Embryonic Epidermal Lectins in Three Amphibian Species, Rana ornativentris, Bufo japonicus formosus, and Cynops pyrrhogaster.

Abstract

Intelectins (Itlns) are secretory lectins found in several chordate species that recognize carbohydrates on the bacterial cell surface depending on Ca2 + . In newly hatched larvae of Rana ornativentris (R. orn), Bufo japonicus formosus (B. jpn), and Cynops pyrrhogaster (C. pyr), an anti-Itln monoclonal antibody (mAb) labeled a subset of epidermal cells in whole-mount immunocytochemical assays. In western blot analyses, the mAb identified protein bands at approximately 33-37 kDa in the larval extracts and concentrated larval culture media. Using RT-PCR and RACE techniques, we isolated cDNAs from newly hatched larvae that encoded proteins of 343 (R. orn), 336 (B. jpn), and 337 (C. pyr) amino acids having 70%, 71%, and 60% identities with that of the Xenopus laevis embryonic epidermal lectin (XEEL), respectively. The proteins, designated REEL, BEEL, and CEEL, showed characteristics conserved among reported Itln proteins, and their amino acid sequences following the signal peptides were identical to those of the N-terminal peptides determined on Itln proteins in the respective larval extracts. Recombinant REEL (rREEL), rBEEL, and rCEEL proteins produced by HEK-293T cells were homo-oligomers of 34-37 kDa subunit peptides, which were similar to the Itlns found in the newly hatched larvae. The rEELs showed carbohydrate-binding specificities similar to that of XEEL and agglutinated Escherichia coli and Staphylococcus aureus cells depending on Ca2 + . These results suggest that REEL, BEEL, and CEEL are Itlns produced and secreted by epidermal cells of R. orn, B. jpn, and C. pyr larvae, respectively, and that Itlns have a conserved role as pathogen recognition molecules in the larval innate immune system.