Microtiter plate cellular assay for human steroid sulfatase with fluorescence readout

Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay.     

Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.     

SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies

Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies.     

Immunohistochemical analysis of steroid sulfatase in human tissues

Steroid sulfatase (EC 3.1.6.2) is an enzyme that removes the sulfate group from 3β-hydroxysteroid sulfates. This enzyme is best known for its role in estrogen production via the fetal adrenal–placental pathway during pregnancy; however, it also has important functions in other physiological and pathological steroid pathways. The objective of this study was to examine the distribution of steroid sulfatase in normal human tissues and in breast cancers using immunohistochemistry, employing a newly developed steroid sulfatase antibody. A rabbit polyclonal antiserum was generated against a peptide representing a conserved region of the steroid sulfatase protein. In Western blotting experiments using human placental microsomes, this antiserum crossreacted with a 65 kDa protein, the reported size of steroid sulfatase. The antiserum also crossreacted with single protein bands in Western blots of microsomes from two human breast cancer cell lines (MDA-MB-231 and MCF-7) and from rat liver; however, there were some size differences in the immunoreactive bands among tissues. The steroid sulfatase antibody was used in immunohistochemical analyses of individual human tissue slides as well as a human tissue microarray. For single tissues, human placenta and liver showed strong positive staining against the steroid sulfatase antibody. ER+/PR+ breast cancers also showed relatively strong levels of steroid sulfatase immunoreactivity. Normal human breast showed moderate levels of steroid sulfatase immunoreactivity, while ER−/PR− breast cancer showed weak immunoreactivity. This confirms previous reports that steroid sulfatase is higher in hormone-dependent breast cancers. For the tissue microarray, most tissues showed some detectable level of steroid sulfatase immunoreactivity, but there were considerable differences among tissues, with skin, liver and lymph nodes having the highest immunoreactivity and brain tissues having the lowest. These data reveal the utility of immunohistochemistry in evaluation of steroid sulfatase activity among tissues. The newly developed antibody should be useful in studies of both humans and rats.     

Complementation of multiple sulfatase deficiency in somatic cell hybrids

Multiple sulfatase deficiency (MSD) is an inherited disorder characterized by deficient activity of seven different sulfatases. Genetic complementation for steroid sulfatase (STS), arylsulfatase A, and N-acetylgalactosamine 6-SO4 sulfatase was demonstrated in somatic cell hybrids between MSD fibroblasts and mouse cells ( LA9 ) or Chinese hamster cells ( CHW ). In an electrophoretic system that separates human and rodent STS isozymes, enzyme from hybrids migrated as human enzyme. We concluded that the rodent cell complemented the MSD deficiency and allowed normal expression of the STS structural gene. Some MSD- LA9 hybrids showed significant levels of human arylsulfatase A activity, as shown by the immunoprecipitation of active enzyme by human-specific antiserum. Complementation was also suggested for N-acetylgalactosamine 6- sulfatate sulfatase (GalNAc-6S sulfatase) in several MSD- LA9 hybrids by the demonstration of a significant increase in activity (10-fold) over that of the GalNAc-6S sulfatase-deficient parental mouse and MSD cells. Thus, it was possible to demonstrate complementation for more than one sulfatase in a single MSD-rodent hybrid. Normal levels of sulfatase activity in hybrids indicate that the sulfatase structural genes are intact in MSD cells.     

Circular dichroism spectroscopic studies of native and turnover-modified sulfatase A

In a recent communication, A. Waheed and R. L. Van Etten (1979, Arch. Biochem. Biophys. 195, 248) showed that the sulfatase A of rabbit liver (arylsulfate sulfohydrolase, EC 3.1.6.1), which becomes inactivated as it catalyzes the hydrolysis of substrate, covalently incorporates ³⁵S from nitrocatechol [³⁵S]sulfate during this reaction and at the same time loses most of its secondary structure in solution. Circular dichroism spectra presented here for the native and turnover-modified forms of the sulfatase A of ox liver indicate no difference in the region of the spectrum below 240 nm associated with polypeptide backbone contributions or in the region from 350-250 nm associated with the side-chain chromophore transitions. In addition no differences were evident for the two forms of the ox liver enzyme from ultraviolet absorbance and fluorescence spectroscopy measurements. From these data we conclude that, in contrast to the situation with the rabbit enzyme, there is no loss of secondary structure associated with inactivation of ox liver sulfatase A in the course of enzymic catalysis.     

