Kazuo Yamamoto

The Macrophage C-type Lectin Specific for Galactose/N-Acetylgalactosamine Is an Endocytic Receptor Expressed on Monocyte-derived Immature Dendritic Cells

Lectins on antigen presenting cells are potentially involved in the antigen uptake and the cellular recognition and trafficking. Serial analysis of gene expression in monocyte-derived dendritic cells (DCs), monocytes, and macrophages revealed that 7 of the 19 C-type lectin mRNA were present in immature DCs. Two of these, the macrophage mannose receptor and the macrophage lectin specific for galactose/N-acetylgalactosamine (MGL), were found only in immature DCs, as confirmed by reverse transcriptase-PCR and flow cytometric analysis. By subcloning and sequencing the amplified mRNA, we obtained nucleotide sequences encoding seven different human MGL (hMGL) subtypes, which were apparently derived from alternatively spliced mRNA. In addition, the hMGL gene locus on human chromosome 17p13 contains one gene. A single nucleotide polymorphism was identified at a position in exon 3 that corresponds to the cytoplasmic region proximal to the transmembrane domain. Of all the splicing variants, the hMGL variant 6C was expressed at the highest levels on immature DCs from all donors tested. Immature DCs could incorporate α-GalNAc-modified soluble acrylamide polymers, and this was significantly inhibited by pretreatment of the cells with an anti-hMGL monoclonal antibody that blocks the lectin-carbohydrate interaction. We propose that hMGL is a marker of imDCs and that it functions as an endocytic receptor for glycosylated antigens.

Interaction of human macrophage C-type lectin with O-linked N-acetylgalactosamine residues on mucin glycopeptides

A fluorescein-labeled synthetic peptide, PTTTPITTTTK, was converted into O-glycosylated glycopeptides with various numbers of attachedN-acetyl-d-galactosamines (GalNAcs) by in vitro glycosylation with UDP-GalNAc and a microsomal fraction of LS174T human colon carcinoma cells. Glycopeptides with 1, 3, 5, and 6 GalNAc residues (G1, G3, G5, and G6) were obtained, and their sizes were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Their sequences were determined by a peptide sequencer to be PTTTGalNAcPITTTTK for G1, PTGalNAcTTPITGalNAcTGalNAcTTK for G3, PTTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G5, and PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G6. A calcium-type human macrophage lectin (HML) was prepared in a recombinant form, and its interaction with these glycopeptides was investigated by surface plasmon resonance (SPR) spectroscopy and fluorescence polarization. The affinity of recombinant HML (rHML) for immobilized glycopeptides increased, as revealed by SPR, in parallel with the number of GalNAc. The highest affinity was obtained when the G6-peptide was immobilized at high density. Fluorescence polarization equilibrium-binding assays also revealed that the affinity of rHML for soluble gly-copeptides increased, depending on the number of attached GalNAcs. Carbohydrate recognition domain (CRD) fragments of HML were prepared, and their affinity for these four glycopeptides was also determined, this affinity was apparently lower than that of rHML. Affinity constants of rHML for the G3- and G5-peptides were 11- and 38-fold higher, respectively, than for the G1-peptide, whereas those of CRD fragments were only 2- and 6-fold higher, respectively. A chemical cross-linking study revealed that rHML but not recombinant CRD forms trimers in an aqueous solution. Thus, preferential binding of densely glycosylatedO-linked glycopeptides should be due to the trimer formation of rHML.

Sugar-binding Properties of VIP36, an Intracellular Animal Lectin Operating as a Cargo Receptor

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of ∼6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.

The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues.

The human non‐classical MHC class I molecule HLA‐E is a ligand for both an inhibitory NK cell receptor (CD94/NKG2A) and an activating receptor (CD94/NKG2C). To identify HLA‐E surface recognized by both receptors, especially to determine if both receptors recognize the same epitope, we made a series of individually Ala‐substituted HLA‐E proteins and analyzed their binding to CD94/NKG2A orCD94/NKG2C. Eight HLA‐E mutations that significantly impaired HLA‐E binding to CD94/NKG2A are all found in the top of α1/α2 domain of HLA‐E. These results suggest that CD94/NKG2A binds a HLA‐E surface equivalent to a NKG2D binding site on MICA. Of the eight mutations that impaired HLA‐E binding to CD94/NKG2A, six significantly impaired HLA‐E binding to CD94/NKG2C suggesting that CD94/NKG2C also binds a similar surface of HLA‐E. Unexpectedly, the two HLA‐E mutations (D69A and H155A) selectively abrogated HLA‐E binding to CD94/NKG2A, not largely affected CD94/NKG2C. These results indicate that a mostly shared, but partly distinct set of HLA‐E residues is discriminated by the two receptors.

The NK Cell MHC Class I Receptor Ly49A Detects Mutations on H-2Dd Inside and Outside of the Peptide Binding Groove

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I molecule H-2Dd and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in α1/α2 domain of H-2Dd. Here we have substituted polymorphic as well as conserved residues of H-2Dd α1/α2 domain (when compared with H-2Kd, which does not interact with Ly49A). We then tested the ability of the H-2Dd mutants to interact with Ly49A by soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition assays. Individual introduction of mutations converting the H-2Dd residue into the corresponding H-2Kd residue (N30D, D77S, or A99F) in H-2Dd partially abrogated the interaction between Ly49A and H-2Dd. Introduction of the three mutations into H-2Dd completely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2Dd that are conserved among murine MHC class I severely impaired the interaction. The crystal structure of H-2Dd reveals that D77 and A99 are located in the peptide binding groove and that N30, D29, and R35 are in the interface of the three structural domains of MHC class I: α1/α2, α3, and β2-microglobulin. These data suggest that Ly49A can monitor mutations in MHC class I inside and outside of the peptide binding groove and imply that inhibitory MHC class I-specific receptors are sensitive to mutations in MHC class I as well as global loss of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.

