Isolation and partial characterization of a calcium-dependent lectin (chiletin) from the haemolymph of the flat oyster, Ostrea chilensis.
A calcium-dependent lectin (chiletin) was isolated from oyster haemolymph by mannose elution from Sepharose CL-6B followed by anion exchange chromatography. Chiletin was predominantly composed of 12 and 24 kDa bands when examined with SDS-PAGE under reducing and non-reducing conditions, respectively. Larger molecular weight bands of 36 and 50 kDa were also variably present under reducing conditions. The NH2-terminal sequence of the 24 kDa band was determined and was not homologous to any known protein from the databases searched. Isolated chiletin was composed of multiple isomers approximately 12 kDa in size and ranging in pI from 5.2 to 6.0. Rabbit antiserum was raised to a synthetic peptide coupled to keyhole limpet hemocyanin and the size of the chiletin subunits was confirmed by Western blot. Two and five different conformational aggregates of chiletin were resolved in oyster haemolymph using size exclusion chromatography in 8 M urea and PBS, respectively. The largest aggregate obtained from size exclusion in 8 M urea was estimated to be greater than 640 kDa. The ability of whole haemolymph and isolated chiletin to agglutinate sheep red blood cells was inhibited by galactose and mannose. Chiletin was identified by immunohistochemistry to be most consistently present in the auricle, followed by the digestive gland, however staining was seen sporadically in haemocytes, gastrointestinal epithelium and interstitial connective tissue cells.
Minamikawa
M
M
Institute of Veterinary, Animal and Biomedical Sciences, Massey University, PB 11-222, Palmerston North, New Zealand.
Hine
M
M
Russell
S
S
Huber
P
P
Duignan
P
P
Lumsden
J S
JS
D000372:Agglutination Tests; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose; D006458:Hemolymph / Q000378:metabolism*; D007150:Immunohistochemistry; D037102:Lectins / Q000097:blood* / Q000235:genetics*; D008358:Mannose; D008969:Molecular Sequence Data; D010125:Ostreidae / Q000378:metabolism*; D020539:Sequence Analysis, Protein
D037102:Lectins; D008358:Mannose; D002118:Calcium; D005690:Galactose