The relationship between ST6Gal I Golgi retention and its cleavage-secretion

Abstract

The ST6Gal I is a sialyltransferase that modifies N-linked oligosaccharides of glycoproteins. Previous results suggested a role for luminal stem and active domain sequences in the efficiency of ST6Gal I Golgi retention. Characterization of a series of STtyr isoform deletion mutants demonstrated that the stem is sensitive to proteases and that preventing cleavage in this region leads to increased cell surface expression. A mutant lacking amino acids 32-104 (STDelta4) is not active or cleaved and secreted like the wild type STtyr, but does exhibit increased cell surface expression. It is probable that the STDelta4 mutant lacks the stem region and some amino acids of the active domain because the STDelta5 mutant lacking amino acids 86-104 is also not active but is cleaved and secreted. In contrast, deletion of stem amino acids between residues 32 and 86 in the STDelta1, STDelta2, and STDelta3 mutants does not inactive these enzyme forms, eliminate their cleavage and secretion, or increase their cell surface expression. Surprisingly, cleavage occurs even though the previously identified Asn63-Ser 64 cleavage site is missing. Further evaluation demonstrated that a cleavage site between Lys 40 and Glu 41 is used in COS cells. Mutagenesis of Lys 40 significantly decreased, but did not eliminate cleavage, suggesting that there are additional secondary sites of cleavage in the ST6Gal I stem.

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“…1A; ref. 22), we analyzed the effects of amino acid substitutions in the region from Leu-37 to Glu-41 on ST6Gal I secretion. COS cells were transiently transfected with a series of mutant ST6Gal I cDNA, metabolically labeled, and chased.…”

“…For transient transfection experiment, ST6Gal I-pSVL and ST6Gal I FLAG-pSVL were constructed as described previously (22). For a stable expression experiment, ST6Gal I FLAG-pcDNA3.1 was constructed by excision of ST6Gal I FLAG from ST6Gal I-pBluescript with BamHI and XhoI, followed by ligation into the BamHI and XhoI sites of pcDNA3.1 (Invitrogen).…”

“…Metabolic labeling of cells and immunoprecipitation of expressed proteins were performed essentially as previously described (22,25). COS cells were cotransfected with rat ST6Gal I and either human BACE1 cDNA or vector alone.…”

Formation of Insoluble Oligomers Correlates with ST6Gal I Stable Localization in the Golgi

“…7). 3 However, it was also not unlikely that the presence of a free Cys would lead to additional disulfide bond formation between adjacent ST monomers or even other Golgi proteins. Another mutant of the STtyr, in which Cys 139 is converted to a Ser (STtyr C139S), also demonstrated reduced cleavage and secretion (Fig.…”

“…The presence of low levels of the STtyr protein at the cell surface also supports the idea that the STtyr is only transiently localized in the Golgi. In addition, it is unlikely that the presence of the Cys at position 123 eliminates cleavage per se because the major cleavage site is found in the stem region between Lys 40 and Glu 41 and because both isoforms are cleaved and secreted from Chinese hamster ovary cells (2,3). This latter observation also suggests that the ST6Gal I isoforms and/or the proteases responsible for cleavage are localized differently in different cell types.…”