Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains.
Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains.
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DOI：
10.4049/jimmunol.152.7.3541
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发表时间：
1994-04
影响因子：
4.4
通讯作者：
Catherine A. Ernst;Yujun Zhang;P. R. Hancock;Barbara J. Rutledge;Christopher L. Corless;B. Rollins
中科院分区：
文献类型：
--
作者：
Catherine A. Ernst;Yujun Zhang;P. R. Hancock;Barbara J. Rutledge;Christopher L. Corless;B. Rollins
单核细胞趋化蛋白-1（MCP-1）是趋化因子-β（或C-C）家族的细胞因子的成员。鼠MCP-1，首先被鉴定为JE基因，在分子大小和糖基化程度上不同于人MCP-1。我们用中国仓鼠卵巢细胞表达重组鼠MCP-1，发现其主要形式是一种M（r）约为25，000的微异质蛋白。大多数MCP-1的微观异质性是由于每个分子末端连接到恒定数量的O-连接寡糖链的唾液酸的量可变。这种碳水化合物，沿着少量N-连接的碳水化合物，占鼠MCP-1表观分子大小的50%，并且不是体外单核细胞趋化活性所需的。突变分析表明，大部分的碳水化合物被添加到一个49个氨基酸的C-末端结构域，这是不存在于人MCP-1，并不需要在体外生物活性，表明鼠MCP-1的N-末端结构域含有单核细胞趋化活性和一个高度糖基化的C-末端结构域的功能还不清楚。COS细胞产生的MCP-1含有少量硫酸盐，而中国仓鼠卵巢产生的MCP-1不含硫酸盐。不存在硫酸盐不会改变MCP-1的体外化学引诱物性质。在体外，高度纯化的鼠MCP-1吸引单核细胞，但不吸引中性粒细胞，其比活性与人MCP-1相似（EC 50约为0.5 nM）。与人单核细胞的平衡结合实验揭示了每个细胞存在约3000个结合位点，Kd为0.77 nM。在体内，在多种鼠品系中注射高达1微克的鼠MCP-1诱导出现稀疏的混合炎性浸润。MCP-1的体外和体内作用之间的差异表明，可能需要其他因素来引发体内单核细胞对MCP-1的完全趋化反应。
Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine-beta (or C-C) family of cytokines. Murine MCP-1, first identified as the JE gene, differs from human MCP-1 in molecular size and extent of glycosylation. We have used Chinese hamster ovary cells to express recombinant murine MCP-1 and find that its predominant form is a microheterogeneous protein of M(r) approximately 25,000. Most of MCP-1's microheterogeneity is due to variable amounts of sialic acid that are terminally attached to a constant number of O-linked oligosaccharide chains per molecule. This carbohydrate, along with a small amount of N-linked carbohydrate, accounts for 50% of the apparent molecular size of murine MCP-1 and is not required for in vitro monocyte chemoattractant activity. Mutational analysis shows that most of the carbohydrate is added to a 49-amino acid C-terminal domain that is not present in human MCP-1 and is not required for in vitro biologic activity, suggesting that murine MCP-1 consists of an N-terminal domain containing monocyte chemoattractant activity and a heavily glycosylated C-terminal domain of as yet unknown function. MCP-1 produced in COS cells contains a small amount of sulfate, but Chinese hamster ovary-produced MCP-1 does not. The absence of sulfate does not alter MCP-1's in vitro chemoattractant properties. In vitro, highly purified murine MCP-1 attracts monocytes, but not neutrophils, with a specific activity similar to human MCP-1 (EC50 approximately 0.5 nM). Equilibrium binding experiments with human monocytes reveal the presence of approximately 3000 binding sites per cell with a Kd of 0.77 nM. In vivo, injection of up to 1 micrograms murine MCP-1 in a variety of murine strains induces the appearance of a sparse mixed inflammatory infiltrate. The disparity between MCP-1's in vitro and in vivo effects suggests that other factors may be required to elicit a full-blown monocyte chemotactic response to MCP-1 in vivo.