Background: CLCA proteins activate CaCCs; CLCAs have roles in cancer and inflammatory lung diseases, but their mechanism of action is unknown. Results: CLCA proteins must undergo self-cleavage via their own novel metalloprotease domain in the N terminus to activate CaCCs. Conclusion: Self-cleavage unmasks the N-terminal fragment, which alone activates CaCCs. Significance: This work identifies a unique ion channel activation mechanism defining framework to understand CLCA functions in diseases.

TREM-2 (triggering receptor expressed on myeloid cells- 2) is an innate immune receptor expressed on dendritic cells, macrophages, osteoclasts, and microglia. Recent genetic studies have reported the occurrence of point mutations in TREM-2 that correlate with a dramatically increased risk for the development of neurodegenerative diseases, including Alzheimer’s disease, frontotemporal dementia, and Parkinson’s disease. Structural and biophysical studies of wild-type and mutant TREM-2 ectodomains are required to understand the functional consequences of these mutations. In order to facilitate these studies, we undertook the production and crystallization of these proteins. Here we demonstrate that, unlike many single Ig domain proteins, TREM-2 could not be readily refolded from bacterially-expressed inclusion bodies. Instead, we developed a mammalian-cell based expression system for the successful production of wild-type and mutant TREM-2 proteins in milligram quantities and a single-chromatography-step purification scheme that produced diffraction-quality crystals. These crystals diffract to a resolution of 3.3 Å and produce data sufficient for structure determination. We describe herein the procedures to produce wild-type and mutant human TREM-2 Ig domains in sufficient quantities for structural and biophysical studies. Such studies are crucial to understand the functional consequences of TREM-2 point mutations linked to the development of neurodegenerative diseases and, ultimately, to develop patient-specific molecular therapies to treat them.

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in the respiratory system and the gastrointestinal tract. Accordingly, human CLCA1 is a critical mediator of hypersecretory diseases that manifest mucus obstruction, such as asthma, COPD, and cystic fibrosis. It has been reported that hCLCA1 modulates calcium-activated chloride channels (CaCCs) in mammalian cell lines (Hamann et al., J Physiol 587: 2255-74; 2009), and that CLCAs are proteolytically processed during secretion (Patel et al., Annu Rev Physiol 71: 425-49; 2009); however, the precise molecular mechanisms of CLCAs remain unclear. To address this, we used a combination of sequence analysis, structure prediction, proteomics, and biochemical, biophysical and electrophysiological assays in HEK293 cells expressing several human and murine CLCA isoforms. We found that CLCAs are metalloproteases capable of both self-cleavage and cross-cleavage of other family members. We identified a novel zincin metalloprotease domain in the N-terminus of CLCA itself that is responsible for the self-proteolysis, and defined a consensus cleavage motif unique to the CLCA family. The activating effect of hCLCA1 on endogenous CaCCs was abolished in cells transfected with mutations that disrupt the metalloprotease activity or the cleavage site, and was recovered in cells transfected with the N-terminal fragment of the proteolysis, but not with the C-terminal fragment. Together, our data indicate that this unique CLCA self-cleavage event is required to unmask the N-terminal fragment of the protein, which is then responsible for the modulation of CaCCs. Our study provides a functional basis for CLCA1 self-cleavage, and a novel mechanism for regulation of chloride channel activity.