Abstract
Human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 2 encoded by ATP2C2 is only expressed in a limited number of tissues, unlike the ubiquitously expressed SPCA1 pump (encoded by ATP2C1, the gene defective in Hailey-Hailey disease). It has not been determined whether there are significant functional differences between SPCA1 and SPCA2 pump enzymes. Therefore, steady-state and transient kinetic approaches were used to characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human SPCA2 enzyme upon heterologous expression in HEK-293 cells. The catalytic turnover rate of SPCA2 was found enhanced relative to SPCA1 pumps. SPCA2 displayed a very high apparent affinity for cytosolic Ca2+ (K0.5 = 0.025 microm) in activation of the phosphorylation activity but still 2.5-fold lower than that of SPCA1d. Our kinetic analysis traced both differences to the increased rate characterizing the E1 approximately PCa to E2-P transition of SPCA2. Moreover, the reduced rate of the E2 to E1 transition seems to contribute in determining the lower apparent Ca2+ affinity and the increased sensitivity to thapsigargin inhibition, relative to SPCA1d. SPCA2 also displayed a reduced apparent affinity for inorganic phosphate, which could be explained by the observed enhanced rate of the E2-P dephosphorylation. The insensitivity to modulation by pH and K+ concentration of the constitutively enhanced E2-P dephosphorylation of SPCA2 is similar to SPCA1d and possibly represents a novel SPCA-specific feature, which is not shared by sarco(endo)plasmic reticulum Ca2+-ATPases.
Highlights
Cations are needed for specific luminal processes including protein sorting and trafficking, endosome fusion, precursor protein processing, and N- and O-linked protein glycosylation [6]
It is not yet clear whether the amino acid differences observed between SPCA1 and SPCA2 protein sequences would translate into different functional properties
The human SPCA2 pump was further analyzed in detail and compared with the newly characterized human SPCA1d paralogue [3] and to the well known SERCA1a enzyme, following their heterologous expression in the HEK
Summary
Cations are needed for specific luminal processes including protein sorting and trafficking, endosome fusion, precursor protein processing, and N- and O-linked protein glycosylation [6]. In addition to ATP2C1, the human genome contains a second SPCA gene (ATP2C2, cytogenetic position 16q24.1), whose protein product (SPCA2 of 946 amino acids) has not been studied until very recently [10, 11]. The two proteins encoded by the respective genes share a 64% amino acid sequence identity and display a different tissue expression pattern. Kinetics of Human SPCA2 transport either Ca2ϩ or Mn2ϩ, but disagreement remained regarding whether SPCA2 could bind Ca2ϩ with high [10] or low [11] affinity. It is not yet clear whether the amino acid differences observed between SPCA1 and SPCA2 protein sequences would translate into different functional properties. Our findings may reflect a specialized physiological role for SPCA2 in a restricted number of cell types
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