RESUMO
BLAST searches against databases for the bullfrog (Rana catesbeiana), using the collectin sequence previously identified in tadpoles, revealed the presence of at least 20 members of the collectin gene family. Phylogenetic analysis demonstrated that the bullfrog possesses expanded gene subfamilies encoding mannose-binding lectin (MBL) and pulmonary surfactant-associated protein D (PSAPD). Two collectins, of 20 kDa (PSAPD1) and 25 kDa (PSAPD6), were purified as a mixture from adult bullfrog plasma using affinity chromatography. These collectins were present as an oligomer of ~400 kDa in their native state, and showed Ca2+-dependent carbohydrate binding with different sugar preferences. Affinity-purified collectins showed weak E. coli agglutination and bactericidal activities, compared with those of plasma. Although both PSAPD1 and PSAPD6 genes were predominantly expressed in the liver, PSAPD1 transcripts were abundant in adults whereas PSAPD6 transcripts were abundant in tadpoles. The findings indicate that two gene subfamilies in the collectin family have diverged structurally, functionally and transcriptionally in the bullfrog. Rapid expansion of the collectin family in bullfrogs may reflect the onset of sub-functionalization of the prototype MBL gene towards tetrapod MBL and PSAPDs, and may be one means of natural adaptation in the innate immune system to various pathogens in both aquatic and terrestrial environments.
