Intracellular degradation of histidine-rich glycoprotein mutants: Tokushima-1 and 2 mutants are degraded by different proteolytic systems

We reported the first case of a congenital histidine-rich glycoprotein defi ciency (HRG; Tokushima) in which substitution of Gly85 with Glu (G85E) in t he first cystatin domain resulted in intracellular degradation and a low pl asma level of HRG; [Shigekiyo, T, et al, (1998) Blood 91, 128-133]. Recentl y, we identified the gene mutation of a second case of HRG deficiency as a Cys223 to Arg (C223R) mutation in the second cystatin domain. To investigat e the molecular and cellular bases of these deficiencies, we expressed thes e HRG; mutants in baby hamster kidney (BHK) cells. Pulse-chase experiments in the absence and presence of various proteinase inhibitors revealed that, while wild-type HRG; was completely secreted during 4-h chase periods, bot h the G85E and C223R mutants were only partially secreted and primarily deg raded within the cells. The intracellular degradation of the C223R mutant w as almost completely inhibited in the presence of a proteasome inhibitor, l actacystin, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-n orleucinal, resulting in increased secretion of the C223R mutant, and thus implicating the proteasome system in this degradation process. In contrast, the sum of the amounts of the G85E mutant inside and outside the cells dec reased during the chase periods even in the presence of the proteasome inhi bitor, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleu cinal, although proteasome-specific inhibitor lactacystin and one of the cy steine protease inhibitors, E-64-d, prevented the intracellular degradation . These results suggested that intracellular degradation of G85E HRG: occur red to some extent through a hitherto unknown mechanism. Similar studies in volving recombinant mutants in which Gly85 or Cys223 was replaced with seve ral other amino acids revealed that proteins with mutations leading to the destruction of the predicted beta-sheet structure of the cystatin domains w ere eliminated by the intracellular quality control system.