Ikuo Yamashina

Accelerated evolution of crotalinae snake venom gland serine proteases

Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein‐coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

The engelbreth-holm-swarm mouse tumor produces undersulfated heparan sulfate and oversulfated galactosaminoglycans

Glycosaminoglycans were prepared from the Engelbreth-Holm-Swarm mouse tumor. Enzymatic analysis demonstrated heparan sulfate (95.8%) and chondroitinase ABC-sensitive galactosaminoglycans (4.2%). HPLC analysis of the disaccharide units showed that heparan sulfate chains were undersulfated on average, comprising approximately 30% nonsulfated and 60% N-sulfated disaccharide units with small proportions of other monosulfated and disulfated disaccharide units. In contrast, galactosaminoglycan chains were oversulfated, containing an appreciable proportion (15%) of a 4,6-disulfated (so-called E-type) disaccharide unit in addition to 51% of a 4-sulfated, 22% of a 6-sulfated, and 11% of a nonsulfated disaccharide unit. The significance of the oversulfated disaccharide structure is discussed in relation to the possible regulation of functions of hybrid proteoglycans from which the galactosaminoglycan chains are derived.

Induction of cyclooxygenase-2 in monocyte/macrophage by mucins secreted from colon cancer cells

Up-regulation of cyclooxygenase-2 (COX-2) and overproduction of prostaglandins have been implicated in the initiation and/or progression of colon cancer. However, it is uncertain in which cells and how COX-2 is induced initially in the tumor microenvironment. We found that a conditioned medium of the colon cancer cell line, LS 180, contained a factor to induce COX-2 in human peripheral blood mononuclear cells. This factor was purified biochemically and revealed to be mucins. A small amount of mucins (≈100 ng of protein per ml) could elevate prostaglandin E2 production by monocytes. The mucins induced COX-2 mRNA and protein levels of monocytes in a dose- and time-dependent manner, indicating a COX-2-mediated pathway. We also have examined immunohistochemically the localization of COX-2 protein and mucins in human colorectal cancer tissues. It is noteworthy that COX-2-expressing macrophages were located around the region in which mucins were detectable, suggesting that COX-2 also was induced by mucins in vivo. These results suggest that mucins produced by colon cancer cells play a critical role in the initial induction of COX-2 in the tumor microenvironment.

A monoclonal antibody directed to Tn antigen

A murine monoclonal antibody, MLS 128, that was assigned to an anti-Tn antibody has been established by immunizing mice with human colonic cancer cells (LS 180). MLS 128 bound to mucin glycopeptides from LS 180 cells and their asialo forms to the same extent as well as to ovine submaxillary mucin (OSM) and asialo OSM. Special non-sialylated GalNAc residue(s) attached to a certain peptide region in the antigens seems to be involved in the binding since N-acetylgalactosaminidase treatment of the antigen abolished the binding and pronase digestion diminished the binding markedly.

The role of syndecan-2 in regulation of actin-cytoskeletal organization of Lewis lung carcinoma-derived metastatic clones.

Syndecans, a family of transmembrane heparan sulphate proteoglycans, contribute to various biological processes, including adhesion, motility, proliferation, differentiation and morphogenesis. We document here the involvement of syndecan-2 acting alone or co-operatively with integrin alpha5beta1, for regulation of actin-cytoskeletal organization on cell adhesion to fibronectin, using fibronectin-recombinant polypeptides containing the ligands for either or both of these receptors as substrata. Lewis lung carcinoma-derived low-metastatic P29 cells binding to the substrata by both receptors formed actin stress fibres, whereas those binding by syndecan-2 or integrin alpha5beta1 alone formed filopodia or cortex actin. In contrast, higher metastatic LM66-H11 cells formed cortex actin even on substrata containing both ligands. Northern-blot and flow-cytometric analyses revealed that syndecan-2 expression in LM66-H11 cells was significantly lower (1/4.5 in mRNA and 1/8 in cell-surface expression) than in P29 cells, whereas expression levels of integrin alpha5beta1 and other syndecans were similar in both cell types. These results suggest that the failure of LM66-H11 to form stress fibres is due to a lower expression of syndecan-2 than that due to a threshold for its function. This was confirmed by the finding that overexpression of syndecan-2 by transfection of its cDNA into LM66-H11 cells caused the formation of stress fibres on the fibronectin substratum. These in vitro cellular responses of the two clones might reflect their in vivo situation in primary tumours in which P29 cells with a stroma-inducing capacity were immediately surrounded by fibronectin-rich matrix formed by the induced stromal cells, whereas LM66-H11 cells without such capacity were not surrounded by a similar matrix.

Purification of β-mannosidase from a snail, Achatina fulica, and its action on glycopeptides

Abstract 1. 1. β-Mannosidase (β- d -mannoside mannohydrolase, EC 3.2.1.25) was extracted and purified from viscera of the snail, Achatina fulica, about 100-fold over the crude extract. The purified β-mannosidase was free from α-mannosidase (α- d -mannoside mannohydrolase, EC 3.2.1.24) and β-acetylglucosamininase (β-2-acetamido-2-deoxy- d -glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) activities. 2. 2. Specific cleavage of β-mannosidic linkage by the purified β-mannosidase was demonstrated using a trimannoside, α-Man-(1 → 4)-β-Man-(1 → 4)-Man, as substrate. This mannoside could be degraded to mannose stepwise by the successive actions of α-mannosidase from hog kidney and the purified β-mannosidase. 3. 3. The purified β-mannosidase released mannose readily from a glycopeptide, Man-(GlcNAc)2-Asn-(Ser), which had been isolated from the α-mannosidase digest of Taka-amylase glycopeptide.

