Structure-function studies of defense molecules found in horseshoe crab hemocytes

我们发现了一种新的马蹄蟹血细胞衍生的凝集素，我们将其命名为速凝素-4。它对人a型红细胞具有更强的血凝活性，比先前发现的与n-乙酰氨基葡萄糖亲和的血细胞凝集素-2更强。纯化的速凝素-4是一个470 kDa的寡聚糖蛋白，由30 kDa和31.5 kDa的亚基组成。10mM Ca2+可使血凝活性提高4倍，EDTA和邻菲罗啉可抑制血凝活性。L-聚焦和n-乙酰神经氨酸在100mm处完全抑制速凝素-4的活性。细菌s型脂多糖（LPS）对酶活性的抑制作用较强，而缺乏o抗原的r型脂多糖对酶活性的抑制作用较弱。s型LPS对人a型红细胞抑制凝集的最低浓度（0.1mg/ml）比明尼苏达沙门氏菌s型LPS低160倍。因此，colitose （3-deoxy-L- focal）是大肠杆菌O111: B4 o抗原中存在的一种独特的糖，结构与L- focal相似，是速凝素-4特异性配体的最有可能的候选者。从血细胞cDNA文库中分离到一个编码速凝素-4的cDNA。1344碱基对cDNA的开放阅读框编码了含有232个氨基酸的成熟蛋白。与其他已知的lps结合凝集素序列无明显相似性，而tachylectin-4与非洲爪蟾戊素1的nh2末端结构域同源，功能未知。

We identified a novel horseshoe crab hemocyte-derived lectin, which we named tachylectin-4. It has more potent hemagglutinating activity against human A-tupe erythrocytes than a previously identified hemocyte lectin with an affinity to N-acetylglucosamine, tachylectin-2. The purified tachylectin-4 is an oligomeric glycoprotein of 470 kDa, composed of subunits of 30 kDa and 31.5 kDa. Ca2+ at 10mM enhanced the hemagglutinatig activity 4-fold and the activity was inhibited by EDTA and o-phenanthroline. L-Fucose and N-acetylneuraminic acid at 100 mM completely inhibited the activity of tachylectin-4. The activity was also inhibited more strongly by bacterial S-type lipopolysaccharides (LPS) but not by R-type LPS lacking O-antigen. The most effective S-type LPS was from Escherichia coil O111 : B4 and the minimum concentration required for inhibiting agglutination against human A-type erythrocytes (0.1mg/ml) was 160-fold lower than those of S-type LPS from Salmonella minnesota. Therefore, colitose (3-deoxy-L-fucose), a unique sugar present in the O-antigen of Escherichia coli O111 : B4 with structural similarity to L-fucose, is the most probable candidate for a specific ligand of tachylectin-4. A cDNA coding for tachylectin-4 was isolated from a hemocyte cDNA library. The open reading frame of the 1344-base pair cDNA coded for the mature protein with 232 amino acids. There is no significant sequence similarity to any other known LPS-binding lectins, whereas tachylectin-4 is homologous to the NH2-terminal domain with unknown functions of Xenopus laevis pentraxin 1.