PURIFICATION AND CHARACTERIZATION OF A LECTIN,BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) 1

When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin–carbohydrate complementary system. Two sugars, N‐acetyl‐d ‐glucosamine and N‐acetyl‐d ‐galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate‐labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N‐acetyl‐d ‐galactosamine and N‐acetyl‐d ‐glucosamine in the crude extract. One‐step column purification using N‐acetyl‐d ‐glucosamine‐agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross‐linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The N‐terminal 15 amino acid sequence of the lectin was Ser–Asp–Leu–Pro–Thr–X–Asp–Phe–Phe–His–Ile–Pro–Glu–Arg–Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa“bryohealin.”     

Purification and characterization of a lectin,BPL-3, from the marine green alga <Emphasis Type="Italic">Bryopsis plumosa</Emphasis>

A novel lectin was isolated and characterized from Bryopsis plumosa (Hudson) Agardh and named BPL-3. This lectin showed specificity to N-acetyl-d-galactosamine as well as N-acetyl-d-glucosamine and agglutinated human erythrocytes of all blood types, showing slight preference to the type A. SDS-PAGE and MALDI-TOF MS data showed that BPL-3 was a monomeric protein with molecular weight of 11.5 kDa. BPL-3 was a non-glycoprotein with pI value of ∼7.0. It was stable in high temperatures up to 70°C and exhibited optimum activity in pH 5.5–10. The N-terminal and internal amino acid sequences of the lectin were determined by Edman degradation and enzymatic digestion, which showed no sequence homology to any other reported proteins. The full sequence of the cDNA encoding this lectin was obtained from PCR using cDNA library, and the degenerate primers were designed from the N-terminal amino acid sequence. The size of the cDNA was 622 bp containing single ORF encoding the lectin precursor. This lectin showed the same sugar specificity to previously reported lectin, Bryohealin, involved in protoplast regeneration of B. plumosa. However, the amino acid sequences of the two lectins were completely different. The homology analysis of the full cDNA sequence of BPL-3 showed that it might belong to H lectin group, which was originally isolated from Roman snails.     

Molecular Cloning of cDNA for Trehalase from the European Honeybee,Apis mellifera L., and Its Heterologous Expression in Pichia pastoris

cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5′ untranslated region (UTR) of 869 bp, and the 3′ UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The M _r of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.     

Isolation and characterization of a new mannose-binding lectin gene from<Emphasis Type="Italic">Taxus media</Emphasis>

In this paper, we report the cloning and characterization of the first mannose-binding lectin gene from a gymnosperm plant species,Taxus media. The full-length cDNA ofT. media agglutinin (TMA) consisted of 676 bp and contained a 432 bp open reading frame (ORF) encoding a 144 amino acid protein. Comparative analysis showed that TMA had high homology with many previously reported plant mannose-binding lectins and thattma encoded a precursor lectin with a 26-aa signal peptide. Molecular modelling revealed that TMA was a new mannosebinding lectin with three typical mannose-binding boxes like lectins from species of angiosperms. Tissue expression pattern analyses revealed thattma is expressed in a tissue-specific manner in leaves and stems, but not in fruits and roots. Phylogenetic tree analyses showed that TMA belonged to the structurally and evolutionarily closely related monocot mannose-binding lectin superfamily. This study provides useful information to understand the molecular evolution of plant lectins.     

A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin.     

光棘球海胆seali基因克隆及表达特性分析

seali基因隶属于PIWI超家族, 其编码的RNA结合蛋白在生殖细胞发育过程中发挥重要作用。研究经同源比对从光棘球海胆(Mesocentrotus nudus)性腺转录组数据库中筛选得到seali基因片段, 随后通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends, RACE), 获得其全长cDNA序列。光棘球海胆seali基因cDNA全长3462 bp, 其中3′UTR长度为416 bp, 5′UTR长度为180 bp, 其中3′UTR的加尾信号并非经典的AAUAAA或AUUAAA, 而是较少见的AAUACA。开放阅读框(Open Reading Frame, ORF)2862 bp, 编码954个氨基酸, 具有保守的PIWI和PAZ结构域, 多重序列比对和系统进化分析结果表明其属于Argonaute家族的PIWI亚家族成员。荧光定量PCR技术检测结果表明, Mnseali基因在光棘球海胆性腺、肠、管足和体腔细胞中均有表达, 在性腺中表达量最高。此外, Mnseali基因为母源因子, 在整个胚胎发育时期均有表达。在卵巢中, 随着卵母细胞的成熟, Mnseali的表达量逐渐升高, 而仅在成熟期的精巢中表达量显著上调。RNA原位杂交结果表明, Mnseali在光棘球海胆性腺的生殖细胞中特异表达, 是光棘球海胆生殖细胞标记基因。该研究为海胆生殖细胞发育相关的研究提供了支撑。     

