Leukodystrophy

Cystic fibrosis (CF) is associated with mutation and abnormal function of the cystic fibrosis transmembrane conductance regulator (CFTR) that affects cellular chloride transport. Clinically, CF of the lung is associated with excessive accumulation of secretions, including the sulfated glycosaminoglycans, chondroitin sulfate and dermatan sulfate (DS), both of which contain sulfated N-acetylgalactosamine residues. The sulfatase enzymes, which are a highly conserved group of enzymes with high specificity for designated sulfate groups, include arylsulfatase B, a lysosomal enzyme. Arylsulfatase B, also known as N-acetyl galactosamine 4-sulfatase, can degrade DS and chondroitin-4 sulfate. Previously reported data demonstrated diminished activity of arylsulfatase B in lymphoid cell lines of patients with CF compared to normal control subjects. Frequent infections with Pseudomonas, a sulfatase-producing organism, occur in patients with CF, whereas infections with Mycobacterium tuberculosis, which lacks sulfatase activity, are infrequent. Additional investigation to determine ff diminished function of arylsulfatase B is a consistent finding in cells of patients with CF may be informative, and may help to correlate the molecular, biochemical, and clinical characteristics of CF.

Key words: arylsulfatase B; chondroitin-4 sulfate; cystic fibrosis; cystic fibrosis transmembrane conductance regulator; dermatan sulfate; endosomes; glycosaminoglycans; lysosomes; N-acetylgalactosamine 4-sulfate

Abbreviations: CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; CS = chondroitin sulfate; DS = dermatan sulfate; ER = endoplasmic reticulum; MLS = Maroteaux-Lamy syndrome; MPS = mucopolysaccharidoses

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Cystic fibrosis (CF) has been associated with mutations of the CF transmembrane conductance regulator (CFTR) gene located on chromosome 7. CFTR is a cyclic adenosine monophosphate-regulated plasma membrane chloride channel, expressed in luminal membranes of both secretory and absorptive epithelia, forming the luminal exit pathway for Cl-. Abnormalities in channels associated with influx of sodium have also been described in CF, for the CFTR also regulates absorption of electrolytes by controlling the activity of the epithelial Na+ channel in epithelial cells of colon, airways, and sweat ducts. (1) The CFTR gene has been found to have > 1,300 mutations, the most common being deletion of a triplet encoding a phenylalanine residue at position 508 (AF508). (2) The sweat duct requires CFTR for activation of epithelial sodium channel, in contrast to the inhibition of epithelial sodium channel by CFTR in colonic and bronchial epithelia. (1) CFTR may also have a role in intracellular chloride trafficking and has been associated with acidification of trans-Golgi network and endosomes. (3,4)

Since clinical variation in the manifestations and severity of CF occurs among patients of the same CFTR genotype and members of the same family, it has been proposed that other genetic and environmental factors may influence disease severity. Up-regulation of a calcium-activated Cl- conductance has been identified in CFTR-deficient mice as such a factor. (5,6)

Increased concentrations of sulfated glycosaminoglycans have been associated with the organ manifestations of CF. (7) These substances consist of sulfated N-acetylglycosaminoglycans and other saccharides that may be sulfated, such as L-iduronic acid. These substances are degraded by glycosidases and sulfatases.

SULFATASES

Arylsulfatase B is one of the family of sulfatase enzymes, a dozen of which are recognized. These represent a highly conserved class of enzymes that include arylsulfatase A (galactose 3-sulfatase, also known as sulfatidase); arylsulfatase B (N-acetylgalactosamine 4-sulfatase); arylsulfatase C (steroid sulfatase); arylsulfatases D, E, and F; glucuronate 2-sulfatase; heparan N-sulfatase (sulfamidase); N-acetylglucosamine 6-sulfatase or glucosamine 6-sulfatase; glucosamine 3-sulfatase; iduronate 2-sulfatase; and N-acetylgalactosamine 6-sulfatase (Table 1). Arylsulfatase A is involved in the desulfation of sulfogalactolipids and is also known as cerebroside sulfatase. Arylsulfatase B desulfates sulfoglycosaminoglycans. Arylsulfatase C is involved in the desulfation of sulfated steroids, such as estrone-3 sulfate and dehydroepiandrosterone-sulfate. Arylsulfatases D, E, and F may comprise a complex of sulfatases that act with arylsulfatase C. The steroid sulfatases are localized in the rough endoplasmic reticulum (ER), whereas the arylsulfatases A and B are found in endosomes and lysosomes. The gene for arylsulfatase B is found on chromosome 5. (8,9)

