Identification, mapping, and genomic structural analysis of an immunoreceptor tyrosine-based inhibition motif-bearing C-type lectin from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Abstract.

Utilizing a spleen-derived cDNA library and rapid amplification of cDNA 5′ ends, we cloned a novel type II C-type lectin from two homozygous clones of rainbow trout. The cDNA is 2535 bp in length, and contains a 1017-bp open reading frame. From this sequence, a protein containing 339 amino acids (aa) was deduced. Using PSI-BLAST to search the GenBank database, the deduced protein is a C-type lectin, belonging to the type II membrane receptors. The protein contains four domains: an 87-aa N-terminal cytoplasmic domain, a 21-aa transmembrane domain, an 82-aa neck domain, and a 149-aa C-terminal C-type lectin domain. Two immunoreceptor tyrosine-based inhibition motifs (ITIMs) were located in the N-terminal cytoplasmic domain. RT-PCR results indicated that this gene is transcribed mainly in peripheral blood lymphocytes, spleen, kidney, and gill, and its expression in liver and intestine is weak. Monoclonal antibody 1.14 was used to isolate B cells from peripheral blood lymphocytes. Analysis revealed that this gene is highly expressed in B cells. Genomic DNA was amplified with long-template PCR and sequenced. The gDNA is 12.0 kb in length and contains nine exons and eight introns. The first intron of the genes from the OSU and AR clones differed in length. Based on this difference, the genotype of 69 doubled-haploid offspring of OSU and AR were screened. Subsequently, this gene was mapped on the rainbow trout linkage map to group XXI. Results of a Southern blot indicated that the gene (TCL-2) exists as a single copy in the rainbow trout genome. The genomic structure, the deduced protein structure, the tissue expression pattern, as well as the phylogenetic analysis of the carbohydrate recognition domain based on the deduced amino acid sequence indicate that TCL-2 resembles CD72; however, the carbohydrate recognition domain sequences of TCL-2 and CD72 are highly diverged.