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From the Department of Pediatrics,* Meyer Hospital, Florence, Italy; the Pediatric Unit, Melegnano Hospital, Milan, Italy; and the Department of Genetics, St. Jude Children’s Research Hospital, Memphis, Tennessee
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Abstract |
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The GLB1 gene gives rise to two alternatively spliced mRNAs that encode the lysosomal GLB1 enzyme and the elastin-binding protein (EBP).2-4 The GLB1 precursor (70 kDa) is proteolytically processed in lysosomes into the 64-kDa mature form.5-7 The active enzyme can be present in a high molecular weight lysosomal complex with protective protein/cathepsin A [PPCA (MIM 256540)], neuraminidase [NEU1 (MIM 608272)], and N-acetyl galactosamine-6-sulfate sulfatase [GALNS (MIM 25300)].8 The alternative spliced EBP is involved in elastic fiber assembly on the cell surface, where it is postulated to be present in complex with PPCA and NEU1.8 EBP acts as a molecular chaperone, protecting tropoelastin from premature degradation and facilitating its assembly into a microfibrillar scaffold that consists of several glycoproteins such as fibrillins and microfibril-associated glyco-proteins (MAGP).9-11 Covalently cross-linked polypeptide chains of soluble tropoelastin are assembled along the microfibrils, providing the extracellular elastic fibers of the tissues.10
Impaired elastogenesis has been previously associated with primary EBP deficiency in patients with either GM1-gangliosidosis or Morquio type B disease.9 EBP contains a galactolectin domain that binds microfibrillar acceptors in the glycosylated microfibrillar scaffold of elastic fibers.9-11 This EBP domain may also interact with galactosugar-bearing moieties, such as chondroitin sulfate and dermatan sulfate, that are pericellularly accumulated in Costello syndrome and Hurler disease. In those cases, EBP undergoes a premature shedding from the cell surface and tropoelastin is dissociated far away from its proper microfibrillar template.9-11 Thus, it has been previously demonstrated that impaired elastogenesis in Costello syndrome and Hurler disease is related to a secondary EBP deficiency caused by chondroitin sulfate and dermatan sulfate accumulation, respectively.10,11 These data have been supported by transduction of skin fibroblasts from Costello syndrome with versican V3 (which lacks chondroitin sulfate). This retrovirally mediated overexpression completely restores normal elastogenesis, suggesting the rescue of EBP to be responsible of the phenotypic reversal.12
Here we report the biochemical and molecular characterization of four new and two known mutations detected in the GLB1 gene of three patients with infantile, juvenile, or adult form of GM1-gangliosidosis phenotypes. This study also focuses on impaired elastogenesis in those patients. In particular, we show that impaired elastogenesis arises from primary and secondary EBP defects, respectively, in the infantile and juvenile patients’ fibroblasts here reported.
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Materials and Methods |
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Patients’ clinical presentations are summarized in Table 1
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All reagents for mRNA extraction from patients’ fibroblasts and lymphocytes were purchased from Eppendorf AG (Hamburg, Germany). RNA integrity was verified by 1% agarose gel electrophoresis. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of GLB1 cDNA was performed by a set of six overlapping amplifications encompassing the entire coding region of the mRNA as previously reported.13 The RT-PCR products were checked on a 1.5% agarose gel, excised, and purified using Nucleospin Extract kit (Macherey-Nagel, Düren, Germany). Sequencing reactions were performed using the ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA) as recommended by the manufacturer. The nomenclature of GLB1 genetic lesions is as designed previously.14 Nucleotide numbering starts at the ATG translation initiation codon.
Analysis of Genomic DNA
The GLB1 mutations were confirmed in DNA samples of patients and their relatives. The oligonucleotides and the amplifying conditions have been described previously.13,15 The PCR products were directly sequenced as described above.
