Jerry M. Troutman

Tools To Analyze Protein Farnesylation in Cells

Farnesylation of proteins is catalyzed by protein farnesyl transferase (FTase) and is obligatory for the function of the oncoprotein Ras and a variety of other physiologically important proteins. The rapid and selective detection of cellular protein farnesylation status is crucial to understanding both the function of farnesylated proteins and FTase inhibitors. The unnatural FPP analogue 8-anilinogeranyl diphosphate (AGPP, 3b) is an effective alternative substrate for mammalian FTase. Using antibodies specific for the anilinogeranyl moiety, we show that the alcohol precursor (AGOH, 5b) of AGPP is incorporated into cellular proteins in an FTase dependent manner competitive with endogenous pools of FPP. Continuous treatment of HEK-293 cells with 100 microM AGOH for up to 3 days is neither cytotoxic or cytostatic. Antibodies to detect the unnatural anilinogeranyl group were raised against bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) bioconjugates of the activated hapten N-hydroxyphthalimido-succinyl-(S-anilinogeranyl)-L-cysteine methyl ester 9a. Polyclonal antisera containing anti-anilinogeranyl antibodies were generated by immunization of rabbits and a monoclonal anti-anilinogeranyl antibody was raised in mice. ELISA and western blotting of anilinogeranyl modified proteins were used to show the selectivity and measure the titer of the antibodies. The unnatural FPP analogue and corresponding antibodies provide a simple and rapid method for monitoring FTase activity in cells and detection of cellular proteins modified by AGOH 5a.

Campylobacter Jejuni PglH is a Single Active Site Processive Polymerase that Utilizes Product Inhibition to Limit Sequential Glycosyl Transfer Reactions

Asparagine-linked protein glycosylation is essential for the virulence of the human gut mucosal pathogen Campylobacter jejuni . The heptasaccharide that is transferred to proteins is biosynthesized via the glycosyltransferase-catalyzed addition of sugar units to an undecaprenyl diphosphate-linked carrier. Genetic studies on the heptasaccharide assembly enzymes have shown that PglH, which transfers three terminal N-acetyl-galactosamine (GalNAc) residues to the carrier polyisoprene, is essential for chick colonization by C. jejuni . While it is now clear that PglH catalyzes multiple transfer reactions, the mechanism whereby the reactions cease after the addition of just three GalNAc residues has yet to be understood. To address this issue, a series of mechanistic biochemical studies was conducted with purified native PglH. This enzyme was found to follow a processive mechanism under initial rate conditions; however, product inhibition and product accumulation led to PglH release of intermediate products prior to complete conversion to the native ultimate product. Point mutations of an essential EX(7)E sequence motif were used to demonstrate that a single active site was responsible for all three transferase reactions, and a homology model with the mannosyltransferase PimA, from Mycobacteria smegmatis , establishes the requirement of the EX(7)E motif in catalysis. Finally, increased binding affinity with increasing glycan size is proposed to provide PglH with a counting mechanism that does not allow the transfer of more than three GalNAc residues. These results provide important mechanistic insights into the function of the glycosyl transfer polymerase that is related to the virulence of C. jejuni .

Protein Farnesyltransferase Catalyzed Isoprenoid Transfer To Peptide Depends On Lipid Size and Shape, not Hydrophobicity

Protein farnesyl transferase (FTase) catalyzes transfer of a 15‐carbon farnesyl group from farnesyl diphosphate (FPP) to a conserved cysteine in the C‐terminal Ca1a2X motif of a range of proteins, including the oncoprotein H‐Ras (“C” refers to the cysteine, “a” to any aliphatic amino acid, and “X” to any amino acid) and the lipid chain interacts with, and forms part of the Ca1a2X peptide binding site. Previous studies have shown that H‐Ras biological function is ablated when it is modified with lipids that are 3–5 orders of magnitude less hydrophobic than FPP. Here, we employed a library of anilinogeranyl diphosphate (AGPP) and phenoxygeranyl diphosphate (PGPP) derivatives with a range of polarities (log P (lipid alcohol)=0.7–6.8, log P (farnesol)=6.1) and shapes to examine whether FTase‐catalyzed transfer to peptide is dependent on the hydrophobicity of the lipid. Analysis of steady‐state transfer kinetics for analogues to dansyl–GCVLS peptide revealed that the efficiency of lipid transfer was highly dependent on both the shape and size, but was independent of the polarity of the analogue. These observations indicate that hydrophobic features of isoprenoids critical for their association with membranes and/or protein receptors are not required for efficient transfer to Ca1a2X peptides by FTase. Furthermore, the results of these studies indicate that the role played by the farnesyl lipid in the FTase mechanism is primarily structural. To explain these results we propose a model in which the FTase active site stabilizes a membrane interface‐like environment.

