title: Effect of Hailey-Hailey Disease mutations on the function of a new variant of human secretory pathway Ca2+/Mn2+-ATPase (hSPCA1).
abstract: ATP2C1, encoding the human secretory pathway Ca2+/Mn2+ ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey Disease (HHD), an autosomal dominant skin disorder characterized by persistent blisters and erosions. To investigate the underlying cause of HHD, we have analyzed the changes in expression level and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into hSPCA1d, a novel splice variant expressed in keratinocytes, described here for the first time. Encoded by the full-length of optional exons 27 and 28, hSPCA1d was longer than previously identified splice variants. The protein competitively transported Ca2+ and Mn2+ with equally high affinity into the Golgi of COS-1 cells. Ca2+- and Mn2+-dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (Pi-fuelled) directions was also demonstrated. HHD mutant proteins L341P, C344Y, C411R, T570I, and G789R showed low levels of expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. P201L had little effect on the enzymatic cycle, whereas I580V caused a block in the E1 approximately P --> E2-P conformational transition. D742Y and G309C were devoid of Ca2+- and Mn2+-dependent phosphoenzyme formation from ATP. The capacity to phosphorylate from Pi was retained in these mutants but with a loss of sensitivity to both Ca2+ and Mn2+ in D742Y and a preferential loss of sensitivity to Mn2+ in G309C. These results highlight the crucial role played by Asp-742 in the architecture of the hSPCA1 ion-binding site and reveal a role for Gly-309 in Mn2+ transport selectivity.
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Dr., United Kingdom.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Fairclough
authors.forename: Leonard
authors.initials: L
authors.lastname: Dode
authors.forename: Jo
authors.initials: J
authors.lastname: Vanoevelen
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
authors.forename: Ludwig
authors.initials: L
authors.lastname: Missiaen
authors.forename: Luc
authors.initials: L
authors.lastname: Raeymaekers
authors.forename: Frank
authors.initials: F
authors.lastname: Wuytack
authors.forename: Alain
authors.initials: A
authors.lastname: Hovnanian
mesh_terms: D017398:Alternative Splicing; D001224:Aspartic Acid / Q000235:genetics; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D005998:Glycine / Q000235:genetics; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016506:Pemphigus, Benign Familial / Q000235:genetics; D010766:Phosphorylation
chemical_list: D001224:Aspartic Acid; C403624:ATP2C1 protein, human; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D005998:Glycine