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. 2023 Oct 28;11(11):2915.
doi: 10.3390/biomedicines11112915.

Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry

Grace T Ramena  1 , Aarushi Sharma  2 , Yan Chang  3   4 , Zui Pan  3   4   5 , Randolph C Elble  2
Affiliations

Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry

Grace T Ramena et al. Biomedicines. .

Abstract

Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conserved HEXXH zinc-binding motif that is proposed to cleave the precursor autoproteolytically. Here, we tested this hypothesis and its link to SOCE. We first studied the conditions for autocleavage in isolated membranes and then in a purified protein system. Cleavage was zinc-dependent and abolished by mutation of the E in the HEXXH motif to Q, E165Q. Cleavage efficiency increased with CLCA2 concentration, implying that it occurs in trans. Accordingly, the E165Q mutant was cleaved by co-transfected wildtype CLCA2. Moreover, CLCA2 precursors with different epitope tags co-immunoprecipitated. In a membrane-free system utilizing immunopurified protease and target, no cleavage occurred unless the target was first denatured, implying that membranes provide essential structural or conformational cues. Unexpectedly, cleavage caused a conformational shift: an N-terminal antibody that immunoprecipitated the precursor failed to precipitate the N-terminal product unless the product was first denatured with an ionic detergent. The E165Q mutation abolished the stimulation of SOCE caused by wildtype CLCA2, establishing that the metalloprotease activity is required for this regulatory function.

