title: Affinity of the heparin binding motif of Noggin1 to heparan sulfate and its visualization in the embryonic tissues.
abstract: 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.
authors.affiliation: 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. Electronic address: comconadin@gmail.com.
authors.forename: Alexey M
authors.initials: AM
authors.lastname: Nesterenko
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.
authors.forename: Eugeny E
authors.initials: EE
authors.lastname: Orlov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.
authors.forename: Galina V
authors.initials: GV
authors.lastname: Ermakova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.
authors.forename: Igor A
authors.initials: IA
authors.lastname: Ivanov
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, 1/40, 119991 Moscow, Russia.
authors.forename: Pavel I
authors.initials: PI
authors.lastname: Semenyuk
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, 1/40, 119991 Moscow, Russia.
authors.forename: Victor N
authors.initials: VN
authors.lastname: Orlov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.
authors.forename: Natalia Y
authors.initials: NY
authors.lastname: Martynova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia. Electronic address: azaraisky@yahoo.com.
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Zaraisky
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D005775:Gastrula / Q000648:ultrastructure; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D008969:Molecular Sequence Data; D014981:Xenopus / Q000196:embryology* / Q000378:metabolism; D029867:Xenopus Proteins / Q000032:analysis / Q000378:metabolism*; D000099176:Noggin Protein
chemical_list: D002352:Carrier Proteins; D006493:Heparin; D006497:Heparan Sulfate; D029867:Xenopus Proteins; D000099176:Noggin Protein
keywords: FRAP; Heparan sulfate; Heparin; Isothermal titration calorimetry; Morphogen; Noggin; Poly-l-lysine; Protein diffusion; Xenopus laevis