D so far. Utilizing the pseudosubstrates, we determined an apparent pH optimum of 4.six for ARSK activity, which strongly suggested a lysosomal localization. This was confirmed by immunofluorescence studies demonstrating colocalization of ARSK using the lysosomal integral membrane protein LAMP-1 upon uptake of partially purified ARSK supplemented to the cell culturemedium. Most lysosomal hydrolases are sorted toward the lysosome by the M6P receptor technique (31), which also mediates uptake of mistargeted M6P-containing proteins in the extracellular space. Accordingly, ARSK was shown to bind effectively to immobilized MPR in an M6P-dependent manner, and, moreover, a robust M6P-signal was detected for ARSK in Western blot analyses utilizing a M6P-specific antibody. Taken with each other, these findings demonstrate a lysosomal localization of ARSK. Interestingly, and in line with our observations, ARSK had already been identified previously in research on the lysosomal subproteome when analyzing the mannose 6-phosphate glycoproteomes from humans, mouse, and rat (324 and reviewed in Ref. 23). In their study, Sleat et al. (34) pinpointed the M6P web site to asparagines Asn-498 and Asn-499 in human and mouse ARSK, respectively. Lysosomal hydrolases are typically synthesized as inactive precursors that undergo restricted proteolysis for the duration of maturation into their active lysosomal forms (35), as applies also to many sulfatases, e.g. arylsulfatase B (N-acetylgalactosamine-4-sulfatase) (36, 37). Inside the case of ARSK, we obtained proof forVOLUME 288 Number 42 OCTOBER 18,30026 JOURNAL OF BIOLOGICAL CHEMISTRYArylsulfatase K, a Novel Lysosomal Sulfataseprocessing from the 68-kDa precursor throughout 24-h pulse-chase experiments for the reason that a stable 23-kDa fragment could possibly be immunoprecipitated by anti-ARSK antibodies from a 2-h chase onwards.IEM-1460 Membrane Transporter/Ion Channel An equivalent His-tagged, i.Merestinib Technical Information e.PMID:23805407 C-terminal, ARSK-derived 23-kDa fragment may 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 might have escaped our analyses on the basis of antibody recognition due to incompatible epitopes just after processing. Further studies on this situation will need expression of larger amounts of ARSK and/or availability of other ARSKspecific antibodies. ARSK is expressed in all tissues examined within this study and was also identified in eight tissues from rat in M6P glycoproteome analyses (33). Its ubiquitous expression pattern may perhaps recommend a typical and widespread sulfated substrate and indicates that ARSK deficiency in all probability 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 identify the physiological substrate of this sulfatase and its overall pathophysiological relevance. Ultimately, the mouse model could enable us to draw conclusions on ARSKdeficient human sufferers who so far escaped diagnosis and may possibly be accessible for enzyme replacement therapy. The presence of M6P on ARSK qualifies this sulfatase for such a therapy, which has established helpful for therapy of several other lysosomal storage problems.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. Lamanna, Jaqueline Alonso Lunar, Kerstin B er, and Claudia Prange for technical help; Markus Damme for initia.