Establishment of strongly neutralizing monoclonal antibody to human interleukin-6 and its epitope analysis

Three monoclonal antibodies against human interleukin-6 were established and characterized. One antibody was shown to strongly neutralize both the Ig-inducing and hybridoma/plasmacytoma growth activity of interleukin-6. The results of its epitope analysis using protease treated interleukin-6 and immobilized antibody indicated that this neutralizing antibody binds to a peptide corresponding to Leu151-Lys171 of interleukin-6 molecule. Further analysis using synthetic peptides showed that a shorter peptide corresponding to Ala153-Thr162 can also inhibit the binding of the antibody to interleukin-6. These results suggest that this carboxyl-terminal region plays a crucial role in interleukin-6 functions.     

Dennd3 Functions as a Guanine Nucleotide Exchange Factor for Small GTPase Rab12 in Mouse Embryonic Fibroblasts

Small GTPase Rab12 regulates mTORC1 (mammalian target of rapamycin complex 1) activity and autophagy through controlling PAT4 (proton/amino acid transporter 4) trafficking from recycling endosomes to lysosomes, where PAT4 is degraded. However, the precise regulatory mechanism of the Rab12-mediated membrane trafficking pathway remained to be determined because a physiological Rab12-GEF (guanine nucleotide exchange factor) had yet to be identified. In this study we performed functional analyses of Dennd3, which has recently been shown to possess a GEF activity toward Rab12 in vitro. The results showed that knockdown of Dennd3 in mouse embryonic fibroblast cells caused an increase in the amount of PAT4 protein, the same as Rab12 knockdown did, and knockdown of Dennd3 and overexpression of Dennd3 were found to result in an increase and a decrease, respectively, in the intracellular amino acid concentration. Dennd3 overexpression was also found to reduce mTORC1 activity and promoted autophagy in a Rab12-dependent manner. Unexpectedly, however, Dennd3 knockdown had no effect on mTORC1 activity or autophagy despite increasing the intracellular amino acid concentration. Further study showed that Dennd3 knockdown reduced Akt activity, and the reduction in Akt activity is likely to have canceled out amino acid-induced mTORC1 activation through PAT4. These findings indicated that Dennd3 not only functions as a Rab12-GEF but also modulates Akt signaling in mouse embryonic fibroblast cells.     

Participation of ezrin in bacterial uptake by trophoblast giant cells

Background  

Trophoblast giant (TG) cells are involved in systematic removal of bacterial pathogens from the maternal-fetal interface of the placenta. In particular, TG cells have the ability to take up extracellular antigens by active phagocytosis induced by interferon-gamma (IFN-gamma). We previously reported that heat shock cognate protein 70 (Hsc70) present on the surface of TG cells mediated the uptake of Brucella abortus. However, the mechanism of bacterial uptake by TG cells is not completely understood. Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.     

Combination of Zinc and All-Trans Retinoic Acid Promotes Protection against Listeria monocytogenes Infection

Zinc (Zn) is the second most abundant transition metal after iron. It plays a vital role in living organisms and affects multiple aspects of the immune system. All-trans retinoic acid (atRA) is an isomeric form of the vitamin A or retinol. It possesses the greatest biological activity of Vitamin A. Vitamin A and related retinoids influence many aspects of immunity. In this study, we demonstrated that treatment with a combination of Zn and atRA contributes to host resistance against infection by Listeria monocytogenes. Pretreatment with Zn and atRA enhanced resistance against L. monocytogenes infection in mice and treatment with both Zn and atRA showed a higher protective effect than treatment with either alone. Supplementation with Zn, atRA or their combination decreased the number of L. monocytogenes present in target organs. In vitro, supplementation increased the bacterial uptake by macrophage cells and reduced the replication of L. monocytogenes. Our results suggest that the combination of Zn and atRA has a great bacteriostatic impact on L. monocytogenes and its infection.     

An alpha-N-acetylgalactosaminylation at the threonine residue of a defined peptide sequence creates the oncofetal peptide epitope in human fibronectin

