␣-Dystroglycan (␣-DG) represents a highly glycosylated cell surface molecule that is expressed in the epithelial cell-basement membrane (BM) interface and plays an essential role in epithelium development and tissue organization. The ␣-DG-mediated epithelial cell-BM interaction is often impaired in invasive carcinomas, yet roles and underlying mechanisms of such an impaired interaction in tumor progression remain unclear. We report here a suppressor function of laminin-binding glycans on ␣-DG in tumor progression. In aggressive prostate and breast carcinoma cell lines, lamininbinding glycans are dramatically decreased, although the amount of ␣-DG and ␤-dystroglycan is maintained. The decrease of lamininbinding glycans and consequent increased cell migration were associated with the decreased expression of ␤3-N-acetylglucosaminyltransferase-1 (␤3GnT1). Forced expression of ␤3GnT1 in aggressive cancer cells restored the laminin-binding glycans and decreased tumor formation. ␤3GnT1 was found to be required for laminin-binding glycan synthesis through formation of a complex with LARGE, thus regulating the function of LARGE. Interaction of the laminin-binding glycans with laminin and other adhesive molecules in BM attenuates tumor cell migratory potential by antagonizing ERK/AKT phosphorylation induced by the components in the ECM. These results identify a previously undescribed role of carbohydrate-dependent cell-BM interaction in tumor suppression and its control by ␤3GnT1 and LARGE.glycosylation ͉ cell adhesion ͉ basement membrane ͉ carcinoma I nteraction of epithelial cells with basement membrane (BM) is mediated by cell adhesion molecules, which operate at the interface of epithelial cell-ECM and regulate cell growth, motility, and differentiation by integrating signals from ECM or soluble factors (1-3). One of the most important epithelial cell-BM interactions is mediated by ␣-dystroglycan (␣-DG) on epithelial cells (4).␣-DG is a cell surface receptor for several major BM proteins, including laminin, perlecan, and agrin. A laminin G-like domain in all these glycoproteins binds to a unique glycan structure attached to ␣-DG, and this interaction has been shown to be critical in assembling BM (5, 6). This unique glycan structure is referred to as laminin-binding glycans hereafter. ␣-DG is not attached directly to the plasma membrane but is bound to it through attachment to the transmembrane protein ␤-dystroglycan (␤-DG), which binds to the cytoplasmic protein dystrophin, which, in turn, binds to the actin cytoskeleton and many adaptor molecules involved in cellular signaling (4,5).␣-DG is highly glycosylated and contains both N-linked glycans and mucin type O-glycans. The mucin type O-glycans are clustered in a mucin-like domain at the N-terminal of mature ␣-DG, which includes unique O-mannosyl glycans and sialic acid ␣233Gal␤134GlcNAc␤132Man␣13Ser/Thr (7). Defects in glycosylation of the O-mannosyl glycans have been shown to cause muscular dystrophy (8). So far, 7 glycosyltransferases or glycosyltransferase-like ...

Lymphocyte homing is mediated by specific interaction between L-selectin on lymphocytes and the carbohydrate ligand 6-sulfo sialyl Lewis X on high endothelial venules. Here we generated mice lacking both core 1 extension and core 2 branching enzymes to assess the functions of O-glycan-borne L-selectin ligands in vivo. Mutant mice maintained robust lymphocyte homing, yet they lacked O-glycan L-selectin ligands. Biochemical analyses identified a class of N-glycans bearing the 6-sulfo sialyl Lewis X L-selectin ligand in high endothelial venules. These N-glycans supported the binding of L-selectin to high endothelial venules in vitro and contributed in vivo to O-glycan-independent lymphocyte homing in wild-type and mutant mice. Our results demonstrate the critical function of N-glycan-linked 6-sulfo sialyl Lewis X in L-selectin-dependent lymphocyte homing and recruitment.

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We have shown that over-sulfated chondroitin sulfate/ dermatan sulfate (CS/DS) chains from various marine organisms exhibit growth factor binding activities and neurite outgrowth-promoting activities in embryonic mouse hippocampal neurons in vitro. In this study we demonstrated that CS/DS hybrid chains purified from embryonic pig brain displayed marked neuritogenic activity and growth factor binding activities toward fibroblast growth factor 2 (FGF2), FGF10, FGF18, pleiotrophin, and midkine, all of which exhibit neuroregulatory activities in the brain. In contrast, the CS/DS preparation from adult pig brain showed considerably less activity to bind these growth factors and no neuritogenic activity. Structural analysis indicated that the average size of the CS/DS chains was similar (40 kDa) between these two preparations, but the disaccharide compositions differed considerably, with a significant proportion of L-iduronic acid (IdoUA)-containing disaccharides (8ϳ9%) in the CS/DS chains from embryos but not in those from adults (<1%). Interestingly, both neurite outgrowth-promoting activity and growth factor binding activities of the CS/DS chains from embryos were abolished by digestion not only with chondroitinase ABC but also with chondroitinase B, suggesting that the IdoUAcontaining motifs are essential for these activities. These findings imply that the temporal expression of CS/DS hybrid structures containing both GlcUA and IdoUA and binding activities toward various growth factors play important roles in neurogenesis in the early stages of the development of the brain. Chondroitin sulfate proteoglycans (CS-PGs)1 are complex macromolecules consisting of a protein core and at least one covalently linked CS glycosaminoglycan (GAG) chain and are ubiquitous components in the extracellular matrices of connective tissues and at the surfaces of many cell types (for reviews, see Refs. 1-3). In the mammalian brain CS-PGs are common extracellular matrix components with a highly regulated spatiotemporal expression (4 -8). Although the CS chains attached to CS-PGs have attracted little attention until recently compared with heparan sulfate (9), recent advances in the structural biology of CS chains have suggested important biological functions in the development of the brain (10). Several studies have demonstrated that the composition of CS chains changes with aging and normal brain maturation and that some CS epitopes are only found in specific sections of the avian and mammalian brain (7,8,11). The developmentally regulated expression and tissue-specific distribution of CS variants suggest that CS chains differing in the degree and profile of sulfation exhibit distinct functions during the development of the brain.The functions of CS-PGs and CS chains in the central nervous system can be categorized as the regulation of cell adhesion and migration, neurite formation, polarization of neurons, synaptic plasticity, survival of neurons, etc. (for reviews, see Ref. 1 and 4). Concerning the neurite formation, studies have sh...

