Protein kinase activity associated with stored messenger ribonucleoprotein particles of Xenopus oocytes

As the oocytes of Xenopus laevis grow and develop they accumulate vast stores of mRNA for use during early embryogenesis. The stored mRNA is stabilized and may be prevented from being translated in oocytes by the binding of a defined set of oocyte-specific proteins to form messenger RNP (mRNP) particles. A key event in the interaction of protein with mRNA is the phosphorylation of those few polypeptides that bind directly to all classes of polyadenylated mRNA. In this study we show that the phosphorylating enzyme (protein kinase), in addition to its target phosphoproteins, is an integral component of the mRNP particles. This association extends through various stages in the formation and use of the mRNP particles. Examination of material from oocytes of an early developmental stage (early stage 1), when the level of accumulated mRNA is low, reveals an excess of protein particles free of RNA, sedimenting at 6-18 S, and containing protein kinase activity and mRNA-binding phosphoproteins. At stages of maximum rate of mRNA accumulation (stages 1 and 2), the phosphoproteins and kinase are found primarily in individual mRNP particles that sediment at 40-80 S. As ribosomes become abundant (stages 2 and 3), the mRNP particles tend to interact with ribosomal subunits, at least in vitro, to form blocked translation initiation complexes that sediment at 80-110 S. These results are compared with observation on stored mRNP in other developmental systems.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Phosphorylation of a 60 kDa polypeptide from Xenopus oocytes blocks messenger RNA translation.

The stored mRNP particles of Xenopus oocytes contain protein kinase activity and two major phosphoproteins of 60 kDa (pp60) and 56 kDa (pp56). These proteins can be phospholabelled in the particles either in vivo or in vitro and then isolated by SDS-PAGE. On renaturing pp60 in the presence of globin mRNA, a stable RNA-protein complex is formed. The complex has a uniform density in Cs salt gradients, corresponding to the binding of about 10 protein molecules to each mRNA, probably at the poly(A) sequence. Compared with uncomplexed mRNA, the RNP complex is translated poorly both in vitro and in vivo. Translation of the complex can be regained after treatment with protein phosphatase. It is shown that dephosphorylation destabilizes the binding of protein to RNA, making the mRNA accessible for translation. Studies with native mRNP particles show that their translation also can be enhanced by dephosphorylation.     

Quantitative Measurement of the Diastereoisomers of CIS Thymidine Glycol in Gamma-Irradiated DNA

A technique for determing the relative content of each of the diastereoisomers of cis thymidine glycol (dTG) in DNA exposed to ionizing radiation has been developed. [³H]thymidine DNA was gamma-irradiated, digested to 2'-deoxyribonucleosides, authentic [¹⁴C] (+, -) cis dTG added to the digestate and the mixture resolved by HPLC. ³H fractions coeluting with [¹⁴C] (+, -) dTG were collected and acetylated.

The acetoxy derivatives.of (+) and (-) cis dTG were easily resolved by a second HPLC analysis and their absolute configuration determined by NMR amd mass spectroscopies. We have constructed a dose-response curve for formation of each isomer in gamma-irradiated DNA and shown that they are formed in equal amounts. This technique may be used to determine the relative formation of cis dTG isomers in DNA resulting from other oxidative stresses and whether repair of these is influenced by their configuration.     

The selectin family of carbohydrate-binding proteins: structure and importance of carbohydrate ligands for cell adhesion.

Protein-carbohydrate interactions have been found to be important in many steps in lymphocyte recirculation and inflammatory responses. A family of carbohydrate-binding proteins or lectins, termed selectins, has been discovered and shown to be involved directly in these processes. The three known selectins, termed L-, E- and P-selectins, have domains homologous to other Ca(2+)-dependent (C-type) lectins. L-selectin is expressed constitutively on lymphocytes, E-selectin is expressed by activated endothelial cells, and P-selectin is expressed by activated platelets and endothelial cells. Here, we review the nature of the carbohydrate determinants in tissues recognized by these selectins. The expression of specific sialylated, fucosylated and sulfated carbohydrates in activated endothelium and high endothelial venules promotes interactions with L-selectin on leukocyte surfaces. In contrast, E- and P-selectins recognize specific carbohydrate determinants related to sialyl Le(x) antigen on neutrophil and monocyte surfaces. The discovery of the selectins has generated excitement among glycoconjugate researchers that other carbohydrate-binding proteins and their cognate ligands will be found to function in regulating many types of cellular interactions.     

Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells

P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.     

A solid-phase assay for beta-1,4-galactosyltransferase activity in human serum using recombinant aequorin

We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), and it was immobilized by adsorption in microtiter plate wells. Serum samples were added to each well along with saturating levels of UDPGal and Mn2+. Galactosylation of the neoglycoprotein acceptor by the serum beta 1,4-GT produces the N-acetyllactosamine derivative Gal beta 1, 4GlcNAc-BSA. The product formed is quantified by adding the biotinylated plant lectin Ricinus communis agglutinin-I, which binds specifically to N-acetyllactosamine, followed by the addition of streptavidin and the biotinylated aequorin. Aequorin produces a flash of light in response to Ca2+ and is detectable to 10(-19) mol in a luminometer. Using this assay, the beta 1,4-GT activity in human serum and the activity of a semipurified beta 1,4-GT are linear with time and serum concentration over a wide range. The reaction is dependent on UDPGal and Mn2+, is highly reproducible with a low background, and can be performed in a few hours. Assays employing aequorin have a wider range of linearity than those employing horseradish peroxidase as an enzyme label. These results demonstrate that the assay for beta 1,4-GT is useful for determining activity in heterogeneous samples and also demonstrate the utility of the recombinant protein aequorin for solid-phase assay methods.     

gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.     

Bipartite Nuclear Localization Signals in the C Terminus of Human Topoisomerase IIα

DNA topoisomerase IIα is the intracellular target for several important chemotherapeutic agents, and drug-resistant human tumor cell lines have been described in which deletions in the C-proximal region of this enzyme are associated with its cytoplasmic localization. We have identified multiple potential bipartite nuclear localization signal (NLS) sequences in this region using a modified definition of the motif, and in the present study, we have expressed five of these as fusion proteins with β-galactosidase. Only one sequence (spanning amino acids 1454 to 1497) was sufficient to cause strong nuclear localization. Subsequent mutation analyses indicated that this NLS sequence was bipartite and that both domains contain more than two basic amino acids. Substitution of the lysine residue at position 1492 in the second basic domain with glutamine resulted in a fusion protein that localized inefficiently to the nucleus, indicating that all three basic residues in this domain are necessary. Our results confirm that a broader definition is required to detect all potential bipartite NLS motifs in a polypeptide sequence, although functional tests are still essential for identification of those sequences actually capable of directing nuclear localization.