Purpose: Angiotensin II is a bioactive peptide of the renin-angiotensin system, acting not only as a vasoconstrictor but also as a growth promoter via angiotensin II type 1 receptors (AT 1 R). The present study examined AT 1 R expression in human ovarian carcinoma and attempted to determine whether AT 1 R blocker could suppress the tumor progression. Experimental Design: Expression of AT 1 R, vascular endothelial growth factor (VEGF), and CD34 was immunohistochemically analyzed in ovarian tumor tissues (n = 99). Effects of AT 1 R blocker on invasive potential and VEGF secretion in ovarian cancer cells were examined in vitro. Effects of AT 1 R blocker in vivo were evaluated in a mouse model of peritoneal carcinomatosis. Results: AT 1 R was expressed in 57 of 67 (85%) invasive ovarian adenocarcinomas and 12 of 18 (66%) borderline malignant tumors but in only 2 of 14 (14%) benign cystadenomas. In invasive carcinomas,VEGF expression intensity and intratumor microvessel density were significantly higher in cases that were strongly positive for AT 1 R (n = 37) compared with those in cases weakly positive (n = 20) or negative (n = 10) for AT 1 R. Angiotensin II significantly enhanced the invasive potential and VEGF secretion in AT 1 R-positive SKOV-3 ovarian cancer cells, both of which were completely inhibited by the AT 1 R blocker candesartan. Administration of candesartan into SKOV-3-transplanted athymic mice resulted in the reduction of peritoneal dissemination, decreased ascitic VEGF concentration, and suppression of tumor angiogenesis. Conclusions: AT 1 R is functionally expressed in ovarian carcinoma and involved in tumor progression and angiogenesis. AT 1 R blockade therapy may become a novel and promising strategy for ovarian cancer treatment.

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.

Serum levels of human placental leucine aminopeptidase/oxytocinase (P-LAP) increase with gestation. cDNA cloning of P-LAP revealed that the enzyme is a type II membrane-bound protein containing the consensus HEXXH(X) 18 E motif found in the M1 family of zinc-metallopeptidase proteins. In this study, a recombinant soluble form of P-LAP found in maternal serum was expressed in Chinese hamster ovary cells, purified to homogeneity and then characterized. Although N-terminal sequencing revealed a four-amino-acid deletion, the purified enzyme was active and was shown to be a zinc-containing homodimeric protein with molecular mass of 280 kDa in solution. Using artificial substrates, it was shown that the enzyme has broad specificity and is inhibited by several compounds known as aminopeptidase inhibitors. Subsequently, sequential N-terminal aminoacid liberation of several peptide hormones by the enzyme was monitored and structures of the products were determined. Among the hormones having a cysteine residue at their N-terminal end and intramolecular disulfide bonds, it was found that vasopressin and oxytocin, but not calcitonin and endothelins, were cleaved by the enzyme. Because the molecular properties of oxytocinase so far reported often conflict, our results provide an initial biochemical and enzymatic characterization of moleculary defined P-LAP/oxytocinase.

In the current study, we report the cloning and initial characterization of a novel human cytosolic aminopeptidase named adipocyte-derived leucine aminopeptidase (A-LAP). The sequence encodes a 941-amino acid protein with significant homology (43%) to placental leucine aminopeptidase (P-LAP)/oxytocinase. The predicted A-LAP contains the HEXXH(X)18E consensus sequence, which is characteristic of the M1 family of zinc-metallopeptidases. Although the deduced sequence contains a hydrophobic region near the N-terminus, the enzyme localized mainly in cytoplasm when expressed in COS-7 cells. Northern blot analysis revealed that A-LAP was expressed in all the tissues tested, some of which expressed at least three forms of mRNA, suggesting that the regulation of the gene expression is complex. When aminopeptidase activity of A-LAP was measured with various synthetic substrates, the enzyme revealed a preference for leucine, establishing that A-LAP is a novel leucine aminopeptidase with restricted substrate specificity. The identification of A-LAP, which reveals strong homology to P-LAP, might lead to the definition of a new subfamily of zinc-containing aminopeptidases belonging to the M1 family of metallopeptidases.

