Structure and distribution of an Alu-type deletion mutation in Sandhoff disease.

Sandhoff disease is a recessively inherited lysosomal storage disease resulting from a deficiency of beta-hexosaminidase activity. The enzyme occurs in two major forms, beta-hexosaminidase A, composed of an alpha- and beta-subunit and beta-hexosaminidase B, composed of two beta-subunits. Both isozyme activities are deficient in Sandhoff disease, owing to mutations of the HEXB gene encoding the common beta-subunit. We have cloned and fully characterized a deletion at the HEXB gene from fibroblasts of a patient with the infantile form of Sandhoff disease. The deletion removes approximately 16 kb of DNA including the HEXB promoter, exons 1-5 and part of intron 5. It most likely arose from recombination between two Alu sequences, with the breakpoints occurring at the midpoint between the left and right arms in each case and regenerating an intact Alu element in the deletion sequence. The deletion allele accounts for 27% of the Sandhoff mutant alleles we analyzed. Two cell lines were shown to be homozygous for the deletion and both had the infantile form of the disease. Four additional patients were compound heterozygotes with other mutations, all of whom displayed a different clinical phenotype. Finally, the mutant allele was present in different ethnic backgrounds, suggesting that it may have been subject to genetic drift.

Characterization of polypeptides serologically and structurally related to hexosaminidase in cultured fibroblasts.

Human fibroblasts synthesize several polypeptides that assort into the various forms of hexosaminidase (hex). We report here the occurrence of three newly identified, hexosaminidase-related polypeptides resolved by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of immunoprecipitates from [35S]methionine-labeled cell extracts. These polypeptides, called band 2 (75,000), band 3 (70,000), and band 4 (63,000), were immunoprecipitated by an antiserum specific to placental hex I2. They are distinct from pre-alpha- (60,000) and pre-beta- (58,000) precursor polypeptides and the alpha- (56,000), beta a- (27,000), and beta b- (27,000) polypeptides of the mature hex A (alpha beta a beta b) and hex B (2[beta a beta b]). When fibroblast extracts were chromatographed on DEAE-Sepharose, bands 2, 3, and 4 were eluted together in fractions before hex A, in a position characteristic of serum and placental hex I2 and serum hex P. This suggests that bands 2, 3, and 4 might represent the polypeptides of a fibroblast hex I. The analysis of partial proteolytic digests of the radioactively labeled polypeptides revealed that bands 2 and 3, pre-beta, and beta a had several peptides in common, suggesting that they are structurally related to each other. However, bands 2, 3, and 4 were present in extracts of Tay-Sachs (pre-alpha and alpha deficiency) and Sandhoff cells (pre-beta, beta a, and beta b deficiency) and appeared later than pre-beta in pulse-chase experiments. These results suggest that bands 2 and 3 occur independently of pre-beta and beta a and are probably specified by different mRNA, whether from the same gene or distinct but homologous genes.

An unusual splicing mutation in the HEXB gene is associated with dramatically different phenotypes in patients from different racial backgrounds.

Sandhoff disease is caused by mutations affecting the beta subunit of lysosomal beta-hexosaminidase (EC 3.2.1.52) and displays a wide spectrum of clinical phenotypes. We report a 57-year-old patient with a very mild phenotype, although residual hexosaminidase A activity in his cultured fibroblasts was less than 3% of normal activity, a level observed in juvenile onset patients. Northern and Western blot analyses confirmed a similar low level of beta subunit-mRNA and mature beta-protein, respectively. Two mutations of the HEXB gene were identified in this patient, a partial 5' gene deletion (a null allele), and a C----T transition 8 nucleotides downstream from the intron 10/exon 11 junction affecting the splicing of the beta subunit-mRNA. In their homozygous forms, the 5' deletion has been previously shown to result in a severe infantile phenotype, and the C----T transition in a juvenile phenotype. The genotype and the low level of residual hexosaminidase A activity would be expected to produce a juvenile Sandhoff phenotype in this patient, as well as in four of his six clinically normal siblings. The biochemical basis of his mild phenotype is uncertain, but may result from genetic variations in the RNA splicing machinery.

Biochemical consequences of mutations causing the GM2 gangliosidoses.