Molecular cloning, characterization and expression of cDNA encoding translationally controlled tumor protein (TCTP) from Jatropha curcas L

A cDNA encoding translationally controlled tumor protein (TCTP) of Jatropha curcas L., JcTCTP, was isolated from an endosperm cDNA library. JcTCTP consisted of a 5?? untranslated region (UTR) of 526 bp, a 3?? UTR of 377 bp and an open reading frame (ORF) of 507 bp, encoding a protein of 168 amino acid residues, which contained two signature sequences of TCTP family. Its deduced amino acid sequence was similar to the other known plants TCTPs in a range of 77.4?C92.3%. Expression of JcTCTP was the highest in the stem, endosperm at embryo formation stage and embryo of J. curcas tissues, and the lowest in the endosperm at seminal leaf embryo stage and flower, demonstrating a pattern of temporal and spatial specific expression.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus)

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5′ untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3′ UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys⁶¹-Cys¹⁵⁸, Cys¹³⁴-Cys¹⁵⁰) and a Ca²⁺/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca²⁺-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca²⁺-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR.     

Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5′-terminal untranslated region (UTR) of 60 bp and a 3′-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys⁵³, Cys¹²⁸, Cys¹⁴⁴, Cys¹⁵²) involved in the formation of disulfides bridges and the potential Ca²⁺/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca²⁺, and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei.     

MOLECULAR CHARACTERIZATION OF AN APOLIPOPHORIN‐III GENE FROM THE CHINESE OAK SILKWORM,Antheraea pernyi (LEPIDOPTERA: SATURNIIDAE)

Apolipophorin‐III (ApoLp‐III) acts in lipid transport, lipoprotein metabolism, and innate immunity in insects. In this study, an ApoLp‐III gene of Antheraea pernyi pupae (Ap‐ApoLp‐III) was isolated and characterized. The full‐length cDNA of Ap‐ApoLp‐III is 687 bp, including a 5′‐untranslated region (UTR) of 40 bp, 3′‐UTR of 86 bp and an open reading frame of 561 bp encoding a polypeptide of 186 amino acids that contains an Apolipophorin‐III precursor domain (PF07464). The deduced Ap‐apoLp‐III protein sequence has 68, 59, and 23% identity with its orthologs of Manduca sexta, Bombyx mori, and Aedes aegypti, respectively. Phylogenetic analysis showed that the Ap‐apoLp‐III was close to that of Bombycoidea. qPCR analysis revealed that Ap‐ApoLp‐III expressed during the four developmental stages and in integument, fat body, and ovaries. After six types of microorganism infections, expression levels of the Ap‐ApoLp‐III gene were upregulated significantly at different time points compared with control. RNA interference (RNAi) of Ap‐ApoLp‐III showed that the expression of Ap‐ApoLp‐III was significantly downregulated using qPCR after injection of E. coli. We infer that the Ap‐ApoLp‐III gene acts in the innate immunity of A. pernyi.     

CLONING AND QUANTITATIVE ANALYSIS OF THE CARBONIC ANHYDRASE GENE FROM PORPHYRA YEZOENSIS1

Carbonic anhydrase (CA), an enzyme that catalyzes the interconversion of CO₂ and HCO₃^?, has a critical role in inorganic carbon acquisition in many kingdoms, including animals, plants, and bacteria. In this study, the full‐length cDNA of the CA gene from Porphyra yezoensis Ueda (denoted as PyCA) was cloned by using an expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE). The nucleotide sequence of PyCA consists of 1,153 bp, including a 5′ untranslated region (UTR) of 177 bp, a 3′ UTR of 151 bp, and an open reading frame (ORF) of 825 bp that can be translated into a 274‐amino‐acid putative peptide with a molecular mass (M) of 29.8 kDa and putative isoelectric point (pI) of 8.51. The predicted polypeptide has significant homology to the β‐CA from bacteria and unicellular algae, such as Porphyridium purpureum. The mRNA in filamentous thalli, leafy thalli, and conchospores was examined, respectively, by real‐time fluorescent quantitative PCR (qPCR), and the levels of PyCA are different at different stages of the life cycle. The lowest level of mRNA was observed in leafy thalli, and the level in filamentous thalli and in the conchospores was 4‐fold higher and 10‐fold higher, respectively.     

