Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.     

Structure and dynamics analysis of a new member heparinase II/III of family 12 polysaccharide lyase from Pseudopedobacter saltans by computational modeling and small-angle X-ray scattering

Abstract

The structure of heparinase II/III belonging to family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was generated by homology modeling. Multiple sequence alignment showed conserved (Asn216, Tyr270 and His400) and semi-conserved active site amino acid residues. The modeled structure of PsPL12a displayed α/α toroid domain at N-terminal and antiparallel β sheets at C-terminal domain. The modeled structure was similar to those of heparinases from polysaccharide lyase 12 and 21 families. Validation of PsPL12a model by Ramachandran plot showed 94.6% of residues in the favored region, 5.2% of residues in the allowed region and only 0.2% of residues in the outlier region. The area and volume computed for PsPL12a displayed nearly a closed conformation of the active site, similar to HepIII from Bacteroides thetaiotaomicron. The charge calculation on the surface of the PsPL12a structure showed the higher distribution of positive charge in the active site cleft as compared with other homologous structures. Molecular docking study of MD-simulated PsPL12a structure with heparin oligosaccharide showed high binding affinity as compared with heparan sulfate oligosaccharides. Comparison of the active site of modeled PsPL12a with other homologous heparinases revealed putative catalytic triad involving the residues Asn216, His400 and Tyr270. Small-angle X-ray scattering analysis of PsPL12a displayed a fully folded and boxing glove-like envelop.

Communicated by Ramaswamy H. Sarma