FTase Activity Research Articles

Effect of Insulin on Farnesyltransferase Gene Transcription and mRNA Stability

Recently, we have shown that hyperinsulinemia increases the activity of farnesyltransferase (FTase) in vitro (1) and in hyperinsulinemic animals (2), stimulates the phosphorylation of the FTase α-subunit (3), increases the amounts of cellular farnesylated p21Ras (4), and potentiates the nuclear effects of other peptide growth factors, such as EGF, IGF-1 and PDGF (5). To further investigate the mechanism by which insulin stimulates FTase activity we tested the effect of insulin on the rate of FTase transcription, the rate of FTase mRNA degradation, and the amounts of FTase protein. Insulin increased the amounts of FTase α- and β-subunit mRNA in 3T3-L1 fibroblasts 2.5-fold to 4-fold after 6 h and 24 h incubation, respectively, but did not increase the rate of FTase transcription over a 24 h period. Insulin did, however, increase the stability of both α- and β-subunit mRNA. The half-life for both FTase α- and β-subunit mRNA was approximately 3 h and 6h in the absence and in the presence of insulin, respectively. Although insulin stabilized the α- and β-subunit mRNA of FTase, there was no increase in amounts of protein of either subunit. These data suggest that although insulin increases the stability of the FTase mRNA, it stimulates FTase enzymatic activity only at the post-translational level.

Insulin stimulates the phosphorylation and activity of farnesyltransferase via the Ras-mitogen-activated protein kinase pathway.

Farnesylation of p21Ras by farnesyltransferase (FTase) is obligatory for anchoring p21Ras to the plasma membrane, where it can be activated by growth factors. Insulin significantly stimulates the phosphorylation of the alpha-subunit of FTase (4-fold) and the enzymatic activity of FTase in 3T3-L1 fibroblasts and adipocytes. FTase activity was assessed by the amount of [3H] mevalonate (a precursor of farnesyl) incorporated into p21Ras in vivo and by quantitating the amount of farnesylated p21Ras before and after insulin administration. Insulin-stimulated phosphorylation of the alpha-subunit of FTase in 3T3-L1 fibroblasts and adipocytes was blocked by the mitogen-activated protein/extracellular-signal regulated kinase-kinase inhibitor, PD98059, but not by wortmannin or bisindolylmaleimide. Additionally, PD98059 blocked insulin-stimulated [3H]mevalonic incorporation and farnesylation of unprocessed p21Ras in both cell lines. Furthermore, expression of the dominant negative mutant of p21Ras precluded insulin-stimulated phosphorylation of the FTase alpha-subunit and activation of its enzymatic activity. In contrast, 3T3-L1 fibroblasts, expressing the constitutively active Raf-1, exhibited enhanced phosphorylation of the FTase alpha-subunit. It seems that insulin's effect on the phosphorylation and activation of FTase in both fibroblasts and adipocytes is mediated via the Ras pathway, resulting in a positive feedback augmentation of the cellular pool of farnesylated p21Ras.

GTP Loading of Farnesylated p21Ras by Insulin at the Plasma Membrane

Insulin promotes the phosphorylation and activation of farnesyltransferase (FTase) in a time- and a dose-dependent manner. Increased FTase activity results in a larger pool of farnesylated p21Ras and allows for enhanced GTP loading. Insulin significantly increases the pool of farnesylated p21Ras from 20–25% in quiescent 3T3-L1 fibroblasts to approximately 70%, most of which is targeted to the plasma membrane. Furthermore, insulin promotes GTP loading of plasma membrane and not cytosolic p21Ras. The half-life of plasma membrane-associated farnesylated p21Ras is approximately 6 hours, and is identical in control and insulin-treated cells. We have also observed a direct correlation between the amounts of farnesylated p21Ras at the plasma membrane and the magnitude of insulin-induced GTP loading of p21Ras.

