Shinji Ihara

A Possible Role of RGS9 in Phototransduction A BRIDGE BETWEEN THE cGMP-PHOSPHODIESTERASE SYSTEM AND THE GUANYLYL CYCLASE SYSTEM

In the current concept of phototransduction, the concentration of cGMP in retinal rod outer segments is controlled by the balance of two enzyme activities: cGMP phosphodiesterase (PDE) and guanylyl cyclase (GC). However, no protein directly mediates these two enzyme systems. Here we show that RGS9, which is suggested to control PDE activity through regulation of transducin GTPase activity (He, W., Cowan, C. W., and Wensel, T. G. (1998) Neuron 20, 95–102), directly interacts with GC. When proteins in the Triton X-100-insoluble fraction of bovine rod outer segments were isolated by two-dimensional gel electrophoresis and binding of GC to these proteins was examined using a GC-specific antibody, proteins (55 and 32 kDa) were found to interact with GC. However, the activity of GC bound to the 55-kDa protein was not detected. This observation was elucidated by the finding that the 55-kDa protein inhibited GC activity in a dose-dependent manner. Amino acid sequence showed that five peptides derived from the 55-kDa protein were identical to corresponding peptides of RGS9. Together with other biochemical characterization of the 55-kDa protein, these observations indicate that the 55-kDa protein is RGS9 and that RGS9 inhibits GC. RGS9 may serve as a mediator between the PDE and GC systems.

Implication of GnT-V in cancer metastasis: a glycomic approach for identification of a target protein and its unique function as an angiogenic cofactor.

It is known that sugar chains have a variety of functions and play a key role in development, differentiation, cell adhesion and cell growth in cells [1,2]. Our group has focused on the underlying mechanism of the biosynthesis of N -glycan branchings in glycoproteins, and reported on various N -glycan branching enzymes that play a pivotal role in various steps [3]. A possible link between β1-6 branched structures of N glycans and cancer metastasis has been proposed by several authors [4–6]. However before the cloning of the UDP-GlcNAc α-mannoside β1-6-N -acetylglucosaminyltransferase (GnT-V) enzyme which catalyzes the formation of β1-6 branching was carried out, it was not clear whether or not β1-6 branching is actually related to the metastatic potential of cancer cells. The enzyme was independently purified from rat kidney [7] by Pierce’s group and from human cancer cells [8] by our group, and both groups subsequently cloned their cDNAs [9,10]. Using the cloned genes several studies have presented evidence for the presence of strong association between β1-6 branching and cancer metastasis [11–13]. Knock out mice developed by Dennis’s group confirmed that the gene is highly linked to cancer development [14]. However, in the case of glycosyl-

A secreted type of β1,6 N-acetylglucosaminyltransferase V (GnT-V), a novel angiogenesis inducer, is regulated by γ-secretase

