A unique primary structure, cDNA cloning and function of a galactose‐specific lectin from ascidian plasma
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The complete amino acid sequence of a galactose‐specific lectin from the plasma of the ascidian Halocynthia roretzi has been determined by sequential Edman degradation analysis of peptide fragments derived by proteolytic fragmentation and chemical cleavage of the reductive S‐pyridylethylated lectin. Peptide fragments were separated by reverse‐phase HPLC. The N‐terminal and C‐terminal amino acid sequences were determined by Edman degradation and enzymatic digestion. The H. roretzi plasma lectin is a single‐chain protein consisting of 327 amino acids and four disulfide bonds, one of which was found to be cross‐linked intramolecularly. A comparison of the amino acid sequence of the H. roretzi plasma lectin with the sequences of other proteins reveals that the H. roretzi lectin has a structure consisting of a twice‐repeated sequence, a fibrinogen‐related sequence and a C‐type lectin‐homologous sequence. The above amino acid sequence was verified by cDNA cloning of this lectin. Three cDNA clones that have single ORFs encoding the lectin precursor were isolated from an H. roretzi hepatopancreas cDNA library. The deduced amino acid sequences in the three cDNA clones contain the same sequence of the mature lectin molecule and the same putative signal sequence. In addition, it was demonstrated that this lectin can enhance phagocytosis by H. roretzi hemocytes. Thus, the plasma lectin is constructed into an oligomer structure via intermolecular disulfide bonds and plays a role in the biological defense of H. roretzi as a defense molecule.Keywords:
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The Dolichos biflorus seed lectin contains two structurally related subunits. A cDNA library was constructed using RNA isolated from D. biflorus seeds actively synthesizing the seed lectin. The library was expressed in Escherichia coli using a lambda Charon 16 vector, and lectin‐specific antiserum was used to isolate a seed lectin cDNA. Hybridization of the D. biflorus seed lectin cDNA to RNA isolated from seeds actively producing both lectin subunits identifies a single‐size RNA of 1100 bases. An oligodeoxyribonucleotide probe, constructed from an amino acid sequence common to both lectin subunits, detects the same size RNA. Translation of seed mRNA in vitro and immunoprecipitation of translation products using a lectin‐specific antiserum yields a single polypeptide of slightly higher molecular mass than the largest seed lectin subunit. This seed lectin precursor is indistinguishable from a polypeptide synthesized from mRNA hybrid selected by the seed lectin cDNA. These data support the existence of a single polypeptide precursor for both subunit types of the D. biflorus seed lectin and suggest that differences between the subunit types arise by posttranslational processing.
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Journal Article A Chimeric Lectin Formed from Bauhinia purpurea Lectin and Lens culinaris Lectin Recognizes a Unique Carbohydrate Structure Get access Kazuo Yamamoto, Kazuo Yamamoto 2 Laboratory of Molecular Medicine, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo7–3–1 Hongo, Bunkyo-ku, Tokyo 113–0033 2 To whom correspondence should be addressed. Tel +81-3-5841-4776, 8843, Fax: +81-03-5841-8923, E-mail:yamamoto@k.u-tokyo.ac.jp Search for other works by this author on: Oxford Academic PubMed Google Scholar Yukiko Konami, Yukiko Konami Laboratory of Molecular Medicine, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo7–3–1 Hongo, Bunkyo-ku, Tokyo 113–0033 Search for other works by this author on: Oxford Academic PubMed Google Scholar Toshiaki Osawa Toshiaki Osawa Laboratory of Molecular Medicine, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo7–3–1 Hongo, Bunkyo-ku, Tokyo 113–0033 Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 127, Issue 1, January 2000, Pages 129–135, https://doi.org/10.1093/oxfordjournals.jbchem.a022573 Published: 01 January 2000 Article history Received: 02 September 1999 Accepted: 25 October 1999 Published: 01 January 2000
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We report here the purification, characterization, and cDNA cloning of a novel N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. The purified lectin showed 19-kDa, 41-kDa, and 60-kDa protein bands on SDS–PAGE, possibly corresponding to a monomer, homodimer, and homotrimer. Interestingly, on 4–20% native PAGE the lectin showed at least nine protein bands, among which oligomers containing six to nine subunits had potent hemagglutination activity for sheep erythrocytes. The hemagglutination activity of the lectin was specifically inhibited by N-acetylgalactosamine, Tn antigen, and blood group A trisaccharide, but not by N-acetylglucosamine, galactose, galactosamine, or blood group B trisaccharide. The specificity of the lectin was further examined using various glycosphingolipids and biotin-labeled lectin. The lectin was found to bind to Gb5Cer, but not Gb4Cer, Gb3Cer, GM1a, GM2, or asialo-GM2, indicating that the lectin specifically binds to the terminal α-GalNAc at the nonreducing end. The hemagglutination activity of the lectin was completely abolished by chelation with EDTA or EGTA and completely restored by the addition of CaCl2. cDNA cloning of the lectin showed that the protein is composed of 168 amino acids, including a signal sequence of 18 residues, and possesses the typical C-type lectin motif. These findings indicate that the protein is a C-type lectin. The recombinant lectin, produced in a soluble form by Escherichia coli, showed binding activity for asialomucin in the presence of Ca2+ but no hemagglutination.
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Several types of lectin domains that specifically recognize chitin have been discovered in plants. One such domain, the hevein domain, also known as CBM18, contains eight cysteine and glycine residues at conserved positions in 40 amino acid residues. It works alone, arranged in tandem, or in combination with other domains. Tomato lectin is a chimeric lectin composed of four hevein domains and extensin-like domains similar to the plant cell wall glycoprotein extensin. It has been used for tissue staining and the fractionation of sugar chains owing to its specificity against poly-N-acetyllactosamine. In this minireview, the author summarizes the current literature on the chitin-binding lectins of plants and discuss the role of tomato lectin.
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Abstract The crystal structures of an L‐type lectin domain from Methanocaldococcus jannaschii in apo and mannose‐bound forms have been determined. A thorough investigation of L‐type lectin domains from several organisms provides insight into the differences in these domains from different kingdoms of life. While the overall fold of the L‐type lectin domain is conserved, differences in the lengths of the carbohydrate‐binding loops and significant variations in the Mn 2+ ‐binding site compared to the Ca 2+ ‐binding site are observed. Furthermore, the sequence and phylogenetic analyses suggest that the archaeal L‐type lectin domain is evolutionarily closer to the plant legume lectins than to its bacterial or animal counterparts. This is the first report of the biochemical, structural, sequence, and phylogenetic analyses of an L‐type lectin domain from archaea and serves to enhance our understanding of the species‐specific differences and evolution of L‐type lectin domains.
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