Kinetics of Protein Farnesyltransferase: Sigmoidal vs Hyperbolic Behavior as a Function of Assay Conditions

Protein farnesyl transferase (FTase) catalyzes the addition of a farnesyl isoprenoid to a conserved cysteine residue in Ras and several other key proteins involved in cell regulation. An assay technique commonly used to measure FTase activity involves vacuum filtration. This assay, which traps precipitated, radiolabeled prenylated proteins on a glass fiber filter for analysis by scintillation counting, was designed to be fast and accurate. In the case of FTase, substrate saturation curves generated by this assay technique using Ras as a substrate often show a lag at low Ras concentrations, resulting in curves with sigmoidal character. We have found that the sigmoidal behavior is due to the use of the filter binding assay and not to any inherent property of FTase. Specifically, the glass fiber filters do not adequately trap precipitated Ras proteins, especially at low concentrations. Addition of cytosol from either bovine brain or liver, or of purified tubulin to the FTase assay mixture prior to the precipitation step, results in the apparent formation of stable complexes of farnesylated Ras protein that can then be optimally trapped on the glass fiber filter. This appears to be at least in part due to the ability of tubulin to bind the prenyl protein reaction product. The ability to obtain accurate kinetics for the FTase using the standard filter binding assay should greatly enhance its use to accurately assess the properties of FTase inhibitors.

Effect of Insulin on Farnesyltransferase Activity in 3T3-L1 Adipocytes

Activation of p21(ras) by GTP loading is a critical step in a cascade of intracellular insulin signaling. Farnesylation of p21(ras) protein is an obligatory event that facilitates Ras migration to the plasma membrane and subsequent activation. Farnesyltransferase (FTase) is a ubiquitous enzyme that catalyzes the lipid modification of p21(ras) by the addition of farnesyl to the C-terminal "CAAX" motif. In vitro and in vivo FTase activities were studied in 3T3-L1 adipocytes in response to insulin challenge. Insulin exerted a biphasic stimulatory effect on FTase activity measured in vitro with a 31% increase at 5 min and a 130% increase at 60 min. Insulin-stimulated farnesylation of p21(ras) pools in vivo correlated with FTase activity seen in vitro by displaying an increase in farnesylated p21(ras) from 40% of total cellular Ras in control cells to 63% by 5 min and 80% by 60 min (p < 0.05) in insulin-treated cells. Insulin challenge of 3T3-L1 adipocytes increased incorporation of tritiated mevalonic acid in p21(ras) in a dose-dependent manner and stimulated a 2-fold increase in phosphorylation of the alpha-subunit of FTase at 5 min and a 4-fold increase at 60 min.

Identification of a Cysteine Residue Essential for Activity of Protein Farnesyltransferase: Cys299 IS EXPOSED ONLY UPON REMOVAL OF ZINC FROM THE ENZYME

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zinc-depleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM. These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [3H]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys299, that is in the beta subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified. While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys299 in the beta subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.

Protection against malignant conversion in SENCAR mouse skin by all trans retinoic acid: inhibition of the ras p21-processing enzyme farnesyltransferase and Ha-ras p21 membrane localization.

Many studies have shown that all trans retinoic acid (RA) exhibits significant protective effects against mouse skin tumor promotion and spontaneous as well as enhanced malignant conversion. In a recently completed study, we showed that under treatments in which papillomas on SENCAR mouse skin are induced at low and high probabilities to convert to malignant carcinomas, RA affords significant protection against both tumor promotion and subsequent malignant conversion. More than 95% of these mouse skin papillomas and carcinomas have been shown to contain point mutation at the 61 codon of Ha-ras oncogene. The ras oncogene encodes a p21 protein that, in its mutated form, transforms mammalian cells only when p21 is at the inner surface of the plasma membrane, by a series of enzymatic reactions in which the initial step is catalyzed by farnesyltransferase (FTase). In this study, we assessed whether the protective effect of RA against malignant conversion involves the inhibition of ras p21 processing in those tumors that contain the activated ras oncogene. The FTase activity and the levels of cytosolic and membrane-bound Ha-ras p21 were determined in all papillomas and carcinomas obtained from acetone- or RA-treated animals. No matter how the data were analyzed and what comparisons were considered, in all the protocols used, compared with controls, papillomas and carcinomas obtained from RA-treated groups showed significantly decreased (P < 0.01-0.001) FTase activity. Furthermore, the tissue samples from RA-treated groups in different protocols also showed significantly diminished membrane localization of Ha-ras p21, with a concomitant increase in cytosolic Ha-ras p21 levels. The analysis of these data also showed that in all the protocols used, the increased FTase activity and membrane localization of Ha-ras p21 were associated with the induction of papillomas and their subsequent malignant conversion to squamous cell carcinomas. Taken together, these results indicate a strong correlation between the inhibition of ras p21 farnesylation because of a decrease in FTase activity by RA and its protective effect against malignant conversion of papillomas to carcinomas. Based on the results of this study, it is tempting to suggest that clinical trials evaluating the preventive or therapeutic potential of retinoids may be directed more toward those clinical malignancies that are known to contain the activated ras oncogene.

