm the se-quence obtained by the modified RACE-PCR. After the screeningof 120,000 colonies, five positive plaques were obtained. Compar-ison of these sequences revealed no significant variation. The full-length cDNA of the protein was 1,095 bp.

Analysis of deduced amino acid sequences. An open readingframe (ORF) of 780 bp within the AOL1 cDNA sequence encoded

FIG 3 Effect of 2-mercaptoethanol on the structural properties of AOL1. M,molecular mass markers; �, protein treated with 2-mercaptoethanol; �, pro-tein without 2-mercaptoethanol.

FIG 4 Full cDNA sequence and deduced amino acid sequence of AOL1. Solid lines represent N-terminal and internal amino acid sequences used for designingdegenerate primers; the dashed line shows the internal amino acid sequence obtained from the protein spots in 2-DE gel. CAF, chemically assisted fragmentation.

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a polypeptide of 259 amino acids. The N-terminal and trypticpeptide amino acid sequences, which are the characteristics ofisolated AOL1, were present in the deduced sequences (Fig. 4).According to the rules of predicting signal peptides (18), a 19-amino-acid-long signal peptide with a signal peptide cleavage sitebetween Thr19 and Ser20 was identified from the deduced proteinsequence. The predicted protein sequence did not contain a po-tential N-linked glycosylation site. From the deduced amino acidsequence, the molecular mass was calculated to be 21.4 kDa andthe theoretical isoelectric point (pI) value was 4.17, which was ingood agreement with that of AOL1 estimated by SDS-PAGE andMALDI-TOF mass spectrometry.

Alignment and Web-based structure prediction of the de-duced amino acid sequence. BLAST search results indicated thatthe amino acid sequence of AOL1 has no significant similarity to

any known proteins. The subcellular location of the protein wasanalyzed using Web-based prediction programs (Targetp v 1.1,plant option [http://www.cbs.dtu.dk/services/TargetP]). AOL1with an N-terminal signal peptide was predicted to be a secretoryprotein targeted to the cell wall or vacuole (prediction value,0.818) rather than to mitochondria (prediction value, 0.063) or tochloroplasts (prediction value, 0.090). Another analysis using amolecular structure prediction program, Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2), showed that AOL1 has a unique molecularstructure with no significant similarity with any known protein.

Determination of the genomic DNA encoding AOL1.Genomic DNA PCR analysis using primers AOL-NSF and AOL-CSR, which span 621 bp of the ORF of AOL1, resulted in a singleproduct of the same size, which indicated that the gene encodingAOL1 contains no intron, and sequences like that obtained byRT-PCR using the same primers (data not shown). The male nu-clear genome also contained the AOL1 gene, and the sequence wasidentical to that of the female.

Sex specificity of the lectin. Sex specificity of the lectin hasbeen analyzed using proteomic and Northern blot analyses. When2-DE images of male and female plants were compared, a female-specific protein was found at the position predicted from the mo-lecular mass (about 22 kDa) and pI (4.82 value) of AOL1 (Fig. 5).MALDI-TOF MS analysis of the internal amino acid sequence ofthe female-specific protein confirmed it as AOL1 (Fig. 4, dashedline, and 5). Northern blot analysis showed that the expression ofthe AOL1 gene was 12 times higher in female plants than in maleplants (Fig. 6).

Inhibition experiment of gamete binding by AOL1. Sperma-tial attachment to trichogynes occurred within a few seconds aftermixing and reached its maximum value within 30 min at 2 � 104

spermatia/ml (data not shown). The percentage of spermatial at-tachment to trichogynes was assayed after preincubation of sper-matia or trichogynes with GalNAc and purified lectin (Table 2).The binding of spermatia to trichogynes was inhibited by AOL1and soybean agglutinin (SBA). However, AOL1 showed almostthe same inhibitory effect as the same concentration of SBA.When AOL1 was heat denatured or preincubated with the com-plementary sugar, GalNAc, the inhibitory effect disappeared (Ta-ble 2). The average number of spermatia attached to a trichogynealso decreased when they were preincubated with AOL1.

DISCUSSION

A novel GalNAc-specific lectin in A. oosumiense was purified andnamed AOL1. AOL1 was monomeric and had a molecular mass of

FIG 5 (A) 2-DE gel images of the female and male plants of Aglaothamnionoosumiense. (B) Enlarged 2-DE gel images. Circles point to the protein spotcorresponding to AOL1. (C) Internal amino acid sequence obtained by chem-ically assisted fragmentation (CAF)-MALDI sequencing.

FIG 6 Northern blot using AOL1 probe showing relative expression level ofAOL1 gene in males and females of Aglathamnion oosumiense. (A) Gel imageand RNA loading control. (B) Relative amount of mRNA in male and femaleplants determined by image analysis system.

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21.4 kDa. Full-length cDNA encoding the lectin was obtained. Thelectin was expressed in a sex-specific manner, in female plants.The lectin reduced spermatial binding to the trichogyne when thespermatia were preincubated with it.