Hydrophilic Anilinogeranyl Diphosphate Prenyl Analogues Are Ras Function Inhibitors

Sequential processing of H-Ras by protein farnesyl transferase (FTase), Ras converting enzyme (Rce1), and protein-S-isoprenylcysteine O-methyltransferase (Icmt) to give H-Ras C-terminal farnesyl-S-cysteine methyl ester is required for appropriate H-Ras membrane localization and function, including activation of the mitogen-activated protein kinase (MAPK) cascade. We employed a Xenopus laevis oocyte whole-cell model system to examine whether anilinogeranyl diphosphate analogues of similar shape and size, but with a hydrophobicity different from that of the FTase substrate farnesyl diphosphate (FPP), could ablate biological function of H-Ras. Analysis of oocyte maturation kinetics following microinjection of in vitro analogue-modified H-Ras into isoprenoid-depleted oocytes revealed that analogues with a hydrophobicity near that of FPP supported H-Ras biological function, while the analogues p-nitroanilinogeranyl diphosphate (p-NO2-AGPP), p-cyanoanilinogeranyl diphosphate (p-CN-AGPP), and isoxazolaminogeranyl diphosphate (Isox-GPP) with hydrophobicities 2-5 orders of magnitude lower than that of FPP did not. We found that although H-Ras modified with FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP was an efficient substrate for C-terminal postprenylation processing by Rce1 and Icmt, co-injection of H-Ras with analogues p-NO2-AGPP, p-CN-AGPP, or Isox-GPP could not activate MAPK. We propose that H-Ras biological function requires a minimum lipophilicity of the prenyl group to allow important interactions downstream of the C-terminal processed H-Ras protein. The hydrophilic FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP are H-Ras function inhibitors (RFIs) and serve as lead compounds for a unique class of potential anticancer therapeutics.

Chemoenzymatic synthesis of an isoprenoid phosphate tool for the analysis of complex bacterial oligosaccharide biosynthesis.

Undecaprenyl Pyrophosphate Synthase (UPPS) is a key enzyme that catalyzes the production of bactoprenols, which act as membrane anchors for the assembly of complex bacterial oligosaccharides. One of the major hurdles in understanding the assembly of oligosaccharide assembly is a lack of chemical tools to study this process, since bactoprenols and the resulting isoprenoid-linked oligosaccharides lack handles or chromophores for use in pathway analysis. Here we describe the isolation of a new UPPS from the symbiotic microorganism Bacteroides fragilis, a key species in the human microbiome. The protein was purified to homogeneity and utilized to accept a chromophore containing farnesyl diphosphate analogue as a substrate. The analogue was utilized by the enzyme and resulted in a bactoprenyl diphosphate product with an easy to monitor tag associated with it. Furthermore, the diphosphate is shown to be readily converted to monophosphate using a common molecular biology reagent. This monophosphate product allowed for the investigation of complex oligosaccharide biosynthesis, and was used to probe the activity of glycosyltransferases involved in the well characterized Campylobacter jejuni N-linked protein glycosylation. Novel reagents similar to this will provide key tools for the study of uncharacterized oligosaccharide assemblies, and open the possibility for the development of rapid screening methodology for these biosynthetic systems.

Farnesyl diphosphate analogues with aryl moieties are efficient alternate substrates for protein farnesyltransferase.

Farnesylation is an important post-translational modification essential for the proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single-turnover rate constant for alkylation does depend on the Ca(1)a(2)X peptide sequence. These results suggest that the isoprenoid transition-state conformation is preferred over the inactive E·FPP·Ca(1)a(2)X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for the design of novel inhibitors.

Species Differences in Alternative Substrate Utilization by the Antibacterial Target Undecaprenyl Pyrophosphate Synthase

Undecaprenyl pyrophosphate synthase (UPPS) is a critical enzyme required for the biosynthesis of polysaccharides essential for bacterial survival. In this report, we have tested the substrate selectivity of UPPS derived from the mammalian symbiont Bacteroides fragilis, the human pathogen Vibrio vulnificus, and the typically benign but opportunistic pathogen Escherichia coli. An anthranilamide-containing substrate, 2-amideanilinogeranyl diphosphate (2AA-GPP), was an effective substrate for only the B. fragilis UPPS protein, yet replacing the amide with a nitrile [2-nitrileanilinogeranyl diphosphate (2CNA-GPP)] led to a compound that was fully functional for UPPS from all three target organisms. These fluorescent substrate analogues were also found to undergo increases in fluorescence upon isoprenoid chain elongation, and this increase in fluorescence can be utilized to monitor the activity and inhibition of UPPS in 96-well plate assays. The fluorescence of 2CNA-GPP increased by a factor of 2.5-fold upon chain elongation, while that of 2AA-GPP increased only 1.2-fold. The 2CNA-GPP compound was therefore more versatile for screening the activity of UPPS from multiple species of bacteria and underwent a larger increase in fluorescence that improved its ability to detect increases in chain length. Overall, this work describes the development of new assay methods for UPPS and demonstrates the difference in substrate utilization between forms of UPPS from different species, which has major implications for UPPS inhibitor development, assay construction, and the development of polysaccharide biosynthesis probes.