α-Subunit of Farnesyltransferase Is Phosphorylatedin Vivo:Effect of Protein Phosphatase-1 on Enzymatic Activity

Farnesyltransferase is a heterodimer consisting of a 49 kDa α-subunit and a 46 kDa β-subunit. In this report, we demonstrate that the endogenous heterodimeric farnesyltransferase protein is phosphorylated at the α-subunitin vivoand phosphorylation plays a role in the regulation of farnesyltransferase activity.In vivo32P-labeling of PC-12 cells followed by immunoprecipitation with specific anti rat α-subunit IgG showed a labeled α-subunit protein band at an expected molecular mass of 49 kDa. Treatment of PC-12 cells with protein phosphatase inhibitor, Calyculin A, resulted in a decrease in FTase activity, and phophoserine/phosphothreonine-specific protein phosphatase-1 treatment of PC-12 and GM37 cell extracts resulted in 100% and 375% increase in farnesyltransferase activity, respectively, compared to untreated extracts.

Farnesyltransferase Activity and mRNA Expression in Human Skin Basal Cell Carcinomas

Ras oncogene encode a protein p21 which in its mutated form transforms mammalian cells only after membrane anchoring by a series of enzymatic reactions where the initial step is catalyzed by farnesyltransferase (FTase). For this reason, FTase has become an attractive target for the development of novel anticancer agents. Virtually nothing is known about FTase activity and the association between the expression of its α and β subunit genes with respect to the processing of Ras p21 in human cancers. In this study, we found that compared to normal skin, FTase activity and levels of both cytosolic and membrane-bound Ha-Ras p21 were significantly higher in human skin basal cell carcinomas (BCCs). In addition, the expression of both α and β subunit genes was significantly higher in BCCs than the normal skin. These results suggest an association between enhanced FTase activity and the processing of overexpressed Ras p21 in such tumor type. This may have a bearing on the pathogenesis of activated Ras oncogene containing human malignancies.

Enzymic characterization of fission yeast farnesyl transferase: recognition of the -CAAL motif at the C-terminus.

The enzyme farnesyl transferase (FTase) catalyzes the posttranslational modification of Ras and other Ras family proteins with a C15 farnesyl group. The target proteins have a consensus -CAAX motif (X, any amino acid except leucine) at the C-terminus. Since proteins that have leucine as the C-terminal amino acid X are modified with a C20 geranylgeranyl group, it is thought that the C-terminal leucine is the signal (-CAAL motif) for selection of isoprenoid molecules. Here, we report the presence of multiple FTase activities in the fission yeast Schizosaccharomyces pombe, each seeming to correspond to a particular protein known to be modified by the farnesyl group in vivo. Using enzymic activities specific to S. pombe Ras1, we found similar affinities for FTases in the wild-type (EVSTKCCVIC) and mutant Ras1 peptide, in which the C-terminal amino acid is replaced by leucine (EVSTKCCVIL). These results suggest that recognition and selection of the correct isoprenoid group by the FTases require other amino acid sequences of the target protein in addition to the C-terminal -CAAX motif.

Characterization of Xenopus laevis oocyte farnesyl transferase.

Farnesyl transferase (FTase) activity, characterized in extracts of Xenopus laevis oocytes using an in vitro filtration assay, was observed to be at least 80% cytosolic with optimal product formation at pH 7.0. Oocyte FTase displayed enzyme activity that was specific for the Ras-CVIM construct but not Ras-CAIL or Ras-SVLS. Labeling of Ras-CVIM using [3H]farnesyl pyrophosphate was inhibited by addition of unlabeled farnesyl pyrophosphate to the assay mixture but not by addition of either geranylgeranyl pyrophosphate or stearic acid. Polyclonal rabbit antibodies against recombinant human FTase subunits were tested for cross-reactivity with oocyte proteins and were used to determine the apparent molecular masses of oocyte FTase enzyme subunits. Anti-alpha subunit antibodies labeled a band of approximately 53 kDa. Anti-beta subunit antibodies bound both a high molecular mass band (140 kDa) and a lower molecular mass band (38 kDa) on immunoblots of cytosolic oocyte proteins. When oocyte FTase activity was resolved by gel filtration, the peak of enzyme activity correlated with immunoblot detection of the 38- and 53-kDa bands as well as a doublet of 86- and 95-kDa. This correlation suggests that Xenopus laevis oocyte FTase is a heterodimer under reducing conditions with subunits of 53 +/- 1 and 38 +/- 2 kDa, and that the holoenzyme migrates on SDS-polyacrylamide gels with an apparent molecular mass of 86-95 kDa.