Multiplex Fluorescent RT-PCR of GLB1 mRNA
Multiplex RT-PCR and quantitation of fluorescent PCR products of insulin receptor and GLB1total mRNA were performed as previously described.16 Primer sets for cDNA synthesis and amplification of human mRNA corresponding to insulin receptor (INSR) were designed as previously reported,16 whereas the GLB1primers used were the following: 1035 cDNA 5'-CACAGTGAGGTCCCCAGCCTCACTCA-3' (1035/1009 nucleotides exon 10); 378 forward 5'-CTGTGCAGAGTGGGAAATGGG-AGGA 3'-(378/402 nucleotides exon 3/4); and 565 reverse 5'-ATTCATTTTCAACCTGCACTGTTAT-3' (565/541 nucleotides exon 6/5). Fluorescent RT-PCR products were amplified and loaded on an ABI Prism 310 gene-tic analyzer (Applied Biosystems) as previously described.16
RT-PCR of EBP
EBPcDNA synthesis was performed by a commercial kit purchased from Eppendorf AG. Primer sets for cDNA synthesis and amplification of human mRNA corresponding to INSR were designed as previously reported,16 whereas the EBPprimers used were the following: cDNA 5'-CACAGTGAGGTCCCCAGCCTCACTCA-3' (644/618 nucleotides exon 10); 185 EBP for 5'-GCTTCTACTGGAAGGACCGGC-3' (185/205 nucleotides exon 2); and 744 EBP Rev 5'-GATGTTGCTGCCTGCACTGTTA-3' (353/342 nucleotides exon 5/7). The amplifying conditions have been previously described.16
Expression Studies
Site-directed mutagenesis and fragment replacement were used to introduce the genetic lesions c161G>A (S54N), c175C>T (R59C), c602G>A (R201H), c689G>A (C230Y), c985A>G (T329A), and c1325G>A (R442Q), detected in the patients, in transient GLB1expression vectors as described previously.15 The integrity of the DNA and the exclusive presence of the expected mutations in the GLB1 cDNA inserts, were verified by sequencing on both strands. The oligonucleotide primers for site-directed mutagenesis have been previously reported15 except for the following: S54N forward 5'-CATCTCAGGAAACATTCACTAC-3' (150/171 nucleotides exon 2); S54N reverse 5'-GTAGTGAATGTTTCCTGAGATG-3' (171/150 nucleotides exon 2); R59C forward 5'-CACTACTCCTGTGTGCCCC-3' (166/184 nucleotides exon 2); R59C reverse 5'-GGGGCACACAGGAGTAGTG-3' (184/166 nucleotides exon 2); R201H forward 5'-GACTACCTGCACTTCCTGC-3' (592/610 nucleotides exon 6); R201H reverse 5'-GCAGGAAGTGCAGGTAGTC-3' (610/592 nucleotides exon 6); C230Y forward 5'-CATTCCTGAAATATGGGGCCC-3' (677/697 nucleotides exon 6); C230Y reverse 5'-GGGCCCCATATTTCAGGAATG-3' (697/677 nucleotides exon 6); T329A forward 5'-GCACAGCCCGCCAGCTACG-3' (976/994 nucleotides exon 10); T329A reverse 5'-CGTAGCTGGCGGGCTGTGC-3' (976/994 nucleotides exon 10); R442Q forward 5'-CCACGATCAAGCATATGTTG-3' (1317/1336 nucleotides exon 13); and R442Q reverse 5'-CAACATATGCTTGATCGTGG-3' (336/1317 nucleotides exon 13). The italized bases correspond to a mispairing with the normal sequence. The mutated transient expression vectors were used to transform the Escherichia coli strain Solo pack gold cells (Stratagene, Amsterdam Zuidoost, The Netherlands), obtaining a great quantity of each recombinant vector.
Transfection into COS-1 Cells
Normal and mutant vectors were transiently overexpressed into African green monkey kidney cells (COS-1) as described previously.15 In each experiment, the value of GLB1 activity in nontransfected COS-1 cells was used to set up the GLB1 intrinsic activity of the expression system.
Cell Cultures and Biochemical Enzymatic Assay
COS-1 cells and patients’ fibroblasts were cultured in Dulbecco’s modified Eagles-Hams F10 medium (1:1, v/v) with fetal bovine serum (10%) and antibiotics. The protein content of transfected COS-1 cells and of patients’ fibroblasts was determined in triplicate by the method of Lowry and colleagues.17 GLB1 enzyme assay in fibroblasts and transfected cells was performed in triplicate in three independent experiments by fluorescence measurement using 4-methylumbelliferyl ß-galactopyranoside artificial substrate.18
Western Blots
Fibroblasts from patients and normal control and transfected COS-1 cells were harvested by scraping in phosphate-buffered saline and sonicated as described previously.19
Approximately 20 µg of total COS-1 cells were used in the blots. Western blots were prepared from 12.5% polyacrylamide gels and probed as described.20
After electrophoresis, proteins were transferred to nitrocellulose (Bio-Rad, Hercules, CA) and immunostaining was performed with the following antibodies: 
85, anti-GLB1 antibody, previously characterized;7,15
Alf1, anti-EBP antibody [the identification of EBP antigenic and hydrophilic peptides was used to obtain the following EBP epitope: NH2-VGSPSAQDEASPLS-COOH (91 to 104 amino acids) that was also previously used.9
This anti-EBP peptide, conjugated with bovine serum albumin and immunized into rabbits by Igtech (Perdifumo, Italy), has been previously reported in a study on a galactosialidosis patient, who showed a reduced EBP amount and in a study on GM1-gangliosidosis.21,22
]; polyclonal human anti-tropoelastin/elastin (Elastin Products Company, Owensville, MO); anti-actin antibody (Sigma, Milan, Italy), used to assay total protein content loaded in the blots. All of the blotted proteins were visualized by reaction with the secondary antibody anti-rabbit IgG (whole molecule) alkaline-phosphatase conjugate (Sigma), using the AP conjugate substrate kit (Bio-Rad).
