Key contributions of a glycolipid to membrane protein integration.

Regulation of membrane protein integration involves molecular devices such as Sec-translocons or the insertase YidC. We have identified an integration-promoting factor in the inner membrane of Escherichia coli called membrane protein integrase (MPIase). Structural analysis revealed that, despite its enzyme-like name, MPIase is a glycolipid with a long glycan comprising N-acetyl amino sugars, a pyrophosphate linker, and a diacylglycerol (DAG) anchor. Additionally, we found that DAG, a minor membrane component, blocks spontaneous integration. In this review, we demonstrate how they contribute to Sec-independent membrane protein integration in bacteria using a comprehensive approach including synthetic chemistry and biophysical analyses. DAG blocks unfavorable spontaneous integrations by suppressing mobility in the membrane core, whereas MPIase compensates for this. Moreover, MPIase plays critical roles in capturing a substrate protein to prevent its aggregation, attracting it to the membrane surface, facilitating its insertion into the membrane, and delivering it to other factors. The combination of DAG and MPIase efficiently regulates the integration of membrane proteins.

Structures, functions, and syntheses of glycero-glycophospholipids.

Biological membranes consist of integral and peripheral protein-associated lipid bilayers. Although constituent lipids vary among cells, membrane lipids are mainly classified as phospholipids, glycolipids, and sterols. Phospholipids are further divided into glycerophospholipids and sphingophospholipids, whereas glycolipids are further classified as glyceroglycolipids and sphingoglycolipids. Both glycerophospholipids and glyceroglycolipids contain diacylglycerol as the common backbone, but their head groups differ. Most glycerolipids have polar head groups containing phosphate esters or sugar moieties. However, trace components termed glycero-glycophospholipids, each possessing both a phosphate ester and a sugar moiety, exist in membranes. Recently, the unique biological activities of glycero-glycophospholipids have attracted considerable attention. In this review, we describe the structure, distribution, function, biosynthesis, and chemical synthetic approaches of representative glycero-glycophospholipids-phosphatidylglucoside (PtdGlc) and enterobacterial common antigen (ECA). In addition, we introduce our recent studies on the rare glycero-glyco"pyrophospho"lipid, membrane protein integrase (MPIase), which is involved in protein translocation across biomembranes.

Identification, Chemical Synthesis, and Sweetness Evaluation of Rhamnose or Xylose Containing Steviol Glycosides of Stevia (Stevia rebaudiana) Leaves.

Steviol glycosides obtained from Stevia rebaudiana leaves are increasingly used in the food industry as natural low-calorie sweeteners. Among them, the sweetness of major glycosides composed of glucose residues (e.g., stevioside and rebaudioside A) has been widely studied. However, the properties of minor natural products containing rhamnose or xylose residues are poorly investigated. In this study, five unreported steviol glycosides containing rhamnose or xylose were extracted from our developing stevia leaves, and their sweetness was evaluated. The highly glycosylated steviol glycosides were identified, and their structures were examined by fragmentation analysis using mass spectrometry. Chemical synthesis of these glycosides confirmed their structures and allowed sensory evaluation of minor steviol glycosides. Our study revealed that a xylose-containing glycoside, rebaudioside FX1, exhibits a well-balanced sweetness, and thus, it is a promising candidate for natural sweeteners used in the food industry.

Structural Requirements of a Glycolipid MPIase for Membrane Protein Integration.

MPIase is a glycolipid involved in membrane protein integration in the inner membrane of Escherichia coli. To overcome the trace amounts and heterogeneity of natural MPIase, we systematically synthesized MPIase analogs. Structure-activity relationship studies revealed the contribution of distinctive functional groups and the effect of the MPIase glycan length on membrane protein integration activity. In addition, both the synergistic effects of these analogs with the membrane chaperone/insertase YidC, and the chaperone-like activity of the phosphorylated glycan were observed. These results verified the translocon-independent membrane integration mechanism in the inner membrane of E. coli, in which MPIase captures the highly hydrophobic nascent proteins via its characteristic functional groups, prevents protein aggregation, attracts the proteins to the membrane surface, and delivers them to YidC in order to regenerate its own integration activity.

Identification of a tomato UDP-arabinosyltransferase for airborne volatile reception.