CAAX Peptidomimetic FTI-244 Decreases Platelet-Derived Growth Factor Receptor Tyrosine Phosphorylation Levels and Inhibits Stimulation of Phosphatidylinositol 3-Kinase but Not Mitogen-Activated Protein Kinase

Cysteine farnesylation of the Ras carboxyl terminal tetrapeptide CAAX motif (where C=cysteine, A=leucine, isoleucine, or valine, and X=methionine or serine) is required for Ras biological activity. In this report, we describe the effects of inhibitors of farnesyltransferase (FTase), the enzyme responsible for this lipid modification, on platelet-derived growth factor (PDGF) signaling in NIH-3T3 cells. In vitro, the CAAX peptidomimetic FTI-232 exhibits potent inhibition of FTase activity (IC 50=150 nM) and its carboxyl-methylated counterpart, FTI-244, inhibits Ras processing in vivo. Treatment of NIH-3T3 cells with FTI-244 inhibits PDGF-induced DNA synthesis but not stimulation of mitogen-activated protein kinase (MAPK). However, FTI-244 significantly reduces PDGF-induced tyrosine phosphorylation levels of PDGF receptor (PDGFR) as well as its association with, and activation of, phosphatidylinositol-3-kinase (PI-3-K), a key enzyme in PDGF-induced mitogenesis.

CAAX Geranylgeranyl Transferase Transfers Farnesyl as Efficiently as Geranylgeranyl to RhoB

RhoB, a small GTP-binding protein, was shown previously to contain farnesyl (C-15) as well as geranylgeranyl (C-20) groups (Adamson, P., Marshall, C. J., Hall, A., and Tilbrook, P. A. (1992) J. Biol. Chem. 267, 20033-20038). The COOH-terminal sequence of the protein is CCKVL. According to current rules of prenylation, the COOH-terminal leucine should render the protein a substrate for CAAX geranylgeranyl transferase (GGTase-1), but not for CAAX farnesyltransferase (FTase). To determine the mechanism of farnesylation, we prepared recombinant RhoB and incubated it with recombinant preparations of either FTase or GGTase-1. RhoB was neither farnesylated nor geranylgeranylated efficiently by FTase, but it was farnesylated as well as geranylgeranylated by GGTase-1. The enzyme attached farnesyl more efficiently than geranylgeranyl to RhoB. Neither farnesylation nor geranylgeranylation required the cysteine at the fifth position from the COOH terminus. However, replacement of the cysteine at the fourth position abolished attachment of both prenyl groups. We conclude that the previously observed farnesylation of RhoB is attributable to the FTase activity of GGTase-1. These data, and other accumulating data, indicate that GGTase-1 is a highly unusual enzyme that efficiently transfers both farnesyl and geranylgeranyl groups and that the choice of prenyl group is dictated by the nature of the protein acceptor.

A novel cellular source for endothelin

Cysteine farnesylation of the Ras carboxyl terminal tetrapeptide CAAX motif (where C=cysteine, A=leucine, isoleucine, or valine, and X=methionine or serine) is required for Ras biological activity. In this report, we describe the effects of inhibitors of farnesyltransferase (FTase), the enzyme responsible for this lipid modification, on platelet-derived growth factor (PDGF) signaling in NIH-3T3 cells. In vitro, the CAAX peptidomimetic FTI-232 exhibits potent inhibition of FTase activity (IC50=150 nM) and its carboxyl-methylated counterpart, FTI-244, inhibits Ras processing in vivo. Treatment of NIH-3T3 cells with FTI-244 inhibits PDGF-induced DNA synthesis but not stimulation of mitogen-activated protein kinase (MAPK). However, FTI-244 significantly reduces PDGF-induced tyrosine phosphorylation levels of PDGF receptor (PDGFR) as well as its association with, and activation of, phosphatidylinositol-3-kinase (PI-3-K), a key enzyme in PDGF-induced mitogenesis.