Fluorescent probes for investigation of isoprenoid configuration and size discrimination by bactoprenol-utilizing enzymes.

Undecaprenyl Pyrophosphate Synthase (UPPS) is an enzyme critical to the production of complex polysaccharides in bacteria, as it produces the crucial bactoprenol scaffold on which these materials are assembled. Methods to characterize the systems associated with polysaccharide production are non-trivial, in part due to the lack of chemical tools to investigate their assembly. In this report, we develop a new fluorescent tool using UPPS to incorporate a powerful fluorescent anthranilamide moiety into bactoprenol. The activity of this analogue in polysaccharide biosynthesis is then tested with the initiating hexose-1-phosphate transferases involved in Capsular Polysaccharide A biosynthesis in the symbiont Bacteroides fragilis and the asparagine-linked glycosylation system of the pathogenic Campylobacter jejuni. In addition, it is shown that the UPPS used to make this probe is not specific for E-configured isoprenoid substrates and that elongation by UPPS is required for activity with the downstream enzymes.

Biosynthetic Assembly of the Bacteroides fragilis Capsular Polysaccharide A Precursor Bactoprenyl Diphosphate-Linked Acetamido-4-amino-6-deoxygalactopyranose

The sugar capsule capsular polysaccharide A (CPSA), which coats the surface of the mammalian symbiont Bacteroides fragilis, is a key mediator of mammalian immune system development. In addition, this sugar polymer has shown therapeutic potential in animal models of multiple sclerosis and other autoimmune disorders. The structure of the CPSA polymer includes a rare stereoconfiguration sugar acetamido-4-amino-6-deoxygalactopyranose (AADGal) that we propose is the first sugar linked to a bactoprenyl diphosphate scaffold in the production of CPSA. In this report, we have utilized a heterologous system to reconstitute bactoprenyl diphosphate-linked AADGal production. Construction of this system included a previously reported Campylobacter jejuni dehydratase, PglF, coupled to a B. fragilis-encoded aminotransferase (WcfR) and initiating hexose-1-phosphate transferase (WcfS). The function of the aminotransferase was confirmed by capillary electrophoresis and a novel high-performance liquid chromatography (HPLC) method. Production of the rare uridine diphosphate (UDP)-AADGal was confirmed through a series of one- and two-dimensional nuclear magnetic resonance experiments and high-resolution mass spectrometry. A spectroscopically unique analogue of bactoprenyl phosphate was utilized to characterize the transfer reaction catalyzed by WcfS and allowed HPLC-based isolation of the isoprenoid-linked sugar product. Importantly, the entire heterologous system was utilized in a single-pot reaction to biosynthesize the bactoprenyl-linked sugar. This work provides the first critical step in the in vitro reconstitution of CPSA biosynthesis.

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A.

Capsular polysaccharide A (CPSA) is a four-sugar repeating unit polymer found on the surface of the gut symbiont Bacteroides fragilis that has therapeutic potential in animal models of autoimmune disorders. This therapeutic potential has been credited to its zwitterionic character derived from a positively charged N-acetyl-4-aminogalactosamine (AADGal) and a negatively charged 4,6-O-pyruvylated galactose (PyrGal). In this report, using a fluorescent polyisoprenoid chemical probe, the complete enzymatic assembly of the CPSA tetrasaccharide repeat unit is achieved. The proposed pyruvyltransferase, WcfO; galactopyranose mutase, WcfM; and glycosyltransferases, WcfP and WcfN, encoded by the CPSA biosynthesis gene cluster were heterologously expressed and functionally characterized. Pyruvate modification, catalyzed by WcfO, was found to occur on galactose of the polyisoprenoid-linked disaccharide (AADGal-Gal), and did not occur on galactose linked to uridine diphosphate (UDP) or a set of nitrophenyl-galactose analogues. This pyruvate modification was also found to be required for the incorporation of the next sugar in the pathway N-acetylgalactosamine (GalNAc) by the glycosyltransferase WcfP. The pyruvate acetal modification of a galactose has not been previously explored in the context of a polysaccharide biosynthesis pathway, and this work demonstrates the importance of this modification to repeat unit assembly. Upon production of the polyisoprenoid-linked AADGal-PyrGal-GalNAc, the proteins WcfM and WcfN were found to work in concert to form the final tetrasaccharide, where WcfM formed UDP-galactofuranose (Galf) and WcfN transfers Galf to the AADGal-PyrGal-GalNAc. This work demonstrates the first enzymatic assembly of the tetrasaccharide repeat unit of CPSA in a sequential single pot reaction.