Characterization of Alternatively Spliced Transcript Variants of CLEC2D Gene

Abstract

Lectin-like transcript 1 (LLT1) encoded by CLEC2D gene is a C-type lectin-like molecule interacting with human CD161 (NKR-P1A) receptor expressed by natural killer cells and subsets of T cells. Using RT-PCR and sequencing, we identified several CLEC2D alternatively spliced transcript variants generated by exon skipping. In addition to the reported transcript variants 1 (LLT1) and 2, we identified a novel splice variant 4 and transcripts coding for putative soluble proteins. CLEC2D transcripts were detected primarily in hematopoietic cell lines and were found to be co-induced by the same activation signals. Although very low amounts of putative soluble CLEC2D protein isoforms could be produced by transfectants, CLEC2D isoforms 2 and 4 were efficiently expressed. By contrast to LLT1, which was detected on the cell surface, isoform 2 and 4 remained in the endoplasmic reticulum where they formed homodimers or heterodimers with LLT1. They failed to interact with CD161, leaving LLT1 as the sole ligand for this receptor. CLEC2D therefore uses gene splicing to generate protein isoforms that are structurally distinct and that have different biological activities.

Lectin-like transcript 1 is a marker of germinal center-derived B-cell non-Hodgkin's lymphomas dampening natural killer cell functions

“…33 IHC and IF stainings with anti-LLT1 mAb revealed a predominant cytoplasmic staining pattern as well as a membranous staining, consistent with our previous data showing both intracellular and cell surface expression of LLT1 32,33 ( Fig. 1).…”

“…Immunofluorescence FFPE sections were deparaffinized, pre-treated and incubated with Mo anti-LLT1 mAb (2F1) 33 or isotype mIgG1 control (R&D) at 1 mg/mL followed by secondary biotinylated Do antiMo IgG as described above. Antibody binding was fluorescently stained using Vectastain and TSA labeled with Alexa-488.…”

“…These samples included 23 cases of Hodgkin's Lymphomas and 81 specimens of NHLs, which were classified according to the WHO classification. Immunohistochemistry IHC staining for LLT1 was performed with mouse (Mo) anti-LLT1 clone 2F1 mAb 33 on FFPE tissue sections (4 mm). FFPE sections were deparaffinized and rehydrated in successive baths of xylene, ethanol and water followed by heat-induced epitope retrieval in Tris/EGTA buffer pH 9.…”

Molecular Basis for LLT1 Protein Recognition by Human CD161 Protein (NKRP1A/KLRB1)

“…4B). This complex structure supports the previous report that PILAR (KACL) cannot bind to CD161 (8,9) because Asp 152 of PILAR (KACL), which corresponds to Lys 169 of LLT1, probably disrupts the interaction with Glu 205 of CD161 (Fig. 4, B and C).…”

“…On the other hand, Aldemir et al (5) and Rosen et al (6) identified one of the KLR family members, the human lectin-like transcript-1 molecule (LLT1) (also referred to as CLEC2D) as a ligand for the human CD161, and this recognition inhibited the CD161 ϩ NK cell cytotoxicity. Another study reported that proliferation-induced lymphocyte-associated receptor (PILAR) (also named KACL/ CLEC2A) is a second ligand of human CD161 (7); however, recent reports using a tetramer staining technique revealed that PILAR cannot bind to CD161 but instead binds to NKp65, which also belongs to the KLR family (8,9).…”