Biosynthesis and Maturation of Arylsulfatase B in Normal and Mutant Cultured Human Fibroblasts*

The biosynthesis of arylsulfatase B in normal and mutant human skin fibroblasts was studied by meta- bolic labeling with radioactive amino acids, monosac-charides, or 32Pi and by specific immunoprecipitation followed by polyacrylamide gel electrophoresis and fluorography. Three major polypeptides with apparent molecular weights of 47,000,40,000, and 31,000 were found intracellularly and one of 64,000 in the medium. Pulse-chase labeling and uptake experiments showed that arylsulfatase B synthesized and secreted as a 64,000 precursor was intracellularly processed within less than 24 h via short lived intermediates to two different forms. Form I (chains of 47,000 and 11,500) was labeled earlier and was about twice as stable as form I1 (chains of 40,000 and 31,000). The secreted 64,000 precursor and the 40,000 chain of form I1 contained oligosaccharides resistant to endo-8-N-ace-tylglucosaminidase H. In the other chains mainly cleavable and phosphorylated oligosaccharides were found. Arylsulfatase B activity was associated with the 64,000 precursor and with form I, but not with form 11. arylsulfatase B activity, 64,000 and

In the present study we have examined the synthesis and maturation as well as some structural properties of arylsulfatase B in normal and mutant skin fibroblasts. Previous studies have established that a large number of lysosomal enzymes are synthesized as larger precursors and subsequently processed to mature forms by a limited proteolysis (for review see Refs. 9 and 10). We extended these findings to arylsulfatase B and report on the biosynthesis and processing of mutant arylsulfatase B in Maroteaux-Lamy fibroblasts.
Cell Culture-Human diploid fibroblasts were maintained at 37 "C in 5% C02 in Eagle's minimal essential medium supplemented with antibiotics, nonessential amino acids, and 7.5% fetal calf serum (Boehringer Mannheim) as described (11). Normal and mutant fibroblasts were obtained from biopsies submitted to our laboratory for diagnosis. The four Maroteaux-Lamy fibroblast lines were derived from patients affected with the severe form of the disease (12).
Labeling of Arylsulfatase B-Radioactive labeling of cells was performed as described by Hasilik and Neufeld (13,14) with minor modifications (14)(15)(16). Cells in 25- (13). The secretions were precipitated with 2.5 g of (NH4),SO,. The precipitate was dissolved in H20 and dialyzed overnight against Eagle's minimal essential medium. After dialysis, 0.25 ml of fetal calf serum and the medium were added to a final volume of 5 ml. The medium containing the labeled secretions (15-30 X IO6 cpm for 35S and 15 X lo6 for 3zP,) was sterilized by filtration and added to confluent cultures in 75-cm2 flasks. Some cultures were harvested after 6 or 24 h, other cultures received the regular cultivation medium and the incubation was continued for up to 7 days.
Treatment with Endo-6-N-acetylglucosaminidase H-Extracts of cells and medium were incubated 36 h at 37 "C with 1 unit/ml of endo-6-N-acetylglucosaminidase H prior to immunoprecipitation as described (16).
Gel Electrophoresis and Fluorography-Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Sigma) with or without dithiothreitol was performed according to Laemmli (17) and fluorography according to Bonner and Laskey (18).
Enzymatic Activity of Arylsulfatase B Precursor-Confluent cultures in 75-cm2 flasks were incubated for 72 h with 15 ml of labeling medium supplemented with 10 mM NH4C1. The media were concentrated 50-fold by ultrafiltration and mixed with either arylsulfatase B antiserum or preimmune serum. After incubation for 30 min at

Biosynthesis of Arylsulfatase B
14323 room temperature and 20 h at 4 "C and centrifugation at 10,000 X g for 10 min, arylsulfatase B activity was determined in the supernatant. Arylsulfatase B activity was defined as the activity precipitable with the antiserum and was calculated from the decrease in the enzyme activity in the soluble fraction.
Enzyme Assay-In cell extracts and secretions, arylsulfatase B activity was mesured with the aid of differential precipitation as described above. During the purification the activity was determined in untreated aliquots. Samples of 30 pl were incubated with 120 pl of 12.5 mM p-nitrocatechol sulfate in 0.5 M sodium acetate, pH 5.5 (20). for 30 min at 37 "C. The reaction was stopped by adding 300 pl of 0.6 N NaOH and absorbance was measured at 510 nm.
One unit is the amount of enzyme catalyzing the formation of 1 pmol of p-nitrocatechol/min. Protein was determined by the method of Lowry et al. (21) with bovine serum albumin as standard.
Purification of Arylsulfatase B-In tissue extraction, frozen placenta, 9.6 kg, was passed through a meat grinder and 1-kg portions were homogenized in 3 volumes of ice-cold 50 mM Tris/HCl, pH 7.5 (buffer A), with an Ultra-Turrax. The homogenate was centrifuged for 30 min at 3000 X g, and the pellet re-extracted with 1 volume of the buffer. At this and following stages, the protein was concentrated by precipitation with (NH4)ZS04, 500 g/liter, collected by centrifugation, and equilibrated with a proper buffer by dialysis. The protein in buffer A was treated with DE52 cellulose (0.08 ml/mg of protein). The unbound protein was concentrated and applied to CM52-cellulose in 25 mM sodium acetate at pH 5.1. The enzyme was eluted with a linear NaCl gradient at 0.15 M. The enzyme re-equilibrated with the acetate buffer was again applied to a CM52-cellulose column and then eluted at pH 7.6 with a linear gradient (by volume) with 25 mM Tris/HCl, pH 8.4, as the terminal buffer. The eluted enzyme preparation was concentrated, equilibrated with 0.1 M sodium acetate, pH 6.0, and applied to concanavalin A-Sepharose 4B (Pharmacia, Freiburg), 0.048 ml of gel/mg of protein. After washing with 1 M NaCl in the buffer, the elution was effected with 10% methyl-a-D-mannoside. At this and the following stages, the enzyme was concentrated by ultrafiltration. After dialysis against 100 mM Tris/HCl, pH 7.5, the preparation was subjected to gel filtration using a Sephadex G-100 column (2.5 X 90 cm) (Pharmacia, Freiburg). The filtration was repeated with a Sephadex G-75 column (2.5 X 92 cm). The enzyme was concentrated, equilibrated with 5 mM Tris/HCl, pH 8.3, passed without binding through a DE52 cellulose column (0.7 X 1 cm), and concentrated. A summary of the purification scheme is given in Table  I. Homogeneity of the preparation was established by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (see Fig.   1).
Preparation of Antiserum-Arylsulfatase B (60 pg) in Freund's complete adjuvant was injected intradermally into a rabbit. The injection was repeated after 4 and 6 weeks using Freund's incomplete adjuvant. Antiserum was obtained 1 week later. One ml precipitated 0.3 mg of enzyme.

Synthesis and Maturation in Human
Fibroblasts-Human skin fibroblasts labeled for 23 h with [3sS]methionine contained three major and several minor radioactive polypeptides precipitable with antiserum against human placental arylsulfatase B (Fig. 1). The apparent molecular weights of the major polypeptides were 47K,' 40K, and 31K, and those of the minor polypeptides 64K, 62 K, and 53K (Fig. lA). Of the minor polypeptides only the 64K and 62K were regularly observed. In the medium, a single polypeptide with an apparent molecular weight of 64K was precipitable. In the presence of 10 mM NH4Cl, which is known to induce the secretion of newly synthesized lysosomal enzymes (13, 22, 23), 70-75% of total labeled arylsulfatase B was secreted. Further, the 64K poly-' 47K, 40K, and 31K, etc. represent M, = 47,000,40,000, and 31,000, for example.  peptide was the most prominent among the intracellular forms. In fibroblasts from a patient with I-cell disease, more than 70% of the newly synthesized arylsulfatase B was secreted as 64K polypeptide. Intracellularly, the 64K polypeptide and smaller polypeptides ( M , = 49-52 kDa) were present.
The purified placental arylsulfatase B which had been used in the immunization contained a 47K polypeptide (Fig. IA). This polypeptide was phosphorylatable in vitro by an extract from rat liver Golgi membranes that transfers N-acetylglucosamine 1-phosphate residues from UDP-N-acetylglucosamine specifically to lysosomal polypeptides (19). The presence of two smaller phosphorylatable polypeptides (Mr = 14.5K and 11.5K) in the purified placental arylsulfatase B was revealed by the phosphorylation assay. Upon close examination traces of the 14.5K polypeptide were detectable on the  1B). Under nonreducing conditions, the arylsulfatase B from normal fibroblasts migrated as two incompletely separated polypeptides of apparent M , = 58K and 56.5K and the arylsulfatase B secreted into the medium as a rather diffuse 60K polypeptide (Fig. 1B).