The monoclonal antibody FDC-6 defines a structure specific to oncofetal fibronectins (onf-FN) isolated from fetal and malignant cells and tissues. The absence of this structure is characteristic of normal fibronectin (nor-FN) isolated from plasma and adult normal tissue (Matsuura, H., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6517-6521). The minimum structure required for FDC-6 reactivity was determined to be Val-Thr-His-Pro-Gly-Tyr (VTHPGY) with alpha-N-acetylgalactosamine (alpha-GalNAc) at Thr, although alpha-GalNAc per se is not involved in the FDC-6 epitope (Matsuura, H., Takio, K., Titani, K., Greene, T., Levery, S. B., Salyan, M. E. K., and Hakomori, S. (1988) J. Biol. Chem. 263, 3314-3322). Thus, a single glycosylation on the normally occurring peptide of FN may induce conformational changes in the peptide to form the specific oncofetal epitope recognized by FDC-6 antibody. The FDC-6-nonreactive synthetic peptide containing the VTHPGY sequence was converted into FDC-6-reactive form on incubation with alpha-N-acetylgalactosaminyltransferase and UDP-[3H]GalNAc in the homogenate of hepatoma cell HUH-7, human fetal fibroblast cell line WI-38, or human epidermoid carcinoma cell line A431. Such a conversion did not take place when the same enzyme fraction of normal adult tissue was incubated with the VTHPGY peptide under the same conditions. Thus, the occurrence of alpha-GalNAc transferase recognizing the VTHPGY peptide sequence (UDP-GalNAc:VTHPGY alpha-GalNAc transferase) is specific for fetal and cancer tissues, and absent in normal adult tissues. However, a similar alpha-GalNAc transferase activity capable of transferring the GalNAc residue to other Ser or Thr hydroxyl groups of nor-FN, and presumably located at the type III connecting segment region, was detectable in homogenate of various normal tissues. Such enzyme activity was determined with the use of enzymatically de-O-glycosylated nor-FN. Thus, the enzymatic basis of FDC-6 epitope formation is a subtle change in the substrate specificity of alpha-GalNAc transferase. The normal enzyme is incapable of transferring alpha-GalNAc from UDP-GalNAc to the Thr residue of the VTHPGY sequence, but is capable of transferring alpha-GalNAc to other Ser or Thr residues of FN. In contrast, alpha-GalNAc transferase of fetal and cancer tissues may have broader specificity and the capability to transfer GalNAc to Thr or Ser residues, including those of the VTHPGY sequence.     

Structural analysis of the major caprine beta-mannosidosis urinary oligosaccharides

Urinary oligosaccharides were studied in beta-mannosidosis, a newly identified, inherited glycoprotein catabolic disorder associated with severe neonatal neurological deficits, widespread lysosomal storage vacuoles and a deficiency of plasma and tissue beta-mannosidase. A preliminary analysis of the oligosaccharides was obtained by gel-permeation chromatography and mass chromatography. The major urinary oligosaccharides were then isolated by gel-permeation chromatography, DEAE-Sephadex column chromatography and preparative paper chromatography, and were analyzed by carbohydrate composition analysis, methylation studies, mass spectrometry and glycosidase digestion. As a result of these studies, beta-mannosyl-(1 leads to 4)-N-acetylglucosamine and beta-mannosyl-(1 leads to 4)-beta-N-acetylglucosaminyl-(1 leads to 4)-N-acetylglucosamine were identified as the major abnormal oligosaccharides. Galactosaminyl-(alpha 1 leads to 3)-[fucosyl-(alpha 1 leads to 2)]-galactose was also found in affected goat urine, while lactose was present in the urine of both control and affected goats.     

O-linked N-acetylglucosamine is present on the extracellular domain of notch receptors

Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as Notch receptors. In the course of mass spectrometric analysis of O-glycans displayed on Drosophila Notch receptors expressed in S2 cells, we found an unusual O-linked N-acetylhexosamine (HexNAc) modification which occurs at a site distinct from those of O-fucose and O-glucose glycosylations. Modification site mapping by mass spectrometry and amino acid substitution studies revealed that O-HexNAc modification occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O-glycosylations. This modification was determined to be O-beta-GlcNAc by galactosyltransferase labeling and beta-N-acetyl-hexosaminidase digestion experiments and by immunoblotting with a specific antibody. O-GlcNAc modification occurs at multiple sites on Notch epidermal growth factor repeats. O-GlcNAc modification was also found on the extracellular domain of Delta, a ligand for Notch receptors. Although the O-GlcNAc modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, the finding of O-GlcNAc modification in extracellular environments predicts a distinct glycosylation process that might be associated with a novel regulatory mechanism for Notch receptor activity.     

One amino acid difference is critical for suppression of the development of experimental autoimmune diabetes (EAD) with intravenous injection of insulinB:9-23 peptide

InsulinB:9-23 peptide (insB:9-23) reactive T cells has been reported as crucial for type 1 diabetes. In this study, experimental autoimmune diabetes (EAD) mice, which subcutaneous immunization of ins1 or 2B:9-23 induced autoimmune diabetes in F1(B7.1B6 × BALB/c), was investigated for antigen specific therapy to delete pathogenic T cells. Intravenous injection of ins1 or 2B:9-23 significantly delayed the development of diabetes on the corresponding peptide-induced EAD (ins1EAD or ins2EAD) concomitant with reduced insulitis and insulin autoantibodies expression. Population of Foxp3⁺ CD4⁺ T cell was unchanged whereas the level of anti-insB:9-23 specific IgG_2a but not IgG₁ were specifically decreased, suggesting reduction of pathogenic insB:9-23 reactive T cells. Most interestingly, intravenous administration of ins2B:9-23, whose amino acid sequence had one amino acid difference at position 9 delayed the development of diabetes in both ins1EAD and ins2EAD whereas ins1B:9-23 administration delayed diabetes in the ins1EAD but not ins2EAD, suggesting that one amino acid difference gives critical influence on the effect of intravenous injection of antigenic peptide for type 1 diabetes.