Purification, characterization, and cDNA cloning of alpha-N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera.
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 alpha-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 CaCl(2). 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 Ca(2+) but no hemagglutination.
Kakiuchi
Mari
M
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Okino
Nozomu
N
Sueyoshi
Noriyuki
N
Ichinose
Sachiyo
S
Omori
Akira
A
Kawabata
Shun-ichiro
S
Yamaguchi
Kuniko
K
Ito
Makoto
M
D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006385:Hemagglutination Inhibition Tests; D037102:Lectins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013215:Starfish / Q000737:chemistry*
D017931:DNA Primers; D018076:DNA, Complementary; D037102:Lectins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D000116:Acetylgalactosamine