Detection, production, modification, and application of arylsulfatases.

Arylsulfatase is a subset of sulfatase which catalyzes the hydrolysis of aryl sulfate ester. Arylsulfatase is widely distributed among microorganisms, mammals and green algae, but the arylsulfatase-encoding gene has not yet been found in the genomes of higher plants so far. Arylsulfatase plays an important role in the sulfur flows between nature and organisms. In this review, we present the maturation and catalytic mechanism of arylsulfatase, and the recent literature on the expression and production of arylsulfatase in wild-type and engineered microorganisms, as well as the modification of arylsulfatase by genetic engineering are summarized. We focus on arylsulfatases from microbial origin and give an overview of different assays and substrates used to determine the arylsulfatase activity. Furthermore, the researches about arylsulfatase application on the field of agar desulfation, soil sulfur cycle and soil evaluation are also discussed. Finally, the perspectives concerning the future research on arylsulfatase are prospected.

Single Systemic Administration of a Gene Therapy Leading to Disease Treatment in Metachromatic Leukodystrophy Arsa Knock-Out Mice.

Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.

Transcription level and phylogeny analyses of Chlamydomonas reinhardtii arylsulfatases.

Sulfur is a required macroelement for all organisms, and sulfate deficiency causes growth and developmental defects. Arylsulfatases (ARS) hydrolyze sulfate from sulfate esters and make sulfate bioavailable for plant uptake. These enzymes are found in microorganisms and animals; however, plant genomes do not encode any ARS gene. Our database searches found nineteen ARS genes in the genome of Chlamydomonas reinhardtii. Among these, ARS1 and ARS2 were studied in the literature; however, the remaining seventeen gene models were not studied. Our results show that putative polypeptide sequences of the ARS gene models all have the sulfatase domain and sulfatase motifs found in known ARSs. Phylogenetic analyses show that C. reinhardtii proteins are in close branches with Volvox carterii proteins while they were clustered in a separate group from Homo sapiens and bacterial species (Pseudomonas aeruginosa and Rhodopirellula baltica SH1), except human Sulf1, Sulf2, and GNS are clustered with algal ARSs. RT-PCR analyses showed that transcription of ARS6, ARS7, ARS11, ARS12, ARS13, ARS17, and ARS19 increased under sulfate deficiency. However, this increase was not as high as the increase seen in ARS2. Since plant genomes do not encode any ARS gene, our results highlight the importance of microbial ARS genes.

Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni.

BACKGROUND: Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176. RESULTS: The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM). CONCLUSIONS: This is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community.

Engineering Pseudomonas aeruginosa arylsulfatase for hydrolysis of α-configured steroid sulfates.

Steroid sulfate esters are important metabolites for anti-doping efforts in sports, pathology and research. Analysis of these metabolites is facilitated by hydrolysis using either acid or enzymatic catalysis. Although enzymatic hydrolysis is preferred for operating at neutral pH, no known enzyme is capable of hydrolyzing all steroid sulfate metabolites. Pseudomonas aeruginosa arylsulfatase (PaS) is ideal for the hydrolysis of ß-configured steroid sulfates but like other known class I sulfatases it is inefficient at hydrolyzing α-configured steroid sulfates. We have used directed evolution with liquid chromatography mass spectrometry screening to find variants capable of hydrolyzing a α-configured steroid sulfate: etiocholanolone sulfate (ECS). After targeting two regions of PaS, four residues were identified and optimized to yield a final variant with a total of seven mutations (DRN-PaS) capable of hydrolyzing ECS ~80 times faster than the best PaS variant previously available. This DRN-PaS also shows improved activity for other α-configured steroid sulfates. Simultaneous mutagenesis was essential to obtain DRN-PaS due to complementarity between targeted residues.

Evidence for high breakpoint variability in 46, XX, SRY-positive testicular disorder and frequent ARSE deletion that may be associated with short stature.

