N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on alpha3beta1 integrin-mediated cell migration

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that alpha3beta1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in beta1,6-GlcNAc-branched N-glycans on integrin alpha3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in beta1,6-GlcNAc-branched N-glycans on the alpha3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the alpha3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions.     

Crystal structure of unsaturated glucuronyl hydrolase complexed with substrate: molecular insights into its catalytic reaction mechanism

Unsaturated glucuronyl hydrolase (UGL), which is a member of glycoside hydrolase family GH-88, is a bacterial enzyme that degrades mammalian glycosaminoglycans and bacterial biofilms. The enzyme, which acts on unsaturated oligosaccharides with an alpha-glycoside bond produced by microbial polysaccharide lyases responsible for bacterial invasion of host cells, was believed to release 4-deoxy-l-threo-5-hexosulose-uronate (unsaturated glucuronic acid, or DeltaGlcA) and saccharide with a new nonreducing terminus by hydrolyzing the glycosidic bond. We detail the crystal structures of wild-type inactive mutant UGL of Bacillus sp. GL1 and its complex with a substrate (unsaturated chondroitin disaccharide), identify active site residues, and postulate a reaction mechanism catalyzed by UGL that triggers the hydration of the vinyl ether group in DeltaGlcA, based on the structural analysis of the enzyme-substrate complex and biochemical analysis. The proposed catalytic mechanism of UGL is a novel case among known glycosidases. Under the proposed mechanism, Asp-149 acts as a general acid and base catalyst to protonate the DeltaGlcA C4 atom and to deprotonate the water molecule. The deprotonated water molecule attacks the DeltaGlcA C5 atom to yield unstable hemiketal; this is followed by spontaneous conversion to an aldehyde (4-deoxy-l-threo-5-hexosulose-uronate) and saccharide through hemiacetal formation and cleavage of the glycosidic bond. UGL is the first clarified alpha(6)/alpha(6)-barrel enzyme using aspartic acid as the general acid/base catalyst.     

Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes

To comprehend the Ras/ERK MAPK cascade, which comprises Ras, Raf, MEK, and ERK, several kinetic simulation models have been developed. However, a large number of parameters that are essential for the development of these models are still missing and need to be set arbitrarily. Here, we aimed at collecting these missing parameters using fluorescent probes. First, the levels of the signaling molecules were quantitated. Second, to monitor both the activation and nuclear translocation of ERK, we developed probes based on the principle of fluorescence resonance energy transfer. Third, the dissociation constants of Ras.Raf, Raf.MEK, and MEK.ERK complexes were estimated using a fluorescent tag that can be highlighted very rapidly. Finally, the same fluorescent tag was used to measure the nucleocytoplasmic shuttling rates of ERK and MEK. Using these parameters, we developed a kinetic simulation model consisting of the minimum essential members of the Ras/ERK MAPK cascade. This simple model reproduced essential features of the observed activation and nuclear translocation of ERK. In this model, the concentration of Raf significantly affected the levels of phospho-MEK and phospho-ERK upon stimulation. This prediction was confirmed experimentally by decreasing the level of Raf using the small interfering RNA technique. This observation verified the usefulness of the parameters collected in this study.     

Deletion of core fucosylation on alpha3beta1 integrin down-regulates its functions

The core fucosylation (alpha1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently alpha1,6-fucosylation has been further reported to be very crucial by the study of alpha1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on alpha3beta1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in alpha3beta1 integrin-mediated events has been studied by using Fut8(+/+) and Fut8(-/-) embryonic fibroblasts, respectively. We found that the core fucosylation of alpha3beta1 integrin, the major receptor for laminin 5, was abundant in Fut8(+/+) cells but was totally abolished in Fut8(-/-) cells, which was associated with the deficient migration mediated by alpha3beta1 integrin in Fut8(-/-) cells. Moreover integrin-mediated cell signaling was reduced in Fut8(-/-) cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of alpha3beta1 integrin.     

Intracellular localization of ClC chloride channels and their ability to form hetero-oligomers

ClC chloride channels (ClCs) can be classified into two groups in terms of their cellular localizations: ClCs present in the plasma membranes and those residing in intracellular organelles. Members of the latter group, including ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7, are often co-expressed in a variety of cell types in many organs. Although the localization of individual channels within cells has been investigated, the degree of overlap between the locations of different ClCs in the same cell has not been clarified. To address this question, different combinations of ClCs, engineered to encode specific epitope tags (FLAG or HA), were either transiently or stably transfected into HEK293 cells, and we then compared the intracellular localization of the expressed channel proteins by immunofluorescence microscopy. Immunofluorescence images of the alternatively labeled channels clearly showed significant co-localization between all pair-wise combinations of ClCs. In particular, ClC-3, ClC-4, and ClC-5 showed a high degree of co-localization. As a significant degree of co-localization between ClCs was observed, we used co-immunoprecipitation to evaluate oligomer formation, and found that each ClC tested could form homo-oligomers, and that any pair-wise combination of ClC-3, ClC-4, and ClC-5 could also form hetero-oligomers. Neither ClC-6 nor ClC-7 was co-precipitated with any other channel protein. These results suggest that within cells ClC-3, ClC-4, and ClC-5 may have combinatorial functions, whereas ClC-6 and ClC-7 are more likely to function as homo-oligomers.     

