Steitz S A, Hasegawa K, Chiang S L, Cobb R R, Castro M A, Lobl T J, Yamada M, Lazarides E, Cardarelli P M
Tanabe Research Laboratories USA, Inc., San Diego, CA 92121, USA.
FEBS Lett. 1998 Jul 3;430(3):158-64. doi: 10.1016/s0014-5793(98)00637-1.
Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13-35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1-10), second loop (amino acids 37-51), and carboxy-terminus (amino acids 56-71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.
单核细胞趋化蛋白-1（MCP-1）是β趋化因子家族的成员，它通过特定的七跨膜受体发挥作用，将单核细胞、嗜碱性粒细胞和T淋巴细胞募集到炎症部位。为了确定人MCP-1蛋白中对其生物学活性重要的区域，我们合成了结构域特异性肽，并测试了它们拮抗MCP-1在THP-1细胞中结合和趋化作用的能力。我们发现，包含氨基酸13 - 35的半胱氨酸间第一环肽可抑制MCP-1的结合和趋化活性，而代表MCP-1氨基末端（氨基酸1 - 10）、第二环（氨基酸37 - 51）和羧基末端（氨基酸56 - 71）的肽则没有作用。此外，我们还发现通过二硫键连接使第一环肽环化并通过酰胺化封闭肽的C末端可增强该肽阻断MCP-1结合和趋化作用的活性。为了具体确定第一环内对MCP-1功能活性至关重要的氨基酸残基，我们在MCP-1重组蛋白中将丙氨酸替代酪氨酸（Y13A）或精氨酸（R18A）。虽然杆状病毒产生的野生型和R18A MCP-1蛋白在诱导THP-1趋化作用的能力上没有区别，并且与市售重组MCP-1蛋白相比在结合活性上显示出适度影响，但Y13A点突变导致功能急剧丧失。MCP-1功能结构域的鉴定将有助于设计MCP-1受体拮抗剂，这在许多炎症性疾病中可能具有临床益处。
