Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells.

Cellular membranes are composed of numerous kinds of glycerophospholipids with different combinations of polar heads at the sn-3 position and acyl moieties at the sn-1 and sn-2 positions, respectively. The glycerophospholipid compositions of different cell types, organelles, and inner/outer plasma membrane leaflets are quite diverse. The acyl moieties of glycerophospholipids synthesized in the de novo pathway are subsequently remodeled by the action of phospholipases and lysophospholipid acyltransferases. This remodeling cycle contributes to the generation of membrane glycerophospholipid diversity and the production of lipid mediators such as fatty acid derivatives and lysophospholipids. Furthermore, specific glycerophospholipid transporters are also important to organize a unique glycerophospholipid composition in each organelle. Recent progress in this field contributes to understanding how and why membrane glycerophospholipid diversity is organized and maintained.

Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2.

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.

Amino acid residues critical for endoplasmic reticulum export and trafficking of platelet-activating factor receptor.

Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro(247), in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.

AML1 enhances the expression of leukotriene B4 type-1 receptor in leukocytes.

Leukotriene B(4) type-1 receptor (BLT1), which plays a role in various inflammatory diseases, is exclusively expressed in peripheral leukocytes, which suggests that its expression is stringently regulated. However, the precise mechanism of BLT1 expression is not fully understood. Here we report that acute myeloid leukemia 1 (AML1/Runx1) is involved in the enhancement of BLT1 expression in leukocytes. In retinoic acid (RA)-stimulated human promyelocytic leukemia (HL-60) cells, the transcription of the BLT1 gene was found to be significantly activated. RA did not directly modulate the BLT1 promoter, suggesting enhancers in other loci. DNase I-hypersensitivity analyses revealed an activated region, termed AE-BLex, at the intron-I:exon-II boundary. AE-BLex acts as an enhancer for the BLT1 promoter and possesses 2 AML1 recognition sites. The importance of AML1 was determined using electrophoretic mobility shift assays, reporter assays, and knockdown experiments. We demonstrated that the enhancement of BLT1 expression during the RA-induced differentiation of HL-60 cells is due to a loosening of the chromatin structure around AE-BLex, which leads to the incremental binding of AML1. The AML1/AE-BLex complex was confirmed in other BLT1- expressing leukemia cell lines and human peripheral leukocytes. Thus, AML1 enhances BLT1 expression by binding to AE-BLex, which is accessible in leukocytes.

Transcriptional regulation of human G2A in monocytes/ macrophages: involvement of c/EBPs, Runx and Pu.1.

G2 accumulation (G2A) is a G-protein coupled receptor, activated by several ligands and stimuli, such as lysophosphatidylcholine (LPC), extracellular low pH and oxidized phospholipids including 9- and 13-hydroxyoctadecadienoic acid, and has been implicated in mediating inflammatory process under oxidative conditions. Recently, it was demonstrated that G2A in monocytes/macrophages plays critical roles in atherosclerosis deterioration, and therefore its transcriptional regulation in monocytes/macrophages is of great interest. Here, we first confirmed the expression of human G2A (hG2A) in lymph nodes, spleen and peripheral blood leukocytes, including monocytes. Thereafter, transcription start site (TSS) of hG2A was determined by 5'-rapid amplification of cDNA ends analysis. In the course of the analysis, we found that two transcriptional variants, hG2A-a and -b, are produced by alternative splicing, resulting in the production of N-terminal different hG2A proteins with similar sensitivity to low pH and LPC. Using a monocytic cell line THP-1 as a model, transcription of hG2A was precisely examined, and we demonstrated that it is dependent both on the chromatin structure around TSS, and on the binding of the transcription factors (c/EBPalpha and beta, Runx1 and Pu.1) to their cis-elements, located at the core promoter just upstream of TSS.

Helix 8 of leukotriene B4 type-2 receptor is required for the folding to pass the quality control in the endoplasmic reticulum.

Many G protein-coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8 (H8), at the proximal region of the C-terminal tail. However, the significance of this domain is not fully understood. Here, we demonstrate the requirement of H8 for the proper folding of GPCRs for passage through the quality control in the endoplasmic reticulum (ER). In the human leukotriene B(4) type-2 receptor (hBLT2), lack of H8 led to an accumulation of the receptor (hBLT2/DeltaH8) in the ER. Similar results were obtained in two representative human GPCRs, dopamine type-1 and lysophosphatidic acid type-2 receptors, which were engineered to lack H8. Treatment with the several ligands, which act as pharmacological chaperones, facilitated the surface expression of hBLT2/DeltaH8. The surface-trafficked hBLT2/DeltaH8 exhibited an agonist-evoked increase in Ca(2+), demonstrating that H8 is not critical for ligand binding and activation of coupled G proteins. Thus, these results suggest that the H8 region of hBLT2 plays an important role in transport-competent receptor folding.

Oligosaccharide specificity of galectins: a search by frontal affinity chromatography.