Volatiles from herbivore-infested plants function as a chemical warning of future herbivory for neighboring plants. (Z)-3-Hexenol emitted from tomato plants infested by common cutworms is taken up by uninfested plants and converted to (Z)-3-hexenyl ß-vicianoside (HexVic). Here we show that a wild tomato species (Solanum pennellii) shows limited HexVic accumulation compared to a domesticated tomato species (Solanum lycopersicum) after (Z)-3-hexenol exposure. Common cutworms grow better on an introgression line containing an S. pennellii chromosome 11 segment that impairs HexVic accumulation, suggesting that (Z)-3-hexenol diglycosylation is involved in the defense of tomato against herbivory. We finally reveal that HexVic accumulation is genetically associated with a uridine diphosphate-glycosyltransferase (UGT) gene cluster that harbors UGT91R1 on chromosome 11. Biochemical and transgenic analyses of UGT91R1 show that it preferentially catalyzes (Z)-3-hexenyl ß-D-glucopyranoside arabinosylation to produce HexVic in planta.

A bacterial glycolipid essential for membrane protein integration.

The proper conformation and orientation of membrane protein integration in cells is an important biological event. Interestingly, a new factor named MPIase (membrane protein integrase) was proven essential in this process in Escherichia coli, besides proteinaceous factors, such as Sec translocons and an insertase YidC. A combination of spectroscopic analyses and synthetic work has revealed that MPIase is a glycolipid despite its enzyme-like activity. MPIase has a long glycan chain comprised of repeating trisaccharide units, a pyrophosphate linker, and a diacylglycerol anchor. In order to determine the mechanism of its activity, we synthesized a trisaccharyl pyrophospholipid termed mini-MPIase-3, a minimal unit of MPIase, and its derivatives. A significant activity of mini-MPIase-3 indicated that it involves an essential structure for membrane protein integration. We also analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate protein using physicochemical methods. The structure-activity relationship studies demonstrated that the glycan part of MPIase prevents the aggregation of substrate proteins, and the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles for interactions with substrate proteins. MPIase serves at an initial step in the Sec-independent integration, whereas YidC, proton motive force, and/or SecYEG cooperatively function(s) with MPIase at the following step in vivo. Furthermore, depletion of the biosynthetic enzyme demonstrated that MPIase is crucial for membrane protein integration and cell growth. Thus, we elucidated new biological functions of glycolipids using a combination of synthetic chemistry, biochemistry, physicochemical measurements, and molecular-biological approaches.

Multimatrix Variation Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry as a Tool for Determining the Bonding of Nitrogen Atoms in Alkaloids.

The reactivity of alkaloids in dehydrogenation was investigated using multimatrix variation matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of over 20 different alkaloids with six matrices. The dehydrogenated molecular ions [M - H]+ generated by in-source decay were detected in the MALDI mass spectra of some types of alkaloids such as reserpine. The dehydrogenation proceeded at the cyclic tertiary amine rather than double-bonded nitrogen atoms and indole rings involved in the electron-delocalized systems. The stable protonated primary amines hindered dehydrogenation. The laser-induced dehydrogenation correlated with the chemical properties and structures of alkaloids. Alkaloids were classified into three types by the ratio of dehydrogenation by comparing the relative abundances of [M - H]+, [M]•+, and [M + H]+ ions in α-cyano-4-hydroxycinnamic acid and 5-formylsalicylic acid matrices. Structural isomers were also discriminated by this method of analyzing the three molecular ions' ratio using multimatrix variation MALDI-MS.

Interaction between glycolipid MPIase and proteinaceous factors during protein integration into the cytoplasmic membrane of E. coli.

Protein integration into biomembranes is an essential biological phenomenon common to all organisms. While various factors involved in protein integration, such as SRP, SecYEG and YidC, are proteinaceous, we identified a glycolipid named MPIase (Membrane Protein Integrase), which is present in the cytoplasmic membrane of E. coli. In vitro experiments using inverted membrane vesicles prepared from MPIase-depleted strains, and liposomes containing MPIase showed that MPIase is required for insertion of a subset of membrane proteins, which has been thought to be SecYEG-independent and YidC-dependent. Also, SecYEG-dependent substrate membrane proteins require MPIase in addition. Furthermore, MPIase is also essential for insertion of proteins with multiple negative charges, which requires both YidC and the proton motive force (PMF). MPIase directly interacts with SecYEG and YidC on the membrane. MPIase not only cooperates with these factors but also has a molecular chaperone-like function specific to the substrate membrane proteins through direct interaction with the glycan chain. Thus, MPIase catalyzes membrane insertion by accepting nascent membrane proteins on the membrane through its chaperone-like function, i.e., direct interaction with the substrate proteins, and then MPIase functionally interacts with SecYEG and YidC for substrate delivery, and acts with PMF to facilitate and complete membrane insertion when necessary. In this review, we will outline the mechanisms underlying membrane insertion catalyzed by MPIase, which cooperates with proteinaceous factors and PMF.

