Rat liver chitobiase: Purification, properties, and role in the lysosomal degradation of Asn-linked glycoproteins

Abstract

Chitobiase, the lysosomal glycosidase responsible for splitting the GlcNAcβ- d-(1–4) GlcNAc moiety in Asn-linked glycoproteins, was purified over 600-fold from frozen rat livers utilizing an assay with di- N-acetylchitobiose as the substrate. The final preparation showed a major polypeptide of M r 43,000 (sodium dodecylsulfate-polyacrylamide gel electrophoresis) that was determined to be the chitobiase by an immunological method. The purified chitobiase also hydrolyzed tri- and tetrasaccharides of chitin, which like di- N-acetylchitobiose were not substrates if first reduced by NaBH 4. The initial products formed during hydrolysis of the tetrasaccharide were trisaccharide and GlcNAc. These results imply that chitobiase is a “reducing-end exohexosaminidase” which cleaves single GlcNAc units only from the reducing end of oligosaccharides. Fucose, typically found linked to the reducing-end GlcNAc in complex oligosaccharide chains, was found to block this reaction. Additional substrates that were hydrolyzed included GlcNAcβ- d-(1–4)MurNAc, the repeating structure from bacterial cell wall peptidoglycan, and the Manβ- d-(1–4)GlcNAcβ- d-(1–4)GlcNAc reducing-end component of glycoproteins. K m and V m for hydrolysis of these substrates were of similar magnitude as for di- N-acetylchitobiose (6.3 m m and 15 μmol/min/mg protein, respectively). Liver tissues from nine mammalian species were surveyed for the presence of chitobiase activity. The activity was found in rat, mouse, rabbit, and guinea pig liver (Stirling [(1974) FEBS Lett. 39, 171–175] previously observed the enzyme in human liver), but not in dog, sheep, pig, cat, and cow liver. The presence or absence of chitobiase so far observed was found to exactly correlate with the type of oligosaccharide fragments found to accumulate in animals containing genetic or inhibitor-induced lysosomal storage pathologies. The presence of the chitobiase corresponds to the occurrence of one GlcNAc unit at the reducing end of stored oligosaccharides, while the absence of this glycosidase yields fragments with an intact GlcNAcβ- d-(1–4)GlcNAc moiety. These results verify our previous proposal that lysosomal disassembly of glycoproteins to free amino acids and sugars is an ordered, bidirectional pathway in which chitobiase (when present) catalyzes the last step during digestion of the protein-oligosaccharide linkage region.