Molecular cloning and expression analysis of a F-type lectin gene from Japanese sea perch (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the fucose-binding lectin (F-type lectin) gene from Japanese sea perch (Lateolabrax Japonicus). The full-length cDNA of sea perch F-lectin (JspFL) contained a 5′ untranslated region (UTR) of 39 bp, an ORF of 933 bp encoding a polypeptide of 310 amino acids with an estimated molecular mass of 10.82 kDa and a 3′ UTR of 332 bp. The searches for nucleotides and protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of JspFL was homological to the Fucose-binding lectin in other fish species. In the JspFL deduced amino acid sequence, two tandem domains that exhibit the eel carbohydrate-recognition sequence motif were found. The temporal expressions of gene in the different tissues were measured by real-time PCR. And the mRNA expressions of the gene were constitutively expressed in tissues including spleen, head-kidney, liver, gill, and heart. The JspFL expression in spleen was different during the stimulated time point, 2 h later the expression level became up-regulated, and 6 h later the expression level became down-regulated. The result indicated that JspFL was constitutive and inducible expressed and could play a critical role in the host-pathogen interaction.     

cDNA cloning and characterization of a mannose-binding lectin fromZingiber officinaleRoscoe (ginger) rhizomes

Using RNA extracted fromZingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA ofZ. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA ofzoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed thatzoa expressed in all the tested tissues ofZ. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed inEscherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae     

Molecular cloning and characterization of a mannose-binding lectin gene from Crinum asiaticum

Full-length cDNA of a mannose-binding lectin or agglutinin gene was cloned from a traditional Chinese medicinal herb Crinum asiaticum var. sinicum through RACE-PCR cloning. The full-length cDNA of C. asiaticum agglutinin (caa) was 820 bp and contained a 528 bp open reading frame encoding a lectin precursor (preproprotein) of 175 amino acid residues with a 22 aa signal peptide. The coding region of the caa gene was high in G/C content. The first 20 bp of the 5' UTR had a dC content of 50%, which was a typical feature of the leader sequence. By cutting away the signal peptide, the CAA proprotein was 15.79 kDa with a pl of 9.27 and contained 3 mannose-binding sites (QDNY). Random coil and extended strand constituted interlaced domination of the main part of the secondary structure. B-lectin conserved domain existed within N24 to G130. Predicted three-dimensional structure of CAA proprotein was very similar to that of GNA (Galanthus nivalis agglutinin). It is significant that besides certain homologies to known monocot mannose-binding lectins from Amaryllidaceae, Orchidaceae, Alliaceae and Liliaceae, caa also showed high similarity to gastrodianin type antifungal proteins. No intron was detected within the region of genomic sequence corresponding to the caa full-length cDNA. Southern blot analysis indicated that the caa gene belonged to a low-copy gene family. Northern blot analysis demonstrated that caa mRNA was constitutively expressed in all the tested tissue types including the root, bulb, leaf, rachise, flower and fruit tissues.     

泥蚶谷胱甘肽过氧化物酶基因的全长克隆与表达分析

为了探讨谷胱甘肽过氧化物酶（Glutathione peroxidase,GPx）基因在泥蚶应激反应中的作用,研究采用RACE技术克隆了泥蚶GPx基因（TgGPx）cDNA全长,其cDNA全长1195 bp,包含45 bp 5’-UTR,639 bp开放阅读框（ORF）和511 bp 3’-UTR。ORF编码212个氨基酸残基,预测蛋白分子量为24.3 kD,理论等电点为8.33,其中,第53个氨基酸U是由密码子202UGA204编码的硒代半胱氨酸（Se-Cys）。在3’-UTR上存在一段序列,形成一种独特的茎环结构,即SECIS元件。SECIS元件在密码子UGA翻译为Se-Cys的过程中起决定性作用。通过序列比对与系统进化分析,发现软体动物中也存在不同种类的GPx基因,TgGPx与GPx1和GPx2的亲缘关系较近。利用qRT-PCR技术对TgGPx在泥蚶的不同组织以及重金属刺激后的表达量进行分析,结果表明,TgGPx在泥蚶的5个组织中都有表达,但存在组织特异性,在外套膜中的表达量最高,在血细胞中的表达量最低。用重金属铅、铜、镉刺激后,TgGPx在肝胰脏中的表达量显著升高,表明TgGPx在维护机体正常功能方面及泥蚶抵御外界刺激的应激反应中发挥作用。     

cDNA cloning, tissues, embryos and larvae expression analysis of Sox10 in half-smooth tongue-sole, Cynoglossus semilaevis