Role of a bacterial glycolipid in Sec-independent membrane protein insertion.

Non-proteinaceous components in membranes regulate membrane protein insertion cooperatively with proteinaceous translocons. An endogenous glycolipid in the Escherichia coli membrane called membrane protein integrase (MPIase) is one such component. Here, we focused on the Sec translocon-independent pathway and examined the mechanisms of MPIase-facilitated protein insertion using physicochemical techniques. We determined the membrane insertion efficiency of a small hydrophobic protein using solid-state nuclear magnetic resonance, which showed good agreement with that determined by the insertion assay using an in vitro translation system. The observed insertion efficiency was strongly correlated with membrane physicochemical properties measured using fluorescence techniques. Diacylglycerol, a trace component of E. coli membrane, reduced the acyl chain mobility in the core region and inhibited the insertion, whereas MPIase restored them. We observed the electrostatic intermolecular interactions between MPIase and the side chain of basic amino acids in the protein, suggesting that the negatively charged pyrophosphate of MPIase attracts the positively charged residues of a protein near the membrane surface, which triggers the insertion. Thus, this study demonstrated the ingenious approach of MPIase to support membrane insertion of proteins by using its unique molecular structure in various ways.

Fragmentation and Ionization Efficiency of Positional and Functional Isomers of Paeoniflorin Derivatives in Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Paeoniflorin and albiflorin, which are functional isomers, are the major constituents of an herbal medicine derived from Paeonia lactiflora. Those functional isomers and their galloylated derivatives, which are positional isomers, were studied by matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS). The resulting mass spectra are discussed based on the fragmentation patterns of the sodium adducts. The product ion spectra of 4-O-galloylalbiflorin and 4'-O-galloylpaeoniflorin differed, even though they were positional isomers. The fragmentations of the ester parts were influenced by the neighboring hydroxyl groups. The ionization efficiency of the sodium adduct of albiflorin was higher than that for paeoniflorin. These results indicate that the carboxylic ester group has a higher affinity for sodium ions than the acetal group, which can be attributed to the carbonyl oxygen being negatively polarized, allowing it to function as a Lewis base.

Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration.

Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.

Alteration of Membrane Physicochemical Properties by Two Factors for Membrane Protein Integration.

After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.

CdsA is involved in biosynthesis of glycolipid MPIase essential for membrane protein integration in vivo.

MPIase is a glycolipid that is involved in membrane protein integration. Despite evaluation of its functions in vitro, the lack of information on MPIase biosynthesis hampered verification of its involvement in vivo. In this study, we found that depletion of CdsA, a CDP-diacylglycerol synthase, caused not only a defect in phospholipid biosynthesis but also MPIase depletion with accumulation of the precursors of both membrane protein M13 coat protein and secretory protein OmpA. Yeast Tam41p, a mitochondrial CDP-diacylglycerol synthase, suppressed the defect in phospholipid biosynthesis, but restored neither MPIase biosynthesis, precursor processing, nor cell growth, indicating that MPIase is essential for membrane protein integration and therefore for cell growth. Consistently, we observed a severe defect in protein integration into MPIase-depleted membrane vesicles in vitro. Thus, the function of MPIase as a factor involved in protein integration was proven in vivo as well as in vitro. Moreover, Cds1p, a eukaryotic CdsA homologue, showed a potential for MPIase biosynthesis. From these results, we speculate the presence of a eukaryotic MPIase homologue.

Syntheses and Activities of the Functional Structures of a Glycolipid Essential for Membrane Protein Integration.