T o establish the relationship between the polypeptides precipitable with antiserum against arylsulfatase B, fibroblasts were labeled for up to 3 h and then subjected to a chase of up to 24 h (Fig. 2). During the first 2 h only the 64K polypeptide was dectectable intracellularly. After a 3-h pulse, in addition to the 64K polypeptide the 47K polypeptide became visible. During a following chase for 3 h, the 40K and 31K polypeptides and a processing intermediate of 62K appeared. The processing intermediate and the 64K precursor disappeared for the most part within the following 21 h of chase, whereas radioactivity in the 47K and, even more prominent, in the 40 and 31K polypeptide increased. Occasionally some processing intermediates (58K, 55K, 53K, and 49K) were seen after labeling for 2 to 3 h (not shown). Further evidence for a precursor-product relationship between the 64K polypeptide and the 47K, 40K, and 31K polypeptide was provided by uptake experiments (Fig. 3). Radioactively labeled secretions obtained from cultures treated with 10 mM NH&l were incubated with fibroblasts for 6 or 24 h and endocytosed arylsulfatase B was isolated. Only processed polypeptides were detectable intracellularly. After incubation for 6 h the 47K polypeptide was visible and after incubation for 24 h the 47K, 40K and 31K polypeptides were visible. After incubation for 3 and 7 days in cultivation medium, 45 and 15% of the endocytosed 47K polypeptides were recovered.
Oligosaccharides in Arylsulfatase B-The 64K precursors   (Fig. 4). More than 90% of the "P label was lost upon treatment with endo-8-N-acetylglucosaminidase H. This endoglycosidase is specific for oligosaccharides of the high mannose and hybrid type. It affected the size and the radioactivity in the polypeptides labeled with ["S]methionine as follows. The secreted 64K precursor was largely converted into a 62K form presumably due to the loss of one sensitive oligosaccharide. The 62K product was still labeled with ['Hlmannose (not shown) indicating the presence of resistant (complex) oligosaccharides. The presence of complex oligosaccharides in the secreted 64K precursor was further indicated by its labeling with [:'H]galactose. The 47K polypeptide was converted into two products smaller in size by 4K and 6K suggesting the loss of two or three sensitive oligosaccharides. Part of the 40K polypeptide was converted into a 38K product. Both the 40K forms resistant to the endoglycosidase and the 38K product were labeled with ["Hlmannose, suggesting that the 40K polypeptide carries a t least two oligosaccharides, of which only one may be cleavable. The 31K form is converted into two products smaller in size by 2K and 3K (in the experiment shown in Fig. 4, the products are incompletely separated). The assignment of the products to the parent polypeptides was facilitated by similar experiments with Maroteaux-Lamy fibroblasts which are deficient in the 47K polypeptide (see below).
After labeling with ['Hlmannose and "PI a polypeptide with an apparent molecular weight of 11.5K was immunoprecipitable which was not detected in immunoprecipitates of cells labeled with ["'S]methionine or ['Hlleucine (not shown). We assume that this fragment is related to the smaller phosphorylatable fragments in placental arylsulfatase B (see Fig.   1, A and B). Synthesis 5). However, the 47K polypeptide was detected only in trace amounts (~1 0 % of normal) in cell line U . and not at all in the other three. In Maroteaux-Lamy fibroblasts, the relative amount of the 40K and 31K polypeptides based on the secreted 64K precursor was about 1.5-fold higher than in normal fibroblasts. Under nonreducing conditions, the apparent M , of intracellular and secreted arylsulfatase B were 56.5-58K and 60K, respectively, in both the normal and Maroteaux-Lamy fibroblasts. Structural alterations in the arylsulfatase B are responsible for the abnormal processing observed in Maroteaux-Lamy fibroblasts. This was proven by endocytosis experiments. Internalization of the mutant arylsulfatase B precursor by normal or Maroteaux-Lamy fibroblasts was followed by processing to 40K and 31K polypeptides. In contrast, arylsulfatase B produced in normal fibroblasts was processed to polypeptides of M , of 47, 40, and 31K. This was observed after endocytosis in either normal or Maroteaux-Lamy fibroblasts (Fig. 6). Under nonreducing conditions, the apparent M , of the internalized mutant and normal arylsulfatase B was 56.5-58K. The products obtained from the 64K precursor and the 31K polypeptides after treatment with endo-P-N-acetylglucosaminidase H were the same as in normal fibroblasts (not shown). In a pulse-chase labeling experiment performed in parallel to that shown in Fig. 2, trace amounts of the 50K processing intermediate and the 47K polypeptide were detected in Maroteaux-Lamy fibroblasts (cell lines G. and M.) after a 3-h pulse. After a chase for 3 h these polypeptides were no longer detectable (Fig. 7). As in normal fibroblasts, the formation of the 40K and 31K polypeptides takes place in the period of 3-24 h after the endocytosis.
