Search

A study of ribosomes was made to define the function of base 530. Cell death was known to result from any base change at 530 position introduced into Escherichia coli on a multiple plasmid. The use of U 530 mutant in a variety of procedures including in vitro protein synthesis along with 30 S subunits reconstituted from 16S RNA synthesized mutant shows the functional defect of the mutant ribosomes. The mutation of G 530 to U interferes with dipeptide synthesis and even abolishes it completely. Mutant 530 ribosomes can effect elongation reactions of protein synthesis under standard assay conditions.

A base substitution of G to U was constructed at position 529 in Escherichia coli 16S rRNA. The U529 mutant ribosomes were functional and present on polysomes but were highly error prone and caused a progressive loss of cell viability. They displayed elevated levels of readthrough of stop codons and frameshifting, and an increase in thermal sensitivity of beta-galactosidase, suggestive of missense errors. These results demonstrate that the university conserved G529 is involved in tRNA selection at the A site during protein synthesis.

While searching for an enzyme capable of breaking epsilon-(gamma-Glu)-Lys isopeptide bonds cross-linking protein chains, we purified a metallo-proteinase which mimics the action of an isopeptidase on the gamma-chain dimers of cross-linked fibrin. The enzyme is present in the growth medium of the bacterium Aeromonas hydrophila, isolated from the intestinal tract of the leech Hirudo medicinalis. It is a 19-kDa protein which specifically hydrolyzes the Gly-Ala peptide bond within the Gly-Gly-Ala sequence, located near the cross-link site in the gamma-chain dimer of fibrin. Substrate specificity studies with a number of synthetic peptides suggest that the enzyme prefers Gly-Gly or acetyl-Gly in the P2 and P1 positions, respectively (Schecter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162). Nonpolar amino acid residues seem to be favored in the P1' and P2' positions. The enzyme contains one atom of zinc and is inhibited by 1,10-phenanthroline, but not by EDTA. Iodoacetate, leupeptin, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, pepstatin, and alpha 2-macroglobulin have no effect on enzyme activity. Disulfide reducing reagents, such as dithiothreitol or 2-mercaptoethanol, inactivate the enzyme completely. The partial amino-terminal sequence shows 46% identity with a zinc metallo-proteinase from a strain of Lysobacter enzymogenes and 69% identity with the LasA protein from Pseudomonas aeruginosa.

Fucosyl residues linked alpha 1 leads to 3 or 4 to N-acetylglucosamine were found in large amounts on glycopeptides from the membranes of human tumor cells of neurectodermal origin but not on membrane glycopeptides from human fibroblasts. The fucosyl residues were detected by release of radioactive fucose from the glycopeptides with an almond alpha-L-fucosidase specific for fucosyl alpha 1 leads to 3(4)-N-acetylglucosamine. In other studies, the linkage was shown to be alpha 1 leads to 3 by nuclear magnetic resonance analysis (U. V. Santer, M. C. Glick, H. van Halbeek, and J. F. G. Vliegenthart. Carbohydr. Res., 118: in press, 1983). Glycopeptides containing these fucosyl residues from four human neuroblastoma cell lines were defined by binding to immobilized lectins. In addition, the glycopeptides from one human neuroblastoma cell line, CHP-134, were further characterized by enzyme degradation and columns calibrated for size and charge. The antennary position of fucosyl alpha 1 leads to 3-N-acetylglucosamine on the glycopeptides was demonstrated by the use of exoglycosidases and endoglycosidase D, since complete degradation to yield fucosyl-N-acetylglucosaminylasparagine was obtained only after treatment with almond alpha-L-fucosidase prior to the sequential degradation. Fucosyl alpha 1 leads to 3-N-acetylglucosamine was present on most size and charge classes of membrane glycopeptides and therefore was not limited to a few glycoproteins. Since the almond alpha-L-fucosidase cleaves fucosyl residues from glycoproteins, the physiological effects of the increased specific fucosylation on human tumors of neurectodermal origin can be examined.

