HUMAN CYSTATIN-C FORMS AN INACTIVE DIMER DURING INTRACELLULAR TRAFFICKING IN TRANSFECTED CHO CELLS

To define the cellular processing of human cystatin C as well as to la y the groundwork for investigating its contribution to Icelandic Hered itary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characte rized the trafficking, secretion, and extracellular fate of human cyst atin C in transfected Chinese hamster ovary (CHO) cells. It is constit utively secreted with an intracellular half-life of 72 min. Gel filtra tion of cell lysates revealed the presence of three cystatin C immunor eactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immuno reactive material, greater than or equal to 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C i ndicating that the cystatin C dimer, formed during intracellular traff icking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large inc rease in intracellular monomer suggesting that dimer dissociation occu rs later in the secretion pathway, after exiting the ER but prior to r elease from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, c ystatin C would be prevented from inhibiting the lysosomal cysteine pr oteases. These results reveal a novel mechanism, transient dimerizatio n, by which cystatin C is inactivated during the early part of its tra fficking through the secretory pathway and then reactivated prior to s ecretion. Similarly, its uptake by the cell also leads to its redimeri zation in the lysosomal pathway. (C) 1997 Wiley-Liss, Inc.