Immunization with a mimotope of GD2 ganglioside induces CD8+ T cells that recognize cell adhesion molecules on tumor cells

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.     

Using size‐controlled multicellular spheroids of murine adenocarcinoma cells to efficiently establish pulmonary tumors in mice

Previous studies demonstrated that multicellular spheroids developed using polydimethylsiloxane‐based microwells exhibited superior functions, such as insulin secretion from pancreatic cells, over suspended cells. To successfully apply these spheroids, the effect of spheroid size on cellular functions must be determined. In this study, using murine adenocarcinoma colon26 cells, the authors examined whether such spheroids were useful for developing tumor‐bearing animal models, which requires the efficient and stable engraftment of cancer cells at implanted sites and/or metastatic sites. The authors prepared microwells with widths of 360, 450, 560, and 770 μm through a micromolding technique, and obtained colon26 spheroids with average diameters of 169, 240, 272, and 341 μm, respectively. Small and medium spheroids were subsequently used. mRNA levels of integrin β1, CD44, and fibronectin, molecules involved in cell adhesion, increased with increasing colon26 spheroid size. Approximately 1.5 × 10⁴ colon26 cells in suspension or in spheroids were intravenously inoculated into BALB/c mice. At 21 days after inoculation, the lung weight of both colon26 spheroid groups, especially the group injected with small spheroids, was significantly higher than that of mice in the suspended colon26 cell group. These results indicate that controlling cancer cell spheroid size is crucial for tumor development in tumor‐bearing mouse models.     

The effect of α2,6-linked sialic acid on anti-IgM antibody-induced apoptosis in Ramos cells

Apoptosis in B cells is induced through the B cell antigen receptor (BCR) and affects the sialic acid recognition molecules on B cells. We investigated the effects of ₁-acid glycoprotein (AGP), which mainly contains 2,6-linked sialic acid, on anti-IgM antibody (Ab)-induced apoptosis in Ramos cells, which are derived from Burkitt's lymphoma. When Ramos cells were incubated with anti-IgM-Ab in plates coated with AGP, neuraminidase-digested AGP (asAGP) or 2,3-sialylated AGP (2,3AGP), apoptosis was suppressed only in those coated with AGP. We also studied the effects of CD22, which is expressed on the surface of mature B cells and binds to sugar chains containing 2,6-linked sialic acid, with anti-CD22 monoclonal antibody (mAb). Anti-CD22mAb enhanced anti-IgM Ab-induced apoptosis in Ramos cells. These contradictory results suggested that the recognition molecules for 2,6-linked sialic acid on AGP, which inhibits B-cell apoptosis, is distinct from CD22, or that different binding domains of CD22 between 2,6-linked sialic acid and anti-CD22 mAb exert opposite functions of suppression or enhancement to anti-IgM Ab-induced B cells.     

Correlation between the activities and the oligomeric forms of pig gastric H/K-ATPase

Membrane-bound H/K-ATPase was solubilized by octaethylene glycol dodecyl ether (C(12)E(8)) or n-octyl glucoside (nOG). H/K-ATPase activity and the distribution of protomeric and oligomeric components were evaluated by high-performance gel chromatography (HPGC) and by single-molecule detection using total internal reflection fluorescence microscopy (TIRFM). As evidenced by HPGC of the C(12)E(8)-solubilized enzyme, the distribution of oligomers was 12% higher oligomeric, 44% diprotomeric, and 44% protomeric species, although solubilization by C(12)E(8) reduced the H/K-ATPase activity to 1.8% of that of the membrane-bound enzyme. The electron microscopic images of the C(12)E(8)-solubilized enzyme showed the presence of protomers and a combination of two and more protomers. While the nOG-solubilized H/K-ATPase retained the same turnover number and 71% of the specific activity as that of the membrane-bound enzyme, 56% higher oligomeric, 34% diprotomeric, and 10% protomeric species were detected. TIRFM analysis of solubilized fluorescein 5'-isothiocyanate (FITC)-modified H/K-ATPase at Lys-518 of the alpha-chain showed a quantized photobleaching of the FITC fluorescence intensity. For the C(12)E(8)-solubilized FITC-enzyme, the fraction of each of the initial relative fluorescence intensity units of 4, 2, and 1 was, respectively, 5%, 44% and 51%. In the case of the nOG-solubilized FITC-enzyme, each fraction of 4 and 2 units was, respectively, 54% and 46% with no detectable 1 unit fraction. This represents the first direct observation of H/K-ATPase in aqueous solution. The correlation between the enzymatic activities and distribution of oligomeric forms of H/K-ATPase by HPGC and the observation of a single molecule of H/K-ATPase and others suggests that the tetraprotomeric form of H/K-ATPase molecules represents the functional species in the membrane.     

Evidence for a relationship between activity and the tetraprotomeric assembly of solubilized pig gastric H/K-ATPase

