Clr-a: A Novel Immune-Related C-Type Lectin-like Molecule Exclusively Expressed by Mouse Gut Epithelium

Abstract

The mouse gut epithelium represents a constitutively challenged environment keeping intestinal commensal microbiota at bay and defending against invading enteric pathogens. The complex immunoregulatory network of the epithelial barrier surveillance also involves NK gene complex (NKC)-encoded C-type lectin-like molecules such as NKG2D and Nkrp1 receptors. To our knowledge, in this study, we report the first characterization of the orphan C-type lectin-like molecule Clr-a encoded by the Clec2e gene in the mouse NKC. Screening of a panel of mouse tissues revealed that Clec2e transcripts are restricted to the gastrointestinal tract. Using Clr-a-specific mAb, we characterize Clr-a as a disulfide-linked homodimeric cell surface glycoprotein. Of note, a substantial fraction of Clr-a molecules are retained intracellularly, and analyses of Clr-a/Clr-f hybrids attribute intracellular retention to both the stalk region and parts of the cytoplasmic domain. Combining quantitative PCR analyses with immunofluorescence studies revealed exclusive expression of Clr-a by intestinal epithelial cells and crypt cells throughout the gut. Challenge with polyinosinic-polycytidylic acid results in a rapid and strong downregulation of intestinal Clr-a expression in contrast to the upregulation of Clr-f, a close relative of Clr-a, that also is specifically expressed by the intestinal epithelium and acts as a ligand of the inhibitory Nkrp1g receptor. Collectively, we characterize expression of the mouse NKC-encoded glycoprotein Clr-a as strictly associated with mouse intestinal epithelium. Downregulation upon polyinosinic-polycytidylic acid challenge and expression by crypt cells clearly distinguish Clr-a from the likewise intestinal epithelium-restricted Clr-f, pointing to a nonredundant function of these highly related C-type lectin-like molecules in the context of intestinal immunosurveillance.

Cellular Mechanisms Controlling Surfacing of AICL Glycoproteins, Cognate Ligands of the Activating NK Receptor NKp80

“…Apart from human CLEC2 family members CD69 and AICL, atypical N-glycosylation sites are also present in mouse CLEC2 family members, including CD69 and most Clr molecules. Of note, evidence for intracellular retention has also been reported for some of these molecules such as CD69 and Clr-a that are predominantly localized intracellularly governed by unknown mechanisms (29,35,60). The extracellular domain of AICL has been shown to mediate intracellular retention (27), whereas the stalk and parts of the cytoplasmic domain were shown to contribute to the impaired cell surface expression of Clr-a (35).…”

“…U937 cells were washed twice with PBS and cultured in RPMI 1640 minimal medium without methionine or cysteine (Sigma-Aldrich) for 30 min at 37˚C prior to labeling with 0.5 mCi/ml EasyTag EXPRESS [ 35…”

“…Peptide arrays (peptides of 18 aa with an offset by 1 aa) covering the entire AICL ED (Lys 26 to His 149 , Fig. 1A) were synthesized by Fmoc chemistry at activated polyethylene glycol spacers on cellulose membranes by automated parallel peptide synthesis on a MultiPep RSi (INTAVIS Bioanalytical Instruments, Cologne, Germany) and used for binding experiments as previously described (34,35). After saturation of unspecific binding sites, peptides were probed with AICL-specific mAb 7F12 or 7G4.…”

“…This atypical N-glycosylation site is located at the base of the CTLD (Fig. 1I) and includes Cys 46 that was predicted to form an intramolecular disulfide bond with Cys 35 (Fig. 1A).…”

“…Depicted is one out of four independent experiments. (B) Proteins of U937 cells were metabolically labeled with35 S-containing amino acids and, subsequently, AICL glycoproteins immunoprecipitated using mAb 7F12. Immunoprecipitates were separated by SDS-PAGE under reducing conditions and visualized by autoradiography.…”