Department of Laboratory Medicine, Division of Medical Microbiology, Lund University, Sölvegatan 23, 223 62 Lund, Sweden.
J Biol Chem. 2011 Sep 30;286(39):33981-91. doi: 10.1074/jbc.M111.251728. Epub 2011 Jul 27.
Sialic acid-binding immunoglobulin-like lectins (Siglecs) are receptors believed to be important for regulation of cellular activation and inflammation. Several pathogenic microbes bind specific Siglecs via sialic acid-containing structures at the microbial surface, interactions that may result in modulation of host responses. Recently, it was shown that the group B Streptococcus (GBS) binds to human Siglec-5 (hSiglec-5), an inhibitory receptor expressed on macrophages and neutrophils, via the IgA-binding surface β protein, providing the first example of a protein/protein interaction between a pathogenic microbe and a Siglec. Here we show that the hSiglec-5-binding part of β resides in the N-terminal half of the protein, which also harbors the previously determined IgA-binding region. We constructed bacterial mutants expressing variants of the β protein with non-overlapping deletions in the N-terminal half of the protein. Using these mutants and recombinant β fragments, we showed that the hSiglec-5-binding site is located in the most N-terminal part of β (B6N region; amino acids 1-152) and that the hSiglec-5- and IgA-binding domains in β are completely separate. We showed with BIAcore(TM) analysis that tandem variants of the hSiglec-5- and IgA-binding domains bind to their respective ligands with high affinity. Finally, we showed that the B6N region, but not the IgA-binding region of β, triggers recruitment of the tyrosine phosphatase SHP-2 to hSiglec-5 in U937 monocytes. Taken together, we have identified and isolated the first microbial non-sialic acid Siglec-binding region that can be used as a tool in studies of the β/hSiglec-5 interaction.
唾液酸结合免疫球蛋白样凝集素（Siglecs）被认为是调节细胞激活和炎症的重要受体。一些致病性微生物通过微生物表面含唾液酸的结构与特定的 Siglecs 结合，这种相互作用可能导致宿主反应的调节。最近，研究表明 B 群链球菌（GBS）通过 IgA 结合表面β蛋白与人 Siglec-5（hSiglec-5）结合，hSiglec-5 是巨噬细胞和中性粒细胞上表达的抑制性受体，这是致病性微生物与 Siglec 之间首次发现的蛋白/蛋白相互作用。在这里，我们表明β蛋白与 hSiglec-5 结合的部分位于该蛋白的 N 端半部分，该区域还包含先前确定的 IgA 结合区域。我们构建了表达β蛋白变异体的细菌突变体，这些变异体在蛋白的 N 端半部分没有重叠缺失。使用这些突变体和重组β片段，我们表明 hSiglec-5 结合位点位于β的最 N 端部分（B6N 区域；氨基酸 1-152），并且β中的 hSiglec-5 和 IgA 结合结构域完全分离。我们通过 BIAcore（TM）分析表明，hSiglec-5 和 IgA 结合结构域的串联变体以高亲和力结合其各自的配体。最后，我们表明 B6N 区域而不是β的 IgA 结合区域可触发 SHP-2 酪氨酸磷酸酶募集到 U937 单核细胞中的 hSiglec-5。总之，我们已经鉴定并分离出第一个微生物非唾液酸 Siglec 结合区域，可作为研究β/hSiglec-5 相互作用的工具。
