Identification of a Golgi GPI-N-acetylgalactosamine transferase with tandem transmembrane regions in the catalytic domain

  • Nat Commun. 2018 Jan 26;9(1):405. doi: 10.1038/s41467-017-02799-0.
Tetsuya Hirata  1  2  3 ,  Sushil K Mishra  4 ,  Shota Nakamura  1 ,  Kazunobu Saito  1 ,  Daisuke Motooka  1 ,  Yoko Takada  2 ,  Noriyuki Kanzawa  1  2 ,  Yoshiko Murakami  1  2 ,  Yusuke Maeda  1  2 ,  Morihisa Fujita  5 ,  Yoshiki Yamaguchi  4 ,  Taroh Kinoshita  6  7
Affiliations
  • 1. Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan.
  • 2. WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan.
  • 3. National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi, 444-8787, Japan.
  • 4. Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN Global Research Cluster, Wako, Saitama, 351-0198, Japan.
  • 5. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
  • 6. Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan. [email protected].
  • 7. WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan. [email protected].
PMID: 29374258 DOI: 10.1038/s41467-017-02799-0
Abstract

Many eukaryotic proteins are anchored to the cell surface via the glycolipid glycosylphosphatidylinositol (GPI). Mammalian GPIs have a conserved core but exhibit diverse N-acetylgalactosamine (GalNAc) modifications, which are added via a yet unresolved process. Here we identify the Golgi-resident GPI-GalNAc transferase PGAP4 and show by mass spectrometry that PGAP4 knockout cells lose GPI-GalNAc structures. Furthermore, we demonstrate that PGAP4, in contrast to known Golgi glycosyltransferases, is not a single-pass membrane protein but contains three transmembrane domains, including a tandem transmembrane domain insertion into its glycosyltransferase-A fold as indicated by comparative modeling. Mutational analysis reveals a catalytic site, a DXD-like motif for UDP-GalNAc donor binding, and several residues potentially involved in acceptor binding. We suggest that a juxtamembrane region of PGAP4 accommodates various GPI-anchored proteins, presenting their acceptor residue toward the catalytic center. In summary, we present insights into the structure of PGAP4 and elucidate the initial step of GPI-GalNAc biosynthesis.