A lectin-carbohydrate complementary binding was proposedfor the gamete recognition in A. oosumiense (11). Fluorescent lec-tin staining on the spermatial surface showed two types of glyco-conjugates, GalNAc and �-methyl-D-mannose, specific to SBAand ConA, respectively. The presence of their complementarylectinlike receptors was shown by inhibition experiment. We chose aGalNAc affinity column to purify AOL1 because the GalNAc moi-eties were located on spermatial appendages and its complemen-tary lectin, SBA, showed a stronger inhibitory effect on gametebinding, while the ConA receptors were involved in developmentof the fertilization canal (11). Our results showed that AOL1 wasonly one of the lectins specific to this sugar in female plants of A.oosumiense. Sex-specific expression of AOL1 also suggests that itmight be a female gamete recognition molecule. Although PCRanalysis using genomic DNA showed that both male and femaleplants have the same AOL1 gene, Northern blot analysis showedthat mRNA of AOL1 was expressed 12 times more in female plantsand 2-DE analysis confirmed the female-specific expression ofAOL1 at the protein level. These results support the idea thatAOL1 is a strong candidate for a female recognition molecule(GalNAc receptor) in A. oosumiense. Recently, another sex-spe-cific lectin, rhodobindin, was reported from a closely related spe-cies, Aglaothamnion callophyllidicola, and was suggested to be in-volved in gamete binding as well (25). Rhodobindin could blockthe spermatial binding to female trichogyne when the spermatiawere preincubated with this lectin. However, rhodobindin did notshow any specificity to the sugar GalNAc. Our previous cytochem-ical study suggested that the gamete binding in A. oosumiensemight be mediated by a double-docking mechanism involvingtwo lectins (11). Purification of both lectins in one species ofAglaothamnion is essential to examine this hypothesis.

Spermatial attachment to trichogynes was inhibited when thespermatia were preincubated with purified AOL1. The inhibitoryeffect of AOL1 disappeared when the protein was preincubatedwith the complementary sugar GalNAc. However, AOL1 blockedgamete binding to almost the same level as the positive control,SBA; 20 �g/ml of AOL1 and SBA was necessary to reduce thegamete binding to 57% and 59% of control, respectively. Thisinhibitory effect seems too low to be a recognition molecule be-cause we expected a more specific and stronger inhibitory effect

from AOL1 than from foreign lectins. As gamete binding of A.oosumiense occurs first between the cell walls of trichogynes andthe spermatial coverings, the female binding proteins are expectedto be embedded in the trichogyne cell wall. The results of primarystructure analysis suggest that AOL1 might be a secretory proteintargeted to the cell wall because AOL1 has no N-terminal signalpeptide and no predicted subcellular location. The molecular sizeof AOL1 (21.4 kDa), however, seems a bit small to mediate bind-ing between male spermatia and trichogynes. It is still too early toconclude that AOL1 is the female gamete recognition molecule.More direct evidence using antibody labeling to localize the dis-tribution of AOL1 on the trichogyne surface would confirm this.

The purified AOL1 did not show agglutinating activity for rab-bit and horse blood cells or for any human blood group. Althoughthe lectins were originally known as agglutinins, which agglutinatecells or precipitate glycoconjugates because of their sugar bindingactivity, a recent definition of lectin is more focused on their re-versible sugar binding activity (19, 23). There are many lectins thathave only one noncatalytic domain and hence do not show agglu-tinating activity. A negative agglutination result does not neces-sarily mean that a sugar binding activity is absent (30). Further-more, agglutination assays detect only lectins having multiplebinding sites (hololectins), whereas other lectins, although able tointeract with specific sugars, do not cause agglutination (20).

Accumulating evidence indicates that the vast majority ofknown terrestrial plant lectins can be classified into four large andthree small families of structurally and evolutionarily related pro-teins (29), but AOL1 did not belong to any of them based on itsprimary structure. AOL1 showed no sequence homology with anyproteins reported in public protein databases. It is not surprising,because very few gene sequences of algal lectins have been re-ported so far (3, 6). The sex-specific nature of AOL1 may alsoexplain the unique primary structure of the protein because thegenes that mediate sexual reproduction are more divergent andrapidly evolving than the genes that are expressed in nonrepro-ductive tissues (26–28).

As a consequence of their sugar binding properties, lectins havebecome a useful tool in many fields of biological research and oneof the most commercially important groups of proteins (for anexample, see reference 23). Although publications on algal lectinsincreased dramatically during the last several years, their biologi-cal roles are still unknown. AOL1 has a characteristic effect ingamete binding, but it is still too early to conclude that it is afemale recognition molecule. Further studies including purifica-tion of possible membrane-bound lectins and antibody labeling tolocalize the distribution of AOL1 on the trichogyne surface arenecessary to confirm this.

ACKNOWLEDGMENTS

We express our sincere thanks to G. C. Zuccarello for his careful review ofand helpful comments on the manuscript.

This study was funded by the National Research Foundation of Korea(grant NRF 20120006718 to G.H.K.). This research was also supported bya grant from the Extreme Genomics Program funded by the Ministry ofLand, Transport and Maritime Affairs of the Korean Government toG.H.K.

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TABLE 2 Inhibition of spermatial attachment to trichogynes by lectinsand carbohydratesa

Treatment

% of trichogyneswith attachedspermatia

Avg no. of attachedspermatia pertrichogyne

Control (enriched seawater) 97.8 � 1.2 7.1Preincubation of spermatia with:

AOL1 56.2 � 4.5 4.6Heat-denatured AOL1 92.8 � 4.7 6.6AOL1 � GalNAc 90.2 � 5.4 5.3SBAb 58.7 � 4.8 4.3SBA � GalNAc 82.6 � 5.4 6.8

a Concentrations used were 20 �g/ml for lectins and 10 mM for carbohydrates.b SBA, soybean agglutinin.

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