Manipulation of β-glucuronidase for use as a reporter in vacuolar targeting studies

It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, -glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of -glucuronidase as a reporter protein in secretory and vacuolar targeting studies, one of the two putative N-linked glycosylation sites within the GUS gene was altered by site-directed mutagenesis. The second N-linked glycosylation site was not altered because sequence analysis of nucleotide sequences around the second putative glycosylation site revealed that the published sequence was incorrect, and that no such site existed.     

Annexin VI is a mannose-6-phosphate-independent endocytic receptor for bovine β-glucuronidase

Endocytosis and transport of bovine liver β-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI–MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine β-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine β-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine β-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine β-glucuronidase.     

Rat β-glucuronidase as a reporter protein for the analysis of the plant secretory pathway

Abstract

E. coliβ-glucuronidase, a cytosolic enzyme, was found not to be a good reporter enzyme for secretion studies in plants. In this study, we chose to test and adapt an animal β-glucuronidase as a better reporter protein for the secretory pathway of plants. We modified rat β-glucuronidase to obtain secreted and vacuolar variants. Five different C-termini were produced: the original C-terminus of the rat enzyme, a 19 codon deletion (Δ19), a 15 codon deletion (Δ15) and fusions of the Δ19 or Δ15 termini with the last 6 or 7 codons of the vacuolar sorting determinant of tobacco chitinase A, respectively. The signal sequence of the rat β-glucuronidase polypeptide was replaced by the sequence encoding the signal peptide of tobacco chitinase A. In a transient expression system, the best enzymatic activity was found with β-glucuronidase having the 15 codons deletion, therefore Δ15 (secRGUS) and Δ15 + Chi (RGUS-Chi) were further evaluated and their efficiency of secretion or vacuolar targeting were tested under different conditions. To determine the correct targeting of reporter genes, we compared the localization of β-glucuronidase and of an endogenous marker, α-mannosidase. Treating cells with drugs that specifically affect different aspects of the secretory pathway also tested the validity of RGUS-based reporters. A non-specific inhibitor such as cytochalasin D and a wide range inhibitor such as BFA were compared with specific inhibitors such as wortmannin and bafilomycin A1. Finally, monensin and NH₄Cl were used to evaluate the role of vacuolar pH in correct RGUS-Chi targeting. The two new reporter proteins proved to be good tools for our studies in the transient expression system in tobacco protoplasts and for further applications.     

Behaviour of the NO-β-d-glucosiduronic acid of N-acetyl-N-phenylhydroxylamine as a substrate for β-glucuronidase

1. Biosynthetic sodium (N-acetyl-N-phenylhydroxylamine NO-beta-d-glucosid)-uronate is hydrolysed completely by purified mouse urinary beta-glucuronidase into the products N-acetyl-N-phenylhydroxylamine and glucuronic acid. The hydrolysis is inhibited by saccharo-(1-->4)-lactone. These results not only confirm the identity and purity of the substrate but also establish it as a substrate for beta-glucuronidase. 2. Mammalian and bacterial beta-glucuronidase preparations hydrolysed the substrate at a rate one-fifth of that for (phenolphthalein beta-d-glucosid)uronic acid under the optimum conditions of hydrolysis for each source. 3. The pH optimum is 4.1 and the Michaelis constant, K(m), is 3.3x10(-4)m with purified mouse urinary beta-glucuronidase as the enzyme source acting on the NO-beta-d-glucosiduronic acid. The aglycone after extraction into chloroform was quantitatively determined spectrophotometrically at its absorption maximum (256mmu). 4. The hydrolysis was studied as a function of time and temperature. 5. From a consideration of the chemical and enzymic properties of this NO-beta-d-glucosiduronic acid it is possible to suggest its catabolism in vivo.     

Non-destructive assay systems for detection of β-glucuronidase activity in higher plants

β-glucuronidase (GUS) can be qualitatively assayed in seedlings and fully grown plants without injury or irreversible damage by short term incubations in X-gluc or by spraying 4-MUG.     

Cloning of a cDNA complementary to rat preputial gland β-glucuronidase mRNA

Messenger RNA was isolated from rat preputial glands by guanidine HCl extraction, ethanol and salt precipitation, followed by chromatography on oligo(dT) cellulose. Double-stranded cDNA was synthesized from the mRNA and inserted into the Pst 1 site of the plasmid pBR322 by the poly(dG)·poly(dC) tailing and annealing procedure. The hybrid plasmids were used to transform $\text{E. coli}$ HB101. Recombinant clones were screened for those containing cDNA inserts complementary to β-glucuronidase mRNA by a hybridization-selection procedure. One clone, containing an insert of about 1.2 kilobases, hybridized to preputial gland mRNA which, when translated $\text{in vitro}$, gave a product that migrated with the β-glucuronidase subunit on polyacrylamide gels.     

