Poster Presentation 51st Lorne Proteins Conference 2026

Computational studies of PA14 of LacdiNAc synthase B4GALNT3 as a lectin domain that binds to sulfated glycan ligands (#404)

Bakhtyar Sepehri 1 , Yuko Tokoro 2 , Takahiro Yamasaki 2 , Hiroaki Tateno 3 4 , Yuji O Kamatari 5 , Tomohiro Sensui 6 , Hiroto Kawashima 6 , Yasuhiko Kizuka 2 , Robert J Doerksen 1
  1. University of Mississippi, University, MS, United States
  2. Institute for Glyco-core Research (iGCORE), Gifu University, Gifu, Japan
  3. Cellular and Molecular Biotechnology Research Institute, Multicellular System Regulation Research Group, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki , Japan
  4. Collaborative Graduate School, University of Tsukuba, Tsukuba, Ibaraki , Japan
  5. Life Science Research Center, Institute for Advanced Study, Gifu University, Gifu, Japan
  6. Laboratory of Microbiology and Immunology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

Mammalian proteins are decorated with a variety of glycans, providing proteins with enormous functional diversity. GalNAcβ1-4GlcNAc (LacdiNAc or LDN), a unique sub-terminal glycan structure regulating the half-life of circulating glycoproteins, is biosynthesized by the dedicated glycosyltransferases, B4GALNT3 and B4GALNT4. We recently reported that B4GALNT3 contains a unique non-catalytic PA14 domain that is necessary for the enzyme activity, while the precise function of PA14 is unclear. Here we performed docking and molecular dynamics (MD) simulations to explore PA14 in B4GALNT3 as a lectin domain required for the activity of B4GALNT3 toward glycoprotein substrates.

Using AlphaFold, we predicted the structure of the PA14 domain of human B4GALNT3 with Ca2+. D189, D190, Q251, N252, and E254 exhibited strong interactions with the Ca2+ ion. We used GlycoTorch Vina to dock L4 or LacNAc with B4GALNT3 PA14, and used selected docked poses as starting points for MD simulations, based on comparison to X-ray crystal structures of langerin (PDB: 3P5I) and sulfatase (PDB: 7OZE) in complex with sulfated glycans. In the MD simulations, LacNAc dissociated rapidly from PA14 as did L4 from one of its docked poses. However, in the other L4 pose, starting with the Gal sulfate interacting with W218 and K226 and the GlcNAc sulfate interacting with N252, stable interaction poses were obtained in three separate 1 microsecond simulations, with the GlcNAc sulfate interaction maintained but the Gal sulfate interaction significantly changed.

In associated research submitted for publication, glycan microarray experiments, surface plasmon resonance and in vitro activity studies of B4GALNT3, including key mutants, and its interactions with and actions on sulfated and nonsulfated glycans, combined with the current computational results, present a comprehensive picture that recognition of sulfated glycan ligands by B4GALNT3’s PA14 domain negatively regulates its activity, highlighting a novel regulation mechanism for LDN synthesis mediated by a lectin domain.