Han J, Mathison J C, Ulevitch R J, Tobias P S
Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1994 Mar 18;269(11):8172-5.
Lipopolysaccharide (LPS) binding protein (LBP), a 58-60 kDa glycoprotein, binds to the lipid A region of LPS. The resulting LPS-LBP complex is recognized by both the membrane-bound (mCD14) and soluble forms of CD14 (sCD14), thereby enhancing the ability of LPS to activate myeloid, endothelial, and epithelial cells. To begin to characterize the structure-function relationships within LBP, we have created and expressed a truncated form of human LBP (herein called NH-LBP) comprising amino acid residues 1-197 of the parent molecule. Experiments were done to characterize the ability of NH-LBP to bind LPS and to promote LPS binding to CD14. We found that NH-LBP efficiently binds LPS but does not transfer the LPS to either mCD14 or sCD14. Additionally, NH-LBP inhibited LPS binding to LBP, inhibited the LBP-promoted binding of LPS to CD14, and inhibited the LBP-dependent activation of rabbit peritoneal exudate macrophages. The apparent dissociation constant for LPS-NH-LBP complexes is less than 1 x 10(-8) M which compares well with the dissociation constant for LPS-LBP complexes of approximately 1 x 10(-9) M. We conclude from these studies that the LPS binding site of LBP resides in the amino-terminal half of LBP and that the CD14 interaction site resides in the carboxyl-terminal half of LBP. These data suggest that appropriately modified fragments of LBP might provide novel reagents with high LPS binding affinity that could be useful in inhibiting LPS-dependent cellular activation in vivo.
脂多糖（LPS）结合蛋白（LBP）是一种58 - 60 kDa的糖蛋白，可与LPS的脂质A区域结合。所形成的LPS - LBP复合物可被膜结合形式（mCD14）和可溶性形式的CD14（sCD14）识别，从而增强LPS激活髓样细胞、内皮细胞和上皮细胞的能力。为了开始表征LBP内的结构 - 功能关系，我们构建并表达了人LBP的截短形式（在此称为NH - LBP），其包含亲本分子的氨基酸残基1 - 197。进行实验以表征NH - LBP结合LPS以及促进LPS与CD14结合的能力。我们发现NH - LBP能有效结合LPS，但不会将LPS转移至mCD14或sCD14。此外，NH - LBP抑制LPS与LBP的结合，抑制LBP促进的LPS与CD14的结合，并抑制LBP依赖的兔腹腔渗出巨噬细胞的激活。LPS - NH - LBP复合物的表观解离常数小于1×10⁻⁸ M，这与LPS - LBP复合物约1×10⁻⁹ M的解离常数相当。我们从这些研究中得出结论，LBP的LPS结合位点位于LBP的氨基末端一半，而CD14相互作用位点位于LBP的羧基末端一半。这些数据表明，LBP经适当修饰的片段可能提供具有高LPS结合亲和力的新型试剂，可用于体内抑制LPS依赖的细胞激活。