Restriction-Site Analysis
The GLB1 gene of 100 normal control DNA samples was analyzed using the PvuI enzyme (Roche, Mannheim, Germany) to screen the R442Q mutation. The PCR fragments were amplified by the genomic primers reported ear-lier.15 A 10 µl aliquot of PCR product was incubated for 1 hour as recommended by the manufacturer in a reaction mixture containing 2 µl of 10x reaction buffer and 1 U of the restriction enzyme. The total volume of the sample was brought up to 20 µl.
Immunostaining
Parallel 10-day-old cultures of normal and patients’ fibroblasts were immunostained as previously reported.4 All fibroblast cultures were confluent and derive from early passage cells. Normal control fibroblasts were cultured with or without keratan sulfate (1 µg/ml). The amount of keratan sulfate added to the medium was based on pathological concentrations detected in the blood of patients affected by Morquio A disease.23 Juvenile patient’s fibroblasts were cultured with or without keratanase (0.1 U/ml/day) as described.10 Normal control fibroblasts were also cultured with or without keratan sulfate (1 µg/ml) and keratanase (0.1 U/ml/day). Polyclonal human anti-tropoelastin/elastin (see above), and anti-fibronectin (Sigma) antibodies, developed in rabbits, were used as primary antibodies as previously described.4 For immunohistochemical assessment of microfibrillar scaffold, normal and juvenile patients’ fibroblasts were incubated with polyclonal primary antibody to fibrillin-1 (Chemicon, Temecula, CA) developed in rabbits, as previously described.4 The only difference with respect to the published method4 concerns the experiment with the fibrillin antibody in which cell lines were directly fixed in methanol 100% instead of paraformaldehyde.
Fibroblast cultures (48 hours old) from juvenile patient and normal control were incubated with a human monoclonal keratan sulfate antibody (Seikagaku Corp., Tokyo, Japan), developed in mouse. Before adding the primary antibody, fibroblasts were permeabilized as described.10 Subconfluent 48-hour cultures of normal fibroblasts and juvenile patient’s fibroblasts were also incubated with Alf1,21,22 anti-EBP antibody. All direct immunofluorescence studies were then performed by secondary fluorescein isothiocyanate-conjugated antibodies purchased from Sigma. To stain nucleic acids, fibroblasts were also incubated with propidium iodide (10 µg/ml), as described previously.21,22
Quantitative Estimate of Keratan Sulfate
Fibroblasts cultures (48 hours and 10 days old) from juvenile patient and normal control were assessed for a chemical quantitative estimate of keratan sulfate by using a keratan sulfate enzyme-linked immunosorbent assay kit (Seikagaku Corp.). Values on normal and juvenile patient’s fibroblasts were normalized by starting from the same amount of protein contents (30 µg).
Insoluble Elastin Assay
To quantify the elastic fiber content in fibroblasts of the juvenile patient, insoluble elastin was measured in confluent fibroblasts obtained from the patient and normal controls as previously described.9 The only difference with respect to the published article9 concerns the metabolic radiolabeling with [3H]-leucine instead of [3H]-valine. The experiments were performed in triplicate and the values are the average of three independent experiments.
Statistical Analysis
GLB1 enzyme activities, measured on patients’ leukocytes and fibroblasts and transfected COS-1 cells, are presented as percentages (Table 2)
coupled with SDs performed in Microsoft Excel 97 SR-2. P values less than 0.05 were considered statistically significant to generate the confidence limits (n = 3, t = 3.18). SDs of keratan sulfate estimate and insoluble elastin assays were also performed by Microsoft Excel 97 SR-2.
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Results |
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The phenotypes of the three GM1-gangliosidosis patients here reported have been summarized in Table 1
. Connective/bone tissue alterations can be noticed in patients with both the infantile and juvenile form of GM1-gangliosidosis. The infantile patient showed cardiac involvement and dysostosis multiplex. Moreover, based on the connective/bone tissue alterations, and on abnormal urinary keratan sulfate excretion, the juvenile patient was first diagnosed as suffering from Morquio type B disease.
