Isolation, purification and characterisation of the pentraxins from Limulus polyphemus and Mustelus canis

Abstract

CRP and SAP form part of the innate immune system in humans, and homologues have been found in mammals, amphibians, fish, elasmobranchs, and invertebrates. The aim of this research was to broaden knowledge of CRP and SAP in horseshoe crabs, and to attempt to determine the structure and function of the CRP and SAP proteins from the elasmobranch smooth dogfish.

In the invertebrate horseshoe crabs, CRP comprises a heterogeneous family of proteins, whilst SAP is present as a single protein. The phylogenetic relationships between the horseshoe crab CRP and SAP sequences were determined, which showed clustering of homologous sequences. The conservation of horseshoe crab CRP and SAP sequences at sites corresponding to functional areas on human CRP was determined. The calcium ion binding site was the most conserved area, and variation at the area corresponding to the ligand binding site suggests a possibility of different binding properties between horseshoe crab pentraxins. Studies on the sequences of horseshoe crab CRP and SAP and the known human CRP and Limulus SAP structures, offers insight into new areas of conservation that may be important in horseshoe crab CRP and SAP function.

The Atlantic horseshoe crab, Limulus polyphemus, CRP and SAP were found to be composed of different subunit and molecular aggregate forms, and also bind to the novel ligands ribose-5-phosphate and AMP. Limulus CRP appeared to have a higher affinity than SAP for ribose-5-phosphate and AMP. The subunit composition and molecular aggregate surface hydrophobicity were analysed for whole Limulus CRP and SAP, and for that isolated using ribose-5-phosphate and AMP.

Attempts were made to isolate the genes in smooth dogfish (Mustelus canis) for CRP and SAP, but these proved unsuccessful primarily due to the availability of only limited amino acid sequence data resulting in primers for PCR that were too short and degenerate.