Cholera toxin (CT) is an AB, hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM, on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GMI pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GMI to cholera toxin thus resembles a 2-fingered grip: the Gal(P1-3)CalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTBs:GMl pentasaccharide complex described here provides a detailed view of a pr0tein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in OM,-mediated signal transduction.Keywords: cholera toxin; crystal structure; ganglioside GM1; sugar binding specificity Cholera is a severe disease that can lead to death within a few hours. The major clinical symptoms are caused by a toxin released after adhesion of the noninvasive Vibrio cholerue bacteria to the proximal small intestine of the host. Cholera toxin (CT) acts intracellularly to catalyze ADP-ribosylation of residue Arg 187 in the a subunit of the trimeric protein G,. The modified G,, loses its GTPase activity and remains constitutively in its GTP-bound state (Cassel & Pfeuffer, 1978), which in turn causes a continuous stimulation of adenylate cyclase. The resulting elevated levels of cyclic AMP lead to massive loss of fluids, the characteristic pathology of enterotoxigenic disease.Cholera toxin is an AB, hexamer consisting of 5 identical B subunits and a single A subunit. It is structurally and functionally related to a larger group of bacterial enterotoxins that includes the closely related Escherichia coli heat-labile enterotoxin (LT) as well as pertussis toxin, diphtheria toxin, shigella toxin, and Pseudomonas aeruginosa exotoxin A. In this class of tox- ins, the biological functions of target cell recognition and enzymatic activity are separated into distinct domains. In the case of CT and LT, cell recognition and binding are carried out by the ...

Pro-MMP2 activation is a two-step process resulting in (1) an intermediate 64 kDa form generated by the MT1-MMP activity, and (2) a mature 62 kDa form. Addition of plasminogen to HT1080 cells cultured under various conditions, or to their membrane preparation, induced a complete conversion of the intermediate MMP-2 form to the mature one, and processing of pro-MMP-9. The pro-MMP-2 activation was inhibited by plasmin inhibitors and anti-uPA antibody. These results provide evidence for involvement of the PA/plasmin system in the second step of MMP-2 activation.

Matrix metalloproteinases (matrixins) constitute a group of extracellular proteinases belonging to the metzincin superfamily. They are involved in both physiological and pathological tissue remodeling processes, including those associated with cancer progression. Stromelysin-3, which is expressed in most invasive human carcinomas, is a matrix metalloproteinase with unusual functional properties. In particular, its mature form does not cleave any of the major extracellular matrix components. To define critical structural determinants involved in controlling stromelysin-3 proteolytic activity, we have used site-directed mutagenesis. We show that the deletion of at least 175 C-terminal aminoacids is sufficient to endow mouse stromelysin-3 with activities against casein, laminin, and type IV collagen. In the case of the human enzyme, however, a further and single Ala-235 3 Pro substitution is necessary to observe similar activities. Ala-235, which characterizes human stromelysin-3 among matrixins, is located immediately after the C terminus of the "Met-turn," which forms a hydrophobic basis for the catalytic zinc atom in the metzincin family. We conclude that human stromelysin-3 has gained specific functional properties during evolution by amino acid substitution in the catalytic zinc environment, and that it represents an attractive target for specific inhibitors that may be used to prevent cancer progression.Matrix metalloproteinases (matrixins) constitute a group of extracellular proteinases of related primary structure, including collagenases, gelatinases, stromelysins, and their activators (1-3). Matrix metalloproteinases are believed to be mediators of physiological and pathological remodeling processes occurring during development, involution, repair, and cancer progression (4 -7). In particular, these enzymes have been shown through both correlative and direct inhibitor studies to be essential for cancer cell invasion (8 -10). However, the demonstration that stromelysin-1 plays a role in morphogenesis during development (11) and the observation that a matrix metalloproteinase-like enzyme is implicated in tumor necrosis factor-␣ processing (12) suggest that the contribution of matrix metalloproteinases to tumor progression may be not limited to a role in cancer invasion and also involve other aspects of the malignant phenotype. In agreement with this possibility, matrix metalloproteinase expression is not specific to invasive carcinomas and is also observed in a number of precursor lesions (Ref. 13 and references therein).Stromelysin-3 (ST3) 1 was identified through its overexpression in fibroblastic cells of invasive breast carcinomas (14), and similar observations were thereafter made in most other human carcinomas (13). ST3-expressing fibroblastic cells are specifically detected in the immediate vicinity of cancer cells, suggesting that ST3 may play a role in stromal-epithelial interactions during carcinoma progression, thereby contributing to tumor growth. Indeed, Engel et al. (15) have found that recurr...

Co‐injection of fibroblasts with human epithelial breast‐tumor MCF7 cells in the presence of Matrigel enhances tumor growth in nude mice. While most of the matrix metalloproteinases (MMPs) have been shown to be produced by stromal cells, tumor cells such as MCF7 cells are unable to produce MMPs. We therefore, hypothesized that the tumor‐promoting effect of fibroblasts could be related to their production of MMPs. In order to inhibit stromal proteases, over‐production of TIMP‐2 was induced in MCF7 cells by in vitro retroviral‐mediated gene transfer. TIMP‐2‐producing MCF7 cells were then co‐injected with fibroblasts into nude mice. Alternatively, we evaluated the effect of Batimastat, a synthetic inhibitor of MMPs, on the tumorigenicity of MCF7 cells co‐inoculated with fibroblasts into nude mice. Both physiological (TIMP‐2) and synthetic (Batimastat) inhibitors of MMPs were able to abolish the tumor‐promoting effect of fibroblasts. On the contrary, they failed to modulate the tumorigenicity of MCF7 cells injected alone. Interestingly, Matrigel from which low‐molecular‐weight proteins or growth factors had been removed failed to favor the tumorigenicity of MCF7 cells inoculated with fibroblasts. These findings emphasize the importance of fibroblasts in cancer progression, and suggest that their role could be related at least in part to production of proteases which can induce the release of factors from the extracellular matrix. Int. J. Cancer 76:267–273, 1998.© 1998 Wiley‐Liss, Inc.

A cDNA library was prepared from poly(A)+RNA extracted from tilapia Oreochromis niloticus anterior pituitaries. The recombinant clones carrying the cDNA sequence of tilapia growth hormone (tiGH) were selected using a fragment of the trout growth hormone (tGH) cDNA as hybridization probe. The nucleotide sequence of the full-length tiGH cDNA was determined. This cDNA encodes a protein of 204 amino acids, including the putative signal peptide of 17 amino acids. Mature tiGH cDNA was inserted in an Escherichia coli expression vector which led to the production of tiGH protein with a yield estimated to be 20% of the total bacterial proteins.