A half-smooth tongue-sole, Cynoglossus semilaevis Sox10 (Accession no.: EU070763) was isolated from brain of tongue sole by using homologous cloning and RACE method. The complete cDNA of the tongue sole Sox10 contains a 35 bp 5′UTR, a 1338 bp open reading frame (ORF) encoding 445 amino acids and a 1155 bp 3′UTR. A condensed phylogenetic tree was constructed based on the amino acid sequences of tongue sole Sox10 and other well-defined vertebrate Sox. The overall topology of the tree showed the tongue sole Sox10 clusters with all Sox10. Alignment of amino acid residues of the tongue sole Sox10 gene with those from other vertebrate indicated high level conservation of amino acid sequence. The RT-PCR analysis demonstrated that the tongue sole Sox10 was highly expressed in brain, gills, skin and eyes, intermediately in spleen, heart, head–kidney and muscles, weakly expressed in kidneys and intestine and no expression in liver and gonad. The Sox10 was also expressed weakly in germ cell and zygote. We cannot detect the expression of the Sox10 in 8-cells stage. However it resumed expression weakly from blastula stage to middle of gastrula. And it expressed highly from neurula stage to 25 dah (day after hatching). It suggested that the Sox10 was involved in the development of embryos and larvae in tongue sole.     

Primary structure of a fucose-specific lectin obtained from a mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) is a protein composed of two identical subunits having no carbohydrate chain and shows sugar-binding specificity for L-fucose. Full-length cDNA encoding for the lectin has been isolated from a lambda gt11 library, screened with an antiserum directed against AAL. The cDNA clone contained 1,370 nucleotides and an open reading frame of 939 nucleotides encoding 313 amino acids. The amino-terminal sequence (residues 1-30) of the lectin isolated from the mushroom coincided with the deduced amino acid sequence starting from proline at the 2nd residue, indicating that the mature AAL consists of 312 amino acids. Its molecular weight is calculated to be 33,398. The deduced amino acid sequence shows that AAL includes six internal homologous regions, and has considerable homology with a hemagglutinin from a Gram-negative bacterium, Myxococcus xanthus, which forms a fruiting body. No significant homology was observed with higher plant or animal lectins. The recombinant AAL produced by Escherichia coli JM109 carrying the AAL expression plasmid pKA-1 [Fukumori, F. et al. (1989) FEBS Lett. 250, 153-156] was purified from the cell lysate by affinity chromatography using a fucose-starch column, and hundreds of milligrams of the lectin was obtained. The recombinant lectin showed the same biochemical characteristics and sugar binding specificity as did the natural AAL.     

Characterization of carbohydrate combining sites of Bryohealin,an algal lectin from <Emphasis Type="Italic">Bryopsis plumosa</Emphasis>

Bryohealin is a lectin involved in the wound-healing process of the marine green alga Bryopsis plumosa. In the previous purification study, it has been shown that lectin was composed of two identical subunits of 27 kDa, cross-linked by disulfide bond, and showed binding specificity to N-acetyl-d-glucosamine and N-acetyl-d-galactosamine (GlcNAc and GalNAc, respectively). To determine if the lectin recognize the two different sugars at the same binding domain, the carbohydrate binding sites of Bryohealin was analyzed using chromatography and chemical modification methods. Results showed that the same binding site of the lectin was responsible for the recognition of two sugars, GalNAc as well as GlcNAc. Chemical modification studies showed that hemagglutinating activities of Bryohealin were not affected by modification of histidine, tryptophan, aspartic acid, and glutamic acid. When arginine residues were modified with 1,2-cyclohexanedione, the activity of Bryohealin rapidly decreased. The sugar binding sites remained intact when the lectin was treated with inhibitory sugars (0.2 M GalNAc and/or GlcNAc) prior to 1,2-cyclohexanedione treatment. The sugar binding domain of Bryohealin was predicted from the MALDI-TOF analysis and the full cDNA sequence of the lectin gene.