CHARACTERIZATION OF MUTANT FORMS OF RECOMBINANT HUMAN PROPERDIN LACKING SINGLE THROMBOSPONDIN TYPE-I REPEATS - IDENTIFICATION OF MODULES IMPORTANT FOR FUNCTION

Properdin is a serum glycoprotein that up-regulates the alternative pa thway of complement by stabilizing the C3b-Bb complex, It also binds s ulfated glycoconjugates, such as sulfatide, in vitro. Properdin is com posed of cyclic dimers, trimers, and tetramers of a 53-kDa monomeric s ubunit. The monomer contains an N-terminal region of no known homology and six thrombospondin type 1 repeats (TSRs) of approximately 60 amin o acids, To identify the regions of properdin important for function, we have expressed human properdin, and mutant forms each lacking a sin gle TSR, in Chinese hamster ovary cells. In addition, limited tryptic digestion yielded ''nicked'' properdin by the cleavage of one peptide bond in TSR5. The structural and functional properties of these altere d forms of properdin were investigated. Properdin ''nicked'' in TSR5 i s unable to bind C3b but retains its overall structure and its ability to bind sulfatide. The removal of TSR5 prevents C3b and sulfatide bin ding. Properdin lacking TSR4 is unable to stabilize the C3b-Bb complex but is able to bind C3b and sulfatide, and shows the presence of mono mers and dimers in an electron microscope. Properdin without TSR3 is a ble to stabilize the C3b-Bb complex, to bind C3b and sulfatide, and fo rms dimers, trimers, and tetramers. Properdin lacking TSR6 is unable t o form oligomers. The N-linked carbohydrate of properdin is not requir ed for oligomerization or stabilization of the C3b-Bb complex, The res ults implicate TSR5 in both C3b and sulfatide binding, and suggest tha t TSR4 may also be involved in stabilization of the C3b-Bb complex.