BACKGROUND: The translocation of SRY onto one of the two X chromosomes results in a 46,XX testicular disorder of sex development; this is supposedly because of non-allelic homologous recombination between the protein kinase X gene (PRKX) and the inverted protein kinase Y pseudogene (PRKY). Although 46,XX SRY-positive men are infertile, the literature data indicate that some of these individuals are of short stature (relative to the general population). We sought to determine whether short stature was linked to additional, more complex chromosomal rearrangements. METHODS: Twelve laboratories gathered detailed clinical, anthropomorphic, cytogenetic and genetic data (including chromosome microarray data) on patients with 46,XX SRY-positive male syndrome. RESULTS: SRY was present (suggesting a der(X)t(X;Y)) in 34 of the 38 cases (89.5%). When considering only the 20 patients with chromosome microarray data, we identified several chromosomal rearrangements and breakpoints, especially on the X chromosome. In the five cases for whom the X chromosome breakpoint was located in the pseudoautosomal region, there was partial duplication of the derivate X chromosome. In contrast, in the 15 cases for whom the breakpoint was located downstream of the pseudoautosomal region, part of the derivate X chromosome had been deleted (included the arylsulfatase E [ARSE] gene in 11 patients). For patients with versus without ARSE deletion, the mean height was, respectively, 167.7 ± 4.5 and 173.1 ± 4.0 cm; this difference was not statistically significant (p = 0.1005). CONCLUSION: Although 46,XX SRY-positive male syndromes were mainly because of imbalanced crossover between the X and Y chromosome during meiosis, the breakpoints differed markedly from one patient to another (especially on the X chromosome); this suggests the presence of a replication-based mechanism for recombination between non-homologous sequences. In some patients, the translocation of SRY to the X chromosome was associated with ARSE gene deletion, which might have led to short stature. With a view to explaining this disorder of sex development, whole exome sequencing could be suggested for SRY-negative patients.

Characterization and substrate-accelerated thermal inactivation kinetics of a new serine-type arylsulfatase.

Arylsulfatase is useful in industrial agar processing by removing sulfate groups. A full-length arylsulfatase gene, designated ArySMA1, was obtained from marine bacteria Serratia sp. SM1. The ArySMA1 gene encoded a novel serine-type arylsulfatase and the enzymatic properties were characterized. The enzyme presented notable capacity of removing sulfate groups from natural algae substrates. Kinetic study suggested that the microscopic thermal inactivation rate of ArySMA1 in free form was smaller than that of the enzyme-substrate complex. The presence of substrate could unexpectedly accelerate ArySMA1 to deactivate at high temperature. Such phenomenon was opposite to many findings that substrate could stabilize enzymes against heat. Molecular dynamics simulation and ANS fluorescent assay indicated the substrate led the hydrophobic regions of the active site more flexible and the sulfate group of the substrate could retard the processivity of ArySMA1 catalysis. This study provides guidance for agar desulfation and down-stream processing industry.

ARSD, a novel ERα downstream target gene, inhibits proliferation and migration of breast cancer cells via activating Hippo/YAP pathway.

Advanced breast cancer (BC), especially basal like triple-negative BC (TNBC), is a highly malignant tumor without viable treatment option, highlighting the urgent need to seek novel therapeutic targets. Arylsulfatase D (ARSD), localized at Xp22.3, is a female-biased gene due to its escaping from X chromosome inactivation (XCI). Unfortunately, no systematic investigation of ARSD on BC has been reported. In this study, we observed that ARSD expression was positively related to ERα status either in BC cells or tissue specimens, which were associated with good prognosis. Furthermore, we found a set of hormone-responsive lineage-specific transcription factors, FOXA1, GATA3, ERα, directly drove high expression of ARSD through chromatin looping in luminal subtype BC cells. Opposingly, ARSD still subjected to XCI in TNBC cells mediated by Xist, CpG islands methylation, and inhibitory histone modification. Unexpectedly, we also found that ectopic ARSD overexpression could inhibit proliferation and migration of TNBC cells by activating Hippo/YAP pathway, indicating that ARSD may be a molecule brake on ERα signaling pathway, which restricted ERα to be an uncontrolled active status. Combined with other peoples' researches that Hippo signaling maintained ER expression and ER + BC growth, we believed that there should exist a regulative feedback loop formation among ERα, ARSD, and Hippo/YAP pathway. Collectively, our findings will help filling the knowledge gap about the influence of ARSD on BC and providing evidence that ARSD may serve as a potential marker to predict prognosis and as a therapeutic target.

Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG).

