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Studies on the proteolysis of the human erythrocyte calcium-pumping ATPase by endogenous calpain

University of British Columbia

It is well known that the erythrocyte plasma membrane Ca²⁺-pumping ATPase can be activated by calmodulin. Limited proteolysis of the Ca²⁺-ATPase with exogenous proteases can also produce a calmodulin-like activation. Because a calcium-dependent cysteine protease (calpain I) is present in the erythrocyte cytosol, it was proposed that this enzyme could proteolytically activate the Ca²⁺-ATPase during a sustained elevation of the cytosolic free Ca²⁺ level. Upon incubation of red blood cell membranes with purified calpain I and Ca²⁺, the membrane-bound Ca²⁺-ATPase activity was increased and its sensitivity to calmodulin was lost. Calmodulin protected the Ca²⁺-ATPase against proteolytic activation by calpain. Both the membrane-bound and the purified Ca²⁺-ATPase (136 kDa) Were transformed by calpain into two phosphoenzyme intermediate-forming fragments of 125 kDa and 124 kDa, followed by the formation of two phosphoenzyme intermediate-forming fragments of 82 kDa and 80 kDa. The 125 kDa and the 82 kDa fragments bound to and were stimulated by calmodulin, whereas, the 124 kDa and the 80 kDa fragments did not bind and were not stimulated by calmodulin and their formation corresponded to the observed proteolytic activation. In the presence of calmodulin, however, the native enzyme was sequentially transformed into two phosphoenzyme intermediate-forming fragments of 127 kDa and 85 kDa. Both of these fragments bound to and were stimulated by calmodulin. Apparently, calmodulin protects the Ca²⁺-ATPase from calpain- mediated activation by preventing the formation of the calmodulin-insensitive 124 kDa and 80 kDa fragments. Smaller inactive fragments were also produced with further proteolysis. Following limited calpain treatment of the phosphatidylcholine liposome-reconstituted Ca²⁺-ATPase, both the initial rates of Ca²⁺ uptake and ATP hydrolysis were increased to near maximal levels, similar to those obtained upon addition of calmodulin. The reconstituted Ca²⁺-ATPase was transformed mainly into 124 kDa and 127 kDa active fragments, in the absence and the presence of calmodulin, respectively. Based on the deduced amino acid sequence of a plasma membrane Ca²⁺-ATPase from a human teratoma, the calmodulin-binding domain was identified to be about 3.5 kDa long and located about 9-10 kDa from the C-terminal end of the enzyme while the acylphosphate site was located about 50 kDa from the N-terminal end. By combining this knowledge with the estimated molecular masses and calmodulin-binding ability of the various fragments, it was postulated that (i) in the absence of calmodulin, calpain I initially cleaves off about 11 kDa from the C-terminal end and about 2 kDa of the calmodulin-binding domain, producing the calmodulinsensitive 125 kDa fragment; (ii) in the absence of calmodulin, a second cleavage by calpain I further removes most (1.5 kDa) of the remaining calmodulin-binding domain and produces the calmodulin-insensitive 124 kDa fragment; (iii) in the presence of calmodulin, calpain cleaves off about 9 kDa of the C-terminal end and produces a calmodulin-sensitive 127 kDa fragment which retained most of the calmodulin-binding domain. A slower cleavage at a site about 42-44 kDa from the N-terminal end was also proposed to generate the 85 kDa, 82 kDa and 80 kDa active fragments from the 127 kDa, 125 kDa and 124 kDa fragments, respectively. Several other calmodulin-binding proteins were also found to be proteolyzed by calpain I, including adducin from human erythrocyte membrane as well as neuromodulin and calcineurin from bovine brain. A literature search further revealed that susceptibility to calpain was in fact a characteristic shared by at least 16 calmodulin-binding proteins. Primary structure analysis showed that the majority of calmodulin-binding proteins sequenced to date have one or more regions enriched in proline (P), glutamate (E), aspartate (D), serine (S) and threonine (T) (PEST sequences). It was proposed that PEST sequences in calmodulin-binding proteins may serve as recognition sites for calpain.

Text

2010-10-18

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http://hdl.handle.net/2429/29314

Pharmaceutical Sciences

University of British Columbia

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