Endoplasmic Reticulum Resident, Immunoglobulin Joining Chain, Can Be Secreted by Perturbation of the Calcium Concentration in the Endoplasmic Reticulum

Abstract

We established a transient human joining (J)-chain gene expression system in the baby hamster kidney (BHK) cell. The J-chain was detected as a 29-kDa single band on Western blotting. Immunofluorescent staining of the transfectant revealed an exclusive localization of the J-chain in the endoplasmic reticulum (ER). Intracellular transport experiment revealed that incubating conditions favorable for vesicular stomatitis virus glycoprotein (VSV-G) transport did not allow the J-chain to exit from the ER. Analysis of glycosylation status of the J-chain in the transfectant was examined by tunicamycin treatment, endoglycosidase H digestion, and also by treatment with brefeldin A. It was found that an N-glycosylation consensus site of the J-chain was functional, and intracellular J-chain was endoglycosidase H sensitive. These results indicate that, in the absence of any immunoglobulin molecules, J-chain localizes exclusively in the ER. We also tested whether the J-chain could be exported from the ER by perturbing the Ca2+ concentration in the ER. Cultivation of the J-chain transfectant in the presence of ionomycin resulted in the time-dependent secretion of the J-chain. The secreted J-chain was modified by the Golgi resident glycosylation enzymes, indicating that the secreted J-chain passed through the normal exocytic pathway.

Nerve Growth Factor Contribution via Transient Receptor Potential Vanilloid 1 to Ectopic Orofacial Pain

“…For Western blotting, 100 -200 g of total protein was subjected to 10% SDS-PAGE. The separated protein was transferred to an Immobilon membrane (Millipore) electrophoretically, and Western blotting was performed as described previously (Asano et al, 2004). The rabbit anti-TRPV1 antibody (1:200 dilution with 1% bovine serum albumin (BSA)-PBST in 0.1% Tween 20-PBS; Alomone) was used as the primary antibody.…”

Functional expression of TLR5 in murine salivary gland epithelial cells

“…The cell lysates were cleared by centrifugation, and protein concentration was measured with a Bio-Rad protein assay kit (Tokyo, Japan). A 100 μg sample of total protein was separated using 10% SDS-PAGE, and Western blotting was performed as described previously (15). Briefly, after 1 h of blocking with 1% BSA-PBST (0.1% Tween 20), the membrane was incubated with rabbit anti-mouse TLR 5 antibody (Ab) (×50 diluted with 1% BSA-PBST) (Santa Cruz, CA, USA) or with rabbit anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ab (×1,000 diluted with 1% BSA-PBST) (Chemicon, Tokyo, Japan) for 1 h at room temperature (RT).…”

Effect of ionomycin on interaction of calnexin with vesicular stomatitis virus glycoprotein is cell type-specific

“…Morphological analysis of VSV-G intracellular transport was performed as previously described (4). BHK cells were plated on coverslips and transfected with a pET11d-GFP-tsO45.…”

“…Drainage of calcium ions from the ER by calcium ionophores can cause rapid secretion of ER-resident proteins (2,3). A previous report found that the ER-resident protein immunoglobulin joining chain (J-chain) was released from ER-retention mechanisms and secreted outside cells by incubating J-chain-transfected fibroblasts with the calcium ionophore ionomycin (4).…”

“…57, No. 4,[305][306][307][308][309][310][311][312]2015 Original Effect of ionomycin on interaction of calnexin with vesicular stomatitis virus glycoprotein is cell type-specific (8,9). Chaperone molecules have a very important role in this system: they form a molecular complex with unfolded proteins in the ER lumen.…”