Highly conserved proteins, the sulfatases require posttranslational modification in order to perform their desulfating function. The posttranslational modification involves alteration of a conserved cysteine residue to an oxo-alanine (or formyl-glycine) in the endoplasmic reticulum, at a late stage of protein translocation. Modification is dependent on protein transport and on the correct positioning of the cysteine residue at position 69 in arylsulfatase A or position 91 in arylsulfatase B. Arylsulfatases A and B do not appear to compete for modification. The posttranslational modification of the sulfatases appears influenced by the sequence CTPSRA, beginning with the cysteine that undergoes posttranslational modification. This motif, adjacent to the active site, is highly conserved. The structure of arylsulfatase B (N-acetylgalatosamine 4-sulfatase) has been determined, revealing that residues conserved among the sulfatase family are involved with stabilization of a calcium ion and positioning of the sulfate ester in the active site. The three-dimensional configuration of the active site resembles the active site of alkaline phosphatase, and the mechanism by which arylsulfatase B breaks the sulfate bond is analogous to the mechanism proposed for alkaline phosphatase, and requires inversion of configuration of the sulfate group. (8,10-13)

The lysosomal enzymes, including arylsulfatases A and B, have been referred to as acid hydrolases, due to their functionality at acid pH. They are synthesized in the ER, transported to the Golgi, and undergo posttranslational modifications within the Golgi complex. Attachment of a terminal mannose-6-phosphate group to some of the oligosaccharide side chains may be recognized by specific receptors in the Golgi, leading to the segregation of the lysosomal enzymes and formation of transport vesicles. These transport vesicles separate from the Golgi and proceed to fuse with the lysosomes. The vesicles recycle back to the Golgi. Absence of formation of the mannose-6-phosphate marker leads to inability of the vesicle to target the lysosome and to extracellular secretion. Transport to the lysosome is accomplished by the mannose-6-phosphate receptor pathway and a prelysosomal acidic compartment. (12,14) If there is deficiency or malfunction of one of the lysosomal enzymes required to metabolize the sulfated glycoconjugate, catabolism is incomplete, leading to accumulation of insoluble intermediates in the lysosome. (7)

Some of the sulfatase deficiencies have well-described clinical characteristics and are recognized as lysosomal storage diseases or mucopolysaccharidoses (MPS) [Table 1]. These disorders include multiple sulfatase deficiency (deficiency of arylsulfarases A, B, C; steroid sulfatase; iduronate sulfatase; and heparan N-sulfatase), metachromatic leukodystrophy (deficiency of arylsulfatase A), Maroteaux-Lamy syndrome (MLS) [deficiency of arylsulfatase B], Hurler syndrome (MPS I, deficiency of [alpha]-L-iduronidase), and Hunter syndrome (MPS II, deficiency of iduronate sulfatase). Complete deficiencies produce marked phenotypic alterations that prove incompatible with prolonged survival, due to organ failure associated with accumulation of unmetabolizable substances in intracellular organelles and derangement of normal cellular metabolism. (9,14)

Arylsulfatase B hydrolyzes the sulfate ester group of N-acetylgalactosamine 4-sulfate residues, such as are found in dermatan sulfate (DS) and chondroitin 4-sulfate. Deficiency of the enzyme in human MPS VI has been associated with intralysosomal accumulation and urinary excretion of DS. Increased urinary excretion of acid mucopolysaccharides has been described as a feature of MLS, and 70 to 95% of the acid mucopolysaccharides are DS. (15,16)

Storage of glycosaminoglycans has been detected histologically and or ultrastructurally in all of the tissues investigated in a mouse model of arylsulfatase B deficiency. These tissues have included liver, heart, lung, kidney, blood vessels, spleen, trachea, esophagus, stomach, small and large intestine, pancreas, salivary gland, testis, skin, adrenal gland and lymph nodes. Storage is found almost exclusively in interstitial, fibroblast-like cells, macrophages, and endothelial cells, and only rarely in parenchymal cells. Genetically altered mice, made homozygous for a mutant arylsulfatase B gene, neither express specific messenger RNA nor have arylsulfatase B activity. Urinary secretion of DS and granular inclusion bodies in leukocytes are observed in the animal model. (16)