The lysosomal degradation of heparan sulfate is mediated by the concerted action of nine different enzymes. Within this degradation pathway, Arylsulfatase G (ARSG) is critical for removing 3-O-sulfate from glucosamine, and mutations in ARSG are causative for Usher syndrome type IV. We developed a specific ARSG enzyme assay using sulfated monosaccharide substrates, which reflect derivatives of its natural substrates. These sulfated compounds were incubated with ARSG, and resulting products were analyzed by reversed-phase HPLC after chemical addition of the fluorescent dyes 2-aminoacridone or 2-aminobenzoic acid, respectively. We applied the assay to further characterize ARSG regarding its hydrolytic specificity against 3-O-sulfated monosaccharides containing additional sulfate-groups and N-acetylation. The application of recombinant ARSG and cells overexpressing ARSG as well as isolated lysosomes from wild-type and Arsg knockout mice validated the utility of our assay. We further exploited the assay to determine the sequential action of the different sulfatases involved in the lysosomal catabolism of 3-O-sulfated glucosamine residues of heparan sulfate. Our results confirm and extend the characterization of the substrate specificity of ARSG and help to determine the sequential order of the lysosomal catabolic breakdown of (3-O-)sulfated heparan sulfate.

Expanding the clinical phenotype in patients with disease causing variants associated with atypical Usher syndrome.

Atypical Usher syndrome (USH) is poorly defined with a broad clinical spectrum. Here, we characterize the clinical phenotype of disease caused by variants in CEP78, CEP250, ARSG, and ABHD12.Chart review evaluating demographic, clinical, imaging, and genetic findings of 19 patients from 18 families with a clinical diagnosis of retinal disease and confirmed disease-causing variants in CEP78, CEP250, ARSG, or ABHD12.CEP78-related disease included sensorineural hearing loss (SNHL) in 6/7 patients and demonstrated a broad phenotypic spectrum including: vascular attenuation, pallor of the optic disc, intraretinal pigment, retinal pigment epithelium mottling, areas of mid-peripheral hypo-autofluorescence, outer retinal atrophy, mild pigmentary changes in the macula, foveal hypo-autofluorescence, and granularity of the ellipsoid zone. Nonsense and frameshift variants in CEP250 showed mild retinal disease with progressive, non-congenital SNHL. ARSG variants resulted in a characteristic pericentral pattern of hypo-autofluorescence with one patient reporting non-congenital SNHL. ABHD12-related disease showed rod-cone dystrophy with macular involvement, early and severe decreased best corrected visual acuity, and non-congenital SNHL ranging from unreported to severe.This study serves to expand the clinical phenotypes of atypical USH. Given the variable findings, atypical USH should be considered in patients with peripheral and macular retinal disease even without the typical RP phenotype especially when SNHL is noted. Additionally, genetic screening may be useful in patients who have clinical symptoms and retinal findings even in the absence of known SNHL given the variability of atypical USH.

Multimodal imaging and genetic findings in a case of ARSG-related atypical Usher syndrome.

Background: Atypical Usher syndrome has recently been associated with arylsulfatase G (ARSG) variants. In these cases, characteristic findings include progressive sensorineural hearing loss (SNHL) without vestibular involvement and ring-shaped late-onset retinitis pigmentosa (RP).Materials and Methods: One patient with atypical Usher syndrome and a novel homozygous ARSG variant was included in this study. The patient underwent a comprehensive ophthalmic examination, including multimodal imaging and genetic testing.Results: A 60-year-old male of Persian decent presented to our clinic with a history of 20 years of progressive SNHL, and 10 years of progressive peripheral vision loss and pigmentary retinopathy. Consistent with previous reports of ARSG-related atypical Usher syndrome, fundus examination revealed ring-shaped retinal hyperpigmentation and fundus autofluorescence (FAF) demonstrated a six-zone pattern of autofluorescence. Optical coherence tomography (OCT) showed extensive cystoid spaces concentrated in the ganglion cell layer. Widefield OCT angiography at the level of the choriocapillaris showed signs of atrophy that corresponded to the FAF hypofluorescent zone. The patient was homozygous for a novel ARSG variant c. 1270 C > T, p. Arg424Cys.Conclusion: We report a novel ARSG variant in a case of atypical Usher syndrome and describe multimodal imaging findings that further characterize the effect of ARSG in the pathogenesis of atypical Usher syndrome.

New clinical and molecular evidence linking mutations in ARSG to Usher syndrome type IV.

In murine and canine animal models, mutations in the Arylsulfatase G gene (ARSG) cause a particular lysosomal storage disorder characterized by neurological phenotypes. Recently, two variants in the same gene were found to be associated with an atypical form of Usher syndrome in humans, leading to visual and auditory impairment without the involvement of the central nervous system. In this study, we identified three novel pathogenic variants in ARSG, which segregated recessively with the disease in two families from Portugal. The probands were affected with retinitis pigmentosa and sensorineural hearing loss, generally with an onset of symptoms in their fourth decade of life. Functional experiments showed that these pathogenic variants abolish the sulfatase activity of the Arylsulfatase G enzyme and impede the appropriate lysosomal localization of the protein product, which appears to be retained in the endoplasmic reticulum. Our data enable to definitely confirm that different biallelic variants in ARSG cause a specific deaf-blindness syndrome, by abolishing the activity of the enzyme it encodes.