The hydrolysis of GM2-ganglioside is unusual in its requirements for the correct synthesis, processing, and ultimate combination of three gene products. Whereas two of these proteins are the alpha- (HEXA gene) and beta- (HEXB) subunits of beta-hexosaminidase A, the third is a small glycolipid transport protein, the GM2 activator protein (GM2A), which acts as a substrate specific co-factor for the enzyme. A deficiency of any one of these proteins leads to storage of the ganglioside, primarily in the lysosomes of neuronal cells, and one of the three forms of GM2-gangliosidosis, Tay-Sachs disease, Sandhoff disease or the AB-variant form. Studies of the biochemical impact of naturally occurring mutations associated with the GM2 gangliosidoses on mRNA splicing and stability, and on the intracellular transport and stability of the affected protein have provided some general insights into these complex cellular mechanisms. However, such studies have revealed little in the way of structure-function information on the proteins. It appears that the detrimental effect of most mutations is not specifically on functional elements of the protein, but rather on the proteins' overall folding and/or intracellular transport. The few exceptions to this generalization are missense mutations at two codons in HEXA, causing the unique biochemical phenotype known as the B1-variant, and one codon in both the HEXB and GM2A genes. Biochemical characterization of these mutations has led to the localization of functional residues and/or domains within each of the encoded proteins.

The GM2 activator protein, its roles as a co-factor in GM2 hydrolysis and as a general glycolipid transport protein.

Although there is only one documented function carried out by the GM2 activator protein in the lysosome, new information suggests that other less obvious roles may also be played by this protein in vivo. This information includes data demonstrating that the GM2 activator is a secretory, as well as a lysosomal protein, and that cells possess a carbohydrate-independent mechanism to re-capture the activator, with or without bound lipid, from the extracellular fluid. Additionally the GM2 activator has been shown to bind, solubilize and transport a broad spectrum of lipid molecules, such as glycolipids, gangliosides and at least one phosphoacylglycerol, between liposomes. At pH 7 the GM2 activator's rate of lipid transport is reduced by only 50% from its maximum rate which is achieved at approx. pH 5, suggesting that the GM2 activator may serve as a general intra- and/or inter-cellular lipid transport protein in vivo. Since the late 1970s the lysosomal form of the GM2 activator has been known to act as a substrate-specific co-factor for the hydrolysis of GM2 ganglioside by beta-hexosaminidase A. Gangliosides are a class of negatively charged glycolipids particularly abundant in neuronal cells which have been linked to numerous in vivo functions, such as memory formation and signal transduction events. Deficiency of the GM2 activator protein results in the storage of GM2 ganglioside and severe neurological disease, the AB-variant form of GM2 gangliosidosis, usually culminating in death before the age of 4 years. The exact mode-of-action of the GM2 activator in its role as a co-factor, and its specificity for various glycolipids are currently matters of debate in the literature.

Two small deletion mutations of the HEXB gene are present in DNA from a patient with infantile Sandhoff disease.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes hexosaminidase A (alpha beta) and B (beta beta). The alpha subunit is encoded by the HEXA gene and the beta subunit by HEXB gene. Defects in the alpha or beta subunits lead to Tay-Sachs or Sandhoff disease, respectively. While many HEXA gene mutations have been reported only three HEXB gene mutations are known. We report the characterization of two rare HEXB mutations present in genomic DNA from a single fibroblast cell line, GM203, taken from a patient with the infantile form of Sandhoff disease. The first is a single base pair deletion in exon 7 changing the codon for Gly-258, GGA, to GA and the second, a two base pair deletion in exon 11 changes the codons for Arg-435/Val-436, AGA/GTC, to AGTC. Each mutation produces a frame shift in the affected allele that results in a premature stop codon 17 or 20 codons downstream, respectively. These mutations also result in the inability to detect beta-mRNA by Northern blot analysis of total mRNA. These data are consistent with the idea that the severe infantile form of Tay-Sachs or Sandhoff disease is associated with a total lack of residual hexosaminidase A activity.

Characterization of the affinity of the G(M2) activator protein for glycolipids by a fluorescence dequenching assay.

