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 catalytic subunit from the inherently labile C3bBb complexes. It is also known to bind sulphated glycoconjugates, such as sulphatides. Properdin has an unusual structure formed by oligomerisation of a rod-like monomer into cyclic dimers, trimers and tetramers. The monomer (approximately 53 kDa) contains an N-terminal region of no known homology, followed by six non-identical repeats of 60 amino acids (based on exon/intron boundaries), called 'thrombospondin type I repeats' or TSR modules. We have expressed and purified the N-terminal region and each of the individual TSR repeats in Escherichia coli. Although the individual recombinant TSRs, after a denaturation-renaturation cycle, appeared to be correctly folded modules, as judged by the one-dimensional (1D)- and 2D-nuclear magnetic resonance spectra of TSR3, they did not show binding to either C3b or sulphatide. Polyclonal antibodies were raised against each TSR and were found to be module-specific. The anti-TSR5 polyclonal antibody was found to inhibit binding of native human properdin to solid-phase C3b, or sulphatides. It could also block properdin-dependent haemolysis of rabbit erythrocytes. These results are consistent with the view that the TSR5 contains the major site in properdin which is involved in both C3b and sulphatide binding. It also suggests that a co-operative intramolecular interaction between TSRs, as found in the native molecule, is required for TSR5 to bind either C3b or sulphatides.
Perdikoulis
M V
MV
Department of Biochemistry, University of Oxford, UK.
Kishore
U
U
Reid
K B
KB
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D002942:Circular Dichroism; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D003179:Complement C3b / Q000378:metabolism; D003170:Complement Pathway, Alternative; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006461:Hemolysis / Q000276:immunology; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011414:Properdin / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D013329:Structure-Activity Relationship; D019700:Thrombospondin 1 / Q000737:chemistry / Q000235:genetics*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D019700:Thrombospondin 1; D011414:Properdin; D003179:Complement C3b; D050577:Complement C3-C5 Convertases