MPIase is the first known glycolipid that is essential for membrane protein integration in the inner membrane of E. coli. Since the amount of natural MPIase available for analysis is limited and it contains structural heterogeneity, precisely designed synthetic derivatives are promising tools for further elucidation of its membrane protein integration mechanism. Thus, we synthesized the minimal unit of MPIase, a trisaccharyl pyrophospholipid termed mini-MPIase-3, and its derivatives. Integration assays revealed that the chemically synthesized trisaccharyl pyrophospholipid possesses significant activity, indicating that it includes the essential structure for membrane integration. Structure-activity relationship studies demonstrated that the number of trisaccharide units and the 6- O-acetyl group on N-acetylglucosamine contribute to efficient integration. Furthermore, anchoring in the membrane by a lipid moiety was essential for the integration. However, the addition of phosphorylated glycans devoid of the lipid moiety in the assay solution modulated the integration activity of MPIase embedded in liposomes, suggesting an interaction between phosphorylated glycans and substrate proteins in aqueous solutions. The prevention of protein aggregation required the 6- O-acetyl group on N-acetylglucosamine, a phosphate group at the reducing end of the glycan, and a long glycan chain. Taken together, we verified the mechanism of the initial step of the translocon-independent pathway in which a membrane protein is captured by a glycan of MPIase, which maintains its structure to be competent for integration, and then MPIase integrates it into the membrane by hydrophobic interactions with membrane lipids.

Effects of domain composition on catalytic activity of human UDP-glucose:glycoprotein glucosyltransferases.

Uridine diphosphate (UDP)-glucose:glycoprotein glucosyltransferase (UGGT) 1 is a soluble protein residing in the endoplasmic reticulum (ER) and partially in ER-Golgi intermediate compartment. Characteristically, it is able to recognize incompletely folded proteins and re-glucosylate their high-mannose-type glycans. By virtue of this, UGGT1 acts as a folding sensor in the glycoprotein quality control system in the ER. On the other hand, human UGGT2 (HUGT2) has been believed to be an inactive homolog of human UGGT1 (HUGT1), whereas our recent study discovered its activity as UGGT. Although the activity of HUGT2 is significantly lower than HUGT1, C-terminal catalytic region, accounting for approximately 20% of the full-length enzyme, shares high amino acid sequence identity (>85%). In this study, we aimed to clarify the contribution of the noncatalytic domains by comparing activities of truncated forms of recombinant HUGT1/HUGT2 and HUGT1/HUGT2 chimeras with full-length enzymes. Our results obtained by using synthetic substrate indicate that the C-terminal catalytic regions of HUGTs are functional as UGGT. While the activity of HUGT1, but not that of HUGT2, was enhanced by the presence of N-terminal domains, activities of catalytic domains are similar between two homologs.

Endoplasmic Reticulum (ER)-Targeted, Galectin-Mediated Retrograde Transport by Using a HaloTag Carrier Protein.

Investigations into metabolic processes within the cell have often relied on genetic methods such as forced expression and knockout or knockdown techniques. An alternative approach would be introducing a molecule into the desired location inside the cell. To translocate compounds from outside cells into the endoplasmic reticulum (ER), we constructed a delivery carrier protein. This comprised N-terminal galectin-1 for cell-surface binding (G1), a protease cleavable sequence (ps), a HaloTag domain for attaching exogenous compounds (Halo), and a C-terminal KDEL sequence for ER retention. Fluorescently labeled G1-ps-Halo-KDEL passed through the Golgi apparatus and reached the ER. By using Man9 GlcNAc2 -BODIPY as a cargo compound, the carrier protein was also delivered into the ER with concomitant processing of mannose to Man5,6, by the ER-resident α1,2-mannosidase. G1-ps-Halo-KDEL might serve as a new type of delivery carrier protein to direct compounds into the ER.

Approaches toward High-Mannose-Type Glycan Libraries.

Asparagine-linked (N-linked) sugar chains are widely found in the rough endoplasmic reticulum (ER), which has attracted renewed attention because of its participation in the glycoprotein quality control process. In the ER, newly formed glycoproteins are properly folded to higher-order structures by the action of a variety of lectin chaperones and processing enzymes and are transported into the Golgi, while terminally misfolded glycoproteins are carried into the cytosol for degradation. A group of proteins related to this system are known to recognize subtle differences in the high-mannose-type oligosaccharide structures of glycoproteins; however, their molecular foundations are still unclear. In order to gain a more precise understanding, our group has established a strategy for the systematic synthesis of high-mannose-type glycans. More recently, we have developed "top-down" chemoenzymatic approaches that allow expeditious access to theoretically all types of high-mannose glycans. This strategy comprehensively delivered 37 high-mannose-type glycans, including G1M9-M3 glycans, and opened up the possibility of the elucidation of structure-function relationships with a series of high-mannose-type glycans.

