Arylsulfatase C is a microsomal membrane-bound enzyme previously thought to be the same as steroid sulfatase, the only X-linked enzyme known to escape from X inactivation in man. We had shown that arylsulfatase C actually consists of two biochemically distinct isozymes, s and f. Only the s form has steroid sulfatase activity. The f and s forms were thought to be related through posttranslational or posttranscriptional modification of the same gene product. In part consistent with this hypothesis, we now report that in a panel of 28 rodent-human somatic cell hybrids, expression of both s and f was concordant only with the human X chromosome, thus showing that the f form is also X linked. In three separate somatic hybrids containing human X chromosomes in an inactive state, the f form was still expressed. Thus, similar to the s form, the f form also escapes from X inactivation. However, contrary to expectations, the s and f forms were not related by posttranslational modification of the same gene product. A full-length cDNA for the s form failed to hybridize to transcripts produced from an f-expressing cell line, showing that there is little sequence identity between the two. They are also not related by differential splicing of a common primary transcript, since fibroblasts from some patients with steroid sulfatase deficiency due to gene deletion of the s form continue to express the f form. Therefore, although the f and s isozymes of arylsulfatase C are X linked and escape from X inactivation, they are products from separate genes, thus providing a unique isoenzyme system to study possible gene duplication and regulation in the part of the human X chromosome that escapes inactivation.

The disadvantages of using human cultured cells for biochemical and genetic studies are their limited lifespan in vitro and their lack of chemical selection markers. These problems are now overcome by transfecting human cultured fibroblasts with the pSV3-gpt and pSV3neo plasmid DNA which carry genes coding for the immortalizing SV40 large T-antigen and dominant selection markers. Transformed human fibroblasts were obtained at a frequency of about 10−5 with both selection systems. These transformed cells showed a twofold increase in growth rate and three to tenfold increase in cell number at confluence. The improved growth characteristics were associated with the expression of the SV40 Tantigen detected with immunoprecipitation. These cell lines also changed from their usual spindle shapes to an epithelioid morphology characteristic of transformed cells. From 60 to 100% of the cells transfected with pSV3 plasmid DNA demonstrated numerical and structural abnormalities in their karyotypes. Cells transfected with DNA from a similar plasmid, pSV2-neo, which differed from the pSV3-neo plasmid only by missing the sequence encoding the complete early region of SV40, neither expressed T-antigen nor showed any change in morphology, improvement in growth characteristics or abnormalities in karyotype. However, they were still selectable with the aminoglycoside G-418. Therefore, by appropriate choice of vector plasmids, dominant selection markers and improved growth characteristics can be imparted separately or simultaneously to human fibroblasts. The morphological, biochemical and chromosomal changes resulting from such transformations must be recognized in using this approach for biochemical and genetic studies.

When arylsulfatase C, a microsomal membrane-bound enzyme, is assayed with its natural substrates, the 3-beta-hydroxysteroid sulfates, it is also known as steroid sulfatase. Whether arylsulfatase C and steroid sulfatase are identical enzymes or not, however, has long been disputed. We now report that two electrophoretic variants of arylsulfatase C occur in normal human fibroblasts: one has a single anodic band of activity, "s," and the other has an additional faster migrating band, "f". The two types, s and "f + s", occur in cells from either sex. When fibroblast strains with the f + s forms of arylsulfatase C were cloned, two types of primary clones were always obtained: s and f + s. A single f band was never seen. When these primary clones were subcloned, however, the arylsulfatase C phenotype remained unchanged: primary s clones gave rise to s subclones and f + s clones to f + s subclones only. Therefore, these forms were clonal in origin and demonstrated a novel inheritance pattern in human cultured cells. The appearance of increasing amounts of the f band was correlated with up to 4-fold increase of arylsulfatase C activity, whereas the steroid sulfatase activity remained constant, thus demonstrating that arylsulfatase C was not identical with steroid sulfatase activity. Polyclonal antibodies raised against the s form immunoprecipitated activities of the s form of arylsulfatase C and steroid sulfatase but not the f form of arylsulfatase C. Therefore, we conclude that only the s form of arylsulfatase C is immunologically related to steroid sulfatase so that arylsulfatase C per se is not necessarily identical with steroid sulfatase. In addition, a novel form of genetic heterogeneity of isozymes in human fibroblasts is demonstrated.

Summary: Deficient arylsulfatase A activity in man has long been associated with the neurodegenerative disease, metachromatic leukodystrophy. However, similar deficiency has been noted in clinically normal individuals, and is referred to as the pseudoarylsulfatase A deficiency condition. Although direct quantitative analysis of arylsulfatase A activity failed to differentiate between these two conditions, analysis of residual arylsulfatase A activity with either Cellogel electrophoresis or isoelectric focusing in polyacrylamide gels now has been shown to distinguish between them unequivocally. With both techniques, cultured fibroblasts from patients with pseudo-arylsulfatase A deficiency showed faint but clear bands of arylsulfatase A activity. Under identical conditions, fibroblasts from patients with metachromatic leukodystrophy showed no trace of activity. These methods can be adapted easily for general laboratory analysis in cases when results from quantitative arylsulfatase A assays are noninformative.

Conventional colorimetric methods for determining arylsulfatase A and B activities are cumbersome and insufficiently sensitive for microassays. A more sensitive fluorogenic technique to determine activities of the two enzymes in human cultured fibroblasts without any prior treatment has been developed. In the assay mixture, lead ions (3 mm) are used to remove endogenous inhibitors and silver ions (0.3 mm) are used to inhibit specifically arylsulfatase A activity. This assay is optimal at pH 5.6 for both enzymes, linear for up to 2 h of incubation at 37° at protein concentrations of 3 to 10 μg for arylsulfatase A and 7 to 40 μg for arylsulfatase B. “Lysis buffer” is the best for extracting arylsulfatase A and 0.15 m sodium acetate for extracting arylsulfatase B. This technique is three- to fivefold more sensitive than the conventional method and applicable to assays of arylsulfatase A or B activity in fibroblasts from patients with various sulfatase deficiencies.

Multiple sulphatase deficiency (MSD) in man is inherited as an autosomal recessive trait and associated with deficient activities of various sulphohydrolases. Cultured fibroblasts from seven different patients were assayed for arylsulphatases-A, -B and -C activities. On the basis of the results, they may be classified into three groups: I, deficient in all three arylsulphatases; II, deficient only in arylsulphatases-A and -C with half or near-normal arylsulphatase-B; electrophoretically, arylsulphatase-A activity bands are undetectable as in metachromatic leukodystrophy; III, same as in II except electrophoretically, the residual arylsulphatase-A is detectable as faint activity bands similar to those in pseudo arylsulphatase-A deficiency. In addition to the variability among different strains, within the same strain of MSD or normal cells, each enzyme activity increased several fold with increasing time in culture. These sources of biochemical variability among and within different cell strains have not been recognized before in the study of this apparently monogenic trait with multiple enzyme deficiencies. They may account for some of the discrepancies reported in the literature on arylsulphatase activities among cultured cells from different multiple sulphatase deficient patients.