Ras p21 Farnesylation in Ultraviolet B Radiation–Induced Tumors in The Skin of SKH-1 Hairless Mice

Cutaneous exposure to solar ultraviolet B (UVB) radiation is well recognized as the major cause of skin cancer in humans; however, the precise molecular mechanisms whereby UVB mediates carcinogenesis remains unclear. The involvement of activated ras oncogenes has been demonstrated extensively in both animal and human skin cancers. Activated ras oncogenes encode mutated ras p21 that exist in the guanosine triphosphate-bound active state and, following localization to the inner side of the plasma membrane, cause cellular transformation. This membrane association requires three post-translational modifications occurring at the C-terminus of the ras p21. The farnesylation of p21 by a cytosolic enzyme known as farnesyltransferase (FTase) is the critical step that triggers biologic functions of the ras p21. In this study, FTase activity was found to be substantially higher (approximately threefold) in UVB radiation-induced tumors in SKH-1 hairless mice compared to epidermis from controls. Western blot analysis showed significantly higher levels of Ha-ras p21 in both cytosolic and membrane fractions prepared from tumors compared to epidermis. Pan ras antibody against mutated p21 at codon 12 showed very strong reactivity for ras val-12p21 in tumors but not in normal epidermis, suggesting a gly to val substitution at 12th position in ras p21 in UVB-induced tumors. Our data indicate that enhanced FTase activity and the processing of overexpressed p21 in UVB-induced tumors are correlated, and predict the role of point mutation at the 12th codon of the ras oncogene during photocarcinogenesis in mice.

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

Protein farnesyltransferase (FTase) catalyses the addition of a farnesyl group to a cysteine within the so-called 'CAAX box' at the C-terminus of various proteins. In the present paper we report purification of Saccharomyces cerevisiae FTase to near-homogeneity. This was accomplished by constructing a yeast strain overproducing FTase approx. 100-fold. The purified enzyme was a heterodimer of approx. 90 kDa and consisted of 43 kDa and 34 kDa subunits. The 43 kDa subunit was shown to be the product of the DPR1 gene by using antibody raised against baculovirus-produced DPR1 polypeptide. The purified enzyme required Mg2+, showed a pH optimum of 7.8 and was most active at 50 degrees C. The Km values for farnesyl pyrophosphate and GST-CIIS (glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein), KmFpp and KmGST CIIS, were 8.1 and 5.1 microM respectively. The enzyme was capable of farnesylating GST-CIIL (the same as GST-CIIS, except that the C-terminal serine is changed to leucine), a substrate protein for the enzyme geranylgeranyltransferase, although with a higher apparent Km than for GST-CIIS. Like its mammalian counterpart, yeast FTase activity was inhibited by peptides containing the C-terminal CAAX sequence (that is, one where C = cysteine, A = aliphatic amino acid and X = any amino acid). These results provide direct evidence for the idea that the yeast and mammalian FTases are structurally and functionally very similar.

Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases.

Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an alpha/beta heterodimer, comprised of subunits having relative molecular masses of approximately 47 (alpha) and 45 kDa (beta). In the yeast Saccharomyces cerevisiae, two unlinked genes, RAM1/DPR1 (RAM1) and RAM2, are required for FTase activity. To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses. cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase alpha-subunit were isolated. Comparison of the amino acid sequences deduced from the alpha-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase alpha-subunit. Antisera raised against the RAM1 gene product reacted specifically with the beta-subunit of bovine FTase, suggesting that the RAM1 gene product is analogous to the bovine FTase beta-subunit. Whereas a ram1 mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to RAM1, specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-II, respectively. In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-II. Antisera that specifically recognized the bovine FTase alpha-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-II activity. Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-II are comprised of different but homologous beta-subunits and that the alpha-subunits of FTase and GGTase-I share common features not shared by GGTase-II.

