Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.     

Somatostatin pretreatment increases the number of somatostatin receptors in GH4C1 pituitary cells and does not reduce cellular responsiveness to somatostatin

The GH4C1 pituitary cell line contains specific plasma membrane receptors for the inhibitory neuropeptide somatostatin (SRIF). Unlike other peptides which bind to cell surface receptors on these cells, SRIF is not rapidly internalized via receptor-mediated endocytosis. Here we examined the effects of chronic SRIF pretreatment on the subsequent ability of GH4C1 cells to bind and respond to this hormone. Treatment of cells with 100 nM SRIF increased [125I-Tyr1]SRIF binding to a maximum value of 220% of control after 20 h. Scatchard analysis demonstrated that the number, but not the affinity, of the receptors was altered. The effect of SRIF was dose-dependent (ED50 = 2.3 +/- 0.4 nM), was not mimicked by an inactive analog, and was specific for the SRIF receptor. Furthermore, pretreatment of cells with other agents, which mimic SRIF's action to decrease intracellular cAMP and free Ca2+ concentrations, did not mimic the SRIF-induced increase in receptor number. Thus, occupancy of the SRIF receptor was required for SRIF receptor up-regulation. Inhibition of protein synthesis with cycloheximide did not prevent the SRIF-induced increase in receptors, consistent with an effect of SRIF to either reduce receptor degradation or cause slow redistribution of preexisting receptors to the plasma membrane. In contrast to the effects on receptor binding, pretreating cells with SRIF did not alter either basal cAMP levels or the potency of SRIF to inhibit cAMP accumulation (ED50 = 0.5 +/- 0.2 nM). However, the maximum cAMP produced by stimulators of adenylyl cyclase was increased. The observation that chronic SRIF exposure did not cause homologous desensitization in GH4C1 cells and increased rather than decreased SRIF receptor number is consistent with the fact that this neuropeptide is not rapidly internalized by receptor-mediated endocytosis.     

Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Bovine mammary gland UDP-GalNAc:GlcNAcbeta-R beta1-->4-N- acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with alpha-lactalbumin

We have identified a novel N -acetylgalactosaminyltransferase activity in lactating bovine mammary gland membranes. Acceptor specificity studies and analysis of products obtained in vitro by 400 MHz1H-NMR spectroscopy revealed that the enzyme catalyses the transfer of N - acetylgalactosamine (GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal, beta-linked N -acetylglucosamine (GlcNAc) residue and establishes a beta1-->4-linkage forming a GalNAcbeta1-->4GlcNAc ( N, N '-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAcbeta-R beta1-->4-N- acetylgalactosaminyltransferase (beta4-GalNAcT). This enzyme resembles invertebrate beta4-GalNAcT as well as mammalian beta4- galactosyltransferase (beta4-GalT) in acceptor specificity. It can, however, be clearly distinguished from the pituitary hormone-specific beta4-GalNAcT by its incapability of acting with an elevated activity on a glycoprotein substrate carrying a hormone-specific peptide motif. Furthermore, the GalNAcT activity appeared not to be due to a promiscuous action of a beta4-GalT as could be demonstrated by comparing the beta4-GalNAcT and beta4-GalT activities of the mammary gland, bovine colostrum, and purified beta4-GalT, by competition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-beta4-GalT polyclonal inhibiting antibody. Interestingly, under conditions where mammalian beta4-GalT forms with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary gland beta4-GalNAcT was similarly induced by alpha-LA to act on Glc with an increased efficiency yielding the lactose analog GalNAcbeta1-->4Glc. This enzyme thus forms the second example of a mammalian glycosyltransferase the specificity of which can be modified by this milk protein. It is proposed that the mammary gland beta4-GalNAcT functions in the synthesis of lacdiNAc- based, complex-type glycans frequently occurring on bovine milk glycoproteins. The action of this enzyme is to be considered when aiming at the production of properly glycosylated protein biopharmaceuticals in the milk of transgenic dairy animals.      

Role of phosphatidylinositol-anchored proteins in T cell activation

A novel class of cell surface proteins are attached to the plasma membrane via a phosphatidylinositol (PI)-glycan anchoring structure, and these proteins can be selectively removed from the cell surface by the enzyme PI-specific phospholipase C (PI-PLC). Enzyme treatment led to a prolonged reduction in cell surface expression of several PI-anchored proteins. Activation of T cells led to a marked decrease in the ability of PI-PLC to remove PI-anchored surface proteins from the activated T cells. This decrease in PI-PLC sensitivity may reflect an alteration in the PI-glycan anchoring structures, or in a general membrane property, which renders the PI-anchored proteins inaccessible to the enzyme. When murine T lymphocytes were treated with PI-PLC and then stimulated with either Con A, the calcium ionophore A23187 and PMA, or an anti-CD3 mAb, the response to Con A stimulation was inhibited by 90%, whereas the responses to ionophore and PMA or anti-CD3 were not affected. Removal of PI-anchored proteins inhibited an early event in the activation process in response to Con A because both IL-2 production and IL-2R expression were inhibited by the PI-PLC treatment. Inhibition of the Con A response was secondary to removal of a PI-linked protein from the responder T cell population because PI-PLC treatment of T-depleted spleen cells did not alter their ability to act as a source of accessory cells. It is unlikely that removal of the known PI-linked proteins on murine T cells, Thy-1 and Ly-6, can fully account for the inhibition of Con A response because the cell line M2B3, that lacks these surface proteins, responded normally to Con A stimulation. These studies demonstrate that one or more PI-anchored T cell proteins play an important role in an early step of Con A activation, perhaps involving T cell-accessory cell interactions. In contrast, the ability to stimulate T cells by direct cross-linking of TCR/CD3 complex is not dependent on the presence of these PI-anchored proteins.