The G(M2) activator protein is a substrate specific cofactor for degradation of G(M2) ganglioside by lysosomal beta-hexosaminidase A. Mutations in the gene encoding the activator result in the AB-variant form of G(M2) gangliosidosis. The activator protein contains at least three functional elements; a hydrophobic binding pocket, an oligosaccharide binding site(s), and an area that interacts with hexosaminidase A. In this report a fluorescence dequenching assay specific for only the hydrophobic binding pocket is evaluated and optimized. It is shown that various glycolipids inhibit the transport between liposomes of a self-quenching fluorescent lipid probe, octadecylrhodamine, by the activator protein. The level of inhibition produced by each glycolipid is then used to characterize the oligosaccharide-binding specificity of the activator. The fluorescence dequenching assay is also used to evaluate the functionality of a truncated form of the activator protein. Our results indicate that this simple assay can be used to determine structure-function relationships within the normal or mutant forms of the activator. The data suggest that the C-terminus of the activator is required to produce a functional hydrophobic binding pocket.

Significance of two point mutations present in each HEXB allele of patients with adult GM2 gangliosidosis (Sandhoff disease) homozygosity for the Ile207-->Val substitution is not associated with a clinical or biochemical phenotype.

The molecular defects in the HEXB gene encoding the common beta-subunit of lysosomal beta-hexosaminidase A (beta-Hex A, alpha beta) and beta-Hex B (beta beta) were investigated in a Portuguese family affected with late onset Sandhoff disease (GM2-gangliosidosis variant 0). This family comprised two unaffected daughters and three affected sibs who developed at about age 17 cerebellar ataxia and mental deficiency. Their parents were consanguineous and clinically asymptomatic. There was no detectable beta-Hex B activity and a profound reduction in the activity of beta-Hex A in the leukocytes and transformed lymphoid cell lines from the affected sibs. The expected intermediate values were observed in the parents as well as in one daughter and her children. Western analysis revealed the presence of reduced, but detectable amounts of mature beta-chain protein in cell lysates from the probands and intermediate levels in the parents. Nucleotide sequencing of amplified, reverse-transcribed beta-chain mRNA demonstrated the presence of two single point mutations: an A619 to G transition in exon 5 (Ile207-->Val), and a G1514 to A transition in exon 13 (Arg505-->Gln). Both of these two mutations have been previously linked to the adult form of Sandhoff disease in compound heterozygote patients. All three affected sibs were found to be homoallelic for both mutations. Interestingly, while the mother was heterozygous for each mutation, the father was homozygote for the A619-->G substitution and heterozygote for the G1514-->A transition. Since the father is homozygote for the A619-->G mutation but expresses a biochemical phenotype consistent with a carrier of Sandhoff disease and is clinically asymptomatic, this substitution is likely a neutral mutation. We confirmed this hypothesis by finding this transition present in 4 of 30 alleles from normal individuals. We conclude that homozygosity for the G1514-->A mutation is exclusively responsible for the adult form of Sandhoff disease in this family, and that the A619-->G substitution is not a deleterious mutation but rather a common HEXB polymorphism.

Characterization of two HEXB gene mutations in Argentinean patients with Sandhoff disease.

Beta-hexosaminidase A (beta-N-acetyl-D-hexosaminidase, EC 3.2.1.5.2) is a lysosomal hydrolase composed of an alpha- and a beta-subunit. It is responsible for the degradation of GM2 ganglioside. Mutations in the HEXB gene encoded beta-subunit cause a form of GM2 gangliosidosis known as Sandhoff disease. Although this is a rare disease in the general population, several geographically isolated groups have a high carrier frequency. Most notably, a 1 in 16-29 carrier frequency has been reported for an Argentinean population living in an area contained within a 375-km radius from Córdoba. Analysis of the genomic DNA of two patients from this region revealed that one was homozygous for a G to A substitution at the 5' donor splice site of intron 2. This mutation completely abolishes normal mRNA splicing. The other patient was a compared of the intron 2 G-->A substitution and a second allele due to a 4-bp deletion in exon 7. The beta-subunit mRNA of this allele is unstable, presumably as a result of an early stop codon introduced by the deletion. Two novel PCR-based assays were developed to detect these mutations. We suggest that one of these assays could be modified and used as a rapid screening procedure for 5' donor splice site defects in other genes. These results provide a further example of the genetic heterogeneity that can exist even in a small geographically isolated population.

