Frank Maley

Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-β-N-acetylglucosaminidase H

The ability of endo-beta-acetylglucosaminidase H (Endo H) from Streptomyces plicatus to hydrolyze high-mannose oligosaccharides from glycoproteins is influenced by numerous factors, including the tertiary structure of the substrate glycoproteins, the amount of Endo H used, the time of incubation, and the presence or absence of reagents that affect protein configuration. Endo H levels below 10 to 20 milliunits/ml may incompletely hydrolyze oligosaccharides, regardless of the incubation time, because even though the enzyme remains active, it becomes trapped or sequestered and is unavailable. Endo H activity can be potentiated by first denaturing substrate glycoproteins in a 1.2-fold weight excess of sodium dodecyl sulfate prior to hydrolysis. However, low levels of Endo H are sensitive to inactivation by sodium dodecyl sulfate, with considerable activity being lost over 4 h when the unbound detergent concentration exceeds protein by 0.02% (0.2 mg/ml). Other denaturants such as the Tritons, the zwittergents, the Brij series, or octylglucoside do not enhance or inhibit Endo H removal of oligosaccharides, but the chaotropic salt sodium thiocyanate at 0.5 M enhances Endo H action on some glycoproteins, particularly bovine thyroglobulin. Under denaturing conditions, proteolytic contaminants are a potential problem. Addition of 1 mM phenylmethylsulfonyl fluoride to Endo H incubations completely inhibits the residual Endo H-associated protease(s). Furthermore, Endo H is unaffected by a wide range of proteolytic inhibitors that may be used to protect substrate glycoproteins.

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

ABSTRACT A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells.

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.

Thymidylate synthase binds to c-myc RNA in human colon cancer cells and in vitro.

Using an immunoprecipitation-reverse transcription-PCR technique, we characterized a thymidylate synthase (TS) ribonucleoprotein complex in cultured human colon cancer cells that consists of TS protein and the mRNA of the nuclear oncogene c-myc. TS protein is complexed in intact cells with the C-terminal coding region of c-myc mRNA that includes nucleotide positions 1625 to 1790. RNA electrophoretic gel mobility shift assays confirm a specific interaction between TS protein and c-myc mRNA and provide additional evidence that the C-terminal coding region represents an important cis-acting regulatory element. Further evidence demonstrates that the in vitro translational efficiency of c-myc mRNA is inhibited as a result of its direct interaction with TS protein. In addition, the presence of exogenous c-myc mRNA specifically relieves the inhibitory effects of TS protein on TS mRNA translation.

A comparison of the substrate specificities of endo-β-N-acetylglucosaminidases from Streptomyces griseus and Diplococcus pneumoniae

Abstract The substrate specificities of the endo-β-N-acetylglucosaminidases from Diplococcus pneumoniae and Streptomyces griseus were compared and found to differ considerably. The enzyme from D. pneumoniae released Asn-GlcNAc-Fuc-containing glycopeptides from exoglycosidase-treated acidic IgM glycopeptides but was limited in its capacity to hydrolyze ovalbumin glycopeptides larger than Asn(GlcNAc)2(Man)5. In contrast, the enzyme from S. griseus hydrolyzed this and larger neutral oligosaccharides but could not hydrolyze the above fucose-containing IgM glycopeptides. Removal of the fucose residue, however, converted the latter to an active substrate for the S. griseus enzyme, thus broadening its substrate range to encompass most of those substrates hydrolyzed by the D. pneumoniae endoglycosidase.

The use of endo-β-N-acetylglucosaminidase H in characterizing the structure and function of glycoproteins

Abstract Endo-β-N-acetylglucosaminidase H from Streptomyces plicatus can be useful in determining both the molecular weight of the protein moiety of glycoproteins and their inherent number of oligosaccharide chains. In the case of carboxypeptidase Y the molecular mass of the carbohydrate free protein was confirmed as 51,000 daltons. The native enzyme was shown to contain 4 oligosaccharide chains each averaging about 14 mannose residues. On treatment of mung bean nuclease I with the endoglycosidase, the molecular mass decreased from 39,000 to 31,000 daltons. The peptides produced on reduction of this enzyme with thiol were 18,700 and 12,500 daltons, indicating that carbohydrate had been present on both. Penicillium nuclease P1 was decreased in size from 40,000 to 30,000 daltons by the endoglycosidase. Although most of the carbohydrate was removed from each of the native enzymes by the endoglycosidase, denaturation of the glycoproteins was necessary to effect complete removal. Enzyme activitywas not affected by carbohydrate depletion of these glycoproteins, a result consistent with similar studies on other oligosaccharide-containing enzymes.

Processing of the intron-containing thymidylate synthase (td) gene of phage T4 is at the RNA level.

The interrupted T4 phage td gene, which encodes thymidylate synthase, is the first known example of an intron-containing prokaryotic structural gene. Analysis of td-encoded transcripts provides evidence in favor of maturation at the RNA level. Northern blotting with T4 RNA and with region-specific probes revealed three classes of RNA: diffuse premessage (ca. 2.5 kb), a low-abundance mature mRNA (ca. 1.3 kb), and an abundant free intron RNA (ca. 1.0 kb). The existence of covalently joined mature mRNA was suggested by hybridization and S1 protection experiments and was confirmed by primer extension analysis of the splice junction. In analogy to expression of interrupted eukaryotic genes, these results are consistent with an RNA processing model that would account for the direct gene transcript serving as precursor for both free intron RNA and a spliced mRNA that is colinear with the thymidylate synthase product.

Factors affecting the oligomeric structure of yeast external invertase

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.

The purification and properties of a β-aspartyl N-acetylglucosylamine amidohydrolase from hen oviduct

Abstract An enzyme capable of hydrolyzing 1- l -β-aspartamido[-2-acetamido]-1,2-dideoxy β- d -glucose (Asn-GlcNAc) and related glycopeptides, such as those from ovalbumin, ribonuclease B, and transferrin, was purified about 1500-fold from hen oviduct. Substitution of the amino or carboxyl groups of the aspartate moiety prevented the enzyme from hydrolyzing the substrate. The products of the reaction were aspartate, ammonia, and N -acetylglucosamine, with 1-amino- N -acetylglucosamine an apparent intermediate. The molecular weight of the protein, determined by sucrose density gradient centrifugation and gel filtration, fell in the range of 101,000–110,000. The enzyme was irreversibly inhibited by 5-diazo-4-oxo- sl -norvaline (DONV), an asparaginase inhibitor, with a K i of 9.5 × 10 −6 m .

Crystal structure of a deletion mutant of human thymidylate synthase Delta (7-29) and its ternary complex with Tomudex and dUMP.

The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 Å and 2.5 Å resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild‐type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 Å away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS ten‐fold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.