Molecular and biochemical analysis of protective protein/cathepsin A mutations: correlation with clinical severity in galactosialidosis
Molecular and biochemical analysis of protective protein/cathepsin A mutations: correlation with clinical severity in galactosialidosisXiao-Yan Zhou+,[sect], Aarnoud van der Spoel+, Robbert Rottier, Greg Hale{, Rob Willemsen1, Gerard T. Berry2, Pietro Strisciuglio3, Generoso Andria4 and Alessandra d'Azzo*
Department of Genetics, St Jude Children's Research Hospital, Memphis, TN 38105, USA, 1Department of Cell Biology and Genetics, Erasmus University, Medical Faculty, 3000 DR Rotterdam, The Netherlands, 2Department of Pediatrics, Division of Biochemical Development and Molecular Diseases, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA, 3Department of Pediatrics, University of Reggio Calabria, Catanzaro 88100, Italy and 4Department of Pediatrics, Federico II University, Napoli 80131, Italy
Received July 25, 1996;Revised and Accepted September 16, 1996
Mutations in the gene encoding lysosomal protective protein/cathepsin A (PPCA) are the cause of the lysosomal disorder galactosialidosis (GS). Depending on age of onset and severity of the symptoms, patients present with either an early infantile (EI), a late infantile (LI), or a juvenile/adult (J/A) form of the disease. To study genotype-phenotype correlation in this disorder, we have analyzed the mutations in the PPCA gene of eight clinically different patients. In two EI and one J/A patient, we have identified four novel point mutations (Val104Met, Leu208Pro, Gly411Ser and Ser23Tyr), that prevent phosphorylation and, hence, lysosomal localization and maturation of the mutant precursors. Two amino acid substitutions (Phe412Val and Tyr221Asn) are shared by five LI patients. These mutations appear to be pathognomonic for this phenotype, and determine the clinical outcome depending on whether they are present together or in combination with other mutations. The latter include a single base deletion and a novel amino acid change (Met378Thr), which generates an additional glycosylation site. Within the LI group, patients carrying the Phe412Val mutation are clinically more severe than those with the Tyr221Asn substitution. This is in agreement with the biochemical behavior of the Asn221-mutant protein, that is, like the Phe412Val protein, phosphorylated, routed to lysosomes and proteolytically processed, but its intralysosomal stability is intermediate between that of wild-type PPCA and Val412-PPCA. Overall, these results may explain the clinical heterogeneity observed in GS patients and may help to correlate mutant allelic combinations with specific clinical phenotypes.
Galactosialidosis (GS), an autosomal recessive lysosomal storage disease found in humans (reviewed in 1 ), is unlike the majority of these disorders in that it is associated with deficiencies of multiple hydrolases, all of which could contribute to the clinical outcome. Three phenotypic subtypes are distinguished according to age of onset and severity of symptoms: early infantile (EI), late infantile (LI) and juvenile adult (J/A). The clinical features are heterogeneous and include dysmorphism, skeletal dysplasia, visceromegaly, cardiac and renal involvement, progressive neurologic manifestations, ocular abnormalities, angiokeratoma and early death. Of particular interest is the small and well-defined group of LI patients, all of Caucasian origin. They develop symptoms within the first two years of life and their disease progression is slow and mild. Specific features of this subtype include visceromegaly, cardiac abnormalities, growth retardation and, most importantly, absence of relevant neurologic symptoms (2 -6 ).
GS is diagnosed as a combined deficiency of lysosomal [beta]-D-galactosidase and N-acetyl-[alpha]-neuraminidase (4 ,7 ,8 ). This deficiency causes lysosomal storage of sialylated oligosaccharides and glycopeptides, and, thereby, oligosacchariduria (7 ,9 -12 ). The primary genetic defect, however, lies in the gene encoding a third lysosomal enzyme, the protective protein/cathepsin A (PPCA), an acid carboxypeptidase that normally associates with the two glycosidases to create a fully functional and stable enzyme complex (13 -18 ). PPCA is active at both acidic and neutral pH and functions as cathepsin A/deamidase/esterase on selected neuropeptides, such as substance P, oxytocin and endothelin I (19 -21 ). Because its protective and catalytic activities are distinct, PPCA may act independently of the two glycosidases to catabolize specific bioactive peptides (22 ). It is not known, however, to what extent loss of its serine carboxypeptidase activity contributes to the GS clinical phenotype.
