A solid-phase substrate of heparanase: its application to assay of human melanoma for heparan sulfate degradative activity

We found a tumor metastasis-associated heparan sulfate (HS)-degrading endoglycosidase in melanoma cells that is a unique endo-beta-glucuronidase (heparanase) capable of specifically cleaving HS at intrachain sites (M. Nakajima, T. Irimura, N. DiFerrante, and G. L. Nicolson, 1984, J. Biol. Chem. 259, 2283-2290). To perform rapid and microscale quantitative assays of heparanase we developed a solid-phase HS substrate by crosslinking radiolabeled HS onto agarose gel beads using one covalent linkage. The HS from bovine lung was partially N-desulfated and labeled with [14C]acetic anhydride. Free HS amino groups were completely acetylated, and reducing terminal saccharides were reductively aminated. The HS derivatives with amino groups at their reducing termini were coupled to amino-reactive agarose beads. Incubation of the solid-phase HS substrates with B16 melanoma cell extracts in the presence of D-saccharic acid 1,4-lactone (a potent exo-beta-glucuronidase inhibitor) resulted in the time- and dose-dependent release of [14C]HS fragments. Human melanoma cell lines were tested for HS-degrading endoglycosidase using the newly developed solid-phase HS substrates. The human malignant melanoma cells tested had high levels of HS-degrading activity that were comparable to those of highly metastatic murine B16-F10 melanoma cells.     

Coordinated binding of sugar, calcium, and antibody to macrophage C- type lectin

Mouse macrophage galactose/N-acetylgalactosamine-specific C-type lectin (MMGL) is a type II transmembrane glycoprotein belonging to the C-type lectin family. Our development of monoclonal antibodies led us to discover that a calcium-dependent conformational change is detected by an antibody (termed mAb LOM-11) and that the antibody's binding to the respective site locks the lectin in an active conformation. These findings correspond to the divalent cation-mediated regulatory mechanisms in a family of cell adhesion molecule integrins that have gained much attention. We now provide direct evidence that mAb LOM-11 increases the affinity of the lectin for calcium ions as a mechanism for the conformational lock using a soluble recombinant form of MMGL (rML) produced in bacteria. Furthermore, we discovered by using an enzyme-linked immunosorbent assay that specific monosaccharides induced a binding site for mAb LOM-11 on the immobilized rML under low calcium environments. We also demonstrated that cell surface MMGL on a transfectant cell line underwent a conformational change upon addition of calcium or ligands, as detected by the binding of mAb LOM-11. These properties are reminiscent of ligand-induced binding sites defined for integrins. The present results suggest a possibility that the mAb LOM- 11 binding site on the lectin may be a site at which protein-protein interaction helps to fine tune the specificity of the C-type lectins by means of coordinated recognition mechanisms.      

The association between major sialoglycoprotein and spectrin of human erythrocyte membranes as detected by fluorescence resonance energy transfer.

The interactions among the major sialoglycoprotein and peripheral proteins of human erythrocyte membranes were investigated by the long range resonance energy transfer between different fluorescent moieties separately conjugated to proteins. Consequently, direct association between the major sialoglycoprotein and spectrin was observed and divalent cations were required for their association.     

Distribution of ferritin-conjugated lectins on sialidase-treated membranes of human erythrocytes.

The labeling of sialidase-treated, human erythrocyte membranes with ferritin-conjugates of four plant lectins, concanavalin A, Ricinus communis hemagglutinin, Bauhinia purpurea hemagglutinin and Arachis hypogoea hemagglutinin, is reported. Among these ferritin-conjugated lectins, ferritin-conjugated concanavalin A and ferritin-conjugated R. communis hemagglutinin were found in clusters on the sialidase-treated membranes, whereas ferritin-conjugated B. purpurea hemagglutinin and ferritin-conjugated A. hypogoea hemagglutinin were found in a random distribution on the membranes. Furthermore, when the membranes were labeled with a mixutre of concanavalin A and ferritin-conjugated B. purpurea hemagglutinin, ferritin particles were found in clusters, indicating that the membrane receptors for B. purpurea hemagglutinin were forced to more together with those for concanavalin A. A method for the quantitative estimation of the clustering of ferritin particles on the membranes was also devised and applied to the labeling of sialidase-treated, human erythrocyte membranes with the above four ferritin-conjugated lectins.     

