Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

Abstract

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 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 indicating that the cystatin C dimer, formed during intracellular trafficking, 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 increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release 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, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.

Removal and Rebound Kinetics of Cystatin C in High-Flux Hemodialysis and Hemodiafiltration

“…There is evidence that cystatin C is dimerized and monomerizes as part of the normal cellular processing pathway (45). There are no data on the relative pool size of dimerized cystatin C. It is likely that the assay that we used measured predominantly monomeric cystatin C, although there are no published data.…”

“…Our model assumed also that transfer across the intracellular-extracellular space was a concentration-driven process akin to diffusion, but the process may be more complex and involve active secretion, which is known to occur for cystatin C in the endoplasmic reticulum-lysosome pathway (45,47). Wallin et al (47) have recently identified a hydrophobic cystatin C region in the cysteine cathepsin binding region of the molecule that seems to be a crucial determinant of cellular uptake.…”

Cystatin M suppresses the malignant phenotype of human MDA-MB-435S cells

“…(n ¼ 8) and compared statistically using one-way ANOVA. Adhesion measurements were normalized to that of the mock clone to clearly identify the differences in tumor cell adhesion undergo N-or O-glycosylation, Ser/Thr-phosphorylation, dimerization, or to bind calcium ions (Bell et al, 1989;Laber et al, 1989;Ekiel et al, 1997;Merz et al, 1997;Taupin et al, 2000). Further studies are required to determine which of these modifications occur in cystatin M and are important for its antiproliferative function.…”

Crystal structure of 12-oxophytodienoate reductase 3 from tomato: Self-inhibition by dimerization

“…Many cases of intramolecular self-inhibition (autoinhibition) are known (29,30), but examples of intermolecular inhibition by homodimerization have been reported as well (31)(32)(33)(34)(35)(36). Proteolytic cleavage, phosphorylation, ligand binding, and even lightinduced conformational changes represent different possibilities to initiate or abrogate self-inhibition.…”