In cultured cells and tissues, the enzyme is synthesized as a 54 kDa precursor/zymogen, which is processed in lysosomes into a mature and active 32/20 kDa two-chain product (13 ,22 ,23 ). Both precursor and mature proteins homodimerize (19 ,24 ). PPCA mRNA levels differ among patients with different clinical phenotypes, as does the quality and quantity of immunoprecipitable protein (13 ,22 ,25 -27 ). Further, severely reduced or absent cathepsin A/deamidase activity has been detected in fibroblasts and tissues of patients (22 ,28 -31 ).
Clinical features of patients with galactosialidosis
Note: A blank box stands for not reported.
A recurrent mutation was identified in the PPCA gene of Japanese patients with the J/A form and mild presentation, but with mental retardation (32 -35 ): a single base substitution at the donor splice site of intron 7 causes aberrant splicing of the mRNA and the omission of exon 7 (SpDEx7). It was postulated that this is a leaky mutation, allowing a small amount of correctly spliced transcript to occur in homozygous patients with adult onset of the disease. In more severe juvenile patients, SpDEx7 occurs in combination with one of three point mutations that result in single amino acid changes: Trp65Arg, Gln49Arg, or Tyr395Cys (35 ). The latter mutation was also found in two EI severe patients, in combination with substitution Ser60Leu (35 ). In a few Caucasian patients with the LI condition, two new point mutations have been identified, Phe412Val and Tyr221Asn (24 ,35 ,36 ). Phe412Val impairs dimerization of the mutant PPCA precursor, causing its partial retention in the endoplasmic reticulum, and its rapid degradation following proteolytic processing in lysosomes (24 ). The second mutation is thought to have a greater effect on the catalytic rather than the protective function of the mutant protein (35 ). Here, we report a comparative analysis of eight GS patients who collectively represent the full range of severity of the disorder. Although we identified novel mutations in patients of all three clinical subtypes, each of the LI cases has at least one of the previously described point mutations. After investigating post-translational processing, subcellular localization and stability of the mutant proteins, we can arrange the mutations relative to the level of functional PPCA they support. From these studies we can begin to correlate specific combinations of mutant alleles with the severity of the clinical symptoms.
We have studied eight patients with galactosialidosis: two patients (NG and VE) were affected by the early infantile type of the disease; one patient (LR) showed so far a phenotype intermediate between the early and the late infantile; four patients (RZ, JC, AW and NT) had a late infantile presentation and one patient (KF) was an example of a classical juvenile/adult type. All patients had coarse facies and most of them presented with inguinal and/or umbilical hernias. Table 1 summarizes other clinical features related to the severity and course of the disease, as obtained from case reports. Some recent unpublished information was given to us by the physicians who care for the patients. Within the LI cases, AW and NT are the mildest affected. They are the only patients free of central nervous system involvement. Next, JC, RZ and LR are all mildly retarded; however, JC does not show dysostosis multiplex, liver/spleen or eye abnormalities. Although patient LR is still too young to foresee her outcome after 10-15 years, she is the most serious LI patient, presenting severely at birth.
