Isolation and Partial Characterization of Oligosaccharides on Beta-Galactosidase (Lysosomal Enzymes).

Abstract

Mannose 6-phosphate residues have been identified on the carbohydrate chains of several lysosomal enzymes. Studies by others suggest that these mannose 6-phosphate residues play a major role in receptor-mediated uptake and intracellular transport of lysosomal enzymes by cultured cells. This dissertation is concerned primarily with the isolation and partial characterization of the oligosaccharides present on purified bovine testicular (beta)-galactosidase that contain covalently-bound mannose 6-phosphate residues. Forty percent of the oligosaccharides present on (beta)-galactosidase were released by endo-(beta)-N-acetylglucosaminidase H. The resistence of the remaining oligosaccharides to enzymatic deglycosylation suggests that 60% of the oligosaccharides on (beta)-galactosidase are of the complex type. The released oligosaccharides were reductively labeled with NaB('3)H(,4), and resolved into seven fractions by sequential gel filtration (P-6) and ion exchange (QAE-Sephadex). Each of the oligosaccharide fractions was characterized with respect to charge, size, number of anionic residues per oligosaccharide and ability to inhibit binding of (beta)-galactosidase to the receptor. Neutral oligosaccharides and those bearing two sialic acid residues did not inhibit the binding of (beta)-galactosidase to the receptor. The most inhibitory oligosaccharides isolated contained two mannose 6-phosphate residues (K(,i) = 4 x 10('-7)M); a similar K(,i) (6 x 10('-7)M) was observed if one of the phosphates was present in diester linkage with N-acetylglucosamine. The inhibition constant for oligosaccharides bearing a single mannose 6-phosphate residue was 14 x 10('-7)M. These results indicate that oligosaccharides bearing two phosphate residues are more inhibitory than those carrying a single phosphate. In addition to the species described above, hybrid oligosaccharides bearing a single sialic acid residue or mannose 6-phosphate and a sialic acid residue were identified. The results of high performance liquid chromatography studies indicated that the oligosaccharides isolated contained predominantly seven mannose residues. High mannose oligosaccharides containing five to nine mannose residues were also present as minor components of each fraction. The above described studies present the isolation and partial characterization of the hybrid and high mannose oligosaccharides which are present on (beta)-galactosidase. These results enhance our underst and ing of the mechanism(s) by which the phosphomannosyl receptor binds to lysosomal enzymes and may ultimately be of use in the study of the intracellular transport of lysosomal enzymes.