Evaluation of Arylsulfatase D (ARSD) and long noncoding RNA ARSD-AS1 gene expression in breast cancer patients and their association with oncogenic transcription factors.

PURPOSE: Long non-coding RNAs (LncRNAs) are thought as tumorigenic factors in cancer progression. We investigated the clinical significance of arylsulfatase D (ARSD) and ARSD antisense in breast cancer patients. METHODS: Eighty breast cancer tumors were obtained from the Tumor Bank of Cancer Institute, Imam Khomeini Hospital. The expression level of ARSD and ARSD-AS1 were examined in breast tumors in comparison to the margin of normal tissues using quantitative real-time PCR. Demographic information and the clinicopathologic characteristics including tumor grade, presence of cell receptors, lymph node and vascular invasion were also evaluated. Bioinformatics databases were used for identification of ARSD and ARSD-AS1 molecular targets and their association with cancer. RESULTS: Significant up-regulation of ARSD was observed in tumor tissues in comparison with its antisense (p<0.05). Both ARSD and ARSD-AS1 expression in tumor specimens were notably lower than those in adjacent normal tissue. High expression of ARSD was associated to lower tumor grade (p<0.05). Bioinformatics results revealed the interaction of ARSD with STS and SUMF1 proteins was attributed to the inhibiting of sulfates activity. Also, ARSD co-expressed genes were associated with oncogenic transcription factors, MAF and GATA. TP53 transcription factor site was identified as a target of ARSD-AS1 mRNA. The interaction of this antisense with microRNA (miR-618) could explain its participation in tumor cell proliferation. CONCLUSION: Low expression of ARSD was associated with higher tumor grade. The evidence from this study enhance our understanding of ARSD and ARSD-AS1 function in cancer gene therapy. Accordingly, they could be introduced as great potential targets for breast cancer treatment.

Arylsulfatase K inactivation causes mucopolysaccharidosis due to deficient glucuronate desulfation of heparan and chondroitin sulfate.

Mucopolysaccharidoses comprise a group of rare metabolic diseases, in which the lysosomal degradation of glycosaminoglycans (GAGs) is impaired due to genetically inherited defects of lysosomal enzymes involved in GAG catabolism. The resulting intralysosomal accumulation of GAG-derived metabolites consequently manifests in neurological symptoms and also peripheral abnormalities in various tissues like liver, kidney, spleen and bone. As each GAG consists of differently sulfated disaccharide units, it needs a specific, but also partly overlapping set of lysosomal enzymes to accomplish their complete degradation. Recently, we identified and characterized the lysosomal enzyme arylsulfatase K (Arsk) exhibiting glucuronate-2-sulfatase activity as needed for the degradation of heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS). In the present study, we investigated the physiological relevance of Arsk by means of a constitutive Arsk knockout mouse model. A complete lack of glucuronate desulfation was demonstrated by a specific enzyme activity assay. Arsk-deficient mice show, in an organ-specific manner, a moderate accumulation of HS and CS metabolites characterized by 2-O-sulfated glucuronate moieties at their non-reducing ends. Pathophysiological studies reflect a rather mild phenotype including behavioral changes. Interestingly, no prominent lysosomal storage pathology like bone abnormalities were detected. Our results from the Arsk mouse model suggest a new although mild form of mucopolysacharidose (MPS), which we designate MPS type IIB.

A Possible Role for Arylsulfatase G in Dermatan Sulfate Metabolism.

Perturbations of glycosaminoglycan metabolism lead to mucopolysaccharidoses (MPS)-lysosomal storage diseases. One type of MPS (type VI) is associated with a deficiency of arylsulfatase B (ARSB), for which we previously established a cellular model using pulmonary artery endothelial cells with a silenced ARSB gene. Here, we explored the effects of silencing the ARSB gene on the growth of human pulmonary artery smooth muscle cells in the presence of different concentrations of dermatan sulfate (DS). The viability of pulmonary artery smooth muscle cells with a silenced ARSB gene was stimulated by the dermatan sulfate. In contrast, the growth of pulmonary artery endothelial cells was not affected. As shown by microarray analysis, the expression of the arylsulfatase G (ARSG) in pulmonary artery smooth muscle cells increased after silencing the arylsulfatase B gene, but the expression of genes encoding other enzymes involved in the degradation of dermatan sulfate did not. The active site of arylsulfatase G closely resembles that of arylsulfatase B, as shown by molecular modeling. Together, these results lead us to propose that arylsulfatase G can take part in DS degradation; therefore, it can affect the functioning of the cells with a silenced arylsulfatase B gene.

A mutant of Pseudoalteromonas carrageenovora arylsulfatase with enhanced enzyme activity and its potential application in improvement of the agar quality.

Enzymatic desulfation using arylsulfatase provides an attractive approach to improve agar quality. We have previously characterized a functional arylsulfatase from Pseudoalteromonas carrageenovora. To further improve its enzymatic performance, we isolated a mutant arylsulfatase of K253Q with improved enzyme activity from a random mutant library. Compared to wild-type arylsulfatase (WT), K253Q showed 33% increase in enzyme activity, with optimal temperature and pH of 55 °C and 8.0, respectively. K253Q demonstrated better substrate binding ability with lower Km value. Structure analysis indicated that a combination of the additional hydrogen bond and the enhanced substrate binding affinity could account for the improved enzyme activity of K253Q. K253Q exhibited about 54% sulfate removal against agar, resulting in additional 8% increase in 3,6-AG content and 20% increase in gel strength compared to WT. Scanning electron microscopy showed that K253Q treatment led to a stronger crosslinking structure of agar.

Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Brønsted coefficient (ßleaving groupobs-Enz = -0.33) than the uncatalyzed reaction (ßleaving groupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. 15N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δßleaving groupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

Lack of Association of the rs11655081 ARSG Gene with Blepharospasm.

Blepharospasm (BSP) is a sub-phenotype of focal dystonia. A few genetic risk factors are considered to be implicated in the risk of developing BSP. There is recent evidence, based on results from GWAS and meta-analyses, to suggest that arylsulfatase G (ARSG), and more specifically rs11655081, is implicated in focal dystonia. The aim of the present study was to evaluate the effect of rs11655081 ARSG on BSP. A Greek cohort, which consisted of 206 BSP patients and an equal number of healthy controls, was genotyped for rs11655081. Only a marginal trend for the association between rs11655081 and the risk of BSP was found in the over-dominant model of inheritance [odds ratio, OR (95% confidence interval, CI): 0.64 (0.38-1.07), p = 0.088]. It is rather unlikely that rs11655081 across ARSG is a major genetic risk contributor for BSP.

Gene Expression Analysis by Arylsulfatase Assays in the Green Alga Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a single-celled green alga, is a powerful microbial experimental system for understanding gene function. As a consequence of a high-quality genome sequence, community-wide efforts for gene model refinement and annotation, resources for strain collections and robust molecular techniques, research with this organism has significantly expanded in the past few decades. In two companion chapters, we outline colorimetric and fluorescence-based methodologies for genetic reporter systems in Chlamydomonas, which can be used to investigate and delineate gene expression and regulatory mechanisms. Here, we describe protocols for arylsulfatase activity assays using ARS2, activity of which can be measured either quantitatively or qualitatively, and in low (individual sample) or high (96-well format) throughput.

A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity.

Kluyveromyces lactis is a common fungal microorganism used for the production of enzyme preparations such as ß-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of ß-galactosidase preparations produced from K. lactis, an unwanted side activity could be the presence of arylsulfatase (EC 3.1.6.1). Due to the action of arylsulfatase, an unpleasant "cowshed-like" off-flavor would occur in the final product. The best choice to avoid this is to use a yeast strain without this activity. Interestingly, we found that certain natural K. lactis strains express arylsulfatases, which only differ in one amino acid at position 139. The result of this difference is that K. lactis DSM 70799 (expressing R139 variant) shows no arylsulfatase activity, unlike K. lactis GG799 (expressing S139 variant). After recombinant production of both variants in Escherichia coli, the R139 variant remains inactive, whereas the S139 variant showed full activity. Mass spectrometric analyses showed that the important posttranslational modification of C56 to formylglycine was not found in the R139 variant. By contrast, the C56 residue of the S139 variant was modified. We further investigated the packing and secondary structure of the arylsulfatase variants using optical spectroscopy, including fluorescence and circular dichroism. We found out that the inactive R139 variant exhibits a different structure regarding folding and packing compared to the active S139 variant. The importance of the amino acid residue 139 was documented further by the construction of 18 more variants, whereof only ten showed activity but always reduced compared to the native S139 variant.