All GS patients included in this study had PPCA mRNA, and in some patients residual cathepsin A activity was measured (Table 2 ). We first asked whether overt differences could be detected in the biosynthesis, post-translational modifications, and processing of the mutant proteins compared with those of the wild-type PPCA. Immunoprecipitation studies were carried out on radiolabelled cell lysates from the patients' fibroblasts using a monospecific anti-PPCA antibody (anti-54), that recognizes both precursor and mature forms of the enzyme. As seen in Figure 1 A, in all GS cells labelled for the 24 h period the level of the various 54 kDa PPCA mutant precursors was higher than that of wild-type precursor, which appeared nearly completely converted to the 32 and 20 kDa mature two-chain protein (Fig. 1 A, lanes 1-9). In cells from both the EI (NG and VE) and J/A (KF) cases, the mutant precursors were not proteolytically cleaved (Fig. 1 A, lanes 1, 7 and 8), whereas the two mature subunits were clearly detected in fibroblasts from all five LI patients, although the amounts and ratios varied from patient to patient (Fig. 1 A, lanes 2-6). In addition, a partially processed intermediate of ~34 kDa was also immunoprecipitated from the latter samples. Among the LI patients, only the sample from LR (Fig. 1 A, lane 3) showed an aberrantly sized band of 56 kDa, which was not proteolytically cleaved, since no processed forms of larger molecular weight were detected. The mutation in this precursor protein had apparently introduced a novel glycosylation site, because deglycosylation of the 56 kDa polypeptide resulted in a protein identical in size to the wild-type precursor free of sugars (Fig. 1 B). Overall these results point to at least partial retention/accumulation of the mutant precursors in an early biosynthetic compartment. Only the LI patients had residual immunoprecipitable amounts of mature PPCA. However, these findings only partially correlate with the level of residual cathepsin A activity measured in all patients' fibroblasts (Table 2 ), and do not explain the apparent lack of mature protein in the adult onset patient.
In contrast to the EI and J/A patients, the five LI patients were genetically more homogeneous. Two point mutations, Phe412Val and Tyr221Asn, appeared to define this clinical phenotype (Table 2 ). We and others have previously identified these two mutations in three of these GS patients (24 ,35 ,36 ). We now find that these mutations can occur in either the homozygous or compound heterozygous state and are diagnostic for the LI phenotype. Three patients had the Tyr221Asn mutation. In patient NT, this amino acid change was encoded by one allele, whereas the second allele carried a deletion of a cytosine at position 118 (C118). The latter caused a frameshift and premature translation termination codon. However, because the deletion was associated with absence of mRNA, no truncated protein was synthesized from this allele, and the expressed mutant PPCA contained the Asn221 change. A similar genotype was found in patient AW, who also carried the Tyr221Asn mutation in one allele. The genomic lesion in the second allele probably caused mRNA instability and was not identified. The Phe412Val mutation was again detected in three of the five LI patients. Patient RZ, described earlier (24 ), was homozygous for this mutation, whereas JC was compound heterozygous for both Tyr221Asn and Phe412Val mutations. We further used allele-specific oligonucleotide hybridization to screen for the presence of either of these two genetic lesions in a recently diagnosed young patient (LR), whose clinical features were suggestive of an LI phenotype. Indeed, we confirmed the presence of the Phe412Val substitution in one of the mutant alleles of this patient. As seen in Figure 2 , the wild-type probe gave a heterozygous signal when hybridized to the patient's DNA, while the mutant oligonucleotide only recognized the patient's sample. The mutation in the second allele of this patient, responsible for the over-glycosylation of the precursor protein, was Met378Thr, which generated a new glycosylation site at amino acid Asn376. From these combined data it is apparent that in the LI patients, who have distinctively milder phenotypes and survive longer, the presence of either of these point mutations, Asn221 and Val412, dictates the clinical outcome. Taking into account their most recent clinical evaluation (see Table 1 ), we can conclude that the Asn221 has a milder effect on the PPCA protein than the Val412 mutation; whereas the combination of both mutations apparently gives rise to an intermediate phenotype. In turn, two copies of the Val412 allele, as seen in patient RZ, seem to confer a less severe condition than only one copy, as in patient LR, who showed clinical manifestations at birth.
