Expression and characterisation of the thrombospondin type I repeats of human properdin

Properdin, an upregulator of the alternative complement pathway, is central to deposition of the activated complement fragment C3b on the surfaces of the pathogens, which it achieves by preventing the dissociation of the Bb c atalytic subunit from the inherently labile C3bBb complexes. It is also kno wn to bind sulphated glycoconjugates, such as sulphatides. Properdin has an unusual structure formed by oligomerisation of a rod-like monomer into cyc lic dimers, trimers and tetramers. The monomer (similar to 53 kDa) contains an N-terminal region of no known homology, followed by six non-identical r epeats of 60 amino acids (based on exon/intron boundaries), called 'thrombo spondin type I repeats' or TSR modules. We have expressed and purified the N-terminal region and each of the individual TSR repeats in Escherichia coh . Although the individual recombinant TSRs, after a denaturation-renaturati on cycle, appeared to be correctly folded modules, as judged by the one-dim ensional (1D)- and 2D-nuclear magnetic resonance spectra of TSR3, they did not show binding to either C3b or sulphatide. Polyclonal antibodies were ra ised against each TSR and were found to be module-specific. The anti-TSR5 p olyclonal antibody was found to inhibit binding of native human properdin t o solid-phase Ob, or sulphatides. It could also block properdin-dependent h aemolysis of rabbit erythrocytes. These results are consistent with the vie w that the TSR5 contains the major site in properdin which is involved in b oth C3b and sulphatide binding. It also suggests that a co-operative intram olecular interaction between TSRs, as found in the native molecule, is requ ired for TSR5 to bind either C3b or sulphatides. (C) 2001 Elsevier Science BN. All rights reserved.