Iwaki D, Kawabata S, Miura Y, Kato A, Armstrong P B, Quigley J P, Nielsen K L, Dolmer K, Sottrup-Jensen L, Iwanaga S
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Eur J Biochem. 1996 Dec 15;242(3):822-31. doi: 10.1111/j.1432-1033.1996.0822r.x.
The American horseshoe crab Limulus polyphemus contains alpha 2-macroglobulin (alpha 2M) in the hemolymph plasma and hemocytes. alpha 2M from Limulus shows many of the typical characteristics of mammalian alpha 2M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian alpha 2M receptor. Additionally, Limulus alpha 2M has the unique property that it regulates the limulin-based hemolytic system of the plasma. A cDNA encoding Limulus alpha 2M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N-terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian alpha 2Ms (28-29% identity) and contains common features found in mammalian alpha 2Ms. a bait region, an internal thiol-ester site, and a receptor-binding domain. However, the N-terminal portion (positions 24-105) has no sequence similarity with those of mammalian alpha 2Ms, and it is structurally related to that of the human complement factor C8 chain, consistent with a role for Limulus alpha 2M in host defense. The component sugar analysis of Limulus alpha 2M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian alpha 2M. However, unlike mammalian alpha 2M, no sialic acid was detected in Limulus alpha 2M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of O-linked sugar chains, which have not been found in mammalian alpha 2M. Expression of alpha 2M was detected in hemocytes, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against alpha 2M indicated the presence of the alpha 2M in large granules. Trypsin-treated Limulus alpha 2M, but not the native alpha 2M, displaced methylamine-treated human 125I-alpha 2M from the human alpha 2M receptor with a Kd of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.
美洲鲎（Limulus polyphemus）的血淋巴血浆和血细胞中含有α2-巨球蛋白（α2M）。来自美洲鲎的α2M具有许多哺乳动物α2M的典型特征，包括存在内部硫酯、与多种内肽酶反应、独特的蛋白酶捕获机制以及与哺乳动物α2M受体反应。此外，美洲鲎α2M具有独特的特性，即它调节血浆中基于鲎试剂的溶血系统。已从血细胞cDNA文库中获得编码美洲鲎α2M的cDNA。开放阅读框编码一个25个氨基酸残基组成的N端信号序列和一个1482个残基的成熟蛋白。整个氨基酸序列与哺乳动物α2M的序列相似（同一性为28 - 29%），并包含在哺乳动物α2M中发现的共同特征，一个诱饵区域、一个内部硫酯位点和一个受体结合结构域。然而，N端部分（第24 - 105位）与哺乳动物α2M的序列没有相似性，并且在结构上与人补体因子C8链相关，这与美洲鲎α2M在宿主防御中的作用一致。美洲鲎α2M 的成分糖分析表明存在一种与哺乳动物α2M类似的复杂型寡糖链。然而，与哺乳动物α2M不同的是，在美洲鲎α2M中未检测到唾液酸，并且它含有约3摩尔/摩尔的N - 乙酰半乳糖胺，表明存在O - 连接糖链，这在哺乳动物α2M中未发现。在血细胞中检测到α-2M的表达，但在肝胰腺、心脏、胃、肠、触角腺、脑和骨骼肌中未检测到。此外，用抗α2M抗血清对血细胞的大颗粒和小颗粒进行免疫印迹表明α2M存在于大颗粒中。经胰蛋白酶处理的美洲鲎α2M，而不是天然α2M，以30 nM的解离常数从人α2M受体上取代了经甲胺处理的人125I-α2M，这表明哺乳动物和节肢动物进化谱系之间蛋白酶清除机制具有保守性。