Keywords: CLCA2; SOCE; metalloprotease; self-cleavage.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Schematic representation of CLCA2 protein structure. The locations of the TVE20, Myc tag, and Flag tag epitopes relative to the cleavage site (scissors) are indicated. ss, signal sequence; CLCA-N, metalloprotease domain; VWA, von Willebrand A domain; bsr, beta sheet domain; fn3, fibronectin-3-like domain; tms, transmembrane segment; C-tail, cytoplasmic tail. Relative location of the zinc-binding HEWAHL motif is indicated; asterisk, site of the E165Q substitution. Lower, apparent sizes of precursor and cleavage products.
Figure 2
Figure 2
Dependence of cleavage on Zn2+ and pH. Immunoblots of crude membrane extracts from CLCA2-transfected HEK293T cells after incubation at 37 °C for 3 h (A) in the presence of different cations or EDTA and (B) at varying pH values. Input control was incubated on ice. The pH of PBS was 7.4. The fluorometric values of the precursor and cleaved product were determined using a Licor Odyssey scanner. Bar graphs represent the ratio of cleaved product to precursor divided by the value of untreated control in lane one; * p-value < 0.05; ** p-value < 0.005; *** p-value < 0.0005. Blot: TVE20. White vertical lines indicate where lanes were omitted or rearranged to remove irrelevant data. All samples were run on one gel. Experiments were repeated three times.
Figure 3
Figure 3
Inhibition of cleavage by E165Q mutation in the HEWAHL amino acid motif of CLCA-N domain. Immunoblot analysis of the wildtype and E165Q mutant forms of Flag-tagged CLCA2 in cell lysates, immunoprecipitates, and membrane extracts from transfected HEK293T cells. Membrane pellets were denatured in SDS sample buffer and loaded directly. Mock refers to cells transfected with the empty pLex vector. Both IP and the blot were with anti-Flag antibody. The band at 50 kDa represents the primary antibody used for IP. WT denotes wildtype CLCA2. EQ denotes the E165Q mutant. Experiment was repeated at least three times.
Figure 4
Figure 4
Effect of substrate concentration on cleavage efficiency. Top, immunoblot analysis of CLCA2-Flag cleavage from lysates of HEK293T cells transfected with increasing amounts of DNA. Bottom, graph relating percent cleavage to quantity of DNA transfected. Percent cleavage was obtained by dividing the signal for product by the sum of signals for product and precursor. Experiment was repeated four times; * p-value < 0.05; ** p-value < 0.005. Blot, anti-Flag.
Figure 5
Figure 5
The E165Q mutant can be cleaved by co-transfected wildtype CLCA2 in a concentration-dependent manner. Left, immunoblot analysis of the co-transfected wildtype (WT) and E165Q (EQ) mutant anti-Flag immunoprecipitates from 1% NP40 lysates. Cleavage of the mutant protein is indicated by the 35 kDa band in lanes 4–6. Lanes 1–2 show level of cleavage product from cells transfected with EQ alone or WT alone; lane 3, nontransfected. Right, relation between percent cleavage of the EQ and amount of co-transfected wildtype CLCA2; * p-value < 0.05; ** p-value < 0.005. The percent cleavage of WT-Flag in lane 2 is 55. Experiment repeated three times.
Figure 6
Figure 6
Co-immunoprecipitation of CLCA2 precursors. (A) Immunoblots of lysates from co-transfected or singly transfected HEK293T cells that were subjected to immunoprecipitation with anti-Flag or anti-Myc antibodies. NT indicates N-terminal insertion of the Myc tag. CT indicates C-terminal insertion of the Flag tag. The Myc antibody immunoprecipitates only the Flag-tagged precursor but not the C-terminal product. (B) E165Q mutation does not affect ability to form a stable complex. Immunoblot analysis of co-immunoprecipitation from lysates of HEK293T cells transfected with Myc-tagged wildtype CLCA2 and/or Flag-tagged CLCA2 E165Q mutant. On both gels, lane 2 was left empty to avoid spillover. Experiments were repeated three times.
Figure 7
Figure 7
Cleavage of immunopurified E165Q mutant (EQ) by immunopurified wildtype CLCA2 (WT) in vitro after denaturation of the mutant protein by SDS. Left, experimental flowchart. Right, immunoblot analysis of cleavage products using the anti-Flag antibody. CLCA2-WT and CLCA2Δ35 lacked a Flag-tag; bands represent the EQ mutant precursor and products. Incubation time is indicated. The lower panel was overexposed relative to the upper panel to generate visible bands. Experiment repeated three times.
Figure 8
Figure 8
Evidence that the N-terminal ectodomain undergoes conformational shift upon cleavage that masks an epitope. Lanes 1 and 2, detection of precursor and N-terminal product on SDS-PAGE immunoblot of HEK293 NP40 lysates. Lane 3, immunoprecipitation of precursor only from NP40 lysate. Lane 4, detection of precursor and product after lysate was first denatured by SDS/heat then renatured by addition of excess NP40 as in Figure 7. Lanes 2 and 5, HEK293 expressing vector only. Experiment repeated three times.
Figure 9
Figure 9
Reduced SOCE in the cells expressing CLCA2 E165Q mutant. (A) Representative traces of intracellular Ca2+ in pLex vector control (HEK293 pLex), wildtype CLCA2 (wt-CLCA2), and E165Q mutant (HEK293-EQ) cells. (B) SOCE activity was calculated as the changes in the ratio of F350 nm/F385 nm (ΔF350/F385). Statistical analyses were vehicle control (0.25 ± 0.089) and wildtype CLCA2 (0.44 ± 0.125) or E165Q mutation (0.12 ± 0.037). Mean ± SD, n > 30 cells, ****: p < 0.0001 (based on one-way ANOVA). Experiment was repeated three times. (C) Immunoblot of wildtype (WT) and E165Q (EQ) cell lines used in the analysis. Blot: anti-Flag and anti-actin antibodies.
Figure 10
Figure 10
Model showing that CLCA2 forms complexes, that cleavage is intermolecular and causes a conformational switch, and that the cleavage activity is necessary for activation of SOCE. The stoichiometry is unknown and is depicted here as a dimer for simplicity. Both the precursor and cleaved forms have been shown to interact with the Orai–STIM complex, but structural details are unknown.
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