Long lived invertebrates are immunoincompetent in the sense that they lack the classical T and B cell arms of the immune system but they respond successfully to challenge with micro-organisms by inflammatory mechanisms involving sensitized phagocytes. This proposal follows from a previous grant (GM 30672) on """"""""animal lectins as recognition molecules"""""""", finding that certain carbohydrate-binding lectins of tunicate species could be identified as members of two families of molecules known to be crucial to immune defense; namely, acute phase proteins of the pentraxin type (e.g. C-reactive proteins) and members of the immunoglobulin superfamily. We focus upon immune-like recognition molecules of tunicates because these protochordate species are ancestral to true vertebrates but are lacking in the sophisticated features of B and T cell immunity. (1) We have devised fractionation schemes for the purification of galactose-binding lectins of the tunicate Didemnum candidum, two lectins specific for sialoconjugates and one for fucose from the tunicate Halocynthia pyriformis, a sialic acid binding lectin from the lamprey and a molecule from the tunicate Boltenia ovipera that is serologically cross-reactive with Igs of lower species and with antibodies to synthetic immunoglobulin joining region peptide. The purified molecules will be characterized physicochemically by determining a) native and subunit molecular weights, b) carbohydrate and amino acid compositions, c) the number and affinity of binding sites, and d) investigations of secondary structure using optical rotatory dispersion and circular dichroism measurements. (2) We will determine the amino acid sequence of these molecules, preparing peptides using proteolytic enzymes of defined specificity and chemical cleavage reagents such as CNBr with the resolution of peptides by gel filtration chromatography using high performance liquid chromatography and by reverse phase peptide chromatography. The peptides will be sequenced using the ABI pulsed liquid phase sequencer. The amino acid sequence will facilitate detailed comparisons with known recognition molecules and enable the synthesis of synthetic oligonucleotide probes to be used in isolation of relevant genes. (3) To ensure that we obtain complete sequence information for the tunicate and lamprey lectins, we will employ the technology of molecular biology. Two strategies will be followed: the first is to produce libraries of genomic DNA and screen these with synthetic oligonucleotide probes predicted from the available sequence of the lectin molecules. A second strategy will be to produce cDNA libraries constructed using total mRNA in expression vectors will then be screened serologically using antibodies to the purified lectins and the synthetic probes. DNA will be sequenced using Sanger's dideoxy chain determination method. We will characterize the genes in terms of their number and types of segments and the arrangements of these elements.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042437-05
Application #
2181376
Study Section
Immunobiology Study Section (IMB)
Project Start
1988-12-01
Project End
1994-11-30
Budget Start
1992-12-01
Budget End
1994-11-30
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Arizona
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Marchalonis, J J; Bernstein, R M; Shen, S X et al. (1996) Emergence of the immunoglobulin family: conservation in protein sequence and plasticity in gene organization. Glycobiology 6:657-63
Shen, S X; Bernstein, R M; Schluter, S F et al. (1996) Heavy-chain variable regions in carcharhine sharks: development of a comprehensive model for the evolution of VH domains among the gnathanstomes. Immunol Cell Biol 74:357-64
Kay, M M; Cover, C; Schluter, S F et al. (1995) Band 3, the anion transporter, is conserved during evolution: implications for aging and vertebrate evolution. Cell Mol Biol (Noisy-le-grand) 41:833-42
Hohman, V S; Schluter, S F; Marchalonis, J J (1995) Diversity of Ig light chain clusters in the sandbar shark (Carcharhinus plumbeus). J Immunol 155:3922-8
Schluter, S F; Schroeder, J; Wang, E et al. (1994) Recognition molecules and immunoglobulin domains in invertebrates. Ann N Y Acad Sci 712:74-81
Marchalonis, J J; Schluter, S F (1994) Development of an immune system. Ann N Y Acad Sci 712:1-12
Marchalonis, J J; Hohman, V S; Kaymaz, H et al. (1994) Cell surface recognition and the immunoglobulin superfamily. Ann N Y Acad Sci 712:20-33
Bernstein, R M; Schluter, S F; Lake, D F et al. (1994) Evolutionary conservation and molecular cloning of the recombinase activating gene 1. Biochem Biophys Res Commun 205:687-92
Marchalonis, J J; Hohman, V S; Kaymaz, H et al. (1993) Shared antigenic determinants of immunoglobulins in phylogeny and in comparison with T-cell receptors. Comp Biochem Physiol B 105:423-41
Hohman, V S; Schuchman, D B; Schluter, S F et al. (1993) Genomic clone for sandbar shark lambda light chain: generation of diversity in the absence of gene rearrangement. Proc Natl Acad Sci U S A 90:9882-6

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