Electrochemical Impedance Spectroscopy Study of Concanavalin A Binding to Self-Assembled Monolayers of Mannosides on Gold Wire Electrodes.

The interactions of the lectin Concanavalin A (Con A) with self-assembled monolayers (SAMs) of thiolated mono-, di-, and tri-mannosides were studied on the surface of gold wires using electrochemical impedance spectroscopy (EIS). The SAMs of mannosides were prepared either pure or along with thiolated triethylene glycol (TEG) at different molar ratios (1:1, 1:2, 1:4, 1:9, and 1:19) to better understand and optimize the interaction conditions. The charge-transfer resistance of the [Fe(CN)6]3-/4- redox probe was compared before and after the interaction at different concentrations of Con A to determine the equilibrium dissociation constant (Kd) and limit of detection (LOD). Values of Kd were found in the nanomolar range showing multivalent interactions between mannosides and Con A, and LOD was found ranging from 4-13 nM depending on the type of mannoside SAM used. Analysis using the Hill equation suggests negative cooperativity in the binding behavior. Peanut agglutinin was used as a negative control, and cyclic voltammetry was used to further support the experiments. We have found that neither the pure nor the widely dispersed monolayers of mannosides provide the conditions for optimal binding of Con A. The binding of Con A to these SAMs is sensitive to the molar ratio of the mannoside used to prepare the SAM and to the structure of the mannoside. A simple cleaning method has also been shown to regenerate the used gold wire electrodes. The results from these experiments contribute to the development of simple, cheap, selective, and sensitive EIS-based bioassays, especially for lectin-carbohydrate interactions.

Construction of a high-mannose-type glycan library by a renewed top-down chemo-enzymatic approach.

A comprehensive method for the construction of a high-mannose-type glycan library by systematic chemo-enzymatic trimming of a single Man9-based precursor was developed. It consists of the chemical synthesis of a non-natural tridecasaccharide precursor, the orthogonal demasking of the non-reducing ends, and trimming by glycosidases, which enabled a comprehensive synthesis of high-mannose-type glycans in their mono- or non-glucosylated forms. It employed glucose, isopropylidene, and N-acetylglucosamine groups for blocking the A-, B-, and C-arms, respectively. After systematic trimming of the precursor, thirty-seven high-mannose-type glycans were obtained. The power of the methodology was demonstrated by the enzymatic activity of human recombinant N-acetylglucosaminyltransferase-I toward M7-M3 glycans, clarifying the substrate specificity in the context of high-mannose-type glycans.

Electrochemical synthesis of nanostructured gold film for the study of carbohydrate-lectin interactions using localized surface plasmon resonance spectroscopy.

Localized surface plasmon resonance (LSPR) spectroscopy is a label-free chemical and biological molecular sensing technique whose sensitivity depends upon development of nanostructured transducers. Herein, we report an electrodeposition method for fabricating nanostructured gold films (NGFs) that can be used as transducers in LSPR spectroscopy. The NGF was prepared by electrodepositing gold from potassium dicyanoaurate solution onto a flat gold surface using two sequential controlled potential steps. Imaging by scanning electron microscopy reveals a morphology consisting of randomly configured block-like nanostructures. The bulk refractive index sensitivity of the prepared NGF is 100±2 nmRIU(-1) and the initial peak in the reflectance spectrum is at 518±1 nm under N2(g). The figure of merit is 1.7. In addition, we have studied the interaction between carbohydrate (mannose) and lectin (Concanavalin A) on the NGF surface using LSPR spectroscopy by measuring the interaction of 8-mercaptooctyl-α-d-mannopyranoside (αMan-C8-SH) with Concanavalin A by first immobilizing αMan-C8-SH in mixed SAMs with 3,6-dioxa-8-mercaptooctanol (TEG-SH) on the NGF surface. The interaction of Con A with the mixed SAMs is confirmed using electrochemical impedance spectroscopy. Finally, the NGF surface was regenerated to its original sensitivity by removing the SAM and the bound biomolecules. The results from these experiments contribute toward the development of inexpensive LSPR based sensors that could be useful for studying glycan-protein interactions and other bioanalytical purposes.