Having identified novel point mutations, we wanted to assess their effects on the subcellular distribution of the mutant proteins. It has been demonstrated that phosphorylation of mannose residues on one of the sugar chains of the wild-type PPCA precursor ensures its correct targeting to the lysosome and subsequent processing (24 ,37 ). Furthermore, we have shown previously that the Val412 mutant, when overexpressed in COS-1 cells, is phosphorylated in part and delivered to the lysosome. Site-directed mutagenized cDNAs, encoding PPCA proteins that carry one of the amino acid changes described above (i.e., Asn221, Val412, Met104, Pro208, Ser411 or Tyr23), were cloned into a mammalian expression vector and transiently transfected into COS-1 cells. Biosynthetic labelling of these cells with [3H]leucine, followed by immunoprecipitation revealed that Met104-, Pro208-, Ser411- and Tyr23-PPCA precursors were not secreted, whereas Asn221-PPCA was present in the culture medium (results not shown). A similar experiment with 32P-labelled cells showed that only the Asn221 precursor was phosphorylated, secreted and processed, albeit in trace amounts, indicating that it might reach the lysosomes (Fig. 3 , lane 3). These results were confirmed by subcellular distribution analysis of the PPCA mutants with indirect immunofluoresence, using an antibody raised against the denatured 32 kDa subunit (anti-32). Wild-type PPCA exhibited the typical lysosomal punctuated staining (Fig. 4 a). In contrast, mutants Tyr23, Met104, Pro208 and Ser411 had an aberrant distribution (Fig. 4 b-e): each of these proteins was confined to the endoplasmic reticulum (ER), distributed throughout the cytoplasm, or restricted to the perinuclear area. To ascertain the extent of lysosomal compartmentalization of Asn221-PPCA, its subcellular distribution was monitored by immunoelectronmicroscopy. Ultrathin sections of cells transfected with the Asn221-cDNA construct were probed with the anti-32 antibody, which revealed a clear lysosomal location for this mutant protein (Fig. 4 g). However, the number of gold particles in the lysosomes of these cells was ~50% of that in cells expressing wild-type PPCA (Fig. 4 f and g; Table 3 ), although a comparable number of grains was counted in the ER of both sets of cells (not shown). Clearly, the amount of Asn221 protein in lysosomes was higher than that of the Val412 mutant, for which we counted ~6% of the grains seen in cells overexpressing the wild-type protein (24 ). Interestingly, in cells cotransfected with Asn221 and Val412 cDNAs (Fig. 4 h; Table 3 ), the lysosomal labelling (15%) was close to that of Val412 transfected cells. Using the same procedure, only ER but no lysosomal labelling was detected in cells overexpressing Met104-, Pro208-, Ser411-, and Tyr23-PPCAs (not shown). Taken together these results suggest that the latter mutations lead to drastic conformational changes in the zymogen that prevent its transport to the Golgi complex, where phosphorylation occurs, and, thereby also prevent its correct compartmentalization and processing in lysosomes. On the other hand, a fraction of both Asn221 and Val412 mutant proteins is correctly modified and routed to the lysosomes, although their intralysosomal fate seems to be different.
In order to explain the relative amounts of Asn221 and Val412 mutants in lysosomes, we first performed a pulse-chase experiment using transfected COS-1 cells. Figure 5 shows that the two mutant precursors were synthesized in similar amounts to the wild-type, although the Asn221 appeared to be more stable in the course of the chase. Both mutant precursors were primarily converted into a `34'/20 kDa intermediate. However, the processed forms of the Val412 mutant were clearly more labile than those of the Asn221 mutant, which were, in turn, less stable than the wild-type mature protein. Consistent with the immunoelectronmicroscopy results, the processed forms in Asn221/Val412-cotransfected cells displayed an intermediate stability, compared with the singly transfected cells. These data suggest that the mutant proteins, alone or in combination, have reduced intralysosomal half-lives when compared with wild-type PPCA. This is most likely due to increased susceptibility of the mutants to proteolytic degradation.
