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...

Selectins recognize ligands containing carbohydrate chains such as sialyl Lewis x (sLe x ) that are mainly presented at the terminus of N-acetyl lactosamine repeats on core 2 O-glycans. Several glycosyltransferases act successively to extend the N-acetyl lactosamine repeats and to synthesize sLe x , and ␤-1,4-galactosyltransferase (␤4GalT) plays a key role in these processes. Recently isolated 6 ␤4GalT genes are candidates, but their individual roles, including those in selectin-ligand biosynthesis, remain to be elucidated. More than 80% of the core 2 O-glycans on the leukocyte membrane glycoproteins of ␤4GalT-I-deficient mice lacked galactose residues in ␤-1,4 linkage, and soluble P-selectin binding to neutrophils and monocytes of these mice was significantly reduced, indicating an impairment of selectin-ligand biosynthesis. ␤4GalT-I-deficient mice exhibited blood leukocytosis but normal lymphocyte homing to peripheral lymph nodes. Acute and chronic inflammatory responses, including the contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses, were suppressed, and neutrophil infiltration into inflammatory sites was largely reduced in these mice. Our results demonstrate that ␤4GalT-I is a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of ␤4GalT-I-deficient mice are impaired because of the defect in selectin-ligand biosynthesis. (Blood. 2003;102: 1678-1685)

BACE1 is a membrane-bound aspartic protease that cleaves the amyloid precursor protein (APP) at the ␤-secretase site, a critical step in the Alzheimer disease pathogenesis. We previously found that BACE1 also cleaved a membrane-bound sialyltransferase, ST6Gal I. By BACE1 overexpression in COS cells, the secretion of ST6Gal I markedly increased, and the amino terminus of the secreted ST6Gal I started at Glu 41 . Here we report that BACE1-Fc chimera protein cleaved the A-ST6Gal I fusion protein, or ST6Gal I-derived peptide, between Leu 37 and Gln 38 , suggesting that an initial cleavage product by BACE1 was three amino acids longer than the secreted ST6Gal I. The three amino acids, Gln 38 -Ala 39 -Lys 40 , were found to be truncated by exopeptidase activity, which was detected in detergent extracts of Golgi-derived membrane fraction. These results suggest that ST6Gal I is cleaved initially between Leu 37 and Gln 38 by BACE1, and then the three-amino acid sequence at the NH 2 terminus is removed by exopeptidase(s) before secretion from the cells.

We previously reported a simple method to analyze the interaction of cell-surface molecules in living cells. This method termed enzyme-mediated activation of radical sources (EMARS) is featured by radical formation of the labeling reagent by horseradish peroxidase (HRP). Herein, we propose an approach to the cell-surface molecular interactome by using combination of this EMARS reaction and MS-based proteomics techniques. In the current study, we employed a novel labeling reagent, fluorescein-conjugated arylazide. The fluorescein-tagged proteins resulting from the EMARS reaction were directly detected in the electrophoresis gels with a fluorescence image analyzer. These products were also purified and concentrated by immunoaffinity chromatography with anti-fluorescein antibody-immobilized resins. The purified fluorescein-tagged proteins were subsequently subjected to an MS-based proteomics analysis. Analysis using HRP-conjugated cholera toxin subunit B, which recognizes a lipid raft marker, ganglioside GM1, revealed 30 membrane and secreted proteins that were candidates for the cell-surface molecules coclustering with GM1. The proposed approach will provide a clue to study functional molecular interactions in a variety of biological events on the cell surface.

Background: Roles of GD3 in gliomas are not well understood. Results: PDGF receptor ␣ was identified as a GD3-associated molecule by enzyme-mediated activation of radical sources and mass spectrometry, and its association with GD3 and Yes leads to increased invasiveness. Conclusion: GD3 enhances invasiveness by forming a molecular complex. Significance: GD3/PDGF receptor ␣⅐Yes complex is a potential target for glioma therapy.

O-linked glycans of secreted and membrane bound proteins play an important role in the pathogenesis of pancreatic cancer by modulating immune responses, inflammation, and tumorigenesis. A critical aspect of O-glycosylation, the position at which proteins are glycosylated with N-acetyl-galactosamine on serine and threonine residues, is regulated by the substrate specificity of UDP-GalNAc: polypeptide N-acetylgalactosaminyl-transferases (GalNAc-Ts). Thus, GalNAc-Ts regulate the first committed step in O-glycosylated protein biosynthesis, determine sites of O-glycosylation on proteins, and are important for understanding normal and carcinoma-associated O-glycosylation. We have found that one of these enzymes, GalNAc-T3, is overexpressed in human pancreatic cancer tissues, and suppression of GalNAc-T3 significantly attenuates growth of pancreatic cancer cells in vitro and in vivo. In addition, suppression of GalNAc-T3 induces apoptosis of pancreatic cancer cells. Our results indicate that GalNAc-T3 is likely to be involved in pancreatic carcinogenesis. Modification of cellular glycosylation occurs in nearly all types of cancer as a result of alterations in the expression levels of glycosyltransferases. We report guanine nucleotide binding protein, alpha transducing activity polypeptide 1 (GNAT1) as a possible substrate protein of GalNAc-T3. GalNAc-T3 is associated with O-glycosylation of GNAT1, and affects the subcellular distribution of GNAT1. Knocking down endogenous GNAT1 significantly suppresses the growth/survival of PDAC cells. Our results imply that GalNAc-T3 contributes to the function of O-glycosylated proteins and thereby affects the growth and survival of pancreatic cancer cells. Thus, substrate proteins of GalNAc-T3 should serve as important therapeutic targets for pancreatic cancers.

Ganglioside GD3 is specifically expressed in human melanomas, and plays a role in the enhancement of malignant phenotypes of melanoma cells. To analyze the mechanisms by which GD3 enhances malignant properties and signals in melanomas, it is essential to clarify how GD3 interacts with membrane molecules on the cell membrane. In this study, we performed proteomics analysis of glycolipid-enriched microdomains (GEM) with current sucrose density gradient ultracentrifugation of Triton X-100 extracts and MS. We also examined GD3-associated molecules using enzyme-mediated activation of radical sources (EMARS) reaction combined with MS. Comparison of molecules identified as residents in GEM/rafts and those detected by EMARS reaction using an anti-GD3 antibody revealed that a relatively low number of molecules is recruited around GD3, while a number of membrane and secreted molecules was defined in GEM/rafts. These results suggested that EMARS reaction is useful to identify actually interacting molecules with gangliosides such as GD3 on the cell membrane, and many other microdomains than GD3-associating rafts exist. Representative examples of GD3-associated molecules such as neogenin and MCAM were shown.

Mammalian sulfoglycolipids are comprised of two major members, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is abundant in the myelin sheath and seminolipid is expressed on the spermatogenic cells. Cerebroside sulfotransferase (CST)-deficient mice generated by gene targeting completely lack sulfatide and seminolipid all over the body. CST-null mice manifest some neurological disorders due to myelin dysfunction, an aberrant enhancement of oligodendrocyte terminal differentiation, and an arrest of spermatogenesis, indicating that sulfation of glycolipids is essential for myelin formation and spermatogenesis. Moreover, CST-deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction, an experimental model of renal interstitial inflammation, indicating that sulfatide is an endogenous ligand of L-selectin. Studies on the molecular mechanisms by which sulfoglycolipids participate in these biological processes are ongoing.