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

Abstract

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.

Keywords: desulfation; glycosaminoglycan degradation; lysosomal storage disorders; lysosomal sulfatases; mucopolysaccharidosis.

Figure 1.. Generation and validation of Arsk-deficient mice.

(A) Schematic representation of the Arsk gene locus in wildtype (WT, upper panel) and Arsk knockout mice (Arsk KO, lower panel). The constitutive Arsk knockout allele (knockout first strategy) was achieved by Cre recombinase expression in B6N(Cg)-Arsk^{tm1b(KOMP)Wtsi}/J mice resulting in the loxP-dependent deletion of exon 3 of the Arsk gene as well as the neomycin phosphotransferase gene (neo) while maintaining the FRT-loxP-flanked β-galactosidase (lacZ) reporter from the targeting cassette. PCR amplicons are indicated for the WT allele (515 bp) and the knockout allele (620 bp). (B) PCR-based genotyping of mice resulted in a 620-bp- and a 515-bp-product in knockout and wildtype, respectively. NTC, no template control. (C) The absence of functional Arsk transcript was verified in various tissues of Arsk knockout mice (Arsk KO) by SYBR-green-based quantitative (q)PCR using Gapdh as reference gene.

Figure 2.. Lysosomes from Arsk-deficient mice do not convert G2A0.

G2A0 (12.5 nmol) was pre-labeled with the fluorescent dye 2-aminoacridone (AMAC). Fifty micrograms of a lysosome-enriched fraction (tritosomes) were incubated with G2A0-substrate at 37°C for 24 h and subsequently analyzed by C18-reversed-phase-HPLC combined to nano-ESI-MS. For disaccharide code also see [32]. (A) G2A0 was treated with Arsk-deficient tritosomes (blue) resulting in unreacted (gray shaded) substrate 1 (m/z 672.17) or was treated with wildtype (WT) tritosomes. (B) resulting in the novel peak 3 (m/z 416.18). (C) Simultaneous treatment of G2A0 (672.17) with Arsk-deficient tritosomes and human recombinant (r)ARSK (20 ng) resulted in peak 2 (m/z 592.21) indicating the loss of a sulfate group and peak 3 (m/z 416.18) representing the AMAC-labelled hexosamine after desulfation and disaccharide cleavage. (D) Molecular structures and calculated m/z-ratios of the G2A0 disaccharide (1), its desulfated product (2) and the resulting AMAC-labelled monosaccharide product (3), which is due to tritosome-mediated glycosidase activity.

Figure 3.. Storage of HS and CS disaccharides in Arsk-deficient mice.

Glycosaminoglycans were extracted from brain, kidney, liver, lung and spleen or from isolated liver lysosomes (tritosomes) of 12-month-old WT or ARSK-deficient mice. The amount of heparan sulfate (HS) was determined in the indicated tissues (A) and tritosomes (B) by LC/MS after glycan reductive isotope labeling (GRIL). Similarly, the amount of chondroitin sulfate (CS) was determined in the various tissues (C) and liver lysosomes (D) (n = 3 per genotype, values represent mean ± SEM, *P < 0.05, **P < 0.01). (E,F) The composition of HS- and CS-derived disaccharides were evaluated regarding acetylation (N-Ac) and sulfation by GRIL-LC-MS indicating a minor increase in 2-O-sulfated HS disaccharides.

Figure 4.. Arsk-deficient mice accumulate 2-O-sulfated non-reducing end (NRE) biomarkers.

HS NREs derived from brain and kidney (A) and isolated liver lysosomes (B) of Arsk KO or WT mice were obtained by GAG-depolymerization using bacterial lyases. Quantification of HS NREs from tissues or liver lysosomes was achieved by GRIL-LC/MS with known amounts of [¹³C₆]aniline-labeled internal standards including the specific G2S0 (A left panel; B) and G2A0 (A right panel) disaccharides. (n = 3 for each genotype, values represent mean ± SEM, *P < 0.05, **P < 0.01). (C) Kidney-derived 2-O-sulfat HS NREs were incubated with recombinant human (rh)ARSK (15 ng), rhIDS (8 ng) or with buffer overnight and quantified as described above. (D) Quantification of CS-derived NREs from brain and kidney of WT and Arsk KO mice.

Figure 5.. Histopathology of kidney in 12-month-old Arsk-deficient mice.

Toluidine blue-stainings of semi-thin sections of Arsk-deficient mice kidneys exhibit dense bodies (arrows) in the thick ascending limbs of the inner stripe of the outer medulla (B,D) as well as in intermediate tubules of the inner medulla (F); the respective WT controls are shown on the left (panels A, C and E). In electron microscopy (G,H), the equivalent of these dense bodies (arrows) appeared as lipofuscin-related material exclusively in the kidney of Arsk-deficient mice. Scale bars: 20 μm (light microscopy); 5 μm (electron microscopy).

Figure 6.. Arsk-deficient mice exhibit no obvious skeletal phenotype.

(A) Representative μCT images of femora from 24-week-old wildtype and Arsk-deficient mice. Mineralized bone appears in red. Quantification of the femoral length and midshaft diameter is given on the right. (B) Representative μCT images of trabecular bone from the same mice. Quantification of the bone volume per tissue volume (BV/TV) and the bone mineral density is given on the right. (C) Representative μCT images of cortical bone from the same mice. Quantification of the cortical thickness is given on the right. (D) Quantification of the maximal force (F_max) to failure in three-point-bending assays of the femoral bones. (E) Representative Kossa-stained sections of vertebral bodies (L3 and L4) from 24-week-old wildtype and Arsk KO mice. Mineralized bone appears in black. (F) Quantification of bone volume per tissue volume (BV/TV) and osteoid volume per bone volume (OV/BV). (G) Quantification of osteoblast (Ob.N./B.Pm.) and osteoclast number (Oc.N./B.Pm.) per bone perimeter. All data represent mean ± SD (n = 3). The asterisk indicates a statistically significant difference (*P < 0.05) between the two groups.

Figure 7.. Behavioral alterations in 12-month-old Arsk-deficient mice.

Social behavior of 12-month-old Arsk knockout (KO) mice and wildtype mice (WT) was assessed in the Sociability and Preference for Social Novelty test (SPSN). Knockout mice (right panel) showed time-dependent changes regarding overall proximity (A) as well as approach behavior (B) towards a caged conspecific in comparison with an empty cage, suggesting changes in sociability. Spatial learning and memory was tested in a Morris water maze set-up (C–F). Total path length during acquisition (C) and direct comparison of time spent in the target quadrant during the first probe trial (D) showed no significant changes. However, in contrast with WT mice, KO mice did not show target quadrant exploration above the coincidence level. This was corroborated by decreased numbers of target area entries (E) and average time spent in the target area (F), suggesting impaired spatial memory. Changes did not persist into the second probe trial. All data are represented as mean ± SEM (wildtype n = 13 females, knockout n = 14 females).