LYSOSOMAL SEGREGATION OF A MANNOSE-RICH GLYCOPROTEIN IMPARTED BY THE PROSEQUENCE OF MYELOPEROXIDASE

The role of the N-terminal sequence of myeloperoxidase in the intracel lular targeting was examined by using glycosylated lysozyme as a repor ter. A fusion protein was constructed in which the presequence residue s -18 through -6 of the lysozyme moiety had been replaced by residues 1-158 of prepromyeloperoxidase. Expression of the fusion protein in Ch inese hamster ovary cells demonstrated its partial secretion and parti al intracellular retention. The latter was accompanied by trimming the myeloperoxidase prosequence off the lysozyme moiety. The rate of the retention of the lysozyme fusion protein was higher than that of glyco sylated lysozyme that had been expressed in cells transfected with cDN A of glycosylated lysozyme. The retention was insensitive to NH4Cl. In the secreted protein, lysozyme contained predominantly complex oligos accharides as demonstrated by a proteolytic fragmentation in vitro and resistance to endo-beta-N-acetylglucosaminidase H. In contrast, when targeted to lysosomes, the lysozyme moiety of the fusion protein conta ined predominantly mannose-rich oligosaccharides. In baby hamster kidn ey cells, the trimming of the oligosaccharides in the lysozyme fragmen t was less vigorous, and a selective targeting of molecules bearing ma nnose-rich oligosaccharides to lysosomes was more apparent than in Chi nese hamster ovary cells. In the presence of monensin, the formation o f complex oligosaccharides in the fusion protein and its secretion wer e strongly inhibited, whereas the intracellular fragmentation was not. We suggest that the prosequence of myeloperoxidase participates in th e intracellular routing of the precursor and that this routing operate s on precursors bearing mannose-rich rather than terminally glycosylat ed oligosaccharides and diverts them from the secretory pathway at a s ite proximal to the monensin-sensitive compartment of the Golgi appara tus. J. Cell. Biochem. 71:158-168, 1998. (C) 1998 Wiley-Liss, Inc.