Calnexin mediates the maturation of GPI-anchors through ER retention

The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.     

Prenatal glucocorticoid administration accelerates the maturation of fetal rat hepatocytes

Molecular Biology Reports - Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for the maturation of cardiopulmonary function. Preterm and...     

Interaction of guanine-nucleotide-binding regulatory proteins with chemotactic peptide receptors in differentiated human leukemic HL-60 cells.

Human leukemic HL-60 cells were differentiated into neutrophil-like cells by treatment with dimethylsulfoxide (Me2SO) or N6,O2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP), and membrane fractions were prepared from the differentiated cells. Receptors for fMLF (fM,N-formylmethionine) and guanine-nucleotide-binding regulatory proteins (G proteins) serving as the substrate for pertussis toxin (islet-activating protein; IAP) were extracted from cell membranes then reconstituted into phospholipid vesicles. The binding of fMLF to the reconstituted vesicles (or the membranes) was determined with 10 nM [3H] fMLF. In both cases, high-affinity binding to vesicle preparations from the Me2SO- and Bt2cAMP-induced cells was abolished following treatment with IAP, suggesting that fMLF receptors were functionally coupled to IAP-sensitive G proteins in each of the two vesicle types. However, the high-affinity fMLF binding was much higher in vesicle preparations originating from Bt2cAMP-induced cells than in those from Me2SO-induced cells, although the amount of IAP-substrate G protein reconstituted into the each phospholipid vesicles preparation was not significantly different from the other. The G proteins of the two differentiated cells were both identified as inhibitory forms (Gi-2) based on their electrophoretic mobilities and immunoblot analyses. When purified Gi-2 from rat brain was reconstituted into the two IAP-treated vesicles, high-affinity fMLF binding was restored in a similar manner in both. IAP-substrate G proteins partially purified from the two differentiated HL-60 cells were also effective in restoring high-affinity fMLF binding to the IAP-treated vesicles. However, a significant difference was observed that the reconstituted binding was higher with the G-protein-rich fraction from Bt2cAMP-induced cells than with that from Me2SO-induced cells, with each of the two IAP-treated vesicle types. These results suggest that the different high-affinity binding of fMLF observed in the two differentiated HL-60 cells are due to a difference in the property of endogenous G proteins rather than fMLF receptors, though the two G proteins are indistinguishable from each other in terms of the subtype of G protein, Gi-2.