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D.1 Humoral immunity in the agnatha: The role of complement-like proteins
The Scientific and Social Program o f the V1~ ISDCI Congress
$77
D.1 HUMORAL IMMUNITY LIKE PROTEINS
IN THE AGNATHA:
THE ROLE
OF COMPLEMENT-
Robert L. Raison ~, Jeffrey Hook 1, Jennifer Coverley~, Rebecca Newton 1, Carolyn Geczy2, David Raftos ~ ~Dept. of Pathology and Immunology, University of Technology Sydney, P.O. Box 123, Broadway, NSW 2007, Australia, and 2Heart Research Institute, Missendon Road, Camperdown, NSW 2050, Australia Hagfish are modern representatives of the earliest evolved vertebrates. While lacking clearly defined lymphoid tissue, hagfish are capable of mounting cellular and inducible humoral responses to foreign antigens. However, extensive investigations in this species have failed to convincingly demonstrate the presence of an immunoglobulin-based adaptive immune response. We have shown that an inducible serum protein, previously described as having an immunoglobulin-like structure, exhibits sequence homology with the mammalian complement proteins C3, C4 and C5. The hagfish complement-like protein (CLP) shares other features in common with the complement protein including intron-exon structure of the gene, size of mRNA and a thioester group on one of its three polypeptide chains. Functional studies indicate that CLP acts as an opsonin with a divalent cation dependent capacity to recognise selected carbohydrate determinants. Using a monoclonal antibody (1BI) specific for hagfish monocytes, we have identified a putative 105kDa receptor which mediates the opsonic activity of CLP. We have recently demonstrated that hagfish large and large-granular leukocytes are capable of directed migration to the classical mammalian chemoattractant, C5a. LPS-activated hagfish serum also acts as a potent chemoattractant, implicating complement related proteins in endogenous chemotaxis. We conclude that hagfish CLP forms part of a non-clonally derived humoral defence mechanism which shares evolutionary origins with the mammalian complement system.
D.2 MOLECULAR CLONING OF A CELL-SURFACE HAGFISH LEUKOCYTES
COMPLEMENT
RECEPTOR
ON
Nicholas J. dos Remedios, Jeffrey W. Hook, David A. Raftos, Robert L. Raison Immunobiology Unit, University of Technology, Sydney, Westbourne Street, Gore Hill, NSW 2065, Australia The hagfish is a modern representative of the earliest evolved group of vertebrates, the jawless fish (ostracoderms). As such it holds a critical evolutionary position between the invertebrates and the vertebrates. The Pacific hagfish (Eptatretus stouti) exihibits a non-anticipatory immune response mediated by a complement-like protein (CLP) present in the serum (Hanley et al., 1992. PNAS 8___?:7910-7914). CLP is structurally and functionally similar to mammalian C3/C4 and is a member of the C3-1ike complement protein family. Functional studies indicate that hagfish leukocytes possess cell surface receptors for CLP. A putative complement receptor has been identified using a phagocyte-specific monoclonal antibody, 1B1. This antibody blocks CLP-mediated phagocytosis of yeast target cells (Raison et al., 1994. Immunol. Cell Biol. In press). In this study we have screened a leukocyte-derived 3,gtl I cDNA expression library using the I B 1 monoclonal antibody. A positive clone was isolated, sub-cloned into pBluescript and sequenced. The nucleic acid and translated amino acid sequences were used to search the Genbank, Swiss-Protein, PIR and GenPeptide computer databases. This partial clone of 106 bp shows low homology with a number of glycoprotein cell-surface receptors. The 106 bp fragment is currently being used as a probe to isolate additional cDNA clones and identify the mRNA species encoding the CLP receptor.
$77
D.1 HUMORAL IMMUNITY LIKE PROTEINS
IN THE AGNATHA:
THE ROLE
OF COMPLEMENT-
Robert L. Raison ~, Jeffrey Hook 1, Jennifer Coverley~, Rebecca Newton 1, Carolyn Geczy2, David Raftos ~ ~Dept. of Pathology and Immunology, University of Technology Sydney, P.O. Box 123, Broadway, NSW 2007, Australia, and 2Heart Research Institute, Missendon Road, Camperdown, NSW 2050, Australia Hagfish are modern representatives of the earliest evolved vertebrates. While lacking clearly defined lymphoid tissue, hagfish are capable of mounting cellular and inducible humoral responses to foreign antigens. However, extensive investigations in this species have failed to convincingly demonstrate the presence of an immunoglobulin-based adaptive immune response. We have shown that an inducible serum protein, previously described as having an immunoglobulin-like structure, exhibits sequence homology with the mammalian complement proteins C3, C4 and C5. The hagfish complement-like protein (CLP) shares other features in common with the complement protein including intron-exon structure of the gene, size of mRNA and a thioester group on one of its three polypeptide chains. Functional studies indicate that CLP acts as an opsonin with a divalent cation dependent capacity to recognise selected carbohydrate determinants. Using a monoclonal antibody (1BI) specific for hagfish monocytes, we have identified a putative 105kDa receptor which mediates the opsonic activity of CLP. We have recently demonstrated that hagfish large and large-granular leukocytes are capable of directed migration to the classical mammalian chemoattractant, C5a. LPS-activated hagfish serum also acts as a potent chemoattractant, implicating complement related proteins in endogenous chemotaxis. We conclude that hagfish CLP forms part of a non-clonally derived humoral defence mechanism which shares evolutionary origins with the mammalian complement system.
D.2 MOLECULAR CLONING OF A CELL-SURFACE HAGFISH LEUKOCYTES
COMPLEMENT
RECEPTOR
ON
Nicholas J. dos Remedios, Jeffrey W. Hook, David A. Raftos, Robert L. Raison Immunobiology Unit, University of Technology, Sydney, Westbourne Street, Gore Hill, NSW 2065, Australia The hagfish is a modern representative of the earliest evolved group of vertebrates, the jawless fish (ostracoderms). As such it holds a critical evolutionary position between the invertebrates and the vertebrates. The Pacific hagfish (Eptatretus stouti) exihibits a non-anticipatory immune response mediated by a complement-like protein (CLP) present in the serum (Hanley et al., 1992. PNAS 8___?:7910-7914). CLP is structurally and functionally similar to mammalian C3/C4 and is a member of the C3-1ike complement protein family. Functional studies indicate that hagfish leukocytes possess cell surface receptors for CLP. A putative complement receptor has been identified using a phagocyte-specific monoclonal antibody, 1B1. This antibody blocks CLP-mediated phagocytosis of yeast target cells (Raison et al., 1994. Immunol. Cell Biol. In press). In this study we have screened a leukocyte-derived 3,gtl I cDNA expression library using the I B 1 monoclonal antibody. A positive clone was isolated, sub-cloned into pBluescript and sequenced. The nucleic acid and translated amino acid sequences were used to search the Genbank, Swiss-Protein, PIR and GenPeptide computer databases. This partial clone of 106 bp shows low homology with a number of glycoprotein cell-surface receptors. The 106 bp fragment is currently being used as a probe to isolate additional cDNA clones and identify the mRNA species encoding the CLP receptor.