The predominant surface glycopeptide from a clone of baby hamster kidney cells transformed by Rous sarcoma virus (C13/B4), metabolically labeled with L-[14C]fucose, has been characterized for the first time. This glycopeptide represents 19% of the total radioactivity removed by trypsin from the cell surface of the transformed fibroblasts and is more abundant in the transformed cells than in the normal counterpart. Purification of the glycopeptide after digestion with Pronase was by successive chromatography on DEAE-cellulose and Sephadex G-50. The monosaccharide content of the glycopeptide was 42, 127, 138, 114, and 243 nmol of fucose, sialic acid, galatose, mannose, and glucosamine, respectively. A partial structure of the glycopeptide was proposed from the results of sequential enzymatic degradation coupled with gas-liquid chromatographic analysis of the resultant monosaccharides. All of the enzymes used were purified and pretested on natural substrates and found to remove terminal monosaccharides of the correct configuration, quantitatively. The purification and properties of an alpha-L-fucosidase from rat testes were described. All of the radioactivity in the glycopeptide, recovered as fucose, was present at the core and was removed by treatment with this alpha-L-fucosidase. The proposed structure is a triantennary, completely sialylated, complex glycopeptide containing a core region of beta-D-mannose, beta-D-N-acetylglucosamine, and alpha-L-fucose.

Phenotypic expression of transformation was inhibited by swainsonine at concentrations which affect the late stages of glycoprotein processing but not growth of cells. In the presence of swainsonine, NIH 3T3 fibroblasts transfected with human tumor DNA (al-l) no longer grew in soft agar or expressed complex type oligosaccharides characteristic of transformed cells. Thus, it appears that glycoproteins with fully processed oligosaccharides are necessary for the maintenance of the transformed phenotype in these cells.

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containing oncogene H-ras were analyzed by 500-MHz 1H-NMR spectroscopy and binding to immobilized lectins. The cells were metabolically labeled with D-[3H]glucosamine or L-[3H]fucose and the glycopeptides included in Bio-Gel P-10 (Mr 5000-3500) were separated into neutral and charged fractions on DEAE-cellulose. The major portion (80%) of these [3H]fucose glycopeptides from the non-transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues. The structure of the predominant neutral glycopeptide from the non-transformed NIH 3T3 cells was determined by 1H-NMR spectroscopy to be tetraantennary containing terminal Gal alpha 1----3. (formula; see text) This structure was verified by binding to the immobilized alpha-Gal-specific lectin, Griffonia simplicifolia I and leukoagglutinating phytohemagglutinin from Phaseolus vulgaris (L-PHA), which binds certain tri- or tetraantennary glycopeptides. In contrast, the structure derived by NMR spectroscopy of one of the predominant charged glycopeptides from the transformed cells was triantennary containing terminal NeuNAc alpha 2----3 in addition to Gal alpha 1----3. (formula; see text) In attempting to verify this structure by lectin-binding properties it was found that removal of NeuNAc alpha 2----3 reduced the affinity to L-PHA - agarose. The other major glycopeptides of the transformed cells which were more charged also cotained NeuNAc alpha 2----3 but no NeuNAc alpha 2----6 or Gal alpha 1----3. A tentative structure was proposed for the major glycopeptide of the first charged class from NIH 3T3 cells on the basis of lectin-binding properties and the NMR spectrum which showed, in addition to NeuNAc alpha 2----3, the presence of NeuNAc alpha 2----6 and Gal alpha 1----3. On the basis of the NMR spectrum and other results, it is concluded that the presence of tetraantennary oligosaccharides are not sufficient for the transformed oligosaccharide phenotype. Rather, the tri- or tetraantennae must be sialylated in alpha 2----3 linkage, on more than one antennae, when properties of transformation are expressed in NIH 3T3 cells. Prior to transformation the tetraantennary oligosaccharides of these cells are terminated in alpha-Gal residues, whereas after transformation alpha-Gal residues appear to be replaced by NeuNAc alpha 2----3.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) activity in fibroblast homogenates was measured by a rapid and simple assay with a synthetic substrate. The activity of neuraminidase in virus transformed hamster fibroblasts was increased over the normal counterpart. In addition, the differential activity seen using the synthetic substrate and fetuin made it possible to detect an enzyme activity hitherto not described. The advantages of this assay for metabolic screening are discussed.

Altered glycosylation of membrane glycoproteins was demonstrated in NIH 3T3 cells transformed by transfection with DNA from human neuroblastoma and bladder carcinoma cell lines. The oncogenes of these two cell lines have been identified as N-ras and c-H-ras-1, respectively. The fucose-labeled membrane glycopeptides of transfection-induced transformants had decreased binding to concanavalin A-Sepharose when compared in dual-isotope experiments to those from NIH 3T3 cells, whereas binding to lentil lectin-Sepharose and leukoagglutinating phytohemagglutinin-agarose was increased. Binding affinities to these immobilized lectins lead to the interpretation of the results as a decrease in biantennary glycopeptides with a simultaneous increase in tri- or tetraantennary glycopeptides. Sephadex G-50 profiles also indicated a size increase of the glycopeptides of the transformants. None of these changes was growth related. This altered glycosylation, representing a heretofore unreported effect of the onc genes, may be necessary for the transformed phenotype.

