Structural, immunological, and biosynthetic studies of a sialic acid-specific O-acetylesterase from rat liver.

We have previously described a membrane-associated intralumenal sialic acid-specific 9-O-acetylesterase (LSE) from rat liver (Higa, H. H., Manzi, A., and Varki, A. (1989) J. Biol. Chem. 264, 19435-19442). Unlike a cytosolic sialate: O-acetylesterase (CSE) with similar specificity, the LSE carries N-linked oligosaccharides. A polyclonal monospecific antibody against homogenous LSE does not cross-react with the CSE. Monoclonal antibodies distinguish between the LSE and another N-glycosylated esterase that tends to partially co-purify with it. Amino-terminal sequencing of the LSE subunits indicates that it is distinct from previously described esterases and shows no homology to any other known proteins. In contrast, the esterase that partially co-purifies is similar but not identical to previously described "microsomal" esterases from rat liver. The LSE is also expressed in several hepatoma cell lines. Pulse-chase studies indicate that the two LSE subunits arise from a single precursor of approximately 65 kDa which yields a core polypeptide of apparent molecular mass approximately 53 kDa upon deglycosylation with peptide: N-glycosidase F. The protein quickly becomes partly resistant to endo-beta-N-acetylglucosaminidase H but remains sensitive to peptide: N-glycosidase F, indicating N-linked oligosaccharide processing during passage through the Golgi. After several hours, the precursor undergoes proteolysis, generating the mature heterodimeric protein of approximately 58 kDa, with subunits of approximately 38 and approximately 28 kDa. A portion of newly synthesized LSE is secreted into the medium intact, indicating that the cleavage normally takes place after diversion from the secretory pathway. These temporal changes and precursor-product distribution are reminiscent of some lysosomal acid hydrolases. In fact, immunofluorescence studies and Triton WR-1339 shift experiments suggest a lysosomal localization for this enzyme. Additional evidence for this, and the role of the LSE in O-acetylated sialic acid turnover are discussed in the accompanying paper (Butor, C., Diaz, S., and Varki, A. (1993) J. Biol. Chem. 268, 10197-10206).