Sphingolipid Engineering Using Marine Resources

本文报道了海星海星中一种新的N-乙酰半乳糖胺特异性凝集素的纯化、鉴定和基因克隆。纯化的凝集素在SDS-PAGE上显示19 kDa、41 kDa和60 kDa的蛋白条带，可能对应于单体、均二聚体和均三聚体。凝集素的血凝活性可被N-乙酰氨基半乳糖、TN抗原和A型血三糖特异性抑制，但不能被N-乙酰氨基半乳糖、半乳糖、半乳糖胺和B型三糖所抑制。克隆结果表明，该蛋白由168个氨基酸组成，含有18个氨基酸残基，具有典型的C型凝集素基序。这些发现表明该蛋白是一种C型凝集素。我们报道了海洋细菌Sewanella alga G8编码该酶的基因的纯化、鉴定、分子克隆和表达。从菌株G8的培养上清液中分离纯化该酶，纯化倍数为1600倍，总收率为24%。纯化的酶经银染后，经SDS-聚丙烯酰胺凝胶电泳法测得其相对分子质量为75 kDa。2,976个核苷酸的开放阅读框编码一个由992个氨基酸组成的多肽，包括一个35个残基的信号序列，其中59个残基与纯化酶的氨基酸序列相匹配。根据推导的氨基酸序列，估计该成熟酶的相对分子质量为105,994。

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. 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. 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.We report here the purification, characterization, molecular cloning, and expression of the gene encoding the enzyme from marine bacterium, Sewanella alga G8. The enzyme was purified by 1,600fold with an overall yield of 24% from a culture supernatant of strain G8. After being stained with a silver staining solution, molecular mass of the purified enzyme was estimated to be 75 kDa on SDS-polyacrylamide gel electrophoresis. The open reading frame of 2,976 nucleotides encoded a polypeptide of 992 amino acids including a signal sequence of 35 residues, 59 residues of which matched the amino acid sequence determined for the purified enzyme. The molecular weight of the mature enzyme was estimated to be 105,994 from the deduced amino acid sequence.