Yukiko Konami

Strong affinity of Maackia amurensis hemagglutinin (MAH) for sialic acid‐containing Ser/Thr‐linked carbohydrate chains of N‐terminal octapeptides from human glycophorin A

The interaction of the Maackia amurensis hemagglutinin (MAH) with various glycopeptides and oligosaccharides was investigated by means of immobilized lectin affinity chromatography. An amino terminal octapeptide obtained from human glycophorin A having three Neu5Acα→3Galβ1→3(Neu5Acα2→6)GalNAc tetrasaccharide chains, designated as CB‐II, was found to have an extremely strong affinity for MAH. Therefore, it is strongly suggested that hemagglutination by MAH was caused by its interaction with Ser/Thr‐linked carbohydrate chains of human glycophorin A on erythrocyte membranes.

The primary structure of the Lotus tetragonolobus seed lectin

The complete amino acid sequence of the Lotus tetragonolobus lectin was determined by a protein sequencer after digestion with endoproteinases of Lys‐C and Asp‐N, and compared with those of other leguminous plant lectins.

Purification and characterization of a carbohydrate-binding peptide from Bauhinia purpurea lectin

In order to examine the correlation between the amino acid sequence and sugar binding specificity of Bauhinia purpurea lectin (BPA), a galactose and lactose binding lectin, a peptide which interacts with lactose was purified from an Asp‐N endoproteinase digest of BPA by means of affinity chromatography on a column of lactose‐Sepharose. The amino acid sequence of this peptide is Asp‐Thr‐Trp‐Pro‐Asn‐Thr‐Glu‐Trp‐Ser. A tryptic, fragment having the ability to interact with lactose was also purified and found to contain the above sequence, consisting of 9 amino acids. The chemical synthesis of this peptide was carried out by the solid‐phase method and the synthetic peptide was found to exhibit lactose binding activity in the presence of calcium.

Correlation between carbohydrate-binding specificity and amino acid sequence of carbohydrate-binding regions of Cytisus-type anti-H(O) lectins

A carbohydrate‐binding peptide of the di‐N‐acetylchitoblose‐binding Cytisus sessilifolius anti‐H(O) lectin I (CSA‐I) was isolated from the endoproteinase Asp‐N digest of CSA‐I by affinity chromatography on a column of N‐acetyl‐d‐glucosamine oligomer‐Sepharose (GIcNAc oligomer‐Sepharose). The amino acid sequence of the carbohydrate‐binding peptide of CSA‐I was determined to be DTYFGKTYNPW using a gas‐phase protein sequencer. This sequence corresponds to the sequence from Asp‐129 to Trp‐139 based on the primary structure of CSA‐I, and shows a high degree of homology to those of the putative carbohydrate‐binding peptide of the Laburnum alpinum lectin I (LAA‐I) (DTYFGKAYNPW) and of the Ulex europaeus lectin II (UEA‐II) (DSYFGKTYNPW). The binding of these three anti‐H(O) lectins is known to be inhibited by di‐N‐acetylchitobiose but not by l‐fucose. These results strongly suggest that there is a good correlation between the carbohydrate‐binding specificity and the amino acid sequence of the carbohydrate‐binding regions of di‐N‐acetylchitobiose‐binding lectins.

The primary structure of the Laburnum alpinum seed lectin.

The complete amino acid sequence of the Laburnum alpinum di‐N‐acetylchitobiose‐binding lectin was determined by using a protein sequencer after digestion with endoproteinases Lys‐C and Asp‐N, and compared with those of other leguminous plant lectins.

Cloning and sequence analysis of theMaackia amurensis haemagglutinin cDNA

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylatedO-linked carbohydrate chains (Konami Y, Yamamoto K, Osawa T, Irimura T (1994)FEBS Lett342:334–38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinatedMaackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.

Determination of the carbohydrate-binding site of Bauhinia purpurea lectin by affinity chromatography

To determine the carbohydrate-binding site of Bauhinia purpurea lectin (BPA), a D-galactose- and lactose-binding lectin, a peptide which interacts with lactose was purified from endoproteinase Asp-N digests of BPA by chromatography on a lactose-Sepharose column. It consists of nine amino acids and its amino acid sequence is Asp-Thr-Trp-Pro-Asn-Thr-Glu-Trp-Ser. A tryptic fragment with the ability to interact with lactose was also purified and found to contain this sequence, consisting of nine amino acids. This nonapeptide was aligned in a part of the metal-binding region conserved in all legume lectins. The chemical synthesis of the nonapeptide was carried out by a solid-phase method and the synthetic peptide showed a lactose-specific binding activity in the presence of calcium. A chimeric lectin gene was constructed using a cDNA coding BPA in which the nonapeptide sequence was replaced by the corresponding region of the alpha-D-mannose binding Lens culinaris lectins. Although BPA is specific for beta-D-galactose, the chimeric lectin expressed in Escherichia coli was found to bind alpha-D-mannosyl-bovine serum albumin and this binding was inhibited by D-mannose.

A putative carbohydrate-binding domain of the lactosebindingCytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of thel-fucose-bindingUlex europaeus anti-H(O) lectin

The complete amino acid sequence of a lactose-bindingCytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of thel-fucose-bindingUlex europaeus lectin I (UEA-I).

Purification and characterization of carbohydrate-binding peptides from Lotus tetragonolobusand Ulex europeus seed lectins using affinity chromatography

Carbohydrate-binding peptides of several anti-H(O) leguminous lectins were obtained from endoproteinase Asp-N or Lys-C digests of L-fucose-binding Lotus tetragonolobus lectin (LTA) and Ulex europeus lectin I (UEA-I) and from that of a di-N-acetylchitobiose-binding Ulex europeus lectin II (UEA-II) by affinity chromatography on columns of Fuc-Gel (for LTA and UEA-I) and on a column of a mixture of several oligomers of N-acetyl-D-glucosamine (GlcNAc) coupled to Sepharose 4B (GlcNAc oligomer-Sepharose 4B) (for UEA-II). These peptides were retained on the Fuc-Gel or GlcNAc oligomer-Sepharose 4B column and were presumed to have an affinity for the columns. The amino acid sequences of the retarded peptides were determined using a protein sequencer.