Activity-oligomeric assembly relationships using octaethylene glycol dodecyl ether (C12E8) solubilized pig gastric H/K-ATPase (unmodified H/K-ATPase) or H/K-ATPase modified with Fluorescein 5'-isothiocyanate (FITC-H/K-ATPase) were examined. The amount of oligomeric species in FITC-H/K-ATPase, which retained little H/K-ATPase activity was estimated by a single-molecule detection technique using total internal reflection fluorescence microscopy. Solubilization of the FITC-H/K-ATPase reduced the potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity to around 5% of the level of the membrane-bound enzyme with the formation of 50% protomer and 40% diprotomer. The solubilization of unmodified H/K-ATPase also reduced both the K-pNPPase and H/K-ATPase activities to around 5%. However, solubilization with increasing concentrations of potassium acetate induced significant and similar increases in K-pNPPase activity (K0.5 = 35 mM) with an increase in the amount of the tetraprotomer of FITC-H/K-ATPase, and the K-pNPPase (K0.5 = 28 mM) and H/K-ATPase (K0.5 = 40 mM) activities of the unmodified H/K-ATPase. The correlation coefficient between the proportion of tetraprotomer and the proportion of the K-pNPPase activity for the same FITC-H/K-ATPase preparation was estimated to be 0.93. Similar coefficients were also obtained between the proportion of tetraprotomer in the FITC-H/K-ATPase and the proportion of K-pNPPase and H/K-ATPase activities in the unmodified H/K-ATPase, with value of 0.85 and 0.86, respectively. Such positive correlations were not obtained between these activities and other oligomeric species. These data, the first direct comparison of oligomeric assembly and enzyme activity both stabilized by K+ in C12E8-solubilized gastric H/K-ATPase, provide strong evidence that the catalytic unit of C12E8-solubilized gastric H/K-ATPase is a tetraprotomer.     

Crystal structure of bovine lipoyltransferase in complex with lipoyl-AMP

Lipoic acid is an essential cofactor of the alpha-ketoacid dehydrogenase complexes and the glycine cleavage system. It is covalently attached to a specific lysine residue of the subunit of the complexes. The bovine lipoyltransferase (bLT) catalyzes the lipoic acid attachment reaction using lipoyl-AMP as a substrate, forming a lipoylated protein and AMP. To gain insights into the reaction mechanism at the atomic level, we have determined the crystal structure of bLT at 2.10 A resolution. Unexpectedly, the purified recombinant bLT contains endogenous lipoyl-AMP. The structure of bLT consists of N-terminal and C-terminal domains, and lipoyl-AMP is bound to the active site in the N-terminal domain, adopting a U-shaped conformation. The lipoyl moiety is buried in the hydrophobic pocket, forming van der Waals interactions, and the AMP moiety forms numerous hydrogen bonds with bLT in another tunnel-like cavity. These interactions work together to expose the C10 atom of lipoyl-AMP to the surface of the bLT molecule. The carbonyl oxygen atom of lipoyl-AMP interacts with the invariant Lys135. The interaction might stimulate the positive charge of the C10 atom of lipoyl-AMP, and consequently facilitate the nucleophilic attack by the lysine residue of the lipoate-acceptor protein, accompanying the bond cleavage between the carbonyl group and the phosphate group. We discuss the structural differences between bLT and the lipoate-protein ligase A from Escherichia coli and Thermoplasma acidophilum. We further demonstrate that bLT in mitochondria also contains endogenous lipoylmononucleotide, being ready for the lipoylation of apoproteins.     

Immobilized α2,6-linked sialic acid suppresses caspase-3 activation during anti-IgM antibody-induced apoptosis in Ramos cells

In Ramos cells, a human Burkitt's lymphoma cell line, stimulation of the B cell antigen receptor with anti-IgM antibody (Ab) induces apoptosis as indicated by a decrease in cell viability and an increase in DNA fragmentation and cell surface exposure of phosphatidylserine. Furthermore, these changes are suppressed by incubating the cells in α₁-acid glycoprotein (AGP)-coated tissue culture plates. Here, we found that, during Anti-IgM Ab-induced apoptosis in Ramos cells, caspase-3 is activated downstream of caspase-8 and the mitochondrial pathway is activated, as indicated by a loss of mitochondrial membrane potential, an increase in the release of cytochrome c to the cytoplasm, and enhanced Bax expression. Anti-IgM Ab-induced apoptosis of neuraminidase-treated Ramos cells was suppressed by incubating the cells on plates coated with AGP, which contains a high concentration of α2,6-linked sialic acid. The incubation on plates coated with AGP also suppressed anti-IgM Ab-stimulated caspase-3 activity and increased the level of X-linked inhibitor of apoptosis protein (XIAP), but it did not affect caspase-8 activity, the mitochondrial membrane potential, cytochrome c release, or Bax expression. The results indicate that the interaction of Ramos cells with immobilized α2,6-linked sialic acid enhances XIAP expression, directly or indirectly suppressing caspase-3 activity and inhibiting anti-IgM Ab-induced apoptosis.     

Absence of cuticular alkenes allows lycaenid larvae to avoid predation by Formica japonica ants

Chemical mimicry and camouflage based on cuticular hydrocarbons (CHCs) are adaptive strategies that are frequently observed in myrmecophilous insects. The larvae of several lycaenid butterfly species that exhibit obligate associations with specific ant species have been reported to use chemical mimicry. However, little is known about the strategies used by the larvae of species that have facultative associations with multiple ant species. We attempted to reveal the effects of larval CHC profiles on interactions with Formica japonica workers, using three lycaenid species, two facultative ant‐associated (Lycaeides argyrognomon and Zizeeria maha) and one non‐ant‐associated (Lycaena phlaeas), which commonly possess n‐alkanes as the major CHCs. In field bioassays, the lycaenid larvae were attacked by ant workers less often than larvae of Papilio polytes (Papilionidae), the CHCs of which were rich in 7‐alkenes. Treating the lycaenid larvae with 7‐heptacosene and 9‐heptacosene significantly activated ant aggression (biting), whereas treating them with n‐heptacosane, n‐octacosane and 13‐methylheptacosane had little effect. Furthermore, larvae of Pieris rapae (Pieridae), possessing n‐alkanes as the dominant CHCs, suffered an intermediate level of ant biting between the lycaenid and Pa. polytes larvae. However, treatments of the P. rapae larvae with 7‐heptacosene and 9‐heptacosene significantly affected the frequency of ant biting. These findings suggest that the absence of alkenes in larval CHC profiles is an effective means of circumventing predation by ants and allows lycaenid larvae to inhabit the foraging territory of predaceous ants, at least to some extent.