MLS (MPS VI) AND CF

MLS, which is attributed to marked deficiency of arylsulfatase B, is associated with impaired cleavage of sulfate from the sulfated N-acetylgalactosamine of DS, resulting in tissue accumulation of unmetabolizable substrate and increased urinary excretion of DS. DS is composed of sulfated N-acetylgalactosamine residues alternating with uronic acid residues, predominantly L-iduronic residues, some of which are sulfated. DS is degraded by three exoglycosidases, as well as by iduronate 2-sulfatase and N-acetylgalactosamine 4-sulfatase. In patients with MLS, deficiency of arylsulfatase B was described in the liver, kidney, spleen, brain, and cultured skin fibroblasts, in peripheral leukocytes, and in a derived lymphoid cell line. This disorder is associated with an autosomal recessive inheritance and specific phenotypic characteristics, including skeletal deformities, growth retardation, hepatosplenomegaly, cardiac valvular disease, growth deficiency, and corneal opacities. (9,15)

Although the clinical characteristics of CF and MLS are not dominated by the same manifestations, some similarities exist between the two conditions. These include growth retardation, occurrence of skeletal deformities such as kyphoscoliosis, developmental dental anomalies, hepatomegaly, normal intellect, and normal psychomotor development. MLS exists in severe, mild, and intermediate forms. (14) Survival to the teens occurs in the severe form. (9) The clinical course of MLS patients appears to be variable; similarly, although most patients with CF have respiratory symptoms and signs in infancy, some individuals may not present with respiratory tract involvement until adulthood. (1) Noisy breathing and expiratory and inspiratory stridor have been reported in published cases of patients with MLS. (17) The relative inactivity of children with MPS has been suggested as a reason that respiratory symptoms are not observed. (18) Tracheal surface area in patients with MLS has been found to be less than in age- and gender-matched control subjects, perhaps attributable to abnormal submucosal storage of mucopolysaccharides. (18)

The clinical picture of MLS is dominated by the skeletal manifestations with growth retardation. (18) The growth failure seen in CF may be multifactorial, related to impaired digestion, malabsorption, and chronic infection. Children with CF may demonstrate low bone mineral density in the first decade of life that worsens with increasing age. Kyphosis and increased risk of fractures may be associated with this. CF has been associated with occurrence of arthropathy of 10 to 15% during a patient's lifetime. (1)

CF and MLS are both associated with dental abnormalities and with hepatomegaly. Developmental dental anomalies, including unerupted and impacted permanent teeth, are seen with MLS. (19) Studies have demonstrated less calcium in the teeth of the population of children with CF for both incisors and total teeth. (20) In experiments with mice with defective CFTR, reduced calcium is found in the enamel layer of growing incisors. (21) The earliest manifestation of liver disease in CF is neonatal cholestasis, and infants can present with hepatomegaly. Hence, overlap exists to some extent between the clinical manifestations of CF and MLS. Further analysis of this interesting question is limited by lack of detailed assessment of specific, pertinent features in the medical literature, such as respiratory function in MLS, and the heterogeneity of the genetic and clinical abnormalities known to occur in each condition.

HYPOTHESIS

Review of experimental data about arylsulfatase B activity and about accumulation of sulfated glycosaminoglycans in CF leads to the hypothesis that deficiency of arylsulfatase B activity may be involved in the pathogenesis of CF. These data include the following: (1) diminished activity of arylsulfatase B in lymphoid cell lines of patients with CF; (2) disease manifestations of CF associated with accumulation of sulfated glycosaminoglycans, including chondroitin 4-sulfate and DS, that are metabolizable by arylsulfatase B; (3) frequent infection in CF by pseudomonas, which has sulfatase activity, and may utilize the sulfated glycosaminoglycans that can accumulate when arylsulfatase B activity is reduced; (4) infrequent infection with Mycobacterium tuberculosis, which lacks sulfatase activity but has a glycolipid sulfotransferase that requires sulfate; (5) associations between S[O.sub.4]- and Cl- and S[O.sub.4]- and Na+ exchange that may be related to the role of CFTR in regulation of chloride movement; (6) possible relation between impaired acidification of intracellular organelles, including endosomes and lysosomes, due to defective CFTR, with subsequent reduction of arylsulfatase B activity; and (7) alteration in viscoelastic properties of respiratory secretions in association with accumulation of chondroitin 4-sulfate and DS, substrates normally metabolized by arylsulfatase B.

