One objective of the proposed research is to examine the interaction between vitamin D-binding protein (i.e., Gc globulin, DBP), the complement (C5)-derived peptide, C5a des Arg, and the Bb fragment of complement factor B. We have established that DBP is the putative C5a des Arg cochemotaxin and that a Bb-like peptide is the inhibitor of complement (C5)-derived chemotactic activity present in serum (and plasma) from some patients with active systemic lupus erythematosus (SLE). DBP forms a complex with C5a des Arg by attaching to sialic acid residues within the oligosaccharide portion of C5a des Arg, while Bb blocks the interaction between DBP and C5a des Arg. The complete sequence of DBP and Bb are known, and both isoforms of DBP (Gc1 and Gc2) have been cloned. We will test DBP isoforms for biologic activity and will generate fragments of DBP and Bb by enzymatic digestion. Fragments of DBP and Bb exhibiting biologic activity will be sequenced to determine specific sites involved in their interactions. Partial (and known) sequences of DBP and Bp will be used to generate synthetic oligonucleotide probes containing all possible substitutions at each codon. These oligonucleotides will be used to amplify relevant fragments of human, rabbit and porcine liver cDNA using the polymerase chain reaction, which will be used in an expression system to generate specific peptides to be tested for biologic activity. Mutants of biologically active peptides, derived from DBP and Bb cDNAa, will be generated to determine specific sites involved in the interaction between DBP, Bb and native C5a des Arg and human polymorphonuclear leukocytes (PMN). Preliminary experiments have demonstrated the radiolabeled BP binds to PMN in a saturable fashion. Experiments will be performed to determine the binding site for DBP on PMN as well as the specific site within /dBP involved in its binding to PMN. Also, we will examine the effect of DBP on the binding to PMN of biologically active, radiolabeled C5a des Arg and on C5a des Arg- induced events involved in PMN activation. The studies out lined in this proposal should yield new information that may explain (in part) why patients with SLE have an increased susceptibility to severe bacterial infections. On a more basic level, these studies should provide information concerning structure-activity relationships and mechanism involved in regulating the biologic activities of complement-derived peptides.
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