Basigin (Bsg) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Bsg knock-out mice exhibit infertility of both sexes. Based on limited results, defective implantation has been considered to be the cause of the female infertility. We demonstrate here that disruption of the Bsg gene produces the failure of female reproductive processes including not only implantation but also fertilization. Bsg mRNA expression in cumulus cells and basolateral localization of the Bsg protein in the endometrial epithelium further support the importance of Bsg in these processes.z 1998 Federation of European Biochemical Societies.

Basigin (Bsg) is a highly glycosylated transmembrane protein with two immunoglobulin (Ig)-like domains. A number of studies, including gene targeting, have demonstrated that Bsg plays pivotal roles in spermatogenesis, implantation, neural network formation and tumor progression. In the present study, to understand the mechanism of action of Bsg, we determined its expression status on the plasma membrane. Cotransfection of Bsg expression vectors with two different tags clarified that Bsg forms homo-oligomers in a cis-dependent manner on the plasma membrane. If the disulfide bond of the more N-terminally located Ig-like domain was destroyed by mutations, Bsg could not form oligomers. In contrast, the mutations of the C-terminal Ig-like domain or N-glycosylation sites did not affect the association. The association of mouse and human Bsgs, which exhibit high homology in the transmembrane and intracellular domains but low homology in the extracellular domain, was very weak as compared with that within the same species, suggesting the importance of the extracellular domain in the association. If the extracellular domain of the human Ret protein was replaced with the N-terminal Ig-like domain of Bsg, the resulting chimera protein was associated with intact wild-type Bsg, but not if the C-terminal Ig-like domain, instead of the N-terminal one, of Bsg was used. No oligomer formation took place between the intact wild-type Ret and Bsg proteins. In conclusion, these data indicate that the N-terminal Ig-like domain is necessary and sufficient for oligomer formation by Bsg on the plasma membrane.Keywords: basigin; EMMPRIN; immunoglobulin superfamily; neurothelin; oligomer.Cell-surface proteins, such as the Ig superfamily, the cadherin superfamily and the integrin superfamily, play important roles in development by regulating cellular differentiation, adhesion and migration [1,2]. Supramolecular structure formation, such as oligomer formation, between these molecules plays crucial roles in exerting their functions. This has been documented by extensive studies of the Ig superfamily. NCAM participates in neural cell adhesion, which is mainly mediated by homophilic binding in a trans-dependent manner through its third Ig-like domain [3,4]. Homodimer formation of ICAM in a cis-dependent manner enhances its binding to a leukocyte b2-integrin [5]. Binding of fibroblast growth factor (FGF) to its receptor (FGFR) leads to dimer formation of FGFR (eventually a complex formation consisting of two FGF, two FGFR and a heparan sulfate chain), which is mediated mainly through homophilic binding at the second Ig-like domain of FGFR [6]. In addition to protein±protein interaction as mentioned above, protein±sugar interaction is also important. P0, a neural adhesion molecule implicated in myelination, harbors an N-glycosylation site, to which the major homophilic binding site is mapped [7]. NCAM and L1, another neural adhesion molecule, associate each other in a cis-dependent manner to reinforce L1 homophilic trans-interaction: in this case...

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A neuropathological hallmark of Alzheimer's disease is the presence of amyloid plaques in the brain. Amyloid-beta peptide (Abeta) is the major constituent of the plaques and is generated by proteolytic cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. Growing evidence shows that lipid rafts are critically involved in regulating the Abeta generation. In support of this, APP, Abeta, and presenilins have been found in lipid rafts. Although cholesterol plays a crucial role in maintaining lipid rafts, functions of other components in the generation of Abeta are unknown. Caveolins (CAVs) and flotillins (FLOTs) are principal proteins related to lipid rafts and have been suggested to be involved in APP processing. Here, we report that FLOT-1 binds to BACE1 (beta-site APP cleaving enzyme 1) and that overexpression of CAV-1 or FLOT-1 results in recruiting BACE1 into lipid rafts and influence on beta-secretase activity in cultured cells. Our results show that both CAV-1 and FLOT-1 may modulate beta-secretase activity by interacting with BACE1.