EXPERIMENTAL DATA

Diminished Arylsulfatase B Enzyme Activity in Lymphoid Cell Lines From Patients With CF

Assay for activity of arylsulfatase B in a lymphoid cell line of a patient with MLS demonstrated arylsulfatase B activity of 0 (activity was expressed in nanomoles of 4-nitrocatechol per milligram of protein per hour). In contrast, determinations in 10 long-term lymphoid cell lines established from normal donors had activity ranging from 15.8 to 31.8, with mean [+ or -] SD of 25.2 [+ or -] 5.6. In two lymphoid cell lines established from patients with CF, arylsulfatase B activity was 11.5 to 14.3 (mean, 12.9). Data for arylsulfatase A for the three groups overlapped. (10) In general, other reports have reported somewhat lower values for sulfatase activity in CF patients than the normal subjects, but not distinguished among the different sulfatases. (22-25)

Increased Sulfated Glycosaminoglycans in CF

Oversulfation of glycoconjugates synthesized by CF epithelial cells of lung, pancreas, and other organs has been noted for many years and is recognized as central to the pathophysiology of CF. (26-31) With regard to the histologic findings in cultured skin fibroblasts from patients with CF, Wiesmann and Neufeld (22) wrote in 1970 that "the observations are sufficiently similar to data obtained on fibroblasts from patients affected with mucopolysaccharidoses to suggest that some disturbance of mucopolysaccharide metabolism might be a primary defect in cystic fibrosis." Disease severity in CF has been associated with increased concentrations of the highly sulfated mucin component of respiratory secretions. (32)

Investigators in the 1960s demonstrated metachromatic granules in fibroblasts from patients with CF. (33) Acid mucopolysaccharides in cultured skin fibroblasts of patients with CF have been found to be as much as 8 to 10 times higher than in normal control subjects and have included chondroitin sulfate (CS) and DS. (27) The metachromatic cytoplasmic granules seen in cultured fibroblasts from patients with CF of the pancreas were lysosomal by electron microscopy. (34) The changes seen in the CF cells appeared to be quantitative, with larger and more numerous lysosomes than control cells. Cultured lymphoid cells from patients with CF were observed to grow in clumps, as did Hurler and Hunter syndrome cells, in contrast to cultures of normal cells, and DS was observed to be increased. (29) Increased sulfation was found in nonpurulent tracheobronchial secretions of patients with CF, compared to patients with chronic bronchitis. (31)

Some studies have not demonstrated increase in glycosaminoglycans, but were not designed to identify N-acetylgalactosamine 4-sulfate-containing substances, such as chondroitin 4-sulfate or DS. For example, comparison of uronic acid levels (33) or [sup.3]H-glucosamine levels (24,35) would not be expected to demonstrate deficiency of arylsulfatase B activity.

High-molecular-weight glycoconjugates identified in viscous mucoid secretions in intrahepatic biliary epithelial cells were identified and compared between cells from patients with CF and normal control subjects. The high-molecular-weight glycoconjugates were identified as CSs, demonstrating that secretion of CS was markedly increased in the CF biliary epithelial cells compared to the normal. Since CS contains N-acetylgalactosamine 4-sulfate residues, as does DS, this is consistent with the hypothesis that diminished activity of arylsulfatase B may be associated with accumulation of these sulfated compounds in CF. (36)

In sputa from patients with CF, CS was identified in 11 of 13 samples from patients with CF vs 1 of 12 samples from noninfected patients with a history of chronic bronchitis. This study utilized infected control subjects in order to assess whether the presence of specific glyeosaminoglycans was related to CF or to infection. CS was found to be predominantly associated with the CF sputa. (37)