The isolation and characterization of glycopeptides and mucopolysaccharides from plasma membranes of normal and regenerating livers of rats.

Isolation of the glycopeptides of the O-glycosidic and N-glycosidic types and of heparan sulfate from plasma membranes of ascites hepatoma cells, AH 66 and AH 130, [1,2], prompted us to investigate the complex carbohydrates occuring in plasma membranes of normal and regenerating livers of rats, as normal control cells. In a preliminary study, we reported the isolation of glycopeptides of the N-glycosidic type from the pronase digest of the plasma membranes of normal rat livers which were acidic (sialic acid-containing) and of relatively low mol. wt [3]. At that time, however, the carbohydrate-containing fractions of relatively high mol. wt, which were excluded from a Sephadex G-50 column, were not investigated. Since the O-glycosidic glycopeptides and the mucopolysaccharides from the hepatoma plasma membranes were obtained in these excluded fractions, we decided to reinvestigate the corresponding fractions from normal livers. Regenerating (dividing) liver cells were also used since they are regarded as more adequate than normal (resting) liver cells for use as a normal control to hepatoma cells which are rapidly dividing. Results reported here show that normal and regenerating livers gave similar glycopeptide patterns, all of which were apparently N-glycosidic and acidic in contrast to the production of both Nand Oglycosidic glycopeptides from the hepatoma membranes. The occurrence of heparan sulfate was, however, common to all the plasma membranes from the livers and hepatomas. 2. Materials and methods

Identification of the mannan-binding protein from rat livers as a hepatocyte protein distinct from the mannan receptor on sinusoidal cells☆

Cellular distribution in rat livers of the mannan-binding protein, a liver lectin specific for mannose and N-acetylglucosamine was investigated. Estimation of mannan binding activity of isolated cells demonstrated that more than 99% of the mannan binding activity recovered from whole livers was derived from hepatocytes. Intracellular localization of the mannan-binding protein disproved its function as a receptor of endocytosis. A small quantity of mannan binding activity detectable in sinusoidal cells seems to represent a receptor for mannan endocytosis. The receptor resembled the mannan-binding protein in its high affinity for mannan, requirement of Ca2+ for binding, and specificity for mannose and N-acetylglucosamine. However, the receptor was clearly distinct from the mannan-binding protein as indicated by the lack of response to antimannan-binding protein antiserum, by the potent inhibition by L-fucose and by the differences of specificity for various glycoproteins. The results in this study confirm the recent report of Maynard and Baenziger (Maynard, Y., and Baenziger, J. (1982) J. Biol. Chem. 257, 3788-3794) with quantitative data using different methodology and ligands, and provide additional evidence indicating the receptor on sinusoidal cells to be distinct from the hepatic binding protein. A possible role of the mannan-binding protein in the intracellular transport of intermediates of glycoprotein biosynthesis is also discussed.

Mucin-carbohydrate directed monoclonal antibody

To raise monoclonal antibodies recognizing cancer‐associated alterations of the carbohydrate structure of glycoproteins, Balb/c mice were immunized with human colonic cancer cells (LS 180 from ATCC). One of the generated hybridomas produced a monoclonal antibody that bound to the carbohydrate moiety of mucin‐type glycoproteins from LS 180. The antibody did not bind to glycoproteins from another colonic cancer cell line, SW 1116, or to glycolipids from any of the colonic cancer cell lines. The antibody bound to ovine and bovine submaxillary mucins (OSM and BSM). NeuAcα2→6Ga1NAc seemed to be involved in the epitope.

Heparan sulphate proteoglycans interact with neurocan and promote neurite outgrowth from cerebellar granule cells

We found that neurocan, a major brain chondroitin sulphate proteoglycan, interacts with HSPGs (heparan sulphate proteoglycans) such as syndecan-3 and glypican-1. Binding of these HSPGs to neurocan was prevented by treatment of the HSPGs with heparitinases I and II, but not by treatment of neurocan with chondroitinase ABC. Scatchard plot analysis indicated that neurocan has two binding sites for these HSPGs with different affinities. It is known that neurocan in the rodent brain is proteolytically processed with aging into N- and C-terminal fragments. When a mixture of whole neurocan and N- and C-terminal fragments prepared from neonatal mouse brains or recombinant N- and C-terminal fragments was applied to a heparin column, the whole molecule and both the N- and C-terminal fragments bound to heparin. A centrifugation cell adhesion assay indicated that both the N- and C-terminal neurocan fragments could interact with these HSPGs expressed on the cell surface. To examine the biological significance of the HSPG-neurocan interaction, cerebellar granule cells expressing these HSPGs were cultured on the recombinant neurocan substrate. A significant increase in the rate of neurite outgrowth was observed on the wells coated with the C-terminal neurocan fragment, but not with the N-terminal one. Neurite outgrowth-promoting activity was inhibited by pretreatment of neurocan substrate with heparin or the addition of heparitinase I to culture medium. These results suggest that HSPGs such as syndecan-3 and glypican-1 serve as the cell-surface receptor of neurocan, and that the interaction of these HSPGs with neurocan through its C-terminal domain is involved in the promotion of neurite outgrowth.