In order to address the latter point, we subjected wild-type and mutant precursors to mild trypsin digest in vitro (Fig. 6 ). We have shown earlier that the normal PPCA precursor can be proteolytically converted into a 32/20 kDa enzyme that has full substrate binding and catalytic activity (38 ). Equal amounts of radiolabelled wild-type, Asn221 and Val412 precursors, derived from the media of transfected COS-1 cells, were incubated for increasing lengths of time with trypsin. Reactions were terminated by the addition of a trypsin inhibitor, and the samples were tested for cathepsin A activity. Already at 2 min incubation time, wild-type PPCA precursor was converted to the 34/20 kDa intermediate and the 32/20 kDa mature enzyme (Fig. 6 , lane 3). Upon prolonged digest, the 34 kDa chain was completely trimmed to a stable protease-resistant 32 kDa molecule (Fig. 6 , lanes 3-6). This conversion of the precursor into the two-chain protein was paralleled by a gradual increase in cathepsin A activity (Fig. 6 , lanes 2-6, lower panel). In contrast, a 2 min digest of the Val412 precursor produced a major 34 kDa band, and minor amounts of 32 and 20 kDa polypeptides (Fig. 6 , lane 15). Prolonged incubation, up to 10 min, caused the rapid disappearance of the 20 kDa chain and the aspecific degradation of the larger proteins (Fig. 6 , lanes 16-18). Protease treatment of this mutant precursor produced no cathepsin A activity (not shown). Instead, trypsin digest of the Asn221 precursor readily yielded a mixture of 34 and 32/20 kDa proteins, that, unlike the Val412 mutant, were stable up to 10 min (Fig. 6 , lanes 9-11). After 5 min, however, the 34 kDa species was not further trimmed to the 32 kDa form, and all three digested polypeptides were slowly degraded at longer incubation points (Fig. 6 , lanes 10-12). Asn221-PPCA clearly acquired cathepsin A activity through trypsin digestion (Fig. 6 , lane 9-10, lower panel), but lost it following extended protease exposure (Fig. 6 , lanes 10-12, lower panel). From these data we may infer that the degree of protease resistance of mature Asn221-PPCA lies between that of wild-type and Val412, which is also consistent with the pulse-chase experiment shown above. Together these results could explain the enhanced stability and higher catalytic activity of the Asn221 mature protein.
We have investigated the properties of PPCA mutants from a group of GS patients that represents the full scale of disease severity. The results indicate that the main factor determining the clinical course of GS patients is the lysosomal level of mutant PPCA. In the two severely affected EI patients, who died in infancy, we identified three novel point mutations that prevent phosphorylation of the PPCA precursor, and, thereby, its transport to the lysosome. In addition, we found that the J/A patient, who gradually deteriorated after the age of 16 until his death at the age of 48, carried the SpDEx7 mutation combined to a new severe point mutation that again caused retention of the mutant precursor in the ER. Thus, the late onset and long survival of this patient can be attributed to very low levels of lysosomal wild-type PPCA, translated from correctly spliced mRNA derived from the SpDEx7 allele.
All five LI GS patients had at least one allele capable of yielding a PPCA protein that could be phosphorylated and, to some extent, transported to the lysosome. The fact that these patients have a relatively mild phenotype indicates that Val412- and Asn221-PPCAs can associate with [beta]-galactosidase and neuraminidase, protecting them to some extent against immediate degradation/inactivation in the lysosome. Interestingly, however, there is a clear gradient of clinical severity among these patients, from the most severe (patient LR) with only one Val412-expressing allele, to the least severe (patients AW and NT) with only Asn221 protein. The combined occurrence of Val412- and Asn221-PPCA in one patient (JC) apparently results in an intermediate phenotype. We can speculate that in the latter case the simultaneous presence of the two mutant proteins in lysosomes may result in a depletion of the more stable Asn221-PPCA due to a `dominant negative' effect of the labile Val412 molecules. Since we have evidence that Asn221 is able to dimerize, it might form heterodimers with Val412-PPCA, that are degraded more rapidly than the Asn221 homodimers.