In a CFTR knock-out mouse model of CF, liver and pancreas of CF mice incorporated higher amounts of [sup.35]S into glycosaminoglycans than the control mice. Liver and ileum incorporated significantly more total sulfate, and increased incorporation was also seen in ileum, jejunum, colon, cecum, spleen, trachea, and gall bladder. In this model, CFTR influenced glycoconjugate sulfation in a manner consistent with the hypothesis of diminished arylsulfatase B activity. Measurement of specific activity of [[sup.35]S]Sulfate in the 4-sulfate disaccharides of CS and DS demonstrated a clear and consistent trend for ileum, cecum, colon, jejunum, liver, spleen, lung, and pancreas, to have a higher mean specific activity of [[sup.35]S]Sulfate incorporation in the 4-sulfate disaccharide from the CF organs than the normal organs. This was not observed in trachea or nasal mucosa. (38)

Cheng et al (39) found in primary cultures of CF cells that the respiratory epithelial cells regulated abnormally the sulfate content of the high-molecular-weight glycoconjugates, and that the [sup.35]S[O.sub.4]/[sup.3]H labeling ratio in CF was greater than in the normal cells or disease control cells. This suggested that oversulfation of a spectrum of high-molecular-weight glycoconjugates was a genetically determined characteristic of CF epithelial cells. (39)

In a CF pancreatic adenocarcinoma cell line of ductal origin from a [DELTA]F508 homozygous patient, glycosaminoglycans synthesized by the CF cells exhibited twofold higher [sup.35]S sulfate: [sup.3]H glucosamine ratios than the control cell line. Although differences in the extent of sulfation of glycosaminoglycans were not detected in this experiment, N-acetylgalactosamine 4-sulfate or disaccharides containing it were not measured. (35)

In a human bronchial xenograft model, the extent of sulfation of secreted mucus glycoproteins was compared between CF and non-CF xenografts. The results demonstrated a significantly higher level of sulfation in the xenografts from the patients with CF. In the bronchial xenograft, primary cultures of human bronchial airway epithelial cells are seeded onto denuded rat tracheas and transplanted into athymic mice. This represents an in vivo differentiated mucociliary airway epithelium and provides an opportunity to generate genetically defined epithelium, free of the effects of bacterial infection and secondary changes related to surface epithelial cell remodeling, such as goblet-cell remodeling. Variation was seen in the extent of mucous sulfation in the CF and control groups studied, but only mucin in excess of 5 million dalton molecular weight was isolated. (40)

CF has been associated with infertility, related to obstructive azoospermia with accumulation of tenacious secretions in the epididymis and vas deferens. It is interesting to note that arylsulfatase B has been found in both seminal plasma and extracellular fluids of the testis and appeared to be secreted by the seminal vesicles in veterinary studies with boars. (41) Human seminal plasma has demonstrated significantly lower activity of arylsulfatase in infertile men than normal subjects. (42) This relationship has not been examined in patients with CF.

Association Between Infection With Pseudomonas and Deficiency of Arylsulfatase B

Pseudomonas organisms, traditionally a source of colonization and infections in patients with CF, appear to possess a glyeosulfatase or carrageenase enzyme that digests sulfated polygalactose molecules. (43) Mucin-sulfatase activity of Pseudomonas aeruginosa and Burkholderia cepacia was determined and found to be present in nine of nine B cepacia strains tested and four of six Pseudomonas strains. The arylsulfatase activities of the Pseudomonas isolates were higher than those of the B cepacia strains? If there is endogenous deficiency of arylsulfatase B activity, sulfated polysaccharides can accumulate, and the environment might favor bacteria, such as Pseudomonas, that can utilize them.

Studies of circulating monocytes in patients with MPS VI (MLS) have demonstrated prominent vacuolar lysosomal cytosomes in CD14+ monocytes, in contrast to findings in age- and sex-matched control subjects. Since the circulating monocytes may be a replacement pool for tissue macrophages, abnormalities in their lysosomes may affect ability to resolve infection. (45) When normal adult neutrophils were preincubated with monoclonal antibodies to CD14, phagocytosis of P aeruginosa strain 808 was impaired. Of 10 different strains of P aeruginosa, only 5 strains were ingested after interaction with CD14 antibodies. Hence, impairment in lysosomal activity in CD14+ monocytes may be related to persistent infection with some strains of P aeruginosa. (46) This finding suggests a possible relation between the lysosomal defect seen in monocytes from patients with arylsulfatase B deficiency and proclivity to persistent infection with some strains of P aeruginosa.

