The PI's long term research interest is the study of innate immunity and its evolution, particularly the role of complement as a mediator of innate immunity and a modulator of adaptive immunity. Studies on shark complement, both at the protein and gene level,, will be a focus of this present application. The broad aim of this proposal is to study the structural and functional features of shark complement proteins and their phylogenetic relationships. The research plan will take a three-pronged experimental approach consisting of (a) functional analysis, (b) protein isolation and characterization, and (c) molecular cloning. The research proposed in this application will test the inter-related hypotheses that 91) shark C3 and factor B are functional analogues and evolutionary ancestors of their mammalian counterparts, (4) a C5 homologue is present in the shark, (3) a functional analogue of mammalian factor H is present in shark serum, and (4) activated shark serum generates complement- derived opsonins and anaphylatoxins.
The specific aims of the proposed research are:
AIM 1 : To perform structural, functional and phylogenetic analysis of shark C3 AIM 2: To purify and partially characterize a shark complement control protein analogous to mammalian factor H AIM 3: To characterize shark anaphylatoxins AIM 4: To further characterize shark factor B and clone its gene(s).
AIM 5 : To clone and shark putative C5 gene AIM 6: To isolate and characterize the membrane attack complex (MAC) in the shark. Research methods will include (a) protein purification by ion exchange, affinity and size exclusion chromatography, (b) amino acid sequence analysis on purified proteins/peptides and tryptic digests, (c) isolation of shark leucocytes by density gradient centrifugation, (d) hemolytic and phagocytosis assays, (e) ATP-release and myeloperoxidase assay for anaphylatoxin, (f) SDS-PAGE and Western blotting, (g) isolation of full length C3 and Bf/C2 clones either by screening a cDNA library constructed from shark liver or employing Rapid Amplification of cDNA ends (RACE) using gene-specific primers, (h) nucleotide sequencing, and (i) isolation of membrane bound MACs employing sucrose density gradient centrifugation. The study will contribute significantly to answering the questions of the origin of C5, the composition of shark MAC, the structure and function of C3 and factor B, and the opsonic activity of shark serum. Information of anaphylatoxins could, potentially, be of medical interest and application.
Showing the most recent 10 out of 146 publications
