Sadamu Kurono

Solid phase synthesis of oligomannopeptoids that mimic the concanavalin A-binding trimannoside

Oligomannopeptoids from the dimer to the hexamer were produced by solid phase synthesis and their abilities to bind to concanavalin A (ConA) were assessed. The assessment indicated similarity between the oligomannopeptoids and the naturally occurring oligomannosides in the enthalpy of the binding and the valence number vs binding strength relationship, encouraging the use of the oligomannopeptoids as oligomannoside mimics.

Syntheses of two trimannose analogs each containing C-mannosyl or 5-thio-C-mannosyl residue: their affinities to concanavalin A

C-Mannosyl residue-containing trimannose ManC alpha(1,6)[Man alpha(1,3)Man] (2) and 5-thio-C mannosyl residue-containing trimannose 5SManC alpha(1,6)[Man alpha(1,3)Man] (3) were synthesized via a glycosyl radical addition to enone derivative of mannose (6). Dissociation constants for the binding of these trisaccharides to concanavalin A (ConA) were determined by a fluorescence anisotropy inhibition assay: Kd = 198 and 31 microM, respectively. The unexpectedly large Kd value for the compound 2 compared with the compound 3 and the natural trimannose 1 demonstrates a characteristic of C-glycoside.

Synthesis of 13C-Labeled iodoacetanilide and application to quantitative peptide analysis by isotope differential mass spectrometry

13C-Labeled and unlabeled iodoacetanilides have been synthesized for covalent modification of the sulfhydryl groups of cysteine residues in proteins or peptides. A combination of these reagents, coupled with mass spectrometry, is a powerful tool for quantitative analysis of peptides and hence proteins.

Proteomics Profiling of the Cone Photoreceptor Cell Line, 661W

Cultured retinal cells are convenient experimental systems offering great advantages in the assessment of numerous retinal processes. The two most important advantages are the ease of evaluation of an isolated cellular function without the effects of other retinal cell types and an avoidance of use of the more costly animal research. The Two obvious disadvantages are the loss of the architecture of the native tissue, and lack of functional influence of other retinal cell types. However, for most of the research applications, the advantages of use of in vitro systems offset the potential limitations. Retinal cell culture can be used to determine effectiveness of promoter fragments from retina-specific genes, the functional role of domains on a retinal protein and how mutations would effect that function and to express retinal proteins for biochemical analyses. Last but not least, retinal cell culture can be used to determine the toxicity of pharmacologic agents, and for studies of cell death and differentiation. Except for Müller cells, all other retinal cells are terminally differentiated, specialized neuronal cells with a limited, if any, capacity for cell division. However, immortalized cell lines currently exist for several retinal cell types including Müller cells (Roque et al., 1997). A cell line expressing retina-specific genes, including the photoreceptor proteins IRBP and cone transducin, has also been isolated from a mouse ocular tumor (Bernstin, Kutty, Wiggert, Albert, & Nickerson, 1994). Furthermore, Y-79 (Reid et al., 1974) and WERI-Rb (McFall, Sery, & Makadon, 1977) are immortalized cell lines derived from human retinoblastoma tumors. It was initially believed that the Y-79 cells had originated from a cone cell lineage (Bogenmann, Lochrie, & Simon, 1988), but later these cells were shown to express rod opsin, rod transducin, rod phosphodiesterase, and recoverin (Di Polo & Farber, 1995;

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 1: Identification of its inhibitory subunit complexes and their roles

Cyclic GMP phosphodiesterase (PDE) in bovine rod photoreceptor outer segments (OS) comprises a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγ) and is regulated by the α subunit of transducin (Tα). Here, we show an overall mechanism for PDE regulation by identifying Pγ complexes in OS homogenates prepared with an isotonic buffer. Before Tα activation, three Pγ complexes exist in the soluble fraction. Complex a, a minor complex, contains Pαβ, Tα, and a protein named Pδ. Complex b, Pαβγγb, has a PDE activity similar to that of membranous Pαβγγ, PαβγγM, and its level, although its large portion is Pδ-free, is estimated to be 20–30% of the total Pαβγγ. Complex c, (Pγ·GDP-Tα)2c, appears to be a dimer of Pγ·GDP-Tα. Upon Tα activation, (1) complex a stays unchanged, (2) Pαβγγb binds to membranes, (3) the level of (Pγ·GDP-Tα)2c is reduced as its GTP-form is produced, (4) complex d, Pγ·GTP-Tαd, is formed on membranes and its substantial amount is released to the soluble fraction, and (5) membranous Pαβγγ, PαβγγM and/or Pαβγγb, becomes Pγ-depleted. These observations indicate that Pγ as a complex with GTP-Tα dissociates from Pαβγγ on membranes and is released to the soluble fraction and that Pγ-depleted PDE is the GTP-Tα-activated PDE. After GTP hydrolysis, both (Pγ·GDP-Tα)2c and Pγ·GDP-Tαd, without liberating Pγ, deactivate Pγ-depleted PDE. The preferential order to be used for the deactivation is membranous Pγ·GDP-Tαd, solubilized Pγ·GDP-Tαd and (Pγ·GDP-Tα)2c. Release of Pγ·GTP-Tα complexes to the soluble fraction is relevant to light adaptation.

Affinity of 5-thio-L-fucose-containing lewis X (LeX) trisaccharide analogs to anti-LeX monoclonal antibody

5-Thiofucose-containing LeX trisaccharide analogs Gal beta(1,4)[5SFuc alpha(1,3)]GlcNAc-OMe (2) and Gal beta(1,4)[5SFuc beta(1,3)]GlcNAc-OMe (4) were synthesized via 5-thiofucosylation of methyl 2-azido-lactoside derivative 6 by the trichloroacetimidate method. Inhibitory activity of these analogs for the binding of LeX to anti-Lex antibody was evaluated by enzyme immunoassay, indicating that anti-LeX strictly recognizes alpha-configuration of the fucose moiety and its binding pocket includes no advantageous region, such as hydrophobic area, for recognizing the ring sulfur atom of 5-thiofucosyl LeX analog 2.

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 2: isolation and characterization of the transducin-activated form.

Rod photoreceptor cGMP phosphodiesterase (PDE6) consists of a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγ). In the accompanying article, using bovine photoreceptor outer segment homogenates, we show that Pγ as a complex with the GTP-bound transducin α subunit (GTP-Tα) dissociates from Pαβγγ on membranes, and the Pαβγγ becomes Pγ-depleted. Here, we identify and characterize the Pγ-depleted PDE. After incubation with or without guanosine 5′-O-(3-thiotriphosphate) (GTPγS), Pαβ complexes are extracted. When a hypotonic buffer is used, Pαβγγ, Pαβγ, and a negligible amount of a Pαβ complex containing Pγ are isolated with GTPγS, and only Pαβγγ is obtained without GTPγS. When an isotonic buffer containing Pδ, a prenyl-binding protein, is used, Pαβγγδ, Pαβγδδ, and a negligible amount of a Pαβ complex containing Pγ and Pδ are isolated with GTPγS, and Pαβγγδ is obtained without GTPγS. Neither Pαβ nor Pαβγγ complexed with GTPγS-Tα is found under any condition we examined. Pαβγ has ~12 times higher PDE activity and ~30 times higher Pγ sensitivity than those of Pαβγγ. These results indicate that the Pγ-depleted PDE is Pαβγ. Isolation of Pαβγγδ and Pαβγδδ suggests that one C-terminus of Pαβ is involved in the Pαβγγ interaction with membranes, and that Pγ dissociation opens another C-terminus for Pδ binding, which may lead to the expression of high PDE activity. Cone PDE behaves similarly to rod PDE in the anion exchange column chromatography. We conclude that the mechanisms for PDE activation are similar in mammalian and amphibian photoreceptors as well as in rods and cones.