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Members of the GalNAc-T family of enzymes utilize distinct Golgi localization mechanisms.

ABSTRACT: Mucin-type O-glycosylation is an evolutionarily conserved and essential post-translational protein modification that is initiated in the Golgi apparatus by a family of enzymes known as the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). GalNAc-Ts are type II membrane proteins which contain short N-terminal tails located in the cytoplasm, a transmembrane domain that crosses the Golgi membrane, to which is connected a stem region that tethers the C-terminal catalytic and lectin domains that reside in the Golgi lumen. Although mucin-type O-glycans have been shown to play critical roles in numerous biological processes, little is known about how the GalNAc-Ts are targeted to their site of action within the Golgi complex. Here, we investigate the essential protein domains required for Golgi localization of four representative members of the GalNAc-T family of enzymes. We find that GalNAc-T1 and -T2 require their cytoplasmic tail and transmembrane domains for proper Golgi localization, while GalNAc-T10 requires its transmembrane and luminal stem domains. GalNAc-T7 can use either its cytoplasmic tail or its luminal stem, in combination with its transmembrane domain, to localize to the Golgi. We determined that a single glutamic acid in the GalNAc-T10 cytoplasmic tail inhibits its ability to localize to the Golgi via a cytoplasmic tail-dependent mechanism. We therefore demonstrate that despite their similarity, different members of this enzyme family are directed to the Golgi by more than one set of targeting signals.

SUBMITTER: Becker JL

PROVIDER: S-EPMC6192461 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Members of the GalNAc-T family of enzymes utilize distinct Golgi localization mechanisms.

Becker Jessica L JL Tran Duy T DT Tabak Lawrence A LA

Glycobiology 20181101 11

Mucin-type O-glycosylation is an evolutionarily conserved and essential post-translational protein modification that is initiated in the Golgi apparatus by a family of enzymes known as the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). GalNAc-Ts are type II membrane proteins which contain short N-terminal tails located in the cytoplasm, a transmembrane domain that crosses the Golgi membrane, to which is connected a stem region that tethers the C-terminal catalytic and lec ...[more]

PMID: 30084948

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Project description:The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na+ or Mg2+, analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity.IMPORTANCE The Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically distinct from other enzyme families; however, to date the structure of just two, physiologically identical, enzymes from this family has been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that members of the CyAH family have the unusual Toblerone fold, albeit with some significant differences.

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Members of the GalNAc-T family of enzymes utilize distinct Golgi localization mechanisms.

Publications

Members of the GalNAc-T family of enzymes utilize distinct Golgi localization mechanisms.

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