PURIFICATION AND CHARACTERIZATION OF A NOVEL STRESS PROTEIN, THE 150-KDA OXYGEN-REGULATED PROTEIN (ORP150), FROM CULTURED RAT ASTROCYTES AND ITS EXPRESSION IN ISCHEMIC MOUSE-BRAIN

As the most abundant cell type in the central nervous system, astrocyt es are positioned to nurture and sustain neurons, especially in respon se to cellular stresses, which occur in ischemic cerebrovascular disea se. In a previous study (Hori, O., Matsumoto, M., Kuwabara, K., Maeda, M., Ueda, H., Ohtsuki, T., Kinoshita, T., Ogawa, S., Kamada, T., and Stern, D. (1996) J. Neurochem., in press), we identified five polypept ide bands on SDS-polyacrylamide gel electrophoresis, corresponding to molecular masses of about 28, 33, 78, 94, and 150 kDa, whose expressio n was induced/enhanced in astrocytes exposed to hypoxia or hypoxia fol lowed by replacement into the ambient atmosphere (reoxygenation). In t he current study, the approximate to 150-kDa polypeptide has been char acterized. Chromatography of lysates from cultured rat astrocytes on f ast protein liquid chromatography Mono Q followed by preparative SDS-p olyacrylamide gel electrophoresis led to isolation of a approximate to 150-kDa band only observed in hypoxic cells and which had a unique N- terminal sequence of 15 amino acids. Antisera raised to either the pur ified approximate to 150-kDa band in polyacrylamide gels or to a synth etic peptide comprising the N-terminal sequence detected the same poly peptide in extracts of cultured rat astrocytes exposed to hypoxia; exp ression was not observed in normoxia but was induced by hypoxia within 24 h, augmented further during early reoxygenation, and thereafter de creased to the base line by 24 h in normoxia. ORP150 expression in hyp oxic astrocytes resulted from de novo protein synthesis, as shown by i nhibition in the presence of cycloheximide. In contrast to hypoxia-med iated induction of the approximate to 150-kDa polypeptide, neither hea t shock nor a range of other stimuli, including hydrogen peroxide, cob alt chloride, 2-deoxyglucose, or tunicamycin, led to its expression, s uggesting selectivity for production of ORP150 in response to oxygen d eprivation, i.e. it was an oxygen-regulated protein (ORP150). Northern and nuclear run-off analysis confirmed the apparent selectivity for O RP150 mRNA induction in hypoxia. Subcellular localization studies show ed ORP150 to be present intracellularly within endoplasmic reticulum a nd only in hypoxic astrocytes, not cultured microglia, endothelial cel ls, or neurons subject to hypoxia. Consistent with these in vitro resu lts, induction of cerebral ischemia in mice resulted in expression of ORP150 (the latter was not observed in normoxic brain). These data sug gest that astroglia respond to oxygen deprivation by redirection of pr otein synthesis with the appearance of a novel stress protein, ORP150. This polypeptide, selectively expressed by astrocytes, may contribute to their adaptive response to ischemic stress, thereby ultimately con tributing to enhanced survival of neurons.