MT1-MMP: a potent modifier of pericellular microenvironment

Cells are regulated by many different means, and there is more and more evidence emerging that changes in the microenvironment greatly affect cell function. MT1-MMP is a type I transmembrane proteinase which participates in pericellular proteolysis of extracellular matrix (ECM) macromolecules. The enzyme is cellular collagenase essential for skeletal development, cancer invasion, growth, and angiogenesis. MT1-MMP promotes cell invasion and motility by pericellular ECM degradation, shedding of CD44 and syndecan1, and by activating ERK. Thus MT1-MMP is one of the factors that influence the cellular microenvironment and thereby affect cell-signaling pathways and eventually alters cellular behavior. As a proteinase, MT1-MMP is regulated by inhibitors, but it also requires formation of a homo-oligomer complex, localization to migration front of the cells, and internalization to become a "functionally active" cell function modifier. Developing new means to inhibit "functional activity" of MT1-MMP may be a new direction to establish treatments for the diseases that MT1-MMP mediates such as cancer and rheumatoid arthritis.     

Application of phosphoinositide-binding domains for the detection and quantification of specific phosphoinositides

In mammals, seven phosphoinositides are known to play crucial roles as signaling molecules in a variety of cellular processes. Their synthesis and degradation are thought to be strictly controlled by metabolic enzymes such as phosphoinositide kinases and phosphatases, and their aberrant activities cause diseases. Thus, there is great interest in convenient and high-throughput measurement of such activities for the screening of drugs that enhance or block them. To date, radioactive labeling and colorimetric detection of released inorganic phosphates are mainly used to measure phosphoinositide kinase and phosphatase activities, respectively. Here, we describe a novel method for detecting and quantifying individual phosphoinositides via phosphoinositide-binding domains that exhibit high specificity and affinity toward this lipid. Enzyme-linked immunosorbent assay wells are modified with alkyl chains (C16), which enables more uniform and quantitative immobilization of phosphoinositide-containing liposomes onto the well surfaces. Phosphoinositides, as the substrate or the product, are detected by pleckstrin homology domains that specifically bind to each phosphoinositide. By this method, phosphoinositide contents are measured with higher sensitivities than those by conventional methods. More importantly, both phosphoinositide kinase and phosphatase activities can be measured for purified enzymes and crude cellular lysates. This assay is easy, sensitive, and quantitative and thus may have a variety of applications in the development of diagnostic tests or the screening of therapeutic treatments for diseases such as cancer and diabetes which may be caused by abnormal phosphoinositide metabolism.     

Methylation of Smad6 by protein arginine N-methyltransferase 1

Signal transduction pathways utilize posttranslational modifications to regulate the activity of their components in a temporal-spatial and efficient fashion. Arginine methylation is one of the posttranslational modifications that can result in monomethylated-, asymmetric dimethylated- and/or symmetric dimethylated-arginine residues in proteins. Here we demonstrate that inhibitory-Smads (Smad6 and Smad7), but not receptor-regulated- (R-)Smads and the common-partner Smad4, can be methylated by protein arginine N-methyltransferase (PRMT)1. Using mass-spectrometric analysis, we found that PRMT1 dimethylates arginine(74) (Arg(74)) in mouse Smad6. PRMT1 interacts with the N-terminal domain of Smad6 in which Arg(74) residue is located. Assays examined so far have shown no significant differences between the functions of Smad6 and those of methylation-defective Smad6 (Smad6R74A). Both wild-type and Smad6R74A were equally efficient in blocking BMP-induced growth arrest upon their ectopic expression in HS-72 mouse B-cell hybridoma cells.     

Normal establishment of epithelial tight junctions in mice and cultured cells lacking expression of ZO-3, a tight-junction MAGUK protein

ZO-1, ZO-2, and ZO-3 are closely related MAGUK family proteins that localize at the cytoplasmic surface of tight junctions (TJs). ZO-1 and ZO-2 are expressed in both epithelia and endothelia, whereas ZO-3 is exclusively expressed in epithelia. In spite of intensive studies of these TJ MAGUKs, our knowledge of their functions in vivo, especially those of ZO-3, is still fragmentary. Here, we have generated mice, as well as F9 teratocarcinoma cell lines, that do not express ZO-3 by homologous recombination. Unexpectedly, ZO-3(-/-) mice were viable and fertile, and rigorous phenotypic analyses identified no significant abnormalities. Moreover, ZO-3-deficient F9 teratocarcinoma cells differentiated normally into visceral endoderm epithelium-like cells in the presence of retinoic acid. These cells had a normal epithelial appearance, and the molecular architecture of their TJs did not appear to be affected, except that TJ localization of ZO-2 was upregulated. Suppression of ZO-2 expression by RNA interference in ZO-3(-/-) cells, however, did not affect the architecture of TJs. Furthermore, the speed with which TJs formed after a Ca(2+) switch was indistinguishable between wild-type and ZO-3(-/-) cells. These findings indicate that ZO-3 is dispensable in vivo in terms of individual viability, epithelial differentiation, and the establishment of TJs, at least in the laboratory environment.