Beta-hexosaminidase: biosynthesis and processing of the normal enzyme, and identification of mutations causing Jewish Tay-Sachs disease.

OBJECTIVES: This report presents an overview of the nearly 100-year history of the study of Tay-Sachs disease in the Ashkenazi Jewish population. DESIGN AND METHODS: Each major step leading to our present understanding of the disease are highlighted. RESULTS: The original interest in the cause of this devastating disease in the late 1800s led to the identification of a novel glycolipid. GM2 ganglioside, stored in the neurons of Tay-Sachs patients in the 1930s, and the elucidation of its structure in the 1960s. The identification of the defective isozyme, beta-hexosaminidase A, followed in 1968-69. Elucidation of the subunit structures of the hexosaminidase A (alpha beta) and B (beta beta) isozymes in 1973 and their purification in 1974-80, led to the characterization of the biosynthesis, assembly, intracellular transport, and posttranslational processing of the two subunits in the 1980s. The ability to purify milligram quantities of the isozymes made possible the isolation of cDNA clones encoding both subunits in 1985, and ultimately the identification of the causes of Jewish Tay-Sachs disease at the genomic DNA level in 1988. CONCLUSIONS: Tay-Sachs disease is the major model for lysosomal storage diseases. Similarly, the work done in the 1980s on hexosaminidase has been used as a model for understanding the cell biology of many other lysosomal proteins. Current research encompassing the fields of enzymology, cell biology, and molecular biology is linking genotypes with the clinical phenotypes of patients with Tay-Sachs and related diseases.

The molecular basis of Tay-Sachs disease: mutation identification and diagnosis.

Tay-Sachs disease is the prototype of lysosomal storage disease. While it was first described over a century ago, the defective enzyme was not identified until 1969, making possible the development of enzyme-based diagnostic and carrier screening techniques. This led to the establishment of the successful international Tay-Sachs screening program, primarily for the high risk Ashkenazi Jewish population. In the past five years the development of recombinant DNA technology has allowed researchers to characterize 95-99% of the mutations causing Tay-Sachs disease in this high risk ethnic group. Knowledge of the exact mutations responsible for the disease coupled with the powerful polymerase chain reaction technique has now made DNA-based screening and diagnosis possible. While the enzyme-based test has proven to be reliable and economical, it cannot differentiate variant phenotypes and requires the presence of specialized testing centers. Although the DNA-based test is presently less economical, it can provide carrier couples with their exact genotype and thus, predict the general phenotype of an unborn child. Furthermore, as the catalogue of mutations leading to human disease increases, more economical DNA methodologies will be developed. In the future it would be expected that a laboratory using a single DNA-based technology could diagnose and screen for a myriad of human diseases including Tay-Sachs disease.

Adult onset motor neuronopathy in the juvenile type of hexosaminidase A and B deficiency.

Two sisters presented with progressive muscle cramps, as well as wasting and weakness of the legs with onset after age 20. They also showed intention tremor of the upper extremities and dysarthria starting during the first decade. The older patient also had fasciculations; the younger, hyperreflexia. Total plasma beta-hexosaminidase (Hex) activity with 4-methylumbelliferyl-acetyl-glucosamine as substrate was reduced to 1.4% and 2.7% of the control in the 2 patients, respectively. Hex A activity measured by 4-methylumbelliferyl-N-acetylglucosamine-6-O-sulphate as substrate was 9.9% and 12.8% of the mean control value in the 2 patients, respectively. Hex B activity was undetectable in both patients. Leukocyte total Hex activity was 7-8% of normal; residual Hex A activity in the 2 patients was 17.8% and 16.3% of normal controls, respectively. Fibroblastic residual Hex A activity in the 2 patients was 9.6% and 22% of normal mean value, respectively. Appendiceal ganglion cells contained membranous cytoplasmic bodies in the younger patient. Thin layer chromatography of the appendiceal extract from one patient (III/2) showed a marked increase of GM2 ganglioside, and some increase of GM3 ganglioside. Northern blots performed on fibroblast cell lines from both patients for the demonstration of alpha and beta locus messenger RNA showed no difference between patients and control. These patients have a rare form of adult-onset progressive motor neuron disease presumably due to abnormal beta subunits, causing severe deficiency of both Hex A and Hex B. The phenotypic expression of this disease is similar to motor neuron disease due to alpha locus mutations, which suggests that the Hex A deficiency, even though only a partial one, may be the important pathogenic factor.