Thiol proteinases participate in the processing of lysosomal enzymes (24, 25) and may be responsible for the degradation of the mutant 47K polypeptide. Therefore, uptake experiments were performed in the presence of leupeptin, an inhibitor of cathepsin B-like thiol proteinases. Leupeptin caused the accumulation of the 64K precursor and several polypep- tides not seen in untreated cells.

DISCUSSION
In human fibroblasts, arylsulfatase B is synthesized as a larger precursor. At least one-third of the precursor is secreted into the medium. The secreted precursor is amenable to uptake in fibroblasts. Within a few hours of synthesis or endocytosis, the precursor matures to smaller polypeptides. Without reduction the precursor and the products behave as molecules of 60K and 56.5-58K3, respectively. After reduction, the precursor has an apparent molecular weight of 64K and the mature products fragment to polypeptides of 47K, 40K, 31K, and 11.5K. The shortest polypeptide can conveniently be labeled with "Pi or [3H]mannose, but not with [3H]leucine or ["S]methionine. We suggest that in fibroblasts, two different products are formed from the 64K precursor, one product (form I) containing the 47K and 11.5K polypeptides and the other (form 11) containing the 40K and 31K polypeptides. The assignment of the polypeptides to the two processing products is based on the following observations. The mutation in Maroteaux-Lamy fibroblasts, which are deficient in arylsulfatase B activity, causes a nearly complete absence of form I, whereas it elevates the amount of form I1 by about 50%. Arylsulfatase B purified from human placenta resembles form I. It contains three polypeptides of 47K, 14.5K, and 11.5K. The two smaller polypeptides can be detected only after subjecting the enzyme to enzymic phosphorylation and to reduction of disulfide bridges. Probably the 47K polypeptide is linked to either of the two smaller polypeptides in a ratio of 1:l. In fibroblasts, form I can be detected 2-3 h after synthesis of the precursor.
The production of form I1 starts 3-6 h after synthesis of the precursor. In endocytosis experiments an even greater temporal displacement in the appearance of two forms was observed (Fig. 3). The delayed production of form I1 might suggest form I to be an intermediate in the processing of the precursor to form 11. Such relationship would require cleavage of the 47K polypeptide of form I into the 40K and 31K polypeptides of form 11. This relation is unlikely because the 40K polypeptide contains largely noncleavable [3H]mannoselabeled oligosaccharides, whereas in the 47K polypeptide all are cleavable. We propose, therefore, that form I and form I1 are products of alternative processing of the precursors. This finding indicates that the 64K precursors while sharing antigenic determinants with arylsulfatase B are heterogeneous at least in respect to their carbohydrate side chains. It should be of interest to find out whether the carbohydrate or another signal is responsible for the specific processing of the precursor to either form I or I1 products.
The enzymic activity of arylsulfatase B is apparently associated with form I. This is based on the following observations. The polypeptides found in form I are similar to those in the purified placental enzyme. Cells deficient in arylsulfatase B activity contain little or no form I polypeptides but normal amounts of form 11. This was observed in all four cell lines from Maroteaux-Lamy patients examined in this study as well as in several multiple sulfatidosis fibroblasts. Whereas the precursor made in Maroteaux-Lamy fibroblasts is deficient in catalytic activity, multiple sulfatase-deficient fibroblasts synthesize catalytically active precursor. As in Maroteaux-Lamy fibroblasts, the form I polypeptides are rapidly degraded in multiple sulfatase-deficient fibroblasts, whereas normal amounts of form I1 polypeptides accumulate.* The precursors secreted in control fibroblasts are endowed with arylsulfatase B activity. It is not known, however, whether the activity is associated with all precursor molecules. If so, processing of precursors to form I1 of arylsulfatase B would be associated with loss of catalytic activity. The mutation in the severe form of Maroteaux-Lamy syndrome has pleiotropic effects on arylsulfatase B. The mutant enzyme is inactive and the mutant form I is unstable. Our results are in agreement with an earlier report on the presence of immunologically cross-reactive material in Maroteaux-Lamy fibroblasts (26).
for providing two of the Maroteaux-Lamy fibroblast lines.