Cloning, binding properties, and tissue localization of rainbow trout (Oncorhynchus mykiss) ladderlectin

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Abstract

The present paper describes the primary structure, expression and immunohistochemical localization of rainbow trout ladderlectin (RTLL), a multimeric serum lectin that binds Sepharose and LPS of Aeromonas salmonicida. Two rainbow trout cDNAs (504 and 546 bp) and a genomic sequence (2 kb) were amplified using ladderlectin-specific primers. The sequences were identified as group VII mannose-binding C-type lectins from predicted amino acid sequences and showed highest identity with the Atlantic salmon mannose-binding lectin. The two cDNA sequences (RTLL-1 and RTLL-2) had 92% identity and encoded 173 and 187 amino acids, respectively. The genomic sequence of RTLL, obtained by PCR, was found to encompass six exons and five introns, with exon 2 encoding 14 amino acids which were exclusive to RTLL-2. The relative expression of both transcripts was highest in the renal kidney, while the intestine, gill and skin exhibited higher relative RTLL-2 expression than RTLL-1. RTLL was immunohistochemically present within cells of the branchial epithelium, hepatic sinusoids, biliary epithelium, renal interstitium, skin, and sub-mucosal granular layer of the intestine. RTLL bound galactan-based Sepharose 6B and Sepharose CL-6B matrices but did not bind unmodified acrylic resin base Toyopearl AF-Epoxy 650M, Toyopearl AF-Amino 650M matrices or N-acetylated Toyopearl AF-Amino 650M acrylic matrices. Two-dimensional SDS-PAGE and Western blots of whole plasma and plasma proteins which bound chitin and intact bacteria demonstrated multiple electrophoretic isoforms of RTLL ranging in size from 16 to 18 kDa and isoelectric points between pH 4.9 and 6.3. These findings show that RTLL is a group VII C-type lectin with multiple isoforms that bind pathogen-associated molecular patterns such as chitin and microbial surfaces.

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