Structure of linker molecules employed for biotin attachment to the stem–loop structures

<p><b>Copyright information:</b></p><p>Taken from "Immobilized stem–loop structured probes as conformational switches for enzymatic detection of microbial 16S rRNA"</p><p>Nucleic Acids Research 2005;33(11):e101-e101.</p><p>Published online 24 Jun 2005</p><p>PMCID:PMC1159122.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> ARP-biotin [-(aminooxyacetyl)--(-biotinoyl)hydrazine] is attached via Schiff base chemistry to an aldehyde group generated by oxidative cleavage of the terminal ribose moiety of cytidin (rC). C3-Biotin contains a C3-ether-glycerol linker attached to the biotin residue via an amide bond. In TEG-biotin, the C3-ether-glycerol linker is extended by a triethyleneglycol (TEG) moiety

Donor Assists Acceptor Binding and Catalysis of Human α1,6-Fucosyltransferase

α1,6-Core-fucosyltransferase (FUT8) is a vital enzyme in mammalian physiological and pathophysiological processes such as tumorigenesis and progress of, among others, non-small cell lung cancer and colon carcinoma. It was also shown that therapeutic antibodies have a dramatically higher efficacy if the α1,6-fucosyl residue is absent. However, specific and potent inhibitors for FUT8 and related enzymes are lacking. Hence, it is crucial to elucidate the structural basis of acceptor binding and the catalytic mechanism. We present here the first structural model of FUT8 in complex with its acceptor and donor molecules. An unusually large acceptor, <i>i.e.</i>, a hexasaccharide from the core of <i>N</i>-glycans, is required as minimal structure. Acceptor substrate binding of FUT8 is being dissected experimentally by STD NMR and SPR and theoretically by molecular dynamics simulations. The acceptor binding site forms an unusually large and shallow binding site. Binding of the acceptor to the enzyme is much faster and stronger if the donor is present. This is due to strong hydrogen bonding between O6 of the proximal <i>N</i>-acetylglucosamine and an oxygen atom of the β-phosphate of GDP-fucose. Therefore, we propose an ordered Bi Bi mechanism for FUT8 where the donor molecule binds first. No specific amino acid is present that could act as base during catalysis. Our results indicate a donor-assisted mechanism, where an oxygen of the β-phosphate deprotonates the acceptor. Knowledge of the mechanism of FUT8 is now being used for rational design of targeted inhibitors to address metastasis and prognosis of carcinomas

Detection of Api m 12 and Ves v 6 in <i>A. mellifera</i> and <i>V. vulgaris</i> venom.

<p>A SDS-PAGE and Coomassie blue staining of the high molecular weight fraction of honeybee venom. The arrow indicates the 200 kDa band that was subjected to MS/MS-based sequencing. B IgE Immunoreactivity of pooled sera from YJV-sensitized patients with the venom of <i>V. vulgaris</i> in Western Blot (AlaBlot™). The arrow indicates the reactive 200 kDa protein band.</p

Recombinant expression and immunoreactivity of Api m 12 and Ves v 6.

<p>A, B SDS-PAGE and immunoblot analyses of Api m 12 and Ves v 6 recombinantly produced in Sf9 insect cells visualized by Coomassie blue staining, monoclonal anti-V5 epitope antibody and <i>Galanthus nivalis</i> agglutinin (GNA), recognizing terminal mannose 1,2-, 1,3-, and 1,6-linked to mannose. C, D Immunoreactivity of recombinant Api m 12 and Ves v 6 in ELISA using the monoclonal anti-V5 epitope antibody and polyclonal anti-HRP antiserum specific for α1,3-fucose residues, the underlying principle of hymenoptera venom cross-reactive carbohydrate determinant (CCD) reactivity.</p

Domain architecture of Api m 12, Ves v 6 and other vitellogenins.

<p>Comparison of the domain architecture of Api m 12 and Ves v 6 with that of the vitellogenins from the hymenoptera species <i>Bombus ignitus</i> (Genbank accession ACM46019) and <i>Nasonia vitripennis</i> (Genbank accession XP_001607388) as well as with that of vitellogenin allergens of the mites <i>Dermatophagoides pteronyssinus</i> (Der p 14, Genbank accession AAM21322) and <i>Euroglyphus maynei</i> (Eur m 14, Genbank accession AAF14270), the fish <i>Oncorhynchus mykiss</i> (Onc m Vg, Genbank accession CAA63421), and <i>Gallus gallus</i> (Gal d 6, Genbank accession AAA49139). DUF, domain of unknown function; VWD, von Willebrand factor type D domain.</p

Grade of concordance of specific IgE (sIgE) in serum and Basophil activation test (BAT).

<p>Statistically significant differences were defined as *P<0.05, **P<0.01 and ***P<0.001.</p><p>Grade of concordance of specific IgE (sIgE) in serum and Basophil activation test (BAT).</p

Recombinant allergens improve the <i>in vitro</i> test specificity for the identification of wasp venom allergic patients.

<p>ROC curves of specific IgE measurement (sIgE) of recombinant Ves v 1, Ves v 2 and Ves v 3 by ELISA (O.D.) (<b>A–C</b>) and recombinant Ves v 5 by ImmunoCAP (KU/L) (<b>D</b>). ROC curves of CD63% trade-off of basophil stimulated with recombinant Ves v 1, Ves v 2, Ves v 3 and Ves v 5 (<b>E–H</b>).</p

Analysis of patients negative to rVes v 5 with rVes v 3, rVes v 1 and natural venom, improves the trade-off between sensitivity and specificity of the Basophil activation test.

<p>(<b>A</b>) Percentage of patients positive to basophil activation test performed with natural venom and recombinant allergens. (<b>B</b>) Analysis of patients negative to recombinant Ves v 5 with natural venom and other recombinant allergens. (<b>C</b>) Specificity and sensitivity of natural venom, recombinant Ves v 5 and recombinant Ves v 3 at cut off 15.7% CD63 expression. (<b>D</b>) Specificity and sensitivity of multistep analysis of BAT considering rVes v 5 alone, rVes v 5 plus rVes v 3, and rVes v 5 plus rVes v 3 plus natural venom.</p

Patient characteristics.

<p>Patient characteristics.</p

Recombinant allergens strongly activate basophils in allergic subjects.

<p>Representative two-color flow cytometric staining dot plots of IgE and CD63 on basophils of (<b>A</b>) five representative allergic subjects responsive to each recombinant allergen, (<b>B</b>) a representative multi-sensitized patient responsive to all the recombinant allergens (<b>C</b>) degree of basophil activation in a representative subject of the control group.</p