Golgi β-glucuronidase of androgen-stimulated mouse kidney

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4-7.9), in solutions of different ionic strength (0.06-0.3) and in the presence of Mg(2+), at 30 degrees C and at 20 degrees C. The values obtained (8.65-11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg(2+)-binding constants were measured spectrophotometrically and the values, 286mm(-1) and 255mm(-1) for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg(2+) concentration and ionic strength there will be little variation in equilibrium constant.     

Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

We have generated transgenic maize seed containing -glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant -glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant -glucuronidase (GUS) was compared to native -glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a K_m of 0.20 mM and a V_max of 0.29 mM MUG per hour, and the native enzyme had a K_m and V_max of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of -glucuronidase, the K_i of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.     

Characterization of a thermostable β-glucuronidase from Thermotoga maritima expressed in Arabidopsis thaliana

TmGUSI, a gene identical to that encoding a thermostable β-glucuronidase in the hyperthermophilic anaerobe Thermotoga maritima, has been synthesized using a PCR-based two-step DNA synthesis and codon optimization for plants, and expressed in both Escherichia coli and Arabidopsis thaliana. TmGUSI expressed in transformed E. coli cells exhibited maximum hydrolytic activity at 65?°C and pH 6.5 and retained more than 80% activity after incubation at 85?°C for 30?min. TmGUSI activity in transgenic A. thaliana plants containing TmGUSI was also stable over the temperature range 65-80?°C. Our data suggest that β-glucuronidase from T. maritima can serve as a useful thermostable marker in higher plants.     

Construction of a Plant Transient Expression Vector which Coexpresses the Marker β-glucuronidase

We have developed a plant transient expression vector that simultaneously expresses -glucuronidase from a Cauliflower mosaic virus promoter, and a test gene from a Figwort mosaic virus promoter. This vector, which is manipulated in E. coli, allows the testing of cell death inducing genes in plant cells. We have demonstrated the capability of this vector by expressing diphtheria toxin.     

Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

We have detected a plant β-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric β-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45°C. It is relatively resistant to proteases and insensitive to-SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by theEscherichia coli β-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay theE. coli β-glucuronidase in transgenic plants without interference from the endogenous plant activity.     

High-performance liquid chromatographic quantification of 4-methylumbelliferyl-β-d-glucuronide as a probe for human β-glucuronidase activity in tissue homogenates

An internally standardized HPLC method to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-β-d-glucuronide by human β-glucuronidase was developed. The assay allows the precise and rapid measurement of specific enzyme activity in human tissue homogenates. Without prior extraction the incubation mixture can be separated using a C₈ column followed by fluorescence detection. The assay showed good accuracy and precision with a detection limit of 20 nM and a limit of quantification of 167 nM. The suitability of the method was shown in enzyme kinetic experiments with human liver homogenates.     

β-glucuronidase as a marker for clonal analysis of tomato lateral roots

In higher plants, the root-shoot axis established during embryogenesis is extended and modified by the development of primary and lateral apical meristems. While the structure of several shoot apical meristems has been deduced by combining histological studies with clonal analysis, the application of this approach to root apical meristems has been limited by a lack of visible genetic markers. We have tested the feasibility of using a synthetic gene consisting of the maize transposable elementActivator (Ac) inserted between a 35S CaMV promoter and the coding region of a -glucuronidase (GUS) reporter gene as a means of marking cell lineages in roots. The GUS gene was activated in individual cells byAc excision, and the resulting sectors of GUS-expressing cells were detected with the histochemical stain X-Gluc. Sectors in lateral roots originated from bothAc excision in meristematic cells and from parent root sectors that bisect the founder cell population for the lateral root initial. Analysis of root tip sectors confirmed that the root cap, and root proper have separate initials. Large sectors in the body of the lateral root encompassed both cortex and vascular tissues. The number of primary initial cells predicted from the size and arrangement of the sectors observed ranged from two to four and appeared to vary between roots. We conclude that transposon-based clonal analysis using GUS expression as a genetic marker is an effective approach for deducing the functional organization of root apical meristems.     

