Structural Biology Research Center, Photon Factory, High Energy Accelerator Research Organization (KEK)^* Institute for Molecular Bioscience, University of Queensland, Australia^** Division of Organic Chemistry, National Institute of Health Sciences (NIHS), Japan^*** Innovative Research Initiatives, Tokyo Institute of Technology, Japan^****
○Tadashi Satoh^* Nathan Cowieson^** Wataru Hakamata^*** Masaaki Kurihara^*** Hiroko Ideo^**** Ryuichi Kato^* Katsuko Yamashita^**** Soichi Wakatsuki^*

VIP36, a vesicular-integral protein of 36 kDa, is a family of Ca²⁺-dependent intracellular animal lectins such as ERGIC-53, and functions as a cargo receptor for trafficking certain glycoproteins in the secretory pathway. To investigate the structural basis for the high mannose-type glycoprotein recognition and transport by VIP36, we have determined crystal structures of the carbohydrate recognition domain (CRD) of VIP36, in the absence and presence of Ca²⁺ ion and a part of the high-mannose type glycan, i.e. α-mannose, mannobiose (Man-α-1,2-Man) and mannotriose (Man-α-1,2-Man-α-1,2-Man). We collected a 2.1 Å resolution data set of metal-free VIP36 and a 1.8 Å resolution data set of Ca²⁺-bound VIP36 on PF-AR-NW12A. Moreover, we collected a 1.8 Å resolution data set of VIP36/mannose and a 1.75 Å resolution data set of VIP36/mannobiose on PF-BL5A. As for VIP36/mannotriose, we collected a 2.0 Å resolution data set on PF-AR-NW12A. The structures were solved by the molecular replacement method using the structure of the Ca²⁺-bound p58/ERGIC-53 CRD as a search model. The CRD is composed of a seventeen-stranded antiparallel β-sandwich, and binds one Ca²⁺ adjoining the carbohydrate-binding site. The binding of Ca²⁺ arranges Asp131, Asn166 and His190 for the proper carbohydrate binding, illustrating its Ca²⁺-dependence mechanism. The mannotriose which corresponds to D1 arm of high-mannose type N-glycan is recognized by eight residues through extensive hydrogen bonds and hydrophobic interactions. Our results clearly show that VIP36 functions as a cargo receptor for high-mannose type glycoproteins, and recognizes their D1 arm of the N-glycans.