Difficulty in clearing P aeruginosa due to impaired phagocytosis could be a factor in the resistance to antibiotics that Pseudomonas develops in the CF lung. It has been reported that the resistant form of P aeruginosa more readily makes biofilms, which may promote antibiotic resistance. (47)

Association Between Viscoelastic Properties of Respiratory Secretions and Increased DS and Chondroitin 4-Sulfate

The viscosity of respiratory secretions in patients with CF has been a subject of debate in the literature. (48-51) Analysis of the viscoelastic properties has been complicated often by the presence of intercurrent infection that may alter the innate properties of the secretions.

Measurements of viscosity of fresh submucosal gland secretions from CF human airways obtained at lung transplantation indicated a twofold increase in viscosity, compared to gland fluid from normal human airways. (52) Since the submucosal gland secretions form part of the airway surface liquid, abnormalities in these secretions may contribute to the pathophysiology of CF, including impaired mucociliary clearance and antimicrobial defense. Although only a linear diffusion was measured, Jayaraman et al (52) noted that the secretions from the CF airways were more sticky, suggesting differences in other properties, such as elasticity and adhesiveness. Since the submucosal gland cells exceed the goblet cells in production of mucus by 40:1, (49) their product contributes significantly to the respiratory secretions, and mucus accumulation due to increased viscosity of the submucosal gland products can contribute to airway obstruction and chronic infection.

Hyaluronic acid is found in pulmonary secretions of patients with CF (53) and is secreted by airway submucosal glands. (54) CS (combined 4-sulfate and 6-sulfate) was shown to markedly increase the viscosity of hyaluronan solutions under physiologic conditions of pH, temperature, ionic strength, and concentrations ranging from 0.5 to 40 mg/mL. (55) These findings suggest that the increase in CS such as would result from deficiency of arylsulfatase B would lead to an increase in the viscosity of submucosal gland secretions, consistent with the observed changes in the viscoelastic properties of secretions in CF.

Association Between CF and Reduced Incidence of M tuberculosis Infections

Several sources have reported the reduced occurrence of M tuberculosis infections in patients with CF. (56-60) It has been hypothesized that the CF genetic defect is responsible for this and confers protection against tuberculosis infection. Reduced incidence of tuberculosis has been reported in parents (obligate heterozygotes) of patients with CF. (61-63)

The [DELTA]F508, the principal CF mutation, varies between population groups and appears to be higher in northwest Europeans than in southeast European populations. (64-65) This distribution is consistent with the higher incidence of M tuberculosis infections in central and eastern Europe (66) than in western Europe. Interestingly, two virulent mycobacterium strains, Mycobacterium fortuitum and Mycobacterium cheilosei, are identifiable by their ability to produce arylsulfatase, in contrast to M tuberculosis, which does not produce arylsulfatase. (67-68)

Recently, gene Rv 1373 of M tuberculosis has been found to encode a novel cytosolic arylsulfotransferase with 24% homology to eucaryotic aryl-sulfotransferases, including the highly conserved sulfotransferase signature sequences that are involved in 3'-phosphoadenosine-5'-phosphosulfate binding and transfer of sulfate. (69-71) This newly identified M tuberculosis sulfotransferase may be involved in production of the sulfated glycolipids of the mycobacterial cell wall. Sulfolipid content of the mycobacterial cell wall has been correlated with virulence. An attenuated form of M tuberculosis (H37Ra) lacks sulfolipid I, whereas it is abundant in the virulent strains H37Rv and Erdman. The M tuberculosis strains have greater expression of several sulfolipids than the nonpathogenic strains. (69)

The survival of M tuberculosis is related to its ability to replicate within the phagosome of host macrophages. (72) Prevention of lysosomal fusion with the phagosome appears to affect survival of M tuberculosis, and sulfation of cell wall glycolipids has been associated with pathogenicity. (73) If sulfotransferase activity is critical to formation of sulfolipid components of the cell wall of virulent mycobacteria, then diminished lysosomal arylsulfatase activity may be a useful defense mechanism, limiting the supply of sulfate available for cell wall biosynthesis through the 3'-phosphoadenosine-5'-phosphosulfate intermediate. (74)

Considering the relationship between mycobacteria and lysosomes as paradoxical, Brown et al (75) wrote, "It may be postulated that the lysosomal enzymes, which apparently fail to inhibit mycobacterial growth, actually provide low molecular weight nutrients beneficial for the growth of these [M tuberculosis and Mycobacterium lepraemurium] two chronic intracellular pathogens." Diminished arylsulfatase B activity in lysosomes of cells of patients with CF may have evolved as a mechanism to retard mycobacterial cell wall biosynthesis by reducing the availability of sulfate, thereby diminishing the potential for proliferation of M tuberculosis.

