Abstract
The critical step in lysosomal targeting of soluble lysosomal enzymes is the recognition by an UDP-N-acetylglucosamine:lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase. The structure of the determinant common to all lysosomal enzymes for proper recognition by the phosphotransferase is not completely understood. Our current knowledge is largely based on the introduction of targeted amino acid substitutions into lysosomal enzymes and analysis of their effects on phosphotransferase recognition. We have investigated the effect of eight anti-arylsulfatase A monoclonal antibodies on the interaction of arylsulfatase A with the lysosomal enzyme phosphotransferase in vitro. We also show that a lysine-rich surface area of arylsulfatases A and B is essential for proper recognition by the phosphotransferase. Monoclonal antibodies bind to at least six different epitopes at different locations on the surface of arylsulfatase A. All antibodies bind outside the lysine-rich recognition area, but nevertheless Fab fragments of these antibodies prevent interaction of arylsulfatase A with the phosphotransferase. Our data support a model in which binding of arylsulfatase A to the phosphotransferase is not restricted to a limited surface area but involves the simultaneous recognition of large parts of arylsulfatase A.
Highlights
The correct lysosomal targeting of soluble lysosomal enzymes mainly depends on the synthesis of mannose 6-phosphate residues on their N-linked oligosaccharide side chains
The abbreviations used are: phosphotransferase, UDP-N-acetylglucosamine:lysosomal enzyme N-acetyl-glucosamine-1-phosphotransferase; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrotransfers N-acetylglucosamine-1-phosphate from UDP-Nacetylglucosamine to mannose residues of high mannose-type oligosaccharide side chains of lysosomal enzymes yielding N-acetylglucosamine-1-phospho-6-mannose residues
Similar conclusions have been reached for aspartylglucosaminidase, in which three lysines distantly spaced on the enzyme surface contribute to the proper recognition by the phosphotransferase (11), and for cathepsin D and L in each of which two lysines approximately 34 Å apart are critical recognition determinants (12)
Summary
Materials—Cell culture media RPMI 1640 for hybridomas and Dulbecco’s modified Eagle’s medium for all other cell types were from Life Technologies, Inc. For production of Fab fragments, purified antibodies were adjusted to a concentration of 1–5 mg in phosphate-buffered saline, 5 mM cysteine. Arylsulfatase A was incubated with a 10-fold molar excess of mAb in 20 mM Tris/HCl, pH 7.4, 0.15 M NaCl. MAb-arylsulfatase A complexes generated in liquid phase were added to the mAb-coated wells and allowed to bind for 2 h at 37 °C. If the antibody coupled to the well and complexed to the arylsulfatase A in the liquid phase compete and cannot bind arylsulfatase A simultaneously, the enzyme cannot be retained in the wells. Purified arylsulfatase A can be labeled via incubation with [35S]cysteine.2 35S-Labeled arylsulfatase A was incubated with a 10-fold molar excess of mAbs. The antibody complexed with [35S]arylsulfatase A was added to the wells at neutral pH when octamerization of arylsulfatase A does not occur. The success of the modification reaction was monitored by alterations in isoelectric focussing pattern
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