As part of our ongoing efforts to solve a complete structure of factor H, the structure of module 13 (fH<sup>~</sup>13) has been solved by NMR spectroscopy. Recombinant fH<sup>~</sup>13 protein was produced in <i>Pichia pastoris</i> in our laboratory and we have prepared unlabelled and <sup>15</sup>N, <sup>13</sup>C labelled samples of this module. Among the 88-indivdual CCP-modules of the regulators of complement activation, module sequence-lengths range from 51 to 67 amino acid residues and fH<sup>~</sup>13 possesses the shortest sequence. The solved solution structure of fH<sup>~</sup>13, reflects this short primary sequence and is unusual amongst the complement control proteins (CCP modules). fH<sup>~</sup>13 possess the expected disulphide-bonding pattern and consensus tryptophan, but lacks many overall 3D-structural features that characterise a “typical” CCP-module. fH<sup>~</sup>13 possesses only two <i>β</i>-strands out of a maximum of eight. The most similar structure of fH<sup>~</sup>13 is fH<sup>~</sup>15, while the most dissimilar CCP module is CR1<sup>~</sup>16. One side of the fH<sup>~</sup>13 domain reveals a highly localised positively charged patch composed of eight residues. To recognize host from non-host cell membranes, factor H binds to polyanions such as sialic acid or heparin sulphate which are bound on to the surface of host cells. There are three putative polyanion binding sites located in modules 7, 13 and 20, whose involvement in this process is, to various extend, supported by experimental evidence. The one in module 13 is the most disputed of the three polyanion binding sites. We have performed binding studies using gel mobility shift assay and tested building of fH<sup>~</sup>13 to a range of heparin derived oligosaccharides from disaccharide to dodecasaccharide with negative results. Similarly, NMR titrations using a fully sulphated heparin-derived tetrasaccharide yielded a negative result. These results point to the importance of an adequate distribution of positively charged residues for the binding of polyanions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:650839 |
| Date | January 2007 |
| Creators | Fenton, Christopher J. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14837 |
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