Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata.
CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-beta-D-galactosaminide (NP-GalNAc) was determined to be 2.3 x 10(4) M(-1), and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment.
Hatakeyama
Tomomitsu
T
Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Japan. thata@net.nagasaki-u.ac.jp
Matsuo
Noriaki
N
Shiba
Kouhei
K
Nishinohara
Shoichi
S
Yamasaki
Nobuyuki
N
Sugawara
Hajime
H
Aoyagi
Haruhiko
H
D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D050260:Carbohydrate Metabolism; D004220:Disulfides; D037102:Lectins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D012616:Sea Cucumbers; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D004220:Disulfides; D037102:Lectins; D000116:Acetylgalactosamine