BIOCHEMICAL-PROPERTIES OF THE MONOMERIC MUTANT OF HUMAN CATHEPSIN-E EXPRESSED IN CHINESE-HAMSTER OVARY CELLS - COMPARISON WITH DIMERIC FORMS OF THE NATURAL AND RECOMBINANT CATHEPSIN-E

Cathepsin E (CE) is the only known aspartic proteinase that exists as a homodimer consisting of two fully catalytically active monomers, whi ch are covalently bound by a disulfide bond between two cysteine resid ues at the NH2-terminal region (Cys(43) in human pro-CE). To understan d the physiological significance of the dimer formation, the monomeric mutant of human CE was constructed by site-directed mutagenesis (Cys( 43)-->Ser(43)) and expressed in Chinese hamster ovary (CHO) cells. Imm unolocalization of the mutant protein at both the light and electron m icroscopic levels revealed the monomeric CE to be associated predomina ntly with the endoplasmic reticulum and the non-lysosomal endocytic or ganelles. The cellular localization of the monomeric protein was compa tible with that of the wild-type (dimeric form) of recombinant human C E expressed in the same cells. The monomeric protein was generated pri marily as the 46-kDa pro-CE with a high-mannose-type oligosaccharide c hain in the cells. In addition to the maximal activation at around pH 3.5, a substantial proportion of the monomeric pro-CE was converted to the mature form by incubation at pH7 and 37 degrees C for 5 min. In c ontrast, the dimeric pro-CE was scarcely activated by treatment at pH7 . Although catalytic properties of the in vitro-activated monomeric CE appeared to be indistinguishable from those of the dimeric forms of n atural and recombinant CE, the monomeric form was more unstable to pH and temperature changes than these dimeric forms. These results indica te that the dimerization of CE is not necessarily required for proper folding to express activity, correct intracellular localization and ca rbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo.