Hese findings demonstrate a lysosomal localization of ARSK. Interestingly, and in line with our observations, ARSK had currently been identified previously in studies in the lysosomal subproteome when analyzing the mannose 6-phosphate glycoproteomes from humans, mouse, and rat (32?4 and reviewed in Ref. 23). In their study, Sleat et al. (34) pinpointed the M6P web page to asparagines Asn-498 and Asn-499 in human and mouse ARSK, respectively. Lysosomal hydrolases are normally synthesized as inactive precursors that undergo limited proteolysis for the duration of maturation into their active lysosomal forms (35), as applies also to various sulfatases, e.g. arylsulfatase B (N-acetylgalactosamine-4-sulfatase) (36, 37). Within the case of ARSK, we obtained proof forVOLUME 288 ?Number 42 ?OCTOBER 18,30026 JOURNAL OF BIOLOGICAL CHEMISTRYArylsulfatase K, a Novel Lysosomal Sulfataseprocessing of your 68-kDa precursor through 24-h pulse-chase experiments mainly because a stable 23-kDa fragment could be immunoprecipitated by anti-ARSK antibodies from a 2-h chase onwards. An equivalent His-tagged, i.e. C-terminal, ARSK-derived 23-kDa fragment might be detected in Western blot analyses of ARSK enriched from conditioned medium of producer cells. Corresponding N-terminal fragment(s) could not be detected. They could possibly have escaped our analyses on the basis of antibody recognition due to incompatible epitopes following processing. Further research on this challenge will demand expression of larger amounts of ARSK and/or availability of other ARSKspecific antibodies. ARSK is expressed in all tissues examined in this study and was also identified in eight tissues from rat in M6P glycoproteome analyses (33). Its ubiquitous expression pattern might recommend a popular and widespread sulfated substrate and indicates that ARSK deficiency most likely results in a lysosomal storage disorder, as shown for all other lysosomal sulfatases. At present, we’re producing an ARSK-deficient mouse model that really should pave the method to recognize the physiological substrate of this sulfatase and its general pathophysiological relevance. Finally, the mouse model could allow us to draw conclusions on ARSKdeficient human individuals who so far escaped diagnosis and could be accessible for enzyme replacement therapy.Methyl 4-hydroxythiophene-3-carboxylate uses The presence of M6P on ARSK qualifies this sulfatase for such a therapy, which has verified valuable for therapy of many other lysosomal storage problems.Y-27632 (dihydrochloride) Price Acknowledgments–We thank Bernhard Schmidt and Olaf Bernhard for mass spectrometry; Nicole Tasch, Annegret Schneemann, Britta Dreier, Martina Balleininger (all from G tingen), William C.PMID:23008002 Lamanna, Jaqueline Alonso Lunar, Kerstin B er, and Claudia Prange for technical assistance; Markus Damme for initial evaluation of subcellular localization; and Jeffrey Esko (San Diego) for critically reading the manuscript. We also thank Kurt von Figura for assistance during the initial phase of this project.Dierks, T. (2007) The heparanome. The enigma of encoding and decoding heparan sulfate sulfation. J. Biotechnol. 129, 290 ?07 Schmidt, B., Selmer, T., Ingendoh, A., and von Figura, K. (1995) A novel amino acid modification in sulfatases that is defective in various sulfatase deficiency. Cell 82, 271?78 von B ow, R., Schmidt, B., Dierks, T., von Figura, K., and Us , I. (2001) Crystal structure of an enzyme-substrate complicated provides insight into the interaction in between human arylsulfatase A and its substrates during catalysis. J. Mol. Biol. 305, 269 ?77 Dierks, T., Lecca, M. R., S.