The transfer of macrophage-secreted arylsulphatase A (ASA) to enzyme-deficient brain cells is part of the therapeutic concept of bone marrow transplantation in lysosomal storage diseases. Here we have investigated this transfer in vitro. The uptake of 125I-labelled recombinant human ASA purified from ASA-overexpressing mouse embryonic fibroblasts deficient for mannose 6-phosphate (M6P) receptors in a mouse ASA-deficient astroglial cell line was completely inhibited by M6P. In contrast, when ASA-deficient astroglial cells were incubated with secretions of [35S]methionine-labelled human macrophages or mouse microglia, containing various lysosomal enzymes, neither ASA nor cathepsin D (CTSD) were detected in acceptor cells. Co-culturing of metabolically labelled macrophages with ASA-deficient glial cells did not result in an M6P-dependent transfer of ASA or CTSD between these two cell types. In secretions of [33P]phosphate-labelled macrophages no or weakly phosphorylated ASA and CTSD precursor polypeptides were found, whereas both intracellular and secreted ASA from ASA-overexpressing baby hamster kidney cells displayed 33P-labelled M6P residues. Finally, the uptake of CTSD from secretions of [35S]methionine-labelled macrophages in rat hepatocytes was M6P-independent. These data indicated that lysosomal enzymes secreted by human macrophages or a mouse microglial cell line cannot be endocytosed by brain cells due to the failure to equip newly synthesized lysosomal enzymes with the M6P recognition marker efficiently. The data suggest that other mechanisms than the proposed M6P-dependent secretion/recapture of lysosomal enzymes might be responsible for therapeutic effects of bone marrow transplantation in the brain.
- mannose 6-phosphate
- metachromatic leucodystrophy
Abbreviations used: ASA, arylsulphatase A; ASOR, asialo orosomucoid; BHK, baby hamster kidney; BMT, bone marrow transplantation; CNS, central nervous system; CTSD, cathepsin D; DMEM, Dulbecco's modified essential medium; M6P, mannose 6-phosphate; MLD, metachromatic leucodystrophy; MPR, M6P-specific receptor; PMP-BSA, pentamannose 6-phosphate coupled to BSA; RT-PCR, reverse transcriptase PCR.
- The Biochemical Society, London ©2002