Cell type–specific glycosylation of Orai1 modulates store-operated Ca ²⁺ entry

Abstract

N-glycosylation of cell surface proteins affects protein function, stability, and interaction with other proteins. Orai channels, which mediate store-operated Ca(2+) entry (SOCE), are composed of N-glycosylated subunits. Upon activation by Ca(2+) sensor proteins (stromal interaction molecules STIM1 or STIM2) in the endoplasmic reticulum, Orai Ca(2+) channels in the plasma membrane mediate Ca(2+) influx. Lectins are carbohydrate-binding proteins, and Siglecs are a family of sialic acid-binding lectins with immunoglobulin-like repeats. Using Western blot analysis and lectin-binding assays from various primary human cells and cancer cell lines, we found that glycosylation of Orai1 is cell type-specific. Ca(2+) imaging experiments and patch-clamp experiments revealed that mutation of the only glycosylation site of Orai1 (Orai1N223A) enhanced SOCE in Jurkat T cells. Knockdown of the sialyltransferase ST6GAL1 reduced α-2,6-linked sialic acids in the glycan structure of Orai1 and was associated with increased Ca(2+) entry in Jurkat T cells. In human mast cells, inhibition of sialyl sulfation altered the N-glycan of Orai1 (and other proteins) and increased SOCE. These data suggest that cell type-specific glycosylation influences the interaction of Orai1 with specific lectins, such as Siglecs, which then attenuates SOCE. In summary, the glycosylation state of Orai1 influences SOCE-mediated Ca(2+) signaling and, thus, may contribute to pathophysiological Ca(2+) signaling observed in immune disease and cancer.

Single-nucleotide polymorphisms in Orai1 associated with atopic dermatitis inhibit protein turnover, decrease calcium entry and disrupt calcium-dependent gene expression

“…Transfection of S218G–Orai1, N223S–Orai1 or the double variant S218G–N223S–Orai1 plasmids all resulted in an increase in Orai1 protein levels above that seen with WT Orai1; the increase was ~ 2-fold at 24 hours and up to 3-fold at 48 hours ( Figures 1A and 1B ). Because N223 on Orai1 is a site for N-linked glycosylation ( 9 ), N223S–Orai1 proteins showed only the lower, non-glycosylated bands. The increase in Orai1–SNP protein expression was not accompanied by an increase in STIM1 levels ( Figure 1A ).…”

“…By contrast, glycosylated WT Orai1 protein (labelled Gly–Orai1–WT) and S218G–Orai1 (Gly–S218G–Orai1) were both relatively stable even in the presence of cycloheximide ( Figure 1G ). Since non-glycosylated Orai1 channels show a slightly larger Ca 2+ flux ( 9 ) and turn over considerably more quickly ( Figure 1 ), we focussed on these channels.…”

Blockage of Store-Operated Ca2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca2+ Selective Orai1 Pore

“… Inhibition of SOCE in GBM cell lines by Synta66: ( A ) Western blot for detection of endogenously expressed Orai1 in lysates of A172, LN-18 and U-87 MG cells. Lysates were deglycosylated in order to achieve a single Orai1 band as in [ 51 ]. ( B ) Western Blot for detection of endogenously expressed STIM1 in lysates of A172, LN-18 and U-87 MG cells.…”

Orai1 channel inhibition preserves left ventricular systolic function and normal Ca2+ handling after pressure overload

“…We further confirmed the overexpression of exogenous Orai1 with the classical multiple band pattern of human Orai1 protein in ventricle tissue by western blot ( Figure 2D). 19,20 In isolated cardiomyocytes, Orai1 immunostaining revealed a sarcolemmal profile in WT cardiomyocytes, which was more pronounced in C-dnO1 cells ( Figure 2E). Any potential compensation by other store-operated Ca 2+ channel components (TRPC1 to -C6, STIM1-2 and Orai3 protein) was observed ( Figure VIII in the onlineonly Data Supplement).…”