Jun Kamishikiryo

Structure‐guided identification of a new catalytic motif of oligosaccharyltransferase

Asn‐glycosylation is widespread not only in eukaryotes but also in archaea and some eubacteria. Oligosaccharyltransferase (OST) catalyzes the co‐translational transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. Here, we report that a thermophilic archaeon, Pyrococcus furiosus OST is composed of the STT3 protein alone, and catalyzes the transfer of a heptasaccharide, containing one hexouronate and two pentose residues, onto peptides in an Asn‐X‐Thr/Ser‐motif‐dependent manner. We also determined the 2.7‐Å resolution crystal structure of the C‐terminal soluble domain of Pyrococcus STT3. The structure‐based multiple sequence alignment revealed a new motif, DxxK, which is adjacent to the well‐conserved WWDYG motif in the tertiary structure. The mutagenesis of the DK motif residues in yeast STT3 revealed the essential role of the motif in the catalytic activity. The function of this motif may be related to the binding of the pyrophosphate group of lipid‐linked oligosaccharide donors through a transiently bound cation. Our structure provides the first structural insights into the formation of the oligosaccharide–asparagine bond.

Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM

Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a β-sheet of the membrane-distal ectodomain of SLAM using the side of its β-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their β-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H–SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

Comparative Structural Biology of Eubacterial and Archaeal Oligosaccharyltransferases

Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes N-glycosylation. We report the 2.8 Å resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria.

Structural analysis for glycolipid recognition by the C-type lectins Mincle and MCL.

Significance Here we report the crystal structures of human C-type lectin receptors Mincle (macrophage inducible C-type “calcium-dependent” lectin; CLEC4E) and MCL (macrophage C-type lectin; CLEC4D), both of which are receptors for mycobacterial glycolipid adjuvant cord factor (also called trehalose-6,6′-dimycolate; TDM). Our structural and functional studies clearly reveal the simultaneous recognition of sugar and lipid moieties by Mincle and MCL, distinct from other C-type lectin receptors. Because better adjuvants are desired for enhancing vaccination effects of medical treatments for infectious diseases, cancer, and so forth, these structures provide a framework for the rational design of more effective adjuvants than TDM. Mincle [macrophage inducible Ca2+-dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6′-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor–TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle–citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca2+-dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.

Molecular Basis for LLT1 Protein Recognition by Human CD161 Protein (NKRP1A/KLRB1)

Human Th17 cells express high levels of CD161, a member of the killer cell lectin-like receptor (KLR) family (also referred to as NK receptor-P1A (NKRP1A) or KLRB1), as a representative marker. CD161 is also expressed on natural killer (NK) cells and NKT cells. Lectin-like transcript 1 (LLT1), another KLR family member, was recently identified as a ligand for CD161. This interaction may play pivotal roles in the immunomodulatory functions of Th17 cells as well as those of NK and NKT cells. However, the molecular basis for the interaction is poorly understood. Here we show that the extracellular domain of CD161 bound directly to LLT1 with a Kd of 48 μm and with the fast kinetics typical of cell-cell recognition receptors. Mutagenesis revealed that the similar membrane-distal β-sheet and loop regions of both CD161 and LLT1 were utilized for the binding, and notably, these regions correspond to the ligand-binding sites for major histocompatibility complex (MHC)-recognizing KLRs. Furthermore, we found a pair of detrimental mutations for both molecules that restored the binding. These results reveal a new template model for the recognition mode between the KLR family members and provide insights into the molecular mechanism underlying Th17/NK/NKT-mediated immune responses.

Purification, crystallization and preliminary X-ray diffraction studies of the soluble domain of the oligosaccharyltransferase STT3 subunit from the thermophilic archaeon Pyrococcus furiosus.