Interestingly, the Met378Thr mutation, present in compound heterozygosity in one of the LI patients, represents the first example among lysosomal proteins of a point mutation that generates a new Asn-linked glycosylation site which is actually utilized. The additional oligosaccharide chain likely affects proper folding and, in turn, compartmentalization of the mutant precursor since no mature protein was found. Several mutations affecting glycosylation sites in lysosomal proteins have been reported, all of which result in the loss of one oligosaccharide side chain (39 -43 ). Elimination of a glycosylation site in arylsulfatase A and [alpha]-glucosidase does not seem to affect the correct functioning of these enzymes and, therefore, these mutations are considered polymorphisms (39 ,40 ,43 ). In contrast, in a number of metachromatic leukodystrophy patients with a saposin B deficiency, the absence of the only oligosaccharide chain of this protein is believed to cause its rapid degradation and, in turn, to contribute to the disease (41 ,42 ).
Human skin fibroblast cultures from a normal individual and patients were obtained from the European Cell Bank, Rotterdam (Dr W. J. Kleijer). Fibroblasts were maintained in Dulbecco's modified Eagle's medium/Ham's F10 medium (1:1 vol/vol) supplemented with antibiotics and 10% fetal bovine serum. COS-1 cells (51 ) were grown in the same medium, supplemented with 5% fetal bovine serum.
Human fibroblasts were grown to confluency in 85 mm dishes and labelled with 350 [mu]Ci [3H]-4,5-leucine (Amersham) per dish for 24 h. Cell extracts were prepared as described (52 ), and preabsorbed with normal rabbit serum and affinity purified anti-fibronectin antiserum (Sigma) for 30 min at room temperature. The samples were precleared by incubating them three times for 15 min with 40 [mu]l formalin-fixed Staphylococcus aureus suspension (Immunoprecipitin, Gibco-BRL), that had been prepared according to manufacturers' instructions, washed six times in phosphate-buffered saline and resuspended in Tris-buffered saline (10 mM Tris-HCl, pH 7.4, 150 mM NaCl) at 20% (w/v). For specific immunoprecipitation cell lysates were incubated with anti-human PPCA 54 kDa precursor antiserum (anti-54; 22 ) for 1 h at RT or overnight at 4oC, followed by absorption to 12.5 [mu]l of the S.aureus suspension for 30 min. Immunocomplexes were collected by centrifugation, washed and prepared for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described (52 ). Polyacrylamide gels (12% and 12.5%) were used (53 ,54 ). After electrophoresis gels were fixed in 40% methanol and 10% glacial acetic acid and treated with Amplify (Amersham) for fluorography of radioactive proteins.
For deglycosylation precipitated proteins were eluted from final washed S.aureus pellets by boiling in 70 [mu]l 0.3% SDS in 50 mM sodium phosphate buffer pH 6.8 (PB) for 5 min. Eluates were supplemented with 140 [mu]l 0.75% NP40 and 0.75% [beta]-mercaptoethanol in PB and denatured for 5 min at 95oC. Samples were split into 100 [mu]l aliquots, of which one received 0.2 U recombinant N-glycosidase F (Boehringer Mannheim). Following incubation overnight at 37oC samples were desalted on Sephadex G50 spin columns (equilibrated in 10 mM Tris-HCl, 1 mM EDTA, pH 7.4), lyophilized and processed for SDS-PAGE.
