SDS-PAGE on the 15 gel. The gel was dried and analyzed by
SDS-PAGE on a 15 gel. The gel was dried and analyzed by phosphorimaging.Final results Endogenous Expression of Arylsulfatase K in Human Tissues– To verify endogenous expression of human ARSK, we initially analyzed its mRNA levels. We looked for tissue-specific expression by RT-PCR of normalized cDNA samples from various human tissues and discovered that ARSK is ubiquitously expressed (Fig. 1). Higher expression levels are located in placenta and pancreas, and minimal expression ranges are found in muscle. Other tissues (lung, brain, heart, liver, and kidney) present intermediate expression ranges. Simply because a certain signal may be discovered in all tissues analyzed, we conclude that ARSK is ubiquitously expressed in most, if not all, human tissues. Expression of Recombinant Arylsulfatase K–The human ARSK-encoding cDNA was obtained by reverse transcription PCR (see “Experimental Procedures”). Its coding sequenceJOURNAL OF BIOLOGICAL CHEMISTRYArylsulfatase K, a Novel Lysosomal SulfataseFIGURE 2. Recombinant expression, N-glycosylation, and stability/processing of ARSK in human cells. A, ARSK was stably expressed in HT1080 and HEK293 cells. Cell lysates (C) and medium (M) samples have been analyzed for ARSK expression by Western blotting making use of an anti-RGS-His6 antibody or an anti-ARSK antiserum, as indicated. Untransfected cells served being a control. The arrow indicates the 68-kDa form of ARSK, as detected within the cell lysates. B, HEK293 cells stably expressing ARSK were lysed, and the cellular protein was handled with endoglycosidases PNGaseF or EndoH, as indicated. In parallel, ARSK secreted by HEK293 cells and enriched by way of HisTrap chromatography was subjected to therapy with endoglycosidases. All samples were analyzed by Western blotting employing the anti-RGS-His6 antibody. The black arrow signifies the completely glycosylated 68-kDa type, whereas the white arrows indicate the partially (64-kDa) or completely deglycosylated types (60-kDa). C, HEK293 cells both overexpressing ARSK or not overexpressing ARSK were metabolically labeled for 1 h with [35S]methionine/cysteine and after that chased for that indicated instances. ARSK was immunoisolated from cell extracts using the anti-ARSK-antibody, separated by SDS-PAGE, and analyzed by autoradiography. ARSK was detected as being a 68-kDa protein (black arrow). In addition, a 23-kDa fragment (white arrow) appeared for the duration of the chase, suggesting processing of the precursor (left panel). A corresponding C-terminal fragment was detected, albeit only weakly, from the anti-RGS-His6 antibody when analyzing ARSK enriched from Met manufacturer conditioned medium of producer cells by Western blotting (suitable panel, showing three elution fractions in the HisTrap column, cf. Fig. 3A).(1608 bp) totally matched GenBankTM accession quantity AY358596. ARSK was stably expressed in HEK293 cells and HT1080 cells like a C-terminally RGS-His6-tagged variant. These cells had been also stably transfected with all the FGE-encoding cDNA since sulfatase activity is determined by posttranslational formylglycine modification. Western blot analyses of untransfected β-lactam manufacturer control and ARSK-expressing HEK293 and HT1080 cells utilizing a His tag-specific antibody (Fig. 2A, left panel) also as an ARSK-specific antibody (appropriate panel) detected a protein with an obvious molecular mass of 68 kDa in transfected cells. The secreted kind of ARSK current in conditioned medium from HT1080 cells exhibited a molecular mass of 70 kDa, i.e. slightly higher than the cellular kind (Fig. 2A, lanes three and eleven). Glycosylation Pattern and Proces.
