This project focuses on molecular aspects of the assembly, function and regulation of human C5b-9, the cytolytic complex of complement proteins C5b, C6, C7, C8, and C9. Emphasis will be on characterizing the structure and function of human C8 (HuC8) as to understand the biological significance of its peculiar structure, identify domains involved in interactions that occur during C5b-9 assembly, determine the role of cysteine- rich modules in mediating these interactions, identify a structural basis for the species-selective recognition of C8 by the C5b-9-regulatory protein CD59, gain insight into hereditary C8 deficiencies and facilitate the design of C8 analogues that might have therapeutic value as cytolytic agents.
Specific Aim 1 is to map domains within HuC8 that are involved in interactions between C5b- 7 and C8-beta, C8-beta and C8-alpha, C8- alpha and C8-gamma, and C8-alpha and C9. Fragments of HuC8-alpha and C8- beta produced by recombinant DNA expression will be tested for their ability to inhibit these interactions.
Specific Aim 2 is to determine the 3-D structure of HuC8-gamma through crystallographic analyses and to characterize its hydrophobic ligand binding properties which are characteristic of the alpha-2u-globulin (lipocalin) family of proteins to which it belongs.
Specific Aim 3 focuses on the mechanism by which human cells are protected from damage by human C5b-9, i.e. the mechanism of homologous restriction of cell lysis. Structural determinants in HuC8-alpha that are recognized in a species-dependent manner by HuCD59 will be localized and fine-mapped. Fragments from human and rabbit C8-alpha will be compared to identify those that interact with HuCD59 in a species-dependent manner.
Specific Aim 4 is to identify the basis of C8 deficiency in the rabbit. What was initially believed to be a C8-alpha/gamma deficiency now appears to be a combined C8-alpha/gamma, C8-beta deficiency analogous to that reported in humans. Experiments will examine the importance of amino acids substitutions already detected in rabbit C8D-alpha and search for additional substitutions in C8D-beta. Such experiments could identify residues critical for C8 subunit interaction and provide insight into the defect in HuC8-alpha/gamma deficiency.
Specific Aim 5 is to determine the intergenic distance between HuC8-alpha and C8- beta loci and their orientation using exon-specific probes and PFGE analysis.
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