Molecular cloning and characterization of a novel 3'-phosphoadenosine 5'-phosphosulfate transporter, PAPST2

Sulfation is an important posttranslational modification associated with a variety of molecules. It requires the involvement of the high energy form of the universal sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Recently, we identified a PAPS transporter gene in both humans and Drosophila. Although human colonic epithelial tissues express many sulfated glycoconjugates, PAPST1 expression in the colon is trace. In the present study, we identified a novel human PAPS transporter gene that is closely related to human PAPST1. This gene, called PAPST2, is predominantly expressed in human colon tissues. The PAPST2 protein is localized on the Golgi apparatus in a manner similar to the PAPST1 protein. By using yeast expression studies, PAPST2 protein was shown to have PAPS transport activity with an apparent Km value of 2.2 microM, which is comparable with that of PAPST1 (0.8 microM). Overexpression of either the PAPST1 or PAPST2 gene increased PAPS transport activity in human colon cancer HCT116 cells. The RNA interference of the PAPST2 gene in the HCT116 cells significantly reduced the reactivity of G72 antibody directed against the sialyl 6-sulfo N-acetyllactosamine epitope and total sulfate incorporation into cellular proteins. These findings indicate that PAPST2 is a PAPS transporter gene involved in the synthesis of sulfated glycoconjugates in the colon.     

C-type lectins do not act as functional receptors for filovirus entry into cells

Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.     

Mucin 21/Epiglycanin Modulates Cell Adhesion

The molecular structure of mouse Mucin 21 (Muc21)/epiglycanin is proposed to have 98 tandem repeats of 15 amino acids and three exceptional repeats with 12 or 13 amino acids each, followed by a stem domain, a transmembrane domain, and a cytoplasmic tail. A cDNA of Muc21 having 84 tandem repeats of 15 amino acids was constructed and transfected using a Venus vector into HEK 293T cells. The fluorescent cells, which were considered to express Muc21, were nonadherent. This antiadhesion effect was lessened when constructs with smaller numbers of tandem repeats were used, suggesting that the tandem repeat domain plays a crucial role. Cells expressing Muc21 were significantly less adherent to each other and to extracellular matrix components than control cells. Antibody binding to the cell surface integrin subunits α5, α6, and β1 was reduced in Muc21 transfectants in a tandem repeat-dependent manner, whereas equal amounts of proteins were detected by Western blot analysis. Muc21 was expressed as a large glycoprotein that was highly glycosylated with O-glycans at the cell surface, as detected by flow cytometry, Western blotting, and lectin blotting. Although at least a portion of Muc21 was glycosylated with sialylated glycans, removal of sialic acid did not influence the prevention of adhesion.     

Distribution and Function of Macrophage Galactose-type C-type Lectin 2 (MGL2/CD301b): EFFICIENT UPTAKE AND PRESENTATION OF GLYCOSYLATED ANTIGENS BY DENDRITIC CELLS*

Dendritic cells (DCs) express cell surface lectins that are potentially involved in the recognition, uptake, and presentation of glycosylated foreign substances. A unique calcium-type (C-type) lectin, the macrophage galactose (Gal)-type C-type lectin (MGL/CD301) expressed on DCs, is thought to participate in the recognition of molecules from both altered self and pathogens due to its monosaccharide specificity for Gal and N-acetylgalactosamine (GalNAc). Although mice have two MGL genes, Mgl1 and Mgl2, their distinct roles have not been previously explored. The present report characterizes the properties of MGL2 by examining its distribution and its role in antigen presentation by DCs. We generated an MGL2-specific monoclonal antibody and examined MGL2 expression in tissues by immunohistochemistry and in isolated cells by flow cytometry. The cells reactive with this antibody were shown to be a portion of MGL1-expressing cells, mostly conventional DCs. Internalization of soluble polyacrylamide polymers (PAA) with α-GalNAc residues (GalNAc-PAA) by bone marrow-derived DCs (BM-DCs) was mediated by MGL2, as revealed by a comparison of Mgl1^−/− and Mgl2^−/− BM-DCs with wild-type BM-DCs. Biotinylated GalNAc-PAA conjugated to streptavidin (SAv) was more efficiently presented to SAv-primed T cells by BM-DCs than β-N-acetylglucosamine-PAA conjugated to SAv or SAv alone as shown by thymidine uptake and cytokine production. This is the first report that demonstrates the involvement of GalNAc residues in antigen uptake and presentation by DCs that lead to CD4⁺ T cell activation.     

Laminin 5 promotes activation and apoptosis of the T cells expressing alpha3beta1 integrin

By introducing an alpha3 gene-containing plasmid into a human T cell line Jurkat, we prepared the T cells, which express a high level of the alpha3beta1 integrin, to assess the role of laminin 5 in the skin immune system. The alpha3beta1-expressing T cells adhered to laminin 5 and exhibited spreading. These adhered T cells showed a significant tyrosine phosphorylation of intracellular proteins including p59(fyn) upon T-cell receptor (TCR) stimulation. Six hours after cross-linking TCR, these cells on laminin 5 secreted a three times higher level of IL-2 than those on a BSA-coated plate. Twenty hours after the stimulation, 48% of the alpha3beta1-expressing T cells on laminin 5 caused apoptosis. The protein level of cyclin D3 and E decreased, while that of p53 increased in these T cells. These data suggest that laminin 5 may play at least two regulatory roles for T cell functions: augmentation of IL-2 production by antigen-stimulated T cells and induction of apoptosis in these T cells.