Amplification and molecular cloning of the hamster tunicamycin-sensitive N-acetylglucosamine-1-phosphate transferase gene. The hamster and yeast enzymes share a common peptide sequence

The first step in the assembly of the dolichol-linked oligosaccharides required for asparagine-linked glycosylation in eukaryotes is catalyzed by a tunicamycin-sensitive, dolichol phosphate-dependent N-acetylglucosamine-1-phosphate transferase (GPT). A fragment of the gene encoding the enzyme from Chinese hamster ovary (CHO) cells was partially cloned and characterized by a novel strategy. By stepwise selection, CHO cells were made 80-fold resistant to tunicamycin and found to have 10-fold elevated levels of GPT activity. Using a cloned segment of the yeast ALG-7 gene, which encodes the putative GPT from yeast, an amplified gene was identified by Southern blotting of the CHO DNA and a 6.6-kilobase segment of the gene was molecularly cloned. A family of RNA molecules in the 2.0-2.2-kilobase range identified with a probe from this gene was overexpressed in the resistant cells. The cloned DNA revealed a 24-amino acid residue sequence that was 92% conserved with the corresponding yeast sequence.     

Small Molecule Agonist of Very Late Antigen-4 (VLA-4) Integrin Induces Progenitor Cell Adhesion

Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin α4β1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via two structural modifications to a previously identified α4β1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4β1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/β subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4β7 as well as α5β1 and αLβ2. When cross-linked to αLβ2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/β subunit interface and not at the ligand-binding site in the inserted (“I”) domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.     

Purification and characterization of avian {beta}1,4 galactosyltransferase: comparison with the mammalian enzyme

Avian ß1,4 galactosyltransferase (GalTase) was purifiedfrom chicken serum, partially characterized, and compared tomammalian GalTase using antibody cross-reactivity, North-ernblot hybridization and amino acid sequence analysis. The enzymewas purified to apparent homogeneity by lactalbumin(LA)-agaroseaffinity chromatography followed by preparative SDS-polyacrylamidegel electrophoresis, and identified as two proteins of apparentmolecular masses of 39 and 46 kD. Chicken serum GalTase hada K_m for UDPGal of 42 µM, for GlcNAc of 10 mM and hadoptimal activity in the presence of 10–20 mM MnCl₂ Substrateand linkage specificity analyses indicated that the purifiedenzyme behaves as a traditional Gal ß1,4 GlcNAc:GalTase,since: (i) the avian ß1,4 GalTase bound to -LA; (ii)terminal GlcNAc residues served as good acceptors for chickenserum GalTase; (iii) the enzyme was inhibited by high concentrationsof GlcNAc; (iv) the galactosylated product was sensitive toß1,4-specific ß-galactosidase. Finally,the disaccharide reaction product comigrated with authenticß1,4 N-acetyllactosamine standard. No other GalTaseactivities were detectable using a battery of defined glycosidesubstrates. Polyclonal antibodies raised against the two gel-purifiedGalTase proteins showed reactivity with avian GalTase by ELISAand immunoprecipitation assays. The antibodies also inhibitedGalTase activity toward both high mol. wt and monosaccharideacceptor substrates. Despite similar kinetics and substratespecificity, the avian and mammalian GalTases showed littleoverall structural similarity, since polyclonal anti-avian GalTaseIgG failed to react with mammalian GalTase purified from bovinemilk, and conversely anti-bovine milk GalTase IgG did not reactwith the avian enzyme. Furthermore, in Northern blot analysis,no hybridization was detected when chicken embryo liver poly(A)⁺RNA was probed with a mouse GalTase cDNA, even under conditionsof reduced stringency. Amino acid sequence analysis identifiedthree of five tryptic peptides that are homologous to the mammaliansequence within a putative substrate binding domain and thecarboxy terminal domain of the enzyme. Their overall structuraldisparity leads us to believe that regions of homology betweenthe avian and mammalian GalTases may represent active sitesof the enzyme. avian ß1,4 galactosyltransferase homology mammalian purification