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

Unlike a cytosolic sia1ate: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 -65 kDa which yields a core polypeptide of apparent molecular mass -53 kDa upon deglycosylation with peptide:Nglycosidase F. The protein quickly becomes partly resistant to endo-B-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 -58 kDa, with subunits of -38 and -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 0-acetylated sialic acid turnover are discussed in the accompanying paper (Butor, C.,