Cleavage site of calcium-dependent protease in human platelet membrane glycoprotein ib

Chicken muscle-derived m-type calcium-dependent protease cleaved purified glycoprotein Ib alpha-chain (GPIb alpha, Mr 130,000) from human platelets into two fragments (Mr 100,000 and Mr 38,000) in the presence of 5 mM calcium. With partially purified glycoprotein Ib (alpha beta-dimer), an appearance of a fragment of Mr 100,000 was also demonstrated after treatment with both the m-type and human platelet-derived mu-type protease. These processes in glycoprotein Ib were inhibited by inhibitors of calcium-dependent proteases, 50 muM E-64-C or 0.2 mM leupeptin and by the chelation of calcium. Using two-dimensional gel electrophoresis system, release of glycocalicin in addition to 100 kDa fragment was demonstrated by calcium-dependent proteases. Then surface-labeled platelets were stimulated with A23187 in the presence of 5mM calcium. Under this condition, endogenous calcium-dependent protease is activated. Of the labeled glycoproteins, glycocalicin and glycoprotein V but not 100 kDa fragment were released from the platelet membrane. The released glycocalicin was further digested into a fragment of Mr 100,000 by the addition of m-type calcium-dependent protease. These results showed (i) that GPIb alpha was hydrolyzed by exogenous calcium-dependent proteases in two points and glycocalicin and 100 kDa fragment were produced and (ii) that endogenous protease cleaved GPIb alpha at one point and released glycocalicin.

Modulating the Actions of NK Cell‐Mediated Cytotoxicity Using Lipid‐PEG (n) and Inhibitory Receptor‐Specific Antagonistic Peptide Conjugates

Regulating the cell surface modulates the actions of the biological cell response, derives practical applications, and is of scientific interest. On the basis of our previous study using dioleylphosphatidylethanolamine poly(ethylene glycol) with multiple units of ethyleneoxide (DOPE‐PEG (n)), we demonstrated the potency of DOPE‐PEG (80) as a cell surface modulator. We prepared conjugates of DOPE‐PEG (80) and two antagonistic peptides (C1, SGGGCLFNLPWLCG; C26, SGGGCPFSFLPWCG), specifically designed for the inhibitory receptor of natural killer (NK) cells. We confirmed that NK cells exhibited cytotoxicity against DOPE‐PEG (80)‐peptides‐incorporated target cells. We further investigated whether the DOPE‐PEG (80)‐peptides could affect the cytotoxicity of NK cells in a concentration‐dependent manner. C1 peptide showed down‐regulation of cytotoxicity at higher concentration, whereas C26 peptide exhibited the saturated cytotoxicity of NK cells at the same concentration. These results suggest that DOPE‐PEG (80) can achieve the role of a cell surface modulator without inhibiting the action of conjugated molecules, despite their relatively small size.

Affinity Chromatography of Oligosaccharides and Glycopeptides with Immobilized Lectins

Lectins are proteins (glycoproteins) that specifically bind to a particular carbohydrate structure and have been shown to be widely distributed among plants, animals, and bacteria. Although the functional role of lectins in living organisms has not been well understood, over 100 kinds of lectins have been isolated and their binding specificities characterized (Goldstein and Hayes, 1978; Liener et al., 1986; Lis and Sharon, 1986; Sharon and Lis, 1989). These lectins have been extensively used for studies on glycoconjugates, especially (1) histochemical detection of cell-surface carbohydrate chains, (2) staining and structural estimation of glycoproteins on Western-blotted membranes or of glycolipids on thin-layer chromatography (TLC) plates, (3) separation of cells with different cell-surface carbohydrate chains, and (4) isolation and fractionation of glycoproteins or oligosaccharides by using affinity chromatography. For the fractionation of glycoproteins and oligosaccharides, a variety of Chromatographic techniques have been employed. These molecules can be separated on the basis of differences in ionic charge, degree of polymerization, or monosaccharide sequences. Affinity chromatography with immobilized lectins seems to be a quite effective technique, because it can achieve not only fractionation of glycoproteins or oligosaccharides but also their structural assessment on the basis of the elution profile from an immobilized lectin column. If affinity columns of several lectins with different binding specificities are serially combined, this technique would be a more powerful tool for the separation of glycoproteins and oligosaccharides. In this chapter, we focus on the separation of glycopeptides and oligosaccharides derived from glycoproteins by affinity chromatography on immobilized lectin columns.

Ribosomal protein S18 identified as a cofilin-binding protein by using phage display library

We previously reported that an actin-binding protein, cofilin, is involved in superoxide production, phagocytosis, and chemotaxis in activated phagocytes through cytoskeletal reorganization. To elucidate the functions of cofilin in greater detail we tried to identify cofilin-binding proteins by using a phage-displayed cDNA library constructed from human brain mRNAs. Several phage clones capable of binding to cofilin were obtained, and the phage with the strongest binding affinity contained the C-terminal half of ribosomal protein S18. To confirm the interaction between the S18 protein and cofilin, we investigated whether cofilin would bind to His-tagged S18 protein immobilized in Ni-NTA-agarose gel. Cofilin and the S18 protein co-eluted with a low pH (4.5) buffer, suggesting that the proteins interact with each other. Preincubation of cofilin with actin abrogated the binding to protein S18, indicating that cofilin interacts with S18 protein at the actin-binding site, and cofilin co-immunoprecipitated with FLAG-tagged S18 protein expressed in COS-7 cells. These results suggest that some cofilin molecules bind the ribosomal S18 protein under physiological conditions. (Mol Cell Biochem 262: 187–193, 2004)

Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK cell cytotoxicity

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