Biochemistry and genetics of Tay-Sachs disease.

Tay-Sachs disease is one of the few neurodegenerative diseases of known causes. It results from mutations of the HEXA gene encoding the alpha subunit of beta-hexosaminidase, producing a destructive ganglioside accumulation in lysosomes, principally in neurons. With the determination of the protein sequence of the alpha and beta subunits, deduced from cDNA sequences, the complex pathway of subcellular and lysosomal processing of the enzyme has been determined. More recently, detailed knowledge of the gene structure has allowed the determination of specific mutations causing Tay-Sachs disease. The high incidence of the disease in Ashkenazi Jews is attributed predominantly to three mutations present in high frequency, while in non-Jews some two dozen mutations have been identified thus far. The cataloguing of mutations has important implications for carrier screening and prenatal diagnosis for Tay-Sachs disease.

Characterization of human placental beta-hexosaminidase I2. Proteolytic processing intermediates of hexosaminidase A.

The lysosomal hydrolase beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) exists as two major isozymes in normal human tissue: an acidic A-form and a basic B-form. There are also minor forms of intermediate pI known as I-forms. Increases in one or more of these intermediates have been associated with various disease states. Although the two major isozymes have been extensively studied, the structure and biosynthetic origins of the I-forms are unknown. Characterization of a placental hexosaminidase I-form, presented in this report, demonstrates that it is composed of two forms of partially processed hexosaminidase A. The major form contains an intact pro-alpha chain and a pro-beta chain lacking 2 residues from its amino terminus (Ala and Arg). The minor form also contains an alpha and a beta subunit, but each has undergone further proteolytic processing. The amino terminus of each of these partially processed polypeptide chains matches one of those previously found on stable processing intermediates in a single normal human fibroblast cell line. These data confirm that similar processing intermediates exist in human placenta, suggesting that this I-form lacks a unique enzymatic function in vivo. A sequence of normal proteolytic processing events is postulated.

Active arginine residues in beta-hexosaminidase. Identification through studies of the B1 variant of Tay-Sachs disease.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isoenzymes, hexosaminidases A (alpha beta) and B (beta beta). The alpha- and beta-subunits are encoded by the HEXA and HEXB genes, respectively. Extensive homology in both the gene structures and deduced primary sequences demonstrate their common evolutionary origin. Defects in the alpha- or beta-subunits lead to Tay-Sachs of Sandhoff disease, respectively. The B1 variant of Tay-Sachs disease is characterized by an unusual phenotype. Patient samples contain both isoenzymes; however, hexosaminidase A lacks activity toward alpha-specific substrates. In a previous report, we analyzed the biochemical consequences of an Arg178----His substitution in the alpha-subunit, causing the B1 phenotype, by in vitro mutagenesis of the homologous codon for Arg211 in the beta-subunit to produce His. We found that the substitution did not affect dimer formation or cellular targeting but caused a near total loss of activity toward a common alpha- and/or beta-substrate. Additional effects were also noted that suggested a perturbation had occurred to the protein's secondary structure. In this report, we investigate further the role of Arg in the catalysis of hexosaminidase substrates. The introduction of more or less conservative amino acid substitutions at the beta-Arg211 site were evaluated in terms of their effects on the protein's catalytic activity and susceptibility to the arginine-specific reagents and on its stability and rate of maturation in the cell's lysosome. These data demonstrate that the changes in the in vivo stability and rate of maturation, previously noted with the Arg211----His substitution, are independent of the loss in enzymatic activity. Whereas treatment of purified normal human placental hexosaminidases A and B with arginine-specific modifying reagents produced a time-dependent loss of enzymatic activity toward both alpha-specific and common substrates, these reagents failed to significantly decrease the residual activities of mutant proteins lacking Arg at position 211. Kinetic analysis of the residual enzyme activity from our most conservative construct, Arg211----Lys, determined an apparent Vmax approximately 400-fold reduced from that of the wild type enzyme but detected no change in the apparent Km. Additionally, the pH optimum of this mutant enzyme was narrower and slightly more basic than that of the normal enzyme. Thus, Arg211 in the beta-subunit and, by extrapolation, the Arg178 in the alpha-subunit of beta-hexosaminidase are "active" residues, i.e. part of the catalytic sites, but do not participate in substrate binding.