Glycosyltransferases are present in the Golgi apparatus in a membrane‐bound form and are released from cells after cleavage by certain proteases. β1,6‐N‐Acetylglucosaminyltransferase V (GnT‐V), which is cleaved and secreted from the cells, is involved in the biosynthesis of β1–6GlcNAc branching on N‐ glycans and has been implicated in tumor progression and metastasis. We recently reported that a secreted type of GnT‐V (soluble GnT‐V) itself could promote angiogenesis, which is completely different from its original function as a glycosyltransferase, and this might play a role in tumor invasion. In this study, to explore the molecular basis for this functional glycosyl‐transferase secretion, its cleavage site was examined and the protease(s) involved in that cleavage were identified. The NH2‐terminal protein sequence of purified soluble GnT‐V (~100 kDa) from GnT‐V‐overex‐pressed cells revealed that its terminus started at His31, located at the boundary position between the transmembrane and stem regions. This secretion was not inhibited by a single amino acid mutation at the cleavage site (Leu29, Leu30 to Asp, His31 to Ala), but specifically inhibited by addition of DFK‐167, a γ‐secretase inhibitor, suggesting that γ‐secretase is a plausible protease for secretion processing. In addition, transfection of the gene of familial Alzheimers disease (FAD)‐ linked presenilin‐1, a component of γ‐secretase, in‐ creased the secretion rate of endogenous GnT‐V; the secretion of soluble GnT‐V (~100 kDa) was completely inhibited in presenilin‐1/2 double‐deficient cells, which have no γ‐secretase activity. Collectively, these results demonstrate that Golgi‐resident GnT‐V is cleaved at the transmembrane region by γ‐secretase, and this might control tumor angiogenesis through a novel pathway.—Nakahara, S., Saito, T., Kondo, N., Moriwaki, K., Noda, K., Ihara, S., Takahashi, M., Ide, Y., Gu, J., Inohara, H., Katayama, T., Tohyama, M., Kubo, T., Taniguchi, N., Miyoshi, E. A secreted type of H1,6 N‐acetylglucosaminyltransferase V (GnT‐V), a novel angiogenesis inducer, is regulated by γ‐secretase. FASEB J. 20, 2451–2459 (2006)

Attachment of Human Colon Cancer Cells to Vascular Endothelium Is Enhanced by N-Acetylglucosaminyltransferase V

Expression of N-acetylglucosaminyltransferase V (GnT-V) in colon cancer has been shown to be related to hematogenous metastasis and poor prognosis. To investigate the mechanism by which cancer cells expressing GnT-V metastasize to distant organs, we established GnT-V-overexpressing DLD-1 and WiDr cells (human colon cancer cell lines) by transfecting them with a GnT-V expression vector. Attachment to endothelial cells expressing E-selectin was studied, and expression of the E-selectin ligand, sialyl Lewis x, in colon cancer cells was investigated. Both of the cell lines showed reduced adhesion to fibronectin as compared with mock transfectants. In contrast, attachment to human umbilical vein endothelial cells expressing E-selectin was significantly enhanced by GnT-V expression (p < 0.01). Sialyl Lewis x is a ligand for E-selectin and a marker for poor prognosis of colon cancer. Its synthesis in cells has been shown to involve GnT-V. We demonstrated that expression of sialyl Lewis x in colon cancer cells was induced by GnT-V expression. These results suggest that GnT-V induces sialyl Lewis x expression and leads colon cancer cells to metastasize by enhancing their ability to attach to vascular endothelium in distant organs, such as liver or lung. Inhibition of GnT-V activity may prevent metastasis in colon cancer patients with high sialyl Lewis x expression.

SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.

Prodomain-dependent tissue targeting of an ADAMTS protease controls cell migration in Caenorhabditis elegans

Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted proteins play important roles in animal development and pathogenesis. However, the lack of in vivo models has hampered elucidation of the mechanisms by which these enzymes are recruited to specific target tissues and the timing of their activation during development. Using transgenic worms and primary cell cultures, here we show that MIG‐17, an ADAMTS family protein required for gonadal leader cell migration in Caenorhabditis elegans, is recruited to the gonadal basement membrane in a prodomain‐dependent manner. The activation of MIG‐17 to control leader cell migration requires prodomain removal, which is suggested to occur autocatalytically in vitro. Although the prodomains of ADAMTS proteases have been implicated in maintaining enzymatic latency, polypeptide folding and secretion, our findings demonstrate that the prodomain has an unexpected function in tissue‐specific targeting of MIG‐17; this prodomain targeting function may be shared by other ADAMTSs including those in vertebrates.

A Glycomic Approach to Hepatic Tumors in N -acetylglucosaminyltransferase III (GnT-III) Transgenic Mice Induced by Diethylnitrosamine (DEN): Identification of Haptoglobin as a Target Molecule of GnT-III

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo. In order to understand the biological effects of N -acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E 4 -PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.

