Abstract
9-O-Acetylation is one of the most common modifications of sialic acids, and it can affect several sialic acid-mediated recognition phenomena. We previously reported a cDNA encoding a lysosomal sialic acid-specific 9-O-acetylesterase, which traverses the endoplasmic reticulum-Golgi pathway and localizes primarily to lysosomes and endosomes. In this study, we report a variant cDNA derived from the same gene that contains a different 5' region. This cDNA has a putative open reading frame lacking a signal peptide-encoding sequence and is thus a candidate for the previously described cytosolic sialic acid 9-O-acetylesterase activity. Epitope-tagged constructs confirm that the new sequence causes the protein product to be targeted to the cytosol and has esterase activity. Using reverse transcription-polymerase chain reaction to distinguish the two forms of message, we show that although the lysosomal sialic acid-specific 9-O-acetylesterase message has a widespread pattern of expression in adult mouse tissues, this cytosolic sialic acid 9-O-acetylesterase form has a rather restricted distribution, with the strongest expression in the liver, ovary, and brain. Using a polyclonal antibody directed against the 69-amino acid region common to both proteins, we confirmed that the expression of glycosylated and nonglycosylated polypeptides occurred in appropriate subcellular fractions of normal mouse tissues. Rodent liver polypeptides reacting to the antibody also co-purify with previously described lysosomal sialic acid esterase activity and at least a portion of the cytosolic activity. Thus, two sialic acid 9-O-acetylesterases found in very different subcellular compartments can be encoded by a single gene by differential usage of a signal peptide-encoding exon at the N terminus. The 5'-rapid amplification of cDNA ends results and the differences in tissue-specific expression suggest that expression of these two products may be differentially regulated by independent promoters.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF156856
We have suggested that the function of the cytosolic Sia 9-O-acetylesterase (Cse) activity is to salvage any 9-O-acetylated molecules that escape the initial action of the lysosomal Sia 9-O-acetylesterase (Lse) enzyme
Two Different Types of Messages Are Encoded by the Lse Gene—During characterization of the cDNA for the lysosomal Sia 9-O-acetylesterase (Lse) [39], we cloned a novel cDNA from a pre B cell line library (70Z/3), in which the signal peptide encoding region of Lse in its 5Ј region was substituted with a novel sequence (Fig. 1)
Summary
Sialic acid; Cse, cytosolic sialic acid 9-O-acetylesterase; Lse, lysosomal sialic acid-specific 9-O-acetylesterase; RT, reverse transcription; PCR, polymerase chain reaction; ConA, sugars typically found at the nonreducing end of sugar chains of animals throughout the deuterostome lineage [1, 2]. Influenza virus C hemagglutinin requires 9-O-acetylated Sia for binding to host cells [11, 26] These examples indicate that O-acetylation may be a key modification regulating Sia-dependent recognition events. Cytosolic Sialic Acid 9-O-Acetylesterase the tissue- and stage-specific expression of this modification [30] These labile Golgi enzymes have so far proven refractory to purification or cloning. It would not have access to free 9-O-acetylated Sias that have been reported in the cytosolic fraction These presumably result from the action of lysosomal sialidases on 9-Oacetylated Sias, followed by export of such molecules to the cytosol by the action of the lysosomal Sia exporter [15, 16]. We have suggested that the function of the cytosolic Sia 9-O-acetylesterase (Cse) activity is to salvage any 9-O-acetylated molecules that escape the initial action of the Lse enzyme.
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