Glycosylation is one of the most common post‐translational modifications, and approximately 50% of all proteins are presumed to be glycosylated in eukaryotes. Branched N‐glycans, such as bisecting GlcNAc, β‐1,6‐GlcNAc and core fucose (α‐1,6‐fucose), are enzymatic products of N‐acetylglucosaminyltransferase III, N‐acetylglucosaminyltransferase V and α‐1,6‐fucosyltransferase, respectively. These branched structures are highly associated with various biological functions of cell adhesion molecules, including cell adhesion and cancer metastasis. E‐cadherin and integrins, bearing N‐glycans, are representative adhesion molecules. Typically, both are glycosylated by N‐acetylglucosaminyltransferase III, which inhibits cell migration. In contrast, integrins glycosylated by N‐acetylglucosaminyltransferase V promote cell migration. Core fucosylation is essential for integrin‐mediated cell migration and signal transduction. Collectively, N‐glycans on adhesion molecules, especially those on E‐cadherin and integrins, play key roles in cell–cell and cell–extracellular matrix interactions, thereby affecting cancer metastasis.
Many plasma membrane-resident molecules cluster with other molecules to collaborate in a variety of biological events. We herein report a sensitive and simple method to identify components of cell surface molecular clusters in living cells. This method includes a recently established reaction, called the enzymemediated activation of radical source (EMARS), to label molecules within a limited distance (Ϸ200 -300 nm) from the probed molecule on which HRP is set. Because the size of this active area is close to that of the reported membrane clusters, it is suggested that the labeled molecules cluster with the probed molecule in the same membrane domain. A combination of the EMARS reaction and antibody array analysis demonstrated that many kinds of receptor tyrosine kinases (RTKs) formed clusters with 1 integrin in HeLa S3 cells. A similar antibody array analysis after the EMARS reaction with three HRP-labeled antibodies against growth factor receptors showed the patterns of biotinylated RTKs to be substantially different from each other. These results suggest that different types of cell surface molecular clusters can thus be distinguished using the EMARS reaction. Therefore, the present ''biochemical visualization'' method is expected to be a powerful tool to elucidate molecular clustering on the cell surface of living cells in various contexts.ganglioside ͉ integrin ͉ microdomain ͉ radicals T he biological events through the plasma membranes, such as signal transmission, cell adhesion, and trafficking require the interactions between receptors, adhesion molecules, and signaling proteins. Recent studies have accumulated a line of evidence in which the functional components are distributed nonrandomly on the plasma membrane and exist as clusters in the nanometerscale domains (1). These membrane domains are formed by the clustering of particular membrane lipids and proteins and display a dynamic property of association and dissociation between interacting molecules that occurs continuously (2). It is therefore essential to identify what functional molecules cocluster in the native membrane, and how they collaborate to create their biological output.Among the many types of membrane domains proposed, the ''lipid rafts'' that are enriched in cholesterol, sphingolipid, GPI-anchored proteins, and the Src kinase family members have so far been most intensively investigated (3-5). It has been assumed that the lipid raft fractions are extracted from the rest of the plasma membrane based on the fact that the membrane domains are resistant to nonionic detergents, whereas the fluid membrane dissolves (6). However, the isolated materials are a mixture of heterogeneous microdomains that could include artificial products extracted during the process of homogenization with detergents. Therefore, it is impossible to identify what molecules cocluster in the same microdomain under the physiological conditions by means of the detergent-resistant membrane fractionation.Until now, four analytical strategies have been developed to analy...
In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lungspecific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for ␣-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and L-xylulose, and were inhibited competitively by nbutyric acid. Therefore, the proteins are designated as dicarbonyl/L-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and L-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC 1.1.1.5) are identical to Lxylulose reductase (EC 1.1.1.10), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.
Background: Molecular mechanisms of the effect of the GOLPH3 oncogenic protein on tumorigenesis remain unclear. Results: GOLPH3 specifically up-regulates sialylation of integrin N-glycans, promotes sialylation-dependent cell migration, and affects AKT signaling. Conclusion: GOLPH3 affects cell biological functions through a specific regulation of sialylation. Significance: The sialylation of N-glycans is important for functions of GOLPH3.
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