Oligosaccharyltransferase catalyzes the transfer of preassembled oligosaccharides onto asparagine residues in nascent polypeptide chains. The STT3 subunit is thought to bear the catalytic site. The C-terminal domain of the STT3 protein of Pyrococcus furiosus was expressed in Escherichia coli cells. STT3 protein prepared from two different sources, the soluble fraction and the inclusion bodies, produced crystals that diffracted to 2.7 A. During crystallization screening, cocrystals of P. furiosus STT3 with an E. coli 50S ribosomal protein, L7/L12, were accidentally obtained. This cross-species interaction is not biologically relevant, but may be used to design a built-in polypeptide substrate for the STT3 crystals.

HLA-G Molecule

Human leukocyte antigen-G (HLA-G) is a non-classical HLA class I molecule, which was first discovered in 1987 by Geraghty and colleagues. While classical HLA class I molecules are expressed on all nucleated cells, the expression of the HLA-G molecule is highly tissue-restricted, such as to placental trophoblast cells. HLA-G binds inhibitory receptors such as leukocyte immunoglobulin-like receptors B1 (LILRB1/ILT2/CD85j) and LILRB2 (ILT4/CD85d), which are widely expressed on immune cells, to suppress a broad range of immune responses. Thus, the expression of HLA-G in placenta protects the fetus from the maternal immune system. On the other hand, emerging studies have shown the relevance of the HLA-G molecule in pathologic conditions, such as transplantation rejection, autoimmunity, and cancer. HLA-G has other unique characteristics, in contrast with classical HLA molecules, including the existence of various forms of HLA-G: several splice variants, subunit-deficient conformations, homodimers, and their combinations have been found. In this review, we highlight the molecular basis for the tolerogenic ability of the HLA-G molecule, especially by LILR recognition of various forms of HLA-G. We also discuss the potential clinical applications of HLA-G molecules.

N-terminal domain of the cholesterol transporter Niemann–Pick C1-like 1 (NPC1L1) is essential for α-tocopherol transport

Both cholesterol and α-tocopherol are essential lipophilic nutrients for humans and animals. Although cholesterol in excess causes severe problems such as coronary heart disease, it is a necessary component of cell membranes and is the precursor for the biosynthesis of steroid hormones and bile acids. Niemann-Pick C1-like 1 (NPC1L1) is a cholesterol transporter that is highly expressed in the small intestine and liver in humans and plays an important role in cholesterol homeostasis. Cholesterol promotes NPC1L1 endocytosis, which is an early step in cholesterol uptake. Furthermore, α-tocopherol is the most active form of vitamin E, and sufficient amounts of vitamin E are critical for health. It has been reported that NPC1L1 mediates α-tocopherol absorption; however, the mechanisms underlying this process are unknown. In this study, we found that treatment of cells that stably express NPC1L1-GFP with α-tocopherol promotes NPC1L1 endocytosis, and the NPC1L1 inhibitor, ezetimibe, efficiently prevents the α-tocopherol-induced endocytosis of NPC1L1. Cholesterol binding to the N-terminal domain (NTD) of NPC1L1 (NPC1L1-NTD) is essential for NPC1L1-mediated cholesterol absorption. We found that α-tocopherol competitively binds NPC1L1-NTD with cholesterol. Furthermore, when cells stably expressed NPC1L1ΔNTD-GFP, α-tocopherol could not induce the endocytosis of NPC1L1ΔNTD. Taken together, these results demonstrate that NPC1L1 recognizes α-tocopherol via its NTD and mediates α-tocopherol uptake through the same mechanism as cholesterol absorption.

Crystal structure of extracellular domain of human lectin‐like transcript 1 (LLT1), the ligand for natural killer receptor‐P1A

Emerging evidence has revealed the pivotal roles of C‐type lectin‐like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor‐P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin‐like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A‐LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor‐binding face of the C‐type lectin‐like domain is flat, and forms an extended β‐sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte‐associated C‐type lectin, which binds to the CTLR member, NKp65. A LLT1‐NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte‐associated C‐type lectin‐NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.

Effect of quercetin on the transport of N-acetyl 5-aminosalicylic acid.

The aim of this study was to investigate the transporter‐mediated transport of N‐acetyl 5‐aminosalicylic acid (Ac‐5‐ASA) and the effect of quercetin on Ac‐5‐ASA transport.