beta-Hexosaminidase isozymes from cells cotransfected with alpha and beta cDNA constructs: analysis of the alpha-subunit missense mutation associated with the adult form of Tay-Sachs disease.

In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the alpha-subunit of beta-hexosaminidase A (alpha beta) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of alpha-chain mutations is not straightforward. We examine three approaches utilizing previously identified mutations affecting alpha-chain folding. These involve transfection of (1) the alpha cDNA alone; (2) a beta cDNA construct encoding a beta-subunit substituted at a position homologous to that of the alpha-subunit, and (3) both alpha and beta cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an "active" alpha Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. We conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some alpha-chain mutations. Using this technique, we demonstrate that the adult-onset Tay-Sachs mutation, alpha Gly --> Ser269, does not directly affect alpha beta dimerization but exerts an indirect effect on the dimer through destabilizing the folded alpha-subunit at physiological temperatures. Two other alpha mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit.

A Cys138-to-Arg substitution in the GM2 activator protein is associated with the AB variant form of GM2 gangliosidosis.

The AB-variant form of GM2 gangliosidosis is an inherited lysosomal storage disease. Biochemical data have linked its cause to the lack of a functional GM2 activator protein (activator). In the present study we identify a mutation in the gene encoding the activator protein of an AB-variant patient. These data represent direct evidence that the disease in the patient described here is a result of mutations at the Activator gene locus. A T412----C transition was found in the homozygous form in cDNA and genomic DNA from the patient. This nucleotide change would result in the substitution of Cys138 by an Arg residue in the activator protein. Whereas the patient's fibroblasts produce apparently normal levels of activator mRNA, they lack a functional activator protein. Transfection of either a construct containing the normal activator cDNA, pAct1, or a cDNA construct containing the T----C transition caused COS-1 cells to transcribe high levels of activator mRNA. Lysates from cells transfected with pAct1 produced an elevated level of both pro- and mature forms of the activator protein, with an accompanying 11-fold enhancement in the ability of purified hexosaminidase A to hydrolyze GM2 ganglioside. However, lysates from cells transfected with the mutant cDNA construct contained only low levels of the pro-activator protein, which failed to enhance hexosaminidase A activity significantly above the endogenous level of mock transfected COS cells. We conclude that the T412----C transition in the GM2 Activator gene of the patient is responsible for the disease phenotype.

Two mechanisms for the recapture of extracellular GM2 activator protein: evidence for a major secretory form of the protein.

The GM2 activator protein is a small monomeric protein containing a single site for Asn-linked glycosylation. Its only proven in vivo function is to act as a substrate specific cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A. However, we and others have shown it can act as a general glycolipid transporter at neutral pH in vitro. Any other possible in vivo functions would require that some of the newly synthesized activator molecules not be targeted to the lysosome. The lysosomal targeting mechanism for the activator has not been conclusively identified. While earlier reports suggested that it is likely through the mannose-6-phosphate receptor, another more recent report demonstrated that deficient human cells could recapture nonglycosylated, bacterially produced activator, suggesting its use of an alternate targeting pathway. Here, we demonstrate that the mannose-6-phosphate pathway is likely the major intracellular, biosynthetic route to the lysosome, as well as a high affinity recapture pathway for the endocytosis of activator protein from extracellular fluids. Additionally, we show that there exists a second lower affinity recapture pathway that requires its native protein structure, is carbohydrate independent, and likely does not involve its ability to bind glycosphingolipids in the plasma membrane. Finally, we document that the pool of newly synthesized precursor activator protein contains a majority of molecules with a complex-type oligosaccharide, which cannot contain a functional mannose-6-phosphate targeting signal. These molecules makeup the secreted forms of the protein in normal human fibroblasts.