Sequence dependence of protein isoprenylation

Several proteins have been shown to be post-translationally modified on a specific C-terminal cysteine residue by either of two isoprenoid biosynthetic pathway metabolites, farnesyl diphosphate or geranylgeranyl diphosphate. Three enzymes responsible for protein isoprenylation were resolved chromatographically from the cytosolic fraction of bovine brain: a farnesyl-protein transferase (FTase), which modified the cell-transforming Ras protein, and two geranyl-geranyl-protein transferases, one (GGTase-I) which modified a chimeric Ras having the C-terminal amino acid sequence of the gamma-6 subunit of heterotrimeric GTP-binding proteins, and the other (GGTase-II) which modified the Saccharomyces cerevisiae secretory GTPase protein YPT1. In a S. cerevisiae strain lacking FTase activity (ram1), both GGTases were detected at wild-type levels. In a ram2 S. cerevisiae strain devoid of FTase activity, GGTase-I activity was reduced by 67%, suggesting that GGTase-I and FTase activities derive from different enzymes but may share a common genetic feature. For the FTase and the GGTase-I activities, the C-terminal amino acid sequence of the protein substrate, the CAAX box, appeared to contain all the critical determinants for interaction with the transferase. In fact, tetrapeptides with amino acid sequences identical to the C-terminal sequences of the protein substrates for FTase or GGTase-I competed for protein isoprenylation by acting as alternative substrates. Changes in the CAAX amino acid sequence of protein substrates markedly altered their ability to serve as substrates for both FTase and GGTase-I. In addition, it appeared that FTase and GGTase-I had complementary affinities for CAAX protein substrates; that is, CAAX proteins that were good substrates for FTase were, in general, poor substrates for GGTase-I, and vice versa. In particular, a leucine residue at the C terminus influenced whether a CAAX protein was either farnesylated or geranylgeranylated preferentially. The YPT1 C terminus peptide, TGGGCC, did not compete or serve as a substrate for GGTase-II, indicating that the interaction between GGTase-II and YPT1 appeared to depend on more than the 6 C-terminal residues of the protein substrate sequence. These results identify three different isoprenyl-protein transferases that are each selective for their isoprenoid and protein substrates.

Sucrose-dependent cell adherence and cariogenicity of serotype c Streptococcus mutans.

Four strains of serotype c Streptococcus mutans differing in glucosyltransferase (GTase) and fructosyltransferase (FTase) activities were examined. These strains had been made resistant to streptomycin. FTase activity of an S. mutans clinical variant, MT6801R, which forms large mucoid colonies on sucrose-containing agar, was considerably higher than that of a typical serotype c strain, MT8148R, which forms small, rough colonies on the same agar. Two mutants, NG14 and NG7183, were induced from strain MT6801R by N-methyl-N'-nitro-N-nitrosoguanidine, and were found to be streptomycin-resistant. GTase and FTase activities of mutant NG14 were similar to those of the typical serotype c strain, while in mutant NG7183 the two enzyme activities were very low. Growing cells of these strains (except NG7183) adhered firmly to a glass surface in sucrose broth. Resting cells of all strains attached in small numbers to saliva-coated hydroxyapatite in the absence of sucrose. On the other hand, the presence of sucrose markedly enhanced the attachment of cells of strains MT8148R, MT6801R and NG14, but not NG7183. Cell-surface hydrophobicity and acid production of all strains were similar. Both strain MT8148R and NG14 colonized tooth surfaces and produced significant dental caries in specific-pathogen-free rats. Strain MT6801R had lower colonization ability and cariogenicity when compared with strains MT8148R and NG14. Furthermore, mutant NG7183 was able to colonize the tooth surfaces in small numbers, but failed to cause dental caries. These results indicate that sucrose-dependent cell adherence mediated by de novo glucan synthesis is necessary for the accumulation of serotype c S. mutans cells on the tooth surface and the induction of dental caries.

Clinical isolates of Streptococcus mutans serotype c with altered colony morphology due to fructan synthesis.

Streptococcus mutans MT6801 , MT6861 , and MT6879 , which form large mucoid colonies on mitis salivarius agar, were isolated from a mother and her two daughters. These isolates were identified as serotype c by immunodiffusion with serotype-specific antisera. The large colonies formed on sucrose-containing agar were found to contain water-soluble fructan . The cell-free fructosyltransferase ( FTase ) activity of the strains which formed large colonies was five to eight times higher than that of serotype c S. mutans which produced small, rough colonies typical of this serotype. Furthermore, greater quantities of fructan were synthesized from sucrose by growing cells of MT6801 when compared with MT8148 , a typical serotype c S. mutans. Glucosyltransferase and FTase could be isolated by chromatofocusing from culture supernatants of MT6801 and MT8148 . The FTase activity of both strains was eluted at pH 4.5, and glucosyltransferase was released by elution with an NaCl linear gradient. The eluted FTase activity of MT6801 was significantly higher than that of MT8148 . Strains MT6861 and MT6879 were also found to possess a similar property in terms of FTase activity. These results suggest that formation of large mucoid colonies by these strains is a consequence of high FTase activity.