For the synthesis and amplification of mutant cDNAs, four sets of oligonucleotide primers were synthesized according to the published cDNA sequence of protective protein (23 ) on an Applied Biosystems 381A oligonucleotide synthesizer. Total RNA was isolated by the method of Auffray and Rougeon (55 ). Four overlapping cDNA fragments, encompassing the entire coding region, were synthesized by reverse PCR (56 ) and amplified on a Perkin-Elmer Cetus thermocycler programmed for 25 cycles. For amplification of genomic DNA, genomic DNA was extracted from cultured fibroblasts and the individual exons of the protective protein gene were amplified by PCR (57 ). Primers were chosen in the intronic sequences surrounding the exon so that it would be possible to determine the complete sequence of the exons. The following pairs of intronic primers were used to verify the mutations at genomic level: 5'-TGCTGCACGGAAGCGCTGAG-3' (sense) and 5'-CTCCCAATCCCGCCCAGAAG-3' (antisense) for nC118 and Ser23Tyr; 5'-TCTCTGAAGTTCTTTCCAGT-3' (sense) and 5'-CAGTCTGACCTTGCTAACTG-3' (antisense) for Val104Met; 5'-GGGTATGCTCGCCTCCTCTG-3' (sense) and 5'-TGTGGTCCTGTTCTCAGGAT-3' (antisense) for Leu208Pro and Tyr221Asn; 5'-GGCTTGTTCCACACCCCTCA-3' (sense) and 5'-CCTGGCCACTCCCAGGCATA-3' (antisense) for Met378Thr; 5'-TCTTTCCTGGTGGGGCAGAT-3' (sense) and 5'-CCATACAGGGGC- CAGATGGT-3' (antisense) for Gly411Ser and Phe412Val. For direct DNA sequence analysis, a portion of above PCR amplified cDNA or genomic DNA was subsequently subjected to asymmetric PCR (58 ), using only one primer, for another 35 cycles. The PCR products were extracted once with phenol/chloroform, followed by filtration through a Centricon-100 membrane (Amicon). The amplified DNAs were sequenced by the dideoxy-chain termination method (59 ).
For ASO hybridization, genomic DNA was amplified by PCR using the appropriate intronic primer pairs as described above. The amplified DNA was blotted on Hybond N+ membranes. Hybridization was performed with appropriate 32P-labelled oligonucleotides. After hybridization the membranes were washed in 2* SSC containing 0.1% SDS for 30 min at 3-4oC below the estimated melting temperature.
Two pairs of allele-specific oligonucleotide probes were synthesized, one pair of normal and mutant sequences for Tyr221Asn and one pair of normal and mutant sequences for Phe412Val mutations: Tyr221Asn, 5'-GTGTAACTTCTATGACAACA-3' (normal) and 5'-GTGTAACTTCAATGACAACA-3' (mutant); Phe412Val, 5'-ATTGCCGGCTTCGTGAAGGAG-3' (normal) and 5'-ATTGCCGGCGTCGTGAAGGAG-3' (mutant).
In vitro mutagenesis of PPCA cDNA was performed as previously described (60 ) using the whole 1.8 kb PPCA cDNA (23 ) as a template. After synthesis by PCR of the 5' 1.5 kb cDNA carrying the complete open reading frame, the presence of the desired mutation was verified by sequencing. The following pairs of mutagenic oligonucleotides were used for site directed mutagenesis: 5'-GTGTAACTTCAATGACAACA-3' (sense) and 5'-TGTTGTCATTGAAGTTACAC-3' (antisense) for mutagenesis of T751 -> A; 5'-TCCCCAGCTGGGATGGGCTT-3' (sense) and 5'-AAGCCCATCCCAGCTGGGGA-3' (antisense) for G400 -> A; 5'-CCAGTACTACGGCTACCTCA-3' (sense) and 5'-TGAGGTAGC- CGTAGTACTGG-3' (antisense) for C158 -> A; 5'-ATTGCCAGCTTCGTGAAGGA-3' (sense) and 5'-TCCTTCTCGAAGCTGGCAAT-3' (antisense) for G1321 -> A; 5'-TCTTCTCCCC- AGACCCACTG-3' (sense) and 5'-CAGTGGGTCTGGGGAGAAGA-3' (antisense) for T713 -> C. The mutagenized PPCA cDNAs were subcloned into a derivative of the mammalian expression vector pCD-X (23 ,61 ).
