Isolation and partial characterisation of a calcium-dependent lectin-like protein from the flat oyster, Ostrea chilensis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Pathology at Massey University, Palmerston North, New Zealand

Abstract

The (Chilean) flat oyster, Ostrea chilensis, is native to New Zealand and the west coast of South America. It is a commercially important species in New Zealand because of its exquisite taste that attracts premium prices. This thesis describes the first isolation and partial charcterisation of an oyster haemolymph calcium-dependent carbohydrate-binding protein. This protein 'chiletin' was originally isolated from oyster haemolymph by binding to the agarose-galactan matrix of a Sepharose column. Chiletin was predominantly composed of a 24 kilodalton (kDa) band when examined with one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis under non-reducing conditions and a 12 kDa band with reduction of disulphide bonds. The N-terminal sequence of the 24 kDa band was determined to be 'IAGPGWEKYN'. This sequence was not homologous to any known protein. Examination of isolated chiletin with two-dimensional protein analysis gel electrophoresis revealed the presence of three (~12 kDa) subunits ranging in isoelectric point from 5.2 to 6.0. The 24 kDa protein was used to immunise rabbits and a separate antiserum was also raised in rabbits using a synthetic peptide (identical to that above) coupled to keyhole limpet haemocyanin. These antisera were used to confirm the size of the chiletin subunits with Western blots and to examine the elution of chiletin in oyster haemolymph with size exclusion chromatography in phosphate buffered saline (PBS) and 8 M urea. There were four or five different sized conformational aggregates of chiletin present in oyster haemolymph under physiological conditions (PBS). The use of 8 M urea produced two separate aggregates. A major characteristic of lectins is the ability to agglutinate sheep red blood cells and both whole oyster haemolymph and isolated chiletin had this property. Chiletin was identified by immunohistochemistry to be present in a number of tissues. Staining intensity was most consistent in the auricular myocardial cells, followed by the digestive gland epithelium. Chiletin was not induced in haemolymph in response to temperature (30??C) stress or injection of turpentine into the adductor muscle. There have been few immunological studies performed with O. chilensis. The results of the project contribute to what is known about comparative immunology. Greater understanding of how oysters respond to stress and deal with pathogens will ultimately be of benefit to the aquaculture industry.