N-acetylgalactosamine-6-sulfate sulfatase in human placenta: purification and characteristics.

N-Acetylgalactosamine-6-sulfate sulfatase from human placenta was purified 33,600-fold using beta-N-acetyl-D-galactosamine 6-sulfate-(1----4)-beta-D-glucuronic acid-(1----3)-N-acetyl-D-[3H]galactosaminitol 6-sulfate as the substrate. This enzyme is an oligomer with a molecular mass of 120 kDa and consists of polypeptides of 40 and 15 kDa. The 15 kDa polypeptide is a glycoprotein. This purified protein has activities of N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. Rabbit antiserum was raised against the purified protein. The antibody titrated N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. The size of the precursor of the enzyme is 60 kDa, as determined by cell-free translation. The optimal pH values of the N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase activities are pH 3.8-4.0, and the Kms are 8 and 13 microM, respectively. Sulfate and phosphate ions are potent competitive inhibitors for the enzyme and their inhibition constants are 35 and 200 microM, respectively. Cross-reactive materials of 40 and 15 kDa were detected by immunoblot analysis, in the placenta, liver, and normal fibroblasts, but not in fibroblasts from a patient with Morquio disease.     

New assay for steroid sulfatase (EC 3.1.6.2) and its application for studies of human placental and skin sulfatase

A new, simple, fast and highly practicable sulfatase assay and its application is described. Sterol sulfatase sulfohydrolase (EC 3.1.6.2) activity is determined by a two-phase scintillation technique separating the unreacted [4-14C]dehydroepiandrosterone sulfate from carbon-14-labeled products. The principle of the separation relies on the limited emulsifying capacity of the dioxane-based scintillation solution for water and the different partition of dehydroepiandrosterone sulfate and sulfate-free steroid products between the scintillation fluid and the aqueous phase as recently applied for determination of aromatase activity [1]. [7-3H]Dehydroepiandrosterone sulfate can also be used as a substrate for this assay. This test was applied to studies of microsomal sulfatase prepared from human term placenta and to the detection of sulfatase activity in human skin biopsies. Using placental microsomes, the Km of dehydroepiandrosterone sulfate was determined to be 5.0 X 10(7)M. Sulfatase activity in frozen scrotal skin was found to be 2-3 fold than with vaginal skin. Using an incubation time of 24h/skin sulfatase can be detected in biopsies as small as 2.5 mm2. The sulfatase assay can be applied for routine detection of human placental sulfatase deficiency and, furthermore, the application of this assay has to be demonstrated for the analysis of sulfatase activity in patients with congenital ichthyosis (X-chromosomal, recessive type).     

Dosage of the Sts gene in the mouse.

In this study we compared steroid sulfatase levels in XO, XX, and XY mice and carried out a clonal analysis in fibroblast cell cultures from mice heterozygous for the steroid sulfatase deficiency gene and heterozygous at the X-linked electrophoretic phosphoglycerate kinase locus. The combined results indicate that the murine steroid sulfatase locus is not dosage compensated and is not subject to X-inactivation. With respect to X-inactivation, it behaves in a somewhat different way from the closely linked sex-reversed gene and the human steroid sulfatase locus.     

Plant cell culture monitoring using an in situ multiwavelength fluorescence probe

A multiwavelength fluorescence probe is proposed for in situ monitoring of Eschscholtzia californica and Catharanthus roseus plant cell cultures. The potential of the probe as a tool for real-time estimation of biomass and production in secondary metabolites has been studied. The probe excitation range is 270-550 nm and the emission range is 310-590 nm, with a step of 20 nm for both excitation and emission filters. Many endogenous fluorophores such as NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine and tryptophan, and fluorescent secondary metabolites were analyzed simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an adequate signal for estimating cells activity. Riboflavins fluorescence signal (450/530 nm) followed C. roseus cell concentration both for the growth phase and after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered with NAD(P)H signal during the production phase. For C. roseus, tryptophan, tryptamine, ajmalicine and serpentine were monitored by the probe. For E. californica, fluorescence from alkaloids overlapped with riboflavins preventing from using the probe to follow cell growth but global alkaloids production could be followed using the probe.     

Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin

In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the fluorophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at beta 93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.     

Detection of pre-mRNA splicing in vitro by an RNA-templated fluorogenic reaction

RNA splicing is an important target for basic research of disease mechanisms and for drug discovery. Here, we report a new method for analysis of the in vitro RNA splicing process that produces fluorescence using a reduction-triggered fluorescence (RETF) probe. The fluorescence signal is produced only when the two probes bind side-by-side with a specific RNA target. Precursor messenger RNA and mature messenger RNA originating from the chicken δ-crystallin (CDC) gene were successfully discriminated in solution using an RETF probe with the assistance of helper oligonucleotide strands. Also, we successfully applied RETF probes to the detection of emerging mature mRNA in an in vitro splicing process.     

Potential-dependent phase partitioning of fluorescent hydrophobic ions in phospholipid vesicles

Summary Fluorescent, dansyl derivatives of triphenylalkylphosphonium ions have been synthesized and exhibit fluorescence intensities in small sonicated phospholipid vesicles that are dependent upon transmembrane potentials. The voltage-dependent fluorescence changes are a result of changes in quantum yield that accompany a voltage-dependent phase partitioning of the probe. This phase partitioning is easily quantitated by calibrating the intensities of totally membrane-associated and aqueous probe. The voltage-dependence is well accounted for by a simple thermodynamic model and allows an estimation of potentials from fluorescence intensities in small vesicle systems.     

Inhibition of steryl sulfatase activity in LNCaP human prostate cancer cells

The enzyme steryl sulfatase may help support the growth of hormone-dependent tumors, including prostate cancers, by facilitating the conversion of circulating precursor steroids to active hormones. We sought to determine the presence of steryl sulfatase activity in the androgen-dependent human prostate cancer cell line LNCaP, and to determine if this activity was inhibited by known steryl sulfatase inhibitors. Intact LNCaP cultures had steryl sulfatase activity, as determined by conversion of [3H]estrone sulfate (E(1)S) to unconjugated steroids. The level of steryl sulfatase activity was relatively low (4.6 pmol/18 h/million cells) compared to MDA-MB-231 breast cancer cells (284.0 pmol/18 h/million cells). The observed activity in both cell lines was blocked by addition of 1 microM estrone sulfamate (EMATE), an active-site-directed, steroidal inhibitor of steryl sulfatase. Steryl sulfatase activity was also inhibited by Danazol, and by (p-O-sulfamoyl)-tetradecanoyl tyramine (C2-14), a non-steroidal inhibitor. Microsomes prepared from LNCaP cultures also showed steryl sulfatase activity, as determined by hydrolysis of [3H]E(1)S and [3H]dehydroepiandrosterone sulfate (DHEAS) to unconjugated forms. LNCaP and MDA-MB-231 microsomes both hydrolyzed E(1)S about two times faster than DHEAS. Hydrolysis of E(1)S in LNCaP and MDA-MB-231 microsomes was blocked by steryl sulfatase inhibitors with the following relative potencies: EMATE>C2-14>Danazol. These data demonstrate that LNCaP prostate cancer cells contain a steryl sulfatase with properties similar to that found in human breast cancer cells, and that the activity of this enzyme can be blocked by known steryl sulfatase inhibitors. Steryl sulfatase inhibitors may be useful as an adjuvant to androgen deprivation therapy for prostate cancer.     

Steroid sulfatase. Biosynthesis and processing in normal and mutant fibroblasts

Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis of this enzyme in human skin fibroblasts. Steroid sulfatase is synthesized as a membrane-bound Mr-63 500 polypeptide with asparagine-linked oligosaccharide chains. Within 2 days, newly synthesized steroid sulfatase is processed to a mature Mr-61 000 form. The decrease in size is due to processing of the oligosaccharide chains, which are cleavable by endoglucosaminidase H in both the early and the mature form of steroid sulfatase. The processing involves mannosidase(s) sensitive to 1-deoxy-manno-nojirimycin. The half-life of the steroid sulfatase polypeptides is 4 days. Synthesis of steroid-sulfatase-related polypeptides and steroid sulfatase activity were not detectable in fibroblasts from four patients with X-linked ichthyosis.     