Chondroitin sulfate (CS) and dermatan sulfate (DS) chains play roles in the central nervous system. Most notably, CS/DS hybrid chains (E-CS/DS) purified from embryonic pig brains bind growth factors and promote neurite outgrowth toward embryonic mouse hippocampal neurons in culture. However, the neuritogenic mechanism is not well understood. Here we showed that pleiotrophin (PTN), a heparin-binding growth factor, produced mainly by glia cells, was the predominant binding partner for E-CS/DS in the membrane-associated protein fraction of neonatal rat brain. The CS/DS chains were separated on a PTN column into unbound, low affinity, and high affinity fractions. The latter two fractions promoted outgrowth of dendrite-and axonlike neurites, respectively, whereas the unbound fraction showed no such activity. The activity of the low affinity fraction was abolished by an anti-PTN antibody or when glia cells were removed from the culture. In contrast, the high affinity fraction displayed activity under both these conditions. Hence, PTN mainly from glia cells mediated the activity of the low affinity but not the high affinity fraction. The anti-CS antibody 473HD neutralized the neuritogenic activities of both fractions. Interaction analysis indicated that the 473HD epitope and PTN-binding domains in the E-CS/DS chains largely overlap. The three affinity subfractions differed in disaccharide composition and the distribution of L-iduronic acid-containing disaccharides along the chains. Oversulfated disaccharides and nonconsecutive iduronic acid-containing units were the requirements for the E-CS/DS chains to bind PTN and to exhibit the neuritogenic activities. Thus, CS subpopulations with distinct structures in the mammalian brain play different roles in neuritogenesis through distinct molecular mechanisms, at least in part by regulating the functions of growth factors. Chondroitin sulfate (CS)1 and dermatan sulfate (DS) proteoglycans (PGs), as well as heparan sulfate PGs (HS-PGs), have been implicated in the processes of neural development in the brain such as neuronal adhesion, migration, and neurite formation (for reviews see Ref. 1-3). CS and DS are synthesized as glycosaminoglycan (GAG) side chains of PGs and consist of repeating disaccharide units of -GlcUA-GalNAc-and -IdoUAGalNAc-, respectively, and also exist as CS/DS hybrid chains composed of both units in varying proportions. Recent studies have demonstrated that the disaccharide composition and the GlcUA/IdoUA ratio of brain CS/DS chains change during development (4 -7) and that certain CS/DS hybrid epitopes are found only in specific regions of the mammalian brain (6, 8), suggesting that CS/DS hybrid chains or their subpopulations play distinct roles in the development of the brain.The effects of CS/DS chains on neurite formation are controversial. During development, strong immunostaining for CS is often localized at the boundaries of brain subregions such as the roof plate and midline dorsal tectum acting as barriers to migrating neurons or extending axons (9...

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SUMMARY Heparan sulfate can bind several adhesion molecules involved in lymphocyte trafficking. However, the in vivo function of endothelial heparan sulfate in lymphocyte homing and stimulation of the immune response has not been elucidated. Here, we generated mutant mice deficient in the enzyme Ext1, which is required for heparan sulfate synthesis, in a Tek-dependent and inducible manner. Chemokine presentation was diminished in the mutant mice, causing the lack of appropriate integrin-mediated adhesion, and resulted in a marked decrease in lymphocyte sticking to high endothelial venules and in recruitment of resident dendritic cells through lymphatic vessels to the lymph nodes. As a consequence, mutant mice displayed a severe impairment in lymphocyte homing and a compromised contact hypersensitivity response. By contrast, lymphocyte rolling was increased due to loss of electrostatic repulsion by heparan sulfate. These results demonstrate critical roles of endothelial heparan sulfate in immune surveillance and immune response generation.

Cancer cells often express surface carbohydrates different from normal cells (1). One such change is expression of sialyl Lewis X and Lewis B blood group antigens in cancer cells (2, 3). These structural elements are seen as capping oligosaccharides attached to the underlying glycan backbone where they likely function as ligands for cell adhesion molecules.The structure of underlying glycans also changes during malignant transformation and differentiation. In particular, there are several reports that an increase in the ␤1,6-N-acetylglucosaminyl branch in N-glycans synthesized by ␤1,6-Nacetylglucosaminyltransferase-V is associated with oncogenic transformation (4 -7). Similar structural changes are seen in mucin-type O-glycans, which have N-acetylgalactosamine at the reducing end linked to polypeptide threonine or serine residues. Addition of different carbohydrate residues to N-acetylgalactosamine confers a variety of backbone structures on mucin-type O-glycans; the most abundant of those are classified as core1, core2, core3, and core4 O-glycans (8) (Fig.