The pCD-constructs, containing in vitro mutagenized cDNAs, were transfected into COS-1 cells using either DEAE-dextran (23 ,24 ,62 ) or the calcium phosphate precipitation method (63 ,64 ). To analyze phosphorylation of overexpressed proteins, 48 h after transfection cells were metabolically labelled with [32P]phosphate (100 [mu]Ci/ml labelling medium) for 7 h and cell extracts and medium samples were immunoprecipitated with anti-54 antiserum. For indirect immunofluorescence, COS-1 cells were reseeded 48 h post-transfection on SuperfrostR/Plus glass slides (Fisher). Next day, cells were processed according to van Dongen et al. (65 ) using an antiserum (anti-32) raised in rabbit against the denatured PPCA 32 kDa chain obtained through overexpression in insect cells (38 ) and FITC-conjugated anti-rabbit IgG antibodies (Sigma). Pulse-chase experiments were performed as described (37 ).
Transfected COS-1 cells in 100 mm dishes were fixed in 0.1 M phosphate buffer pH 7.3, containing 1% acrolein and 0.4% glutaraldehyde. Further embedding in gelatin, preparation for ultracryotomy and methods for immunoelectronmicroscopy were as previously reported (66 ). For these experiments we used antibodies (anti-32) raised against the 32 kDa denatured chain of human protective protein, isolated from human placenta (23 ).
Cos-1 cells seeded in 85 mm dishes were transfected with pCD-PPCA-WT, -751A (Asn221) and -1324G (Val412). Eighty-four hours post-transfection, cells were metabolically labelled with [3H]-4,5-leucine (50 [mu]Ci/ml labelling medium) in the absence or presence of 2.5% dialyzed fetal bovine serum for 16-18 h. Serum-free media containing radiolabelled PPCA precursors were used for limited trypsin digests, as described (24 ,37 ). Immunoprecipitation was performed with anti-54 antiserum as above. For uptake experiments serum-containing COS-1 media were concentrated as described (52 ). Since the secreted PPCA precursors are the major labelled proteins in the medium concentrates, aliquots of these preparations containing equal c.p.m. were added to the media of recipient EI GS fibroblasts (23 ), grown to confluency in 35 mm dishes. After 4 days of uptake, internalized PPCAs were recovered by immunoprecipitation as above.
Cathepsin A activity was measured with the synthetic N-blocked dipeptide carbobenzoxy-phenyalanyl-alanine as substrate according to Galjart et al. (22 ) and Kleijer et al. (31 ). Total protein was quantitated with bicinchoninic acid (67 ) following manufacturers' guidelines (Pierce Chemical Co.).
The authors are indebted to Prof. H. Galjaard and the Foundation of Clinical Genetics, Rotterdam (The Netherlands), for support during the initial stages of this project. We would like to thank Drs G. V. Watters (Department of Pediatrics, Montreal Children's Hospital, McGill University, Quebec, Canada), D. A. Applegarth (British Columbia Children's Hospital, Vancouver, Canada) and G. Carpenter (Department of Pediatrics, Jefferson Medical College, Thomas Jefferson University Hospital, Philadelphia, PA) for providing us with recent clinical evaluations of galactosialidosis patients; Dr A. C. Sewell (Department of Pediatrics, University Children's Hospital, Frankfurt, Germany), Drs D. A. Applegarth, E. Zammarchi and M. A. Donati (Department of Pediatrics, University of Florence, Italy) for making patient fibroblasts available. Special thanks to Dr A. Morrone (Department of Pediatrics, University of Florence, Italy) for participating in the initial analysis of the mutations in patient NG. We are grateful to Peggy Burdick for editing and typing this manuscript. These studies were supported in part by the American Lebanese Syrian Associated Charities of St Jude Children's Research Hospital and the Assisi Foundation.
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*To whom correspondence should be addressed
+Both authors contributed equally to this work
Present addresses: [sect]Pediatric Research Institute, St Louis School of Medicine, St Louis, MO 63110, USA and [dagger]Department of Pediatrics, Markey Cancer Center, University of Kentucky Medical Center, Lexington, KY 40536, USA
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