Inhibitors of sialyltransferases: potential roles in tumor growth and metastasis.

For over thirty years it has been evident that there is altered glycosyltransferase activity in neoplastic tissue when compared to healthy tissue. It has also long been speculated that disruption of the neoplastic expression of sialic acid on cellular glycoconjugates, is a valid target in anti-metastatic therapeutic development. Over the years attempts have been made to synthesize inhibitors of sialyltransferases in a effort to assist in the validation or dissolution of these enzymes as potential therapeutic targets.

A rapid, semi-automated method for detection of Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) activity using the lectin Sambucus nigra agglutinin.

Sialyltransferase activity has traditionally been studied by determining the rate at which the enzyme transfers a labeled donor sugar to an acceptor substrate. These types of assays can be difficult to quantitate, and the separation of untransfered donor sugar from the sialylated acceptor is time-consuming. The biosensor-based method described here is both rapid and semi-automated. The NeuAc-alpha2-6Gal-R-specific lectin Sambucus nigra agglutinin (SNA) immobilized to the carboxymethyl dextran surface of a BIAcore sensor chip was used to detect and measure the formation of the NeuAc-alpha2-6Gal-R moieties. The sialyltransferase assays were carried out using modified protocols based on the method described in Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of betaD-galactoside alpha2-3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444-4451. The complete assay mixture was simply diluted before injection into the instrument. All injections were performed automatically using the robotics of the BIAcore instrument. Using this technique it is possible to detect product from 0.4 microU of commercial Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I). One unit of sialyltransferase is defined as the quantity that will transfer 1 micromol of N-acetylneuraminic acid from cytidine monophosphate (CMP)-N-acetylneuraminic acid to asialofetuin per min at pH 6.5 and 37 degrees C. The method described here requires as little as 10 microl total assay volume, thus reducing the consumption of reagents. In addition, the sample is completely recoverable from the sensor chip surface, which allows for downstream analysis of the reaction product if desired. This method eliminates the need for labeled donor and acceptor molecules and does not require the separation of the substrates from the product before analysis. Although some kinetic properties of the enzyme can be estimated using this method, further development and validation is required. The method is most useful in determining qualitative estimates of ST6Gal I activity in tissue extracts and in characterizing the production of enzymes in cultured cell systems. The use of a microtiter plate assay format enables the rapid screening of multiple fractions for sialyltransferase activity.

Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface.

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by approximately 40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.

Identification of an immunodominant IgE epitope of the Parietaria judaica major allergen.

Par j 1.0101 is one of the two major allergens of the Parietaria judaica (Pj) pollen, and its three-dimensional structure was built by three-dimensional structural homology modeling. The resultant model was used to identify putative IgE binding regions. Western blot analysis of gene fragmentation products showed that the 1 to 30 region was capable of binding specific IgE from a pool of sera (n = 30) of patients allergic to Pj pollen. Using the structural model as a guide, deletion and site-directed mutagenesis of the 1 to 30 region was performed, and the amino acids involved in IgE binding were identified. In addition, a synthetic peptide covering the 1 to 30 region was capable of binding human IgE without triggering histamine release from basophils of Pj allergic patients (n = 6) and thus represents a haptenic molecule with potential use as an immunotolerant agent. This epitope is also present on the Par j 2.0101 major allergen representing a common IgE epitope. It is an immunodominant epitope, since it was capable of inhibiting 30% of all specific IgE against the Pj major allergens, and therefore, it might be a candidate for the future development of immunotherapeutics.

An RNA recognition motif in Wilms' tumour protein (WT1) revealed by structural modelling.

The Wilms' tumour suppressor gene 1 (WT1) (1,2) encodes four C2H2 zinc finger-containing proteins (3) critical for normal mammalian urogenital development (4). Mutations in this gene are observed in the childhood kidney cancer, Wilms' tumour (WT) (5). WT1 can bind specific DNA targets within the promoters of many genes (6-9) and both transcriptional repression and activation domains have been identified (10). On this basis, it has been assumed that regulation of transcription is the basis of WT1 tumour suppressor activity. However, subnuclear localization studies have revealed an association between WT1 proteins and 'speckled bodies' within the nucleus. Degradation of nuclear RNA in cells expressing WT1 abolishes this speckled localization and WT1 co-immunoprecipitates with a number of spliceosomal proteins, suggesting that it may also bind to RNA (11). Using structural rather than sequence comparison, we have now identified an evolutionarily conserved N-terminal RNA recognition motif (RRM) in all known WT1 isoforms similar to that in the constitutive splicing factor U1A. Given the association between WT1 mutations and Wilms' tumours, this study, together with other recent findings, may suggest a novel tumour suppression mechanism.

Binding kinetics and bioassay of RRE mRNA fragments to a peptide containing the recognition domain of HIV-1 Rev.

Surface plasmon resonance techniques have been used to examine the kinetics of binding for two RNA fragments to an RNA binding domain of HIV-1 REv. RBE3 RNA elicited an apparent dissociation constant (KD) of 121 nM while RREIIB41-79 RNA exhibited an apparent dissociation constant (KD) of 2.5 nM. The dissociation rates for both RNA fragments were comparable. However, the shorter sequence, RBE3, exhibited considerably slower association kinetics. A series of known inhibitors were assayed against these RNA' and the derived K1's were consistent with those reported in the literature, validating the method for routine inhibitor assays.

Identification of a mouse orthologue of the human ras-GAP-SH3-domain binding protein and structural confirmation that these proteins contain an RNA recognition motif.

Human ras-GTPase-activating protein (GAP120) SH3-domain-binding protein (G3BP) has recently been identified on the basis of its specific binding to the GAP120 SH3 binding domain. Here we report the identification of a mouse G3BP cDNA and the confirmation by three dimensional modelling of an RNA recognition motif (RRM) in the encoded protein. Mouse G3BP also contains an RGG domain, an acid-rich amino acid domain, and several SH3 domain-binding consensus sequences, indicating that mammalian G3BPs represent a new family of signal transduction proteins which connect tyrosine kinase-linked receptors to cellular RNA metabolism.

Verification of the interaction between peptide T and CD4 using surface plasmon resonance.

Peptide T is currently in phase II clinical trials for the treatment of AIDS-associated dementia. Its putative mode of action is inhibition of binding of the HIV envelope protein (gp120) to its cellular receptor (CD4), thus preventing viral infectivity and gp120-induced neuronal toxicity. However, a number of reports have appeared in the literature which have failed to observe any inhibitory activity of Peptide T on CD4-gp120 binding, thus casting doubt on this hypothesis. This study uses a novel biosensor technique to demonstrate that Peptide T does bind to CD4 and that this binding can be specifically inhibited by an anti-CD4 monoclonal antibody. A detailed analysis of the kinetics of the interaction is presented.