A dual fluorochrome probe for imaging proteases

Near-infrared fluorescence (NIRF) optical probes have been able to provide a noninvasive assessment of enzyme activity for a number of different enzymes and types of pathology. Here we describe a dual fluorochrome enzyme-activatable probe featuring one NIRF fluorochrome that is activated by protease activity and a second fluorochrome that is protease resistant and serves as an internal standard. The probe was prepared by attaching Cy7 directly to an amino-CLIO, an amine functional cross-linked iron oxide (CLIO) nanoparticle carrier, in a protease resistant manner. Cy5.5 was attached to a protease sensitive polyarginine peptide spacer, also attached to amino-CLIO. In vitro and in vivo the ratio of the Cy5.5 to Cy7 fluorescence was increased by protease, reflecting the increase in Cy5.5 fluorescence by protease in the vicinity of the probe. In vitro and in vivo the absolute values of the Cy5.5 and Cy7 fluorescence reflected lesion size and the distance of lesions from the surface, while the ratio of Cy5.5 to Cy7 fluorescence obtained was constant and independent of lesion size and depth. The dual fluorochrome probe, and related dual wavelength imaging method, represents a novel approach for imaging protease activity in vivo.     

Scanning near-field fluorescence resonance energy transfer microscopy

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.     

A multiwavelength fluorescence probe: is one probe capable for on-line monitoring of recombinant protein production and biomass activity?

Monitoring cell culture performance requires maximizing the number and the quality of measured parameters and in situ 2D fluorescence spectroscopy could allow intensification of simultaneous data acquisition. The use of a multiwavelength fluorescence probe is proposed for monitoring GFP-producing cultures in bioreactor. The yeast Pichia pastoris and NSO mammalian cells were studied as model systems. Tryptophan, NAD(P)H and riboflavins (riboflavin, FMN, FAD) signals were effective for on-line yeast biomass estimation during the growth phase. During the GFP production phase, in situ measurements of the GFP concentration from the fluorescence probe were well correlated with off-line analyses. Tryptophan and NAD(P)H signals diverged from that of biomass during GFP production. With NSO mammalian cells, results showed that the culture parameters have to be optimized for the use of a fluorescence probe. The use of serum and phenol-red interfered with NAD(P)H and riboflavins fluorescence signals. Nevertheless, it appears that a multiwavelength probe could be useful for culture monitoring of biomass, cell activity and recombinant protein expression in an optimized culture medium.     

Steroid sulfatase activities in human leukocytes: biochemical and clinical aspects.

Steroid sulfatase is a membrane-bound microsomal enzyme, present in various tissues. In this report, data on sulfatase activity in peripheral blood leukocytes isolated from normal women and the characterization of its enzyme are studied. In addition, sulfatase activities in placental sulfatase deficiency (PSD) and ichthyosis patients including ichthyosis vulgaris (IV) and recessive X-linked ichthyosis (RXLI) were analysed and were compared with normal subjects. Steroid sulfatase activity was measured by using tritium labeled steroid sulfate as the reaction substrate. It is demonstrated that human leukocytes contain a sulfatase activity for pregnenolone sulfate (P5-S), dehydroepiandrosterone sulfate (DHA-S) and estrone sulfate (E1-S) respectively. This enzyme has a greatest affinity for P5-S, but the activity for E1-S was the highest among the three substrates. The steroid sulfatase activity in female leukocytes is significantly stronger than that in normal males (p less than 0.001) as determined by the cleavage of DHA-S. Sulfatase in leukocytes obtained from the PSD babies and RXLI patients had lower sensitivity. In the case of the mother affected with PSD, the activity was less than half of that in normal men (p less than 0.001) and the levels did not overlap with that in normal women. In patients with IV, the activities were in the normal ranges for both males and females. The measurement of leukocyte sulfatase activity would be a clinically useful tool for the diagnosis of PSD carriers and pedigree analysis.     

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.