An atypical hyperacidic Grp78/BiP isoform is upregulated by TLR2 agonism or bacterial infection but not by endoplasmic reticulum stress

CRISTIAN JORGE ALEJANDRO ASENSIO; LI JUN TANG; STEFANIA ZAMPIERI; MARÍA G PITTIS; RODOLFO C GARCÍA

CELL BIOLOGY INTERNATIONAL

ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD

Lugar: Amsterdam; Año: 2008 vol. 32 p. 50 - 52

1065-6995

We have used an in vitro protein phosphorylation assay to detect changes in the levels of phosphorylatable proteins of low abundance with sensitivity higher than that of silver, Sypro Ruby or Pro-Q Diamond staining. THP-1 macrophage-like cells were infected with Mycobacterium avium, Borrelia burgdorferi or heat-killed E. coli, or treated with agonists of TLR2 and TLR4 for different times. We screened for proteome differences induced by the different agents by comparing with non-infected, untreated cells. One of the proteins up-regulated after infection or treatment with TLR2 agonists was identified as a novel Grp78/BiP isoform. This in vitro phosphorylated isoform (haGrp78) was less abundant and had a very low isoelectric point compared with the typical, more basic isoforms of Grp78 (bGrp78) that are not phosphorylated in vitro. haGrp78 up-regulation was apparent from 6 hours after infection and peaked at 24 hours regardless of the bacterial type or the TLR2 agonist. LPS did not affect the level of haGrp78, nor did 2-deoxyglucose-induced ER stress or thapsigargin-induced UPR. At day 4 post-infection, haGrp78 levels were higher when macrophages had ingested live as opposed to heat-killed M. avium. This suggests that live, intracellular M. avium continuously synthesizes TLR2 agonistic molecules while residing and proliferating in the phagosome. All forms of Grp78 were found both in cytosolic and membrane cell fractions. The up-regulation of haGrp78 has the potential to become a novel and useful marker of delayed and sustained effects of TLR2 agonists and of bacterial infections.