Sulfate Ion and Cl- and Na+ Exchange

A sulfate/chloride antiporter that is a membrane anion exchanger has been described in the mutations associated with the skeletal dysplasias known as achondrogenesis 1B, atelosteogenesis 2 diastrophic dysplasia, and multiple epiphyseal dysplasia. (76) The antiporter has been designated as the diastrophic dysplasia sulfate transporter. The exchanger appears to bring S[O.sub.4]- into cells in exchange for Cl-. The sulfate transport mechanisms appear to function as either sulfate/chloride antiporters or anion exchangers or as sodium/sulfate cotransporters. (77)

Although the human S[O.sub.4]/Cl antiporter gene has been identified, a coordinated model of sulfate metabolism and possible relationship with CFTR has not been considered. However, we hypothesize that disruption of the human S[O.sub.4]/Cl antiporter due to reduced production of S[O.sub.4] because of arylsulfatase B deficiency may have an influence on other Cl-transport mechanisms.

The diagnosis of CF has been made in at least one case with a pathologic sweat test and clinical features, but without abnormality of the CFTR gene and unremarkable intestinal current measurement or nasal potential difference. (78) Hence, it is possible that manifestations consistent with the diagnosis of CF may arise without abnormality of the CFTR gene.

Arylsulfatases A and B have been reported to be inhibited by sulfate and phosphate and stimulated by chloride ions. (79) Another report indicated that arylsulfatase B from horse leukocytes was inhibited by chloride, sulfate, sulfite, and silver ions. (80) Therefore, impaired chloride secretion due to abnormality of CFTR might lead to alteration of intracellular arylsulfatase B activity, although it is unclear if this would be associated with increased or reduced activity.

Role of CFTR in Acidification of Intracellular Organelles

CFTR, by acting as an intracellular chloride channel, has been associated with function in the trans-Golgi network and in endosomes. Mutations in the CFTR have been associated not only with plasma membrane Cl- channels, but also defective acidification of intracellular organelles that are involved in the intracellular transport of Cl-. (4) Impaired acidification in CF cells of the trans-Golgi network, of prelysosomes, and of lysosomes would be anticipated to be associated with reduced activity of arylsulfatase B, since arylsulfatase B functions optimally at pH 5.6. (8)

Some reports have suggested that trans-Golgi, endosomal, and lysosomal pH were not regulated by CFTR. (81-84) Conflicting reports with functional and immunolocalization studies have demonstrated the presence of CFTR in the endosomal compartment. (3,85)

SUMMARY

Several lines of evidence converge to suggest that there may be a role for arylsulfatase B deficiency in the clinical manifestations of CF. These are summarized in Table 2. Table 3 summarizes some of the pertinent research questions that emerge from consideration of this hypothesis.

The observations demonstrating reduced activity of arylsulfatase B and possible relation to impairment in metabolism of sulfated glycosaminoglycans require confirmation and elaboration. It is possible that the alterations in the mucus of patients with CF and the well-characterized defects in chloride transport may be associated with deficiency of arylsulfatase B activity. Increased levels of chondroitin 4-sulfate and DS, which are substrates for metabolism by arylsulfatase B, may be responsible for alterations in the characteristics of the respiratory secretions in patients with CF. Today, we have available the tools to answer questions raised by review of previous work.

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* From the Department of Internal Medicine, University of Iowa Health Care, Iowa City, IA.

Manuscript received March 18, 2002; revision accepted August 2, 2002.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

Correspondence to: Joanne K Tobacman, MD, Department of Internal Medicine, University of Iowa, Iowa City, IA 52242-1081; e-mail: joanne-tobacman@uiowa.edu

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