Nesterenko Alexey M, Orlov Eugeny E, Ermakova Galina V, Ivanov Igor A, Semenyuk Pavel I, Orlov Victor N, Martynova Natalia Y, Zaraisky Andrey G
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, 1/40, 119991 Moscow, Russia.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.
Biochem Biophys Res Commun. 2015;468(1-2):331-6. doi: 10.1016/j.bbrc.2015.10.100. Epub 2015 Oct 23.
Heparin binding motifs were found in many secreted proteins and it was suggested that they are responsible for retardation of the protein diffusion within the intercellular space due to the binding to heparan sulfate proteoglycanes (HSPG). Here we used synthetic FITC labeled heparin binding motif (HBM peptide) of the Xenopus laevis secreted BMP inhibitor Noggin1 to study its diffusion along the surface of the heparin beads by FRAP method. As a result, we have found out that diffusivity of HBM-labeled FITC was indeed much lesser than those predicted by theoretical calculations even for whole protein of the Noggin size. We also compared by isothermal titration calorimetry the binding affinity of HBM and the control oligolysine peptide to several natural polyanions including heparan sulfate (HS), heparin, the bacterial dextran sulfate and salmon sperm DNA, and demonstrated that HBM significantly exceeds oligolysine peptide in the affinity to HS, heparin and DNA. By contrast, oligolysine peptide bound with higher affinity to dextran sulfate. We speculate that such a difference may ensure specificity of the morphogen binding to HSPG and could be explained by steric constrains imposed by different distribution of the negative charges along a given polymeric molecule. Finally, by using EGFP-HBM recombinant protein we have visualized the natural pattern of the Noggin1 binding sites within the X. laevis gastrula and demonstrated that these sites forms a dorsal-ventral concentration gradient, with a maximum in the dorsal blastopore lip. In sum, our data provide a quantitative basis for modeling the process of Noggin1 diffusion in embryonic tissues, considering its interaction with HSPG.
在许多分泌蛋白中发现了肝素结合基序，有人认为它们通过与硫酸乙酰肝素蛋白聚糖（HSPG）结合，导致蛋白在细胞间空间内的扩散受阻。在这里，我们使用非洲爪蟾分泌的骨形态发生蛋白抑制剂Noggin1的合成荧光素异硫氰酸酯（FITC）标记的肝素结合基序（HBM肽），通过荧光漂白恢复（FRAP）方法研究其沿肝素珠表面的扩散。结果，我们发现即使对于Noggin大小的完整蛋白，HBM标记的FITC的扩散系数确实比理论计算预测的要小得多。我们还通过等温滴定量热法比较了HBM和对照寡聚赖氨酸肽与几种天然聚阴离子的结合亲和力，这些聚阴离子包括硫酸乙酰肝素（HS）、肝素、细菌硫酸葡聚糖和鲑鱼精DNA，并证明HBM在与HS、肝素和DNA的亲和力方面显著超过寡聚赖氨酸肽。相比之下，寡聚赖氨酸肽与硫酸葡聚糖的结合亲和力更高。我们推测这种差异可能确保形态发生素与HSPG结合的特异性，并且可以由给定聚合物分子上负电荷的不同分布所施加的空间限制来解释。最后，通过使用EGFP - HBM重组蛋白，我们可视化了非洲爪蟾原肠胚中Noggin1结合位点的天然模式，并证明这些位点形成了背腹浓度梯度，在背侧胚孔唇处达到最大值。总之，我们的数据为在考虑Noggin1与HSPG相互作用的情况下，对其在胚胎组织中的扩散过程进行建模提供了定量基础。