Analysis of T-DNA-mediated translational β-glucuronidase gene fusions

Three random translational -glucuronidase (gus) gene fusions were previously obtained in Arabidopsis thaliana, using Agrobacterium-mediated transfer of a gus coding sequence without promoter and ATG initiation site. These were analysed by IPCR amplification of the sequence upstream of gus and nucleotide sequence analysis. In one instance, the gus sequence was fused, in inverse orientation, to the nos promoter sequence of a truncated tandem T-DNA copy and translated from a spurious ATG in this sequence. In the second transgenic line, the gus gene was fused to A. thaliana DNA, 27 bp downstream an ATG. In this line, a large deletion occurred at the target site of the T-DNA. In the third line, gus is fused in frame to a plant DNA sequence after the eighth codon of an open reading frame encoding a protein of 619 amino acids. This protein has significant homology with animal and plant (receptor) serine/threonine protein kinases. The twelve subdomains essential for kinase activity are conserved. The presence of a potential signal peptide and a membrane-spanning domain suggests that it may be a receptor kinase. These data confirm that plant genes can be tagged as functional translational gene fusions.     

Stimulation of hepatic microsomal β-glucuronidase by calcium

Hydrolysis of 3-methylumbelliferyl glucuronide by liver microsomal β-glucuronidase is enhanced about 2-fold by micromolar concentrations of Ca²⁺; half-maximal stimulation occurs with 0.35 μM Ca²⁺. Dissociation of the enzyme from microsomal membranes by various treatments increases basal β-glucuronidase activity and markedly decreases the sensitivity of the enzyme to Ca²⁺. Under similar conditions, the soluble lysosomal form of the enzyme is insensitive to Ca²⁺. Ca²⁺ stimulation was unaltered by addition of calmodulin inhibitors or exogenous calmodulin. Thus, interaction of cytosolic Ca²⁺ with membrane bound β-glucuronidase may modulate glucuronidation in intact hepatocytes via a novel, calmodulin-independent mechanism.     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Purification and characterization of a highly selective glycyrrhizin-hydrolyzing β-glucuronidase from Penicillium purpurogenum Li-3

A novel β-glucuronidase from filamentous fungus Penicillium purpurogenum Li-3 was purified to electrophoretic homogeneity by ultrafiltration, ammonium sulfate precipitation, DEAE-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration with an 80.7-fold increase in specific activity. The purified β-glucuronidase is a dimeric protein with an apparent molecular mass of 69.72 kDa (m/z = 69,717), determined by MALDI/TOF-MS. The optimal temperature and pH of the purified enzyme are 40 °C and 6.0, respectively. The enzyme is stable within pH 5.0–8.0, and the temperature up to 45 °C. Mg²⁺ ions enhanced the activity of the enzyme, Ca²⁺ and Al³⁺ showed no effect, while Mn²⁺, Zn²⁺, Hg²⁺ and Cu²⁺ substantially inhibited the enzymatic activity. The K_m and V_max values of the purified enzyme for glycyrrhizin (GL) were evaluated as 0.33 mM and 59.0 mmol mg^?1 min^?1, respectively. The purified enzyme displayed a highly selective glycyrrhizin-hydrolyzing property and converted GL directly to glycyrrhetic acid mono-glucuronide (GAMG), without producing byproduct glycyrrhetic acid (GA). The results suggest that the purified enzyme may have potential applications in bio-pharmaceutical and biotechnological industry.     

A thermostable β-glucuronidase obtained by directed evolution as a reporter gene in transgenic plants

A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter.     

Detection of endogenous β-glucuronidase activity in Aspergillus niger

 An endogenous β-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous β-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uidAβ-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7–8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous β-glucuronidase activity, was achieved by staining at this pH. Received : 22 March 1995/Received last revision : 17 August 1995/Accepted : 22 August 1995     

Rabbit β-glucuronidase. Subcellular distribution and immunochemical properties

1. The subcellular distribution of beta-glucuronidase and other hydrolases in rabbit liver was investigated. beta-Glucuronidase was found in both microsomal and lysosomal fractions. 2. Multiple forms of beta-glucuronidase were present in extracts of microsomal and lysosomal fractions. All forms were common to both fractions. 3. A specific antiserum against beta-glucuronidase was raised, and characterized by immunoprecipitation and affinity-chromatography procedures. 4. The immunological identity of the multiple forms in the pure beta-glucuronidase preparation, and the immunological identity of the beta-glucuronidase complement of lysosomal extracts with that of microsomal extracts, were demonstrated by means of the antiserum. The presence of inactive enzyme in various enzyme preparations was shown.