Biochemical and Genetic Analysis
The diagnosis of GM1-gangliosidosis was confirmed by the absence or reduction of GLB1 enzyme activity in patients’ leukocytes and fibroblasts (Table 2)
coupled with normal NEU1 activity. Quantitative analysis of RNA by multiplex fluorescent RT-PCR showed similar levels of GLB1 total mRNA in all control and patient samples. Normal amounts of EBP mRNA were present in all patients, compared with normal controls (Figure 1)
. Thus, synthesis of mRNA corresponding to GLB1and to EBP was unaltered in all patients reported here. The full-length GLB1 cDNA of the patients and the exon/intron boundaries of the corresponding GLB1 genes were amplified and directly sequenced on both strands. Four new and two known missense mutations were identified in the patients’ samples (Table 2
, Figure 2
).
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. The juvenile patient was found to be compound heterozygous for the novel C230Y and the known R201H24
amino acid changes; both mutations affecting only the GLB1 protein and not the EBP (Figure 2B)
. The adult patient was found to be compound heterozygous for the new T329A and R442Q mutations (Figure 2C)
, mapping to the central region of the protein. The mutations detected in the patients’ cDNA were confirmed in the patients’ and parents’ genomic DNA. A possible benign polymorphism for each new point mutation was excluded by transient expression studies and/or restriction site analysis (Table 2
, Figure 3
).
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Expression vectors with mutated GLB1 cDNA containing the individual point mutations were created by in vitro site-directed mutagenesis and fragment replacement. The pcD-GLB1-mutated vectors were transiently expressed in COS-1 cells. As predicted from the clinical phenotype, no residual GLB1 activity was detected for the S54N and R59C amino acid substitutions present in the infantile patient (Table 2)
. Absence of GLB1 activity was also observed in COS-1 cells expressing the GLB1 variants with the new C230Y and T329A amino acid substitutions identified in the juvenile and adult patients, respectively (Table 2)
. By contrast, the R201H and R442Q mutations, expressed in COS-1 cells resulted in 33% and 6% of residual GLB1 activity, respectively, compared with normal levels. These values indicate that these mutations are responsible for the residual enzyme activity measured in these patients’ fibroblasts (Table 2)
. Given the high residual activity of the new R442Q GLB1 variant, the exclusion of its eventual polymorphic nature was performed by restriction site analysis of 100 control DNAs (Figure 3)
.
Western Blot Analysis
Immunoblot analysis performed on mutant fibroblasts showed the presence of GLB1 and EBP proteins in all three patients. However, the amount of EBP was significantly reduced in the juvenile patient’s fibroblasts (Figure 4A)
. Western blot analysis of transfected COS-1 cells, demonstrated that the R442Q/T329A mutations did not significantly affect the levels of the GLB1 precursor protein of 85 kDa. Rather, a partial reduction of the precursor was detected in cells expressing the R201H and R59C mutant proteins, while a complete absence of the GLB1 protein was detected in cells expressing the S54N mutation (Figure 4B)
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To exclude the variability of elastin deposition and cell vitality on cell numbers and confluence, each cell culture was assayed at the same passage number. The amount of fibronectin detected in the patients’ fibroblasts was equal to that produced by normal fibroblasts (data not shown); these data are similar to those produced previously in GM1-gangliosidosis patients.9
Condensed cytoplasmic accumulation of keratan sulfate was detected in the juvenile patient’s fibroblasts compared with normal controls (Figure 5, A and B)
. Direct immunofluorescence showed a substantial reduction of extracellular elastin deposition in the infantile and juvenile patients’ fibroblasts compared with normal fibroblasts (Figure 5
; C, F, and H). The addition of keratanase (an enzyme capable of keratan sulfate degradation) to the culture medium of the patient’s fibroblasts restored the assembly of elastic fibers in juvenile patients’ fibroblasts while impaired elastic fiber assembly was still observed in infantile patient’s fibroblasts (Figure 5, G and I)
. Also, the addition of keratan sulfate to the culture medium of normal controls led to a remarkable decrease in the assembly of elastic fibers, as revealed by reduced elastin content (Figure 5D)
. A second control established by combined addition of keratan sulfate and keratanase in the culture medium of normal control fibroblasts resulted in a normal elastogenesis (Figure 5E)
. In contrast to the decreased elastin immunoreactivity of the juvenile patient’s fibroblasts, the microfibrillar scaffold, represented by fibrillin-1 protein, was normal in that patient (Figure 6, A and B)
. Moreover, the juvenile patient’s fibroblasts immunostained with antibody to EBP showed irregular and punctate immunogenic materials (Figure 6D)
, