Side reactions in the synthesis of phosphotyrosine-containing peptides

Summary A series of phosphopeptides Tyr(PO₃H₂)-Val-Pro-Xxx-Leu (Xxx=Met, Met(O), Nle, Dab or Cys), derived from the native platelet-derived growth factor- receptor (PDGF-) sequence, has been prepared to study their interaction with the src-homology 2 (SH2) domains of the p85 subunit of PI3 kinase. The phosphopeptides were synthesized using Fmoc methodology incorporating N-Boc dibenzyl-protected phosphotyrosine (Boc-Tyr[PO₃(Bzl)₂]) as the N-terminal amino acid, since the benzyl groups can be removed during resin cleavage with TFA. Only peptides containing methionine were found to exist partially as S-benzyl sulfonium salts after TFA cleavage from the resin. The desired peptide could be obtained from the S-benzyl sulfonium salt by hydrogenolysis.     

Progressive determination of cell fates along the dorsoventral axis in the sea urchin Heliocidaris erythrogramma

In the direct-developing sea urchin Heliocidaris erythrogramma the first cleavage division bisects the dorsoventral axis of the developing embryo along a frontal plane. In the two-celled embryo one of the blastomeres, the ventral cell (V), gives rise to all pigmented mesenchyme, as well as to the vestibule of the echinus rudiment. Upon isolation, however, the dorsal blastomere (D) displays some regulation, and is able to form a small number of pigmented mesenchyme cells and even a vestibule. We have examined the spatial and temporal determination of cell fates along the dorsoventral axis during subsequent development. We demonstrate that the dorsoventral axis is resident within both cells of the two-celled embryo, but only the ventral pole of this axis has a rigidly fixed identity this early in development. The polarity of this axis remains the same in half-embryos developing from isolated ventral (V) blastomeres, but it can flip 180° in half-embryos developing from isolated dorsal (D) blastomeres. We find that cell fates are progressively determined along the dorsoventral axis up to the time of gastrulation. The ability of dorsal half-embryos to differentiate ventral cell fates diminishes as they are isolated at progressively later stages of development. These results suggest that the determination of cell fates along the dorsoventral axis in H. erythrogramma is regulated via inductive interactions organized by cells within the ventral half of the embryo.     

Interactions of HIV-1 and HIV-2 envelope glycoproteins with sulphated polysaccharides and mannose-6-phosphate

Envelope glycoproteins of human immunodeficiency viruses (HIV-1and HIV-2) can interact with high-mannose glycans and with themannosyl or N-acetylglucosaminyl core of complex-type oligosaccharidicstructures. HIV-1 glycoproteins also specifically bind sulphatedpolysaccharides such as dextran sulphate (DS) and heparin. Here,we show that the latter property is shared by HIV-2 recombinantgp140 (rgpl40) precursor glycoprotein. Binding of rgpl40 andof corresponding rgp160 of HIV-1 to heparin- and DS-substituted(sulphated dextran beads; SDB) affinity matrices was inhibitedby the soluble specific ligand and also by fetuin, asialofetuinor the anionic simple carbohydrate derivative manncsse-6-phosphate(M6P). Interaction of HIV-1 rgpl20 subunit with the two affinitymatrices was also inhibited by M6P, but only rgpl20 bindingto heparin-agarose, and not that to SDB, was affected by fetuinand asialofetuin. These results suggest that HIV-1 and HIV-2envelope glycoproteins presumably display different sulphatedpolysaccharide and carbohydrate recognition sites. Some of thesemay be common or in close proximity: with respect to rgpl60,for example, the sites may be common on the gp41 moiety and/orin a region of gp120 which would be more accessible when expressedon rgpl60 than on processed gpl20, while they may be distincton the cleaved gpl20 subunit. Finally, because M6P is a markerof lysosomal enzymes, we verified that HIV-1 and HIV-2 envelopeglycoproteins could specifically bind in a M6P-inhibitable mannerto a representative lysosomal enzyme, bovine liver ß-glucuronidasecoupled to agarose, suggesting that they may possibly interferewith lysosomal enzyme sorting in HIV-infected cells. env glycoproteins HIV lectin mannose-6-phosphate sulphated polysaccharides     

Potato lectin: a modular protein sharing sequence similarities with the extensin family, the hevein lectin family, and snake venom disintegrins (platelet aggregation inhibitors)

Potato (Solanum tuberosum) lectin, is a chimeric chitin-binding protein comprised of a lectin domain fused to a hydroxyproline-rich glycoprotein domain. Here peptide sequence information from both domains is presented. A partial sequence of a major tryptic peptide T2: Leu-Pro-Ser-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-(His)-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp- was similar to the ‘P3’ type extensin major repetitive sequence: Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-suggesting common evolutionary origins for the extensins and the hydroxyproline-rich glycoprotein (HRGP) domain of potato lectin. Furthermore, alignment of three chymotryptic peptides from potato lectin, C1: Cys-Gly-Thr-Thr-Ser-Asp-Tyr, C2: Cys-Ser-Pro-Gly-Tyr, and C8: Thr-Gly-Glu-Cys-Cys-Ser-Ile with similar sequences from the hevein lectin family indicates that they have homologous chitin-binding domains, and hence have common evolutionary origins. Finally, all plant chitin-binding domains examined bore a remarkable sequence similarity, particularly in the spacing of Cys residues, to the disintegrins (platelet aggregation inhibitors) which occur in crotalid and viperid snake venoms. As such, sequence similarities not only identify potato lectin as a member of both the hevein and extensin families of plant proteins, but also suggest that an archetypal polypeptide module gave rise to both the plant chitin-binding domain and the reptile disintegrins.     

The forever young gene encodes an oxidoreductase required for proper development of the Arabidopsis vegetative shoot apex

In plant development, leaf primordia are formed on the flanks of the shoot apical meristem in a highly predictable pattern. The cells that give rise to a primordium are sequestered from the apical meristem. Maintenance of the meristem requires that these cells be replaced by the addition of new cells. Despite the central role of these activities in development, the mechanism controlling and coordinating them is poorly understood. These processes have been characterized in the Arabidopsis mutant forever young (fey). The fey mutation results in a disruption of leaf positioning and meristem maintenance. The predicted FEY protein shares significant homology to a nodulin and limited homology to various reductases. It is proposed that FEY plays a role in communication in the shoot apex through the modification of a factor regulating meristem development.     

Monte Carlo study of the effect of β2-microglobulin on the binding cleft of the HLA-A2 complex

Peptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β₂-microglobulin. We present results of Monte Carlo simulations of the β-pleated sheet floor of the human class I MHC molecule, HLA-A2, with and without β₂-microglobulin. We find a significant effect of β₂-microglobulin on the side chains of residues near a region that would accommodate the C-terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β₂-microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.     

Visualization and ablation of phenylethanolamineN-methyltransferase producing cells in transgenic mice

We cloned and sequenced the mouse phenylethanolamineN-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5 flanking sequences from the cloned mouse PNMT gene can direct expression of theEscherichia coli -galactosidase (lacZ) gene to predicted regions of the adrenal, eye can direct in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT_{8 kb}-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.     

Diversity of siderophore genes encoding biosynthesis of 2,3-dihydroxybenzoic acid in Aeromonas spp.

Most species of the genus Aeromonas produce the siderophore amonabactin, although two species produce enterobactin, the siderophore of many enteric bacteria. Both siderophores contain 2,3-dihydroxybenzoic acid (2,3-DHB). Siderophore genes (designated aebC, -E, -B and -A, for aeromonad enterobactin biosynthesis) that complemented mutations in the enterobactin genes of the Escherichia coli 2,3-DHB operon, entCEBA(P15), were cloned from an enterobactin-producing isolate of the Aeromonas spp. Mapping of the aeromonad genes suggested a gene order of aebCEBA, identical to that of the E. coli 2,3-DHB operon. Gene probes for the aeromonad aebCE genes and for amoA (the entC-equivalent gene previously cloned from an amonabactin-producing Aeromonas spp.) did not cross-hybridize. Gene probes for the E. coli 2,3-DHB genes entCEBA did not hybridize with Aeromonas spp. DNA. Therefore, in the genus Aeromonas, 2,3-DHB synthesis is encoded by two distinct gene groups; one (amo) is present in the amonabactin-producers, while the other (aeb) occurs in the enterobactin-producers. Each of these systems differs from (but is functionally related to) the E. coli 2,3-DHB operon. These genes may have diverged from an ancestral group of 2,3-DHB genes.