Evolution of the Neural Immunoglobulin Supergene Family and Functional Studies of One of its Members

Citation

Lane, Robert P. (1997) Evolution of the Neural Immunoglobulin Supergene Family and Functional Studies of One of its Members. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/rgww-db14. https://resolver.caltech.edu/CaltechTHESIS:07162025-220110244

Abstract

The immunoglobulin supergene family is a diverse set of molecules that share in common the immunoglobulin (Ig) domain. In the nervous system, a large subfamily of these proteins has been characterized that contain, in addition to N-terminal Ig-like domains, numerous fibronectin (Fn) type III repeats. One group of these neural homologs has been well characterized and includes Neuroglian, Bravo/Nr-CAM, Neurofascin, L1, Ng-CAM, Contactin/ F11, Axonin-1/ Tag-1, Big-1/Tag-like, and Big-2. Each of these proteins have six Ig-like domains and either four or five fibronectin type III repeats, and various developmental functions have been attributed to this group, including neurite outgrowth, fasciculation, cell adhesion and axon guidance.

Using structural modeling and cladistic analyses, the evolutionary relationships among these homologous neural Ig superfamily proteins were investigated. This study reinforces the idea that individual Ig-like and Fn domains are probably not distinct functional modules that can be shuffled in evolution, but rather that they may act in tandem. Patterns of conservation and divergence of specific residues along the various phylogenetic branches of the evolutionary tree suggest a model whereby important interactions may predominantly map between domains, with the "top" loops of one domain, the "bottom" loops of the adjacent domain, and the interdomain residues forming part of a ligand "pocket". The evolutionary analyses also permits an evaluation of the controversial identification of Ng-CAM and L1 as species orthologs, and in light of avian-mammalian speciation events, it appears these proteins are orthologous but perhaps not functionally identical.

A new member of this neural Ig subfamily has been cloned and identified as the human ortholog to the chicken Bravo/Nr-CAM protein. The complete coding sequence was determined, and like its chicken homolog, it is composed of six V-like Ig-like domains, five fibronectin type III repeats, as well as a transmembrane and intracellular domain. Overall, the human protein is 82% identical to the chicken homolog, although the trans-membrane and intracellular domains are 100% conserved at the amino acid level. Independent cDNA's encoding four distinct isoforms were identified, all of which contain alternatively spliced variants around the fifth fibronectin repeat, including one isoform previously identified in chicken in which- the entire 93 amino acid domain is spliced. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult brain. Fluorescence in situ hybridization maps the human Bravo/NrCAM gene to human chromosome 7q31.1-31.2, a locus previously identified to contain a tumor suppressor gene.

Although the cell adhesion (CAM) nomenclature implies that Bravo/ Nr-CAM and its family members function merely as a sort of indiscriminate cell-cell "glue", evidence has mounted that these proteins participate in receptor-like intracellular signalling functions with cell behavioral consequences. Of particular interest with regard to the Bravo/Nr-CAM protein is the conserved alternative splicing of the membrane-near fibronectin domain, as well as the striking sequence conservation C-terminal to this alternative exon that extends through the membrane and inside the cell. To explore the function of these sequences, both the 93 amino acid alternative Fn5 exon and the 100% conserved intracellular domains of Bravo/Nr-CAM were separately produced in heterologous expression systems and purified by various biochemical techniques. Affinity chromatography and expression library screening were used in an attempt to identify putative ligands to these presumably important protein regions.

The significance of the fifth fibronectin alternative exon usage was also investigated by using the expressed domain to raise domain-specific monoclonal antibodies, and using the antibodies in a histological study of spatial and temporal regulation of these splicing events. Using double labeling and confocol microscopy, as well as PCR analysis, in all tissues and across all stages of development, both the domain-containing and domain-lacking isoforms appear to be uniformly expressed in the same cells. Therefore, the developmental function of the complex array of alternatively spliced variants around the fifth fibronectin domain is subtle. A model is discussed whereby isoform diversity may provide a means to integrate multiple ligandbinding events involving the same protein on the same cell that interact with distinct ligands and co-receptors.

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