Galectins are widely distributed sugar-binding proteins whose basic specificity for beta-galactosides is conserved by evolutionarily preserved carbohydrate-recognition domains (CRDs). Although they have long been believed to be involved in diverse biological phenomena critical for multicellular organisms, in only few a cases has it been proved that their in vivo functions are actually based on specific recognition of the complex carbohydrates expressed on cell surfaces. To obtain clues to understand the physiological roles of diverse members of the galectin family, detailed analysis of their sugar-binding specificity is necessary from a comparative viewpoint. For this purpose, we recently reinforced a conventional system for frontal affinity chromatography (FAC) [J. Chromatogr., B, Biomed. Sci. Appl. 771 (2002) 67-87]. By using this system, we quantitatively analyzed the interactions at 20 degrees C between 13 galectins including 16 CRDs originating from mammals, chick, nematode, sponge, and mushroom, with 41 pyridylaminated (PA) oligosaccharides. As a result, it was confirmed that galectins require three OH groups of N-acetyllactosamine, as had previously been denoted, i.e., 4-OH and 6-OH of Gal, and 3-OH of GlcNAc. As a matter of fact, no galectin could bind to glycolipid-type glycans (e.g., GM2, GA2, Gb3), complex-type N-glycans, of which both 6-OH groups are sialylated, nor Le-related antigens (e.g., Le(x), Le(a)). On the other hand, considerable diversity was observed for individual galectins in binding specificity in terms of (1) branching of N-glycans, (2) repeating of N-acetyllactosamine units, or (3) substitutions at 2-OH or 3-OH groups of nonreducing terminal Gal. Although most galectins showed moderately enhanced affinity for branched N-glycans or repeated N-acetyllactosamines, some of them had extremely enhanced affinity for either of these multivalent glycans. Some galectins also showed particular preference for alpha1-2Fuc-, alpha1-3Gal-, alpha1-3GalNAc-, or alpha2-3NeuAc-modified glycans. To summarize, galectins have evolved their sugar-binding specificity by enhancing affinity to either "branched", "repeated", or "substituted" glycans, while conserving their ability to recognize basic disaccharide units, Galbeta1-3/4GlcNAc. On these bases, they are considered to exert specialized functions in diverse biological phenomena, which may include formation of local cell-surface microdomains (raft) by sorting glycoconjugate members for each cell type.

Glyco-catch method: A lectin affinity technique for glycoproteomics.

Protein glycosylation is a critical issue of post-genome science not only because it is one of the major post-translational modifications but also because it has significant effects on protein properties and functions. The glyco-catch method was recently developed as a novel affinity technique for comprehensive analysis of glycoproteins in the context of glycomics, which is defined as research targeting the whole set of glycans produced in an organism (Hirabayashi J, Kasai K, Trends Glycosci Glycotechnol 2000;12:1-5). This method enables us to identify possible glycoprotein genes as well as glycosylation sites in a systematic manner by combining conventional lectin affinity chromatography and concurrent in silico database searching (Hirabayashi J, Kasai K, J Chromatogr B 2002; 771:67-87). Application of the strategy to a simple organism, Caenorhabditis elegans, has already proved its practical validity (Hirabayashi J, Kaji H, Isobe T, Kasai K, J Biochem (Tokyo) 2002;132:103-114). Accumulation of data on protein glycosylation in a variety of organisms for which entire genome information is available should thus reveal the biological meaning of glycans in complex carbohydrates from a global viewpoint, that is, under the concept of "genome-proteomeglycome." In this article, we briefly review the issues of protein glycosylation and demonstrate the usefulness of the glyco-catch method for identification of complex-type N-glycoproteins of mouse liver that were captured by galectin-1, which is a major galectin in mammals. Future plans for technical improvement and construction of a glycome database are also described.

Specific recognition of Leishmania major poly-beta-galactosyl epitopes by galectin-9: possible implication of galectin-9 in interaction between L. major and host cells.

Leishmania parasites are the causative agents of leishmaniasis, manifesting itself in a species-specific manner. The glycan epitopes on the parasite are suggested to be involved in the Leishmania pathogenesis. One of such established species-unique glycan structures is the poly-beta-galactosyl epitope (Galbeta1-3)n found on L. major, which can develop cutaneous infections with strong inflammatory responses. Interestingly, the polygalactosyl epitope is also suggested to be involved in the development of the parasites in its host vector, sand fly. Thus, the recognition of the galactosyl epitope by lectins expressed in host or sand fly should be implicated in the species-specific manifestations of leishmaniasis and in the parasite life cycle, respectively. We recently reported that one host beta-galactoside-binding protein, galectin-3, can distinguish L. major from the other species through its binding to the poly-beta-galactosyl epitope, proposing a role for galectin-3 as an immunomodulator that could influence the L. major-specific immune responses in leishmaniasis. Here we report that galectin-9 can also recognize L. major by binding to the L. major-specific polygalactosyl epitope. Frontal affinity analysis with different lengths of poly-beta-galactosyllactose revealed that the galectin-9 affinity for polygalactose was enhanced in proportion to the number of Galbeta1-3 units present. Even though both galectins have comparable affinities toward the polygalactosyl epitopes, only galectin-9 can promote the interaction between L. major and macrophages, suggesting distinctive roles for the galectins in the L. major-specific development of leishmaniasis in the host.

Functional analysis of the carbohydrate recognition domains and a linker peptide of galectin-9 as to eosinophil chemoattractant activity.

Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.