This study reveals a potential role for MAIT cells in patients with AS and is the first linking IL-7 to the elevated IL-17 profile in patients through the AS-associated risk gene IL7R.

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2–4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD–exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

Mannosylphospho dolichol synthase (DPMS) is a critical enzyme in the biosynthesis of lipid-linked oligosaccharide (LLO; Glc3Man9GlcNAc2-PP-Dol), a pre-requisite for asparagine-linked (N-linked) protein glycosylation. We have shown earlier that DPMS is important for angiogenesis, i.e., endothelial cell proliferation. This is true when cAMP is used for intracellular signaling. During cAMP signaling, DPMS is activated and ER stress is reduced. To understand the activation of DPMS at the molecular level we have isolated a cDNA clone for the DPMS gene (bDPMS) from the capillary endothelial cells of bovine adrenal medulla. DNA sequencing and the deduced amino acid sequence have established that bDPMS has a motif to be phosphorylated by cAMP-dependent protein kinase (PKA). Based on the sequence information Serine 165 has been found to be the phosphorylation target in bDPMS. Hydropathy Index when plotted against amino acid number indicates the presence of a hydrophobic region around the amino acid residues 120–160, supporting that bDPMS has one membrane spanning region. The recombinant bDPMS has now been purified as His-tag protein with an apparent molecular weight of Mr 33 kDa. Additionally, we show here that overexpression of DPMS is indeed angiogenic. The capillary endothelial cells proliferate at a higher rate carrying the DPMS overexpression plasmid over the parental cells or the vector.

Mannosylphospho dolichol synthase (DPMS) plays a critical role in Glc3Man9GlcNAc2-PP-Dol (lipid-linked oligosaccharide, LLO) biosynthesis, an essential intermediate in asparagine-linked (N-linked) protein glycosylation. We have observed earlier that phosphorylation of DPMS increases the catalytic activity of the enzyme by increasing the Vmax as well as the enzyme turnover (kcat) without significantly changing the Km for GDP-mannose. As a result, LLO biosynthesis, turnover and protein N-glycosylation are increased. This is manifested in increased proliferation of capillary endothelial cells, i.e., angiogenesis. We have then asked if the phosphorylation event or the up-regulation of the DPMS due to over production of the enzyme is a key factor in up-regulating angiogenesis? This question has been answered by isolating a stable capillary endothelial cell clone overexpressing the DPMS gene. Our results indicate that the DPMS overexpressing clone has a high level DPMS mRNA judged by QRT-PCR. The clone also expresses nearly four-times higher DPMS protein over the clone transfected with pEGFP-N1 vector only (i.e., control) as analyzed by western blotting. Most importantly, the overexpressing DPMS clone has ~108% higher DPMS activity than that of the vector control. Immunofluorescence microscopy with Texas-Red conjugated WGA indicates a high level expression of GlcNAc-β-(1,4)-GlcNAc)1-4-β-GlcNAc-NeuAc glycans on the external surface of the capillary endothelial cells overexpressing DPMS. Increased cellular proliferation and accelerated healing of the wound induced by a mechanical stress of the DPMS overexpressing clone unequivocally supports DPMS for angiogenesis.