Stage-specific activation of MIG-17⁄ADAMTS controls cell migration in Caenorhabditis elegans

The activation of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family proteases depends on removal of the prodomain. Although several studies suggest that ADAMTS activities play roles in development, homeostasis and disease, it remains unclear when and where the enzymes are activated in vivo. MIG‐17, a Caenorhabditis elegans glycoprotein belonging to the ADAMTS family, is secreted from the body wall muscle cells and localizes to the gonadal basement membrane to control the migration of gonadal distal tip cells. Here, we developed a monoclonal antibody that recognizes the N‐terminal neo‐epitope of the activated MIG‐17. In western blotting, the antibody specifically detected the activated form, the signal for which dramatically increased during the third and fourth larval stages, when MIG‐17 is required to direct distal tip cell migration. In in situ staining, the monoclonal antibody recognized the activated form in the basement membrane, whereas it failed to detect a processing‐resistant mutant form localized to the basement membrane. MIG‐17 was activated in the basement membranes of the muscle, intestine and gonad in the third larval stage, and downregulated in nongonadal basement membranes in young adults and in gonadal basement membranes in older adults. Thus, the activation of MIG‐17 is regulated in a spatiotemporal manner during C. elegans development. This is the first report demonstrating the regulated activation of an ADAMTS protein in vivo. Our results suggest that monoclonal antibodies against neo‐epitopes have potential as powerful tools for detecting activation of ADAMTSs during development and in disease pathogenesis.

The Novel Secreted Factor MIG-18 Acts with MIG-17/ADAMTS To Control Cell Migration in Caenorhabditis elegans

The migration of Caenorhabditis elegans gonadal distal tip cells (DTCs) offers an excellent model to study the migration of epithelial tubes in organogenesis. mig-18 mutants cause meandering or wandering migration of DTCs during gonad formation, which is very similar to that observed in animals with mutations in mig-17, which encodes a secreted metalloprotease of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family. MIG-18 is a novel secreted protein that is conserved only among nematode species. The mig-17(null) and mig-18 double mutants exhibited phenotypes similar to those in mig-17(null) single mutants. In addition, the mutations in fbl-1/fibulin-1 and let-2/collagen IV that suppress mig-17 mutations also suppressed the mig-18 mutation, suggesting that mig-18 and mig-17 function in a common genetic pathway. The Venus-MIG-18 fusion protein was secreted from muscle cells and localized to the gonadal basement membrane, a tissue distribution reminiscent of that observed for MIG-17. Overexpression of MIG-18 in mig-17 mutants and vice versa partially rescued the relevant DTC migration defects, suggesting that MIG-18 and MIG-17 act cooperatively rather than sequentially. We propose that MIG-18 may be a cofactor of MIG-17/ADAMTS that functions in the regulation of the gonadal basement membrane to achieve proper direction of DTC migration during gonadogenesis.

Identification of various types of α2-HS glycoprotein in sera of patients with pancreatic cancer: Possible implication in resistance to protease treatment

α2-Heremans-Schmid glycoprotein (human fetuin) is one of numerous serum proteins produced in the liver. Recently, the biological functions of fetuin, such as calcification and insulin resistance, have been clarified. However, these effects appear to be indirect, occurring through binding to other molecules. When equal amounts of fetuin in sera were treated with chymotrypsin, resistance to the protease treatment was observed in patients with pancreatic cancer, but not in normal volunteers. To investigate the molecular mechanism behind this resistance, gel-filtration chromatography was performed. The results revealed that high molecular types of fetuin showed a resistance to protease treatment. When fetuin was purified from sera of patients with pancreatic cancer and normal volunteers, certain types of proteins, including haptoglobin (which binds to fetuin derived from pancreatic cancer patients), were identified using mass spectrometry. Furthermore, the oligosaccharide structures of fetuin analyzed with lectin microarray differed between pancreatic cancer patients and normal volunteers. This macro/micro heterogeneity of fetuin might contribute to pancreatic cancer resistance to chymotrypsin treatment.