Human tumor cells of neurectoderm origin contain a high proportion of alpha-L-fucosyl linkages were determined by high-resolution, 500-MHz, 1H-n.m.r. spectroscopy which gave signals characteristic for alpha-L-Fucp-(1 leads to 3)-D-GlcNAc residues these L-fucosyl residues. This was shown by use of a specific alpha-L-fucosidase from almond emulsin and a broad-spectrum alpha-L-fucosidase from rat testes. The exact alpha-L-fucosyl linkages were determined by high-resolution, 500-MHz, 1H-n.m.r. spectroscopy which gave signals characteristic for alpha-L-Fucp-(1 leads to 3)-D-GlcNAc residues linked to branches and for alpha-L-Fucp-(1 leads to 6)-D-GlcNAc residues linked to the core. More than 95% of the asparagine-linked GlcNAc residues were substituted with (1 leads to 6)-alpha-L-fucosyl groups. Further definition of the range of neutral glycopeptides was obtained with immobilized lectins. Binding to E-PHA-agarose suggested the presence of a beta-D-mannopyranosyl residue substituted at O-4 by a 2-acetamido-2-deoxy-D-glucopyranosyl group. alpha-L-Fucp-(1 leads to 3)-GlcNAc interfered with this binding since removal of alpha-L-fucosyl groups by almond emulsin alpha-L-fucosidase increased the binding by 100%. These studies demonstrate the ability of a combination of high-resolution 1H-n.m.r., enzyme degradation, and lectin-binding affinities to delineate structural elements of small amounts of oligosaccharide residues.

The presence of fucosyl residues linked alpha 1----3(4) to N-acetylglucosamine was demonstrated on the oligosaccharides from glycoproteins of 11 human neuroblastoma tumors from ten different patients. This finding is in complete agreement with the previous report that human neuroblastoma cell lines contained an unusually large proportion of metabolically incorporated L-[3H]fucose in this specific linkage (U. V. Santer and M. C. Glick, Cancer Res., 43:4159-4166, 1983). Furthermore, the glycopeptides derived from the neuroblastoma tumors had a low percentage of fucose-containing biantennary oligosaccharides as determined by affinity to concanavalin A-Sepharose and in this characteristic were similar to glycopeptides from virus transformed and other tumor cells. To obtain these results, the tumor cells were labeled metabolically for 48 h with L-[3H]fucose. The cells were harvested and digested with Pronase, and the glycopeptides were isolated and treated with alpha-L-fucosidase from almonds, specific for the release of fucose linked alpha 1----3(4) to N-acetylglucosamine. A portion of the glycopeptides was characterized by serial affinity chromatography on immobilized concanavalin A and lentil lectin. The phenotypic similarity of the tumor cells to the cell lines, particularly CHP-134, included the paucity of biantennary oligosaccharides and the presence of fucosyl residues on the multiantennae of the glycopeptides.

Glycoproteins from NIH 3T3 transformed by transfection with human tumor DNA had altered glycosylation. That is, the glycoproteins have a decrease in biantennary oligosaccharides with a concomitant increase in tri- and tetraantennary oligosaccharides. After swainsonine treatment, glycosylation of oncogene transfected NIH 3T3 was more similar to the normal counterpart. Thus, this inhibitor of glycoprotein processing is able to reverse at least one phenotypic expression of transformation, the formation of more highly branched oligosaccharides. The mechanism may be by inhibition of a specific glycosyl transferase.

Two large ribonucleic acid (RNA) fragments have been obtained from T1-RNase-treated 30S ribosomes of Escherichia coli. One fragment, about 475 nucleotides long, contains all the unique oligonucleotides found by Fellner and associates in sections of 16S RNA designated P, E, E', and K, and one-half the large oligonucleotides of section A. The other large fragment is about 300 nucleotides long and contains the oligonucleotides found in sections C, C', C''. The isolation of these large fragments seems to confirm the arrangement of sections within 16S RNA. There are also recovered from nuclease-treated ribosomes three small fragments, one (120 nucleotides long) from the 5' end, one (26 nucleotides long) from the 3' OH end of the chain, and another section (66 nucleotides long) from the middle of the 16S RNA chain. Small molecular weight material is also generated by nuclease treatment, and about half this material is derived from a region close to the 3' OH end of the 16S RNA chain. This indicates that the most accessible part of the rRNA of E. coli 30S ribosomes is a region 100 to 150 nucleotides long near the 3' end of the chain. A general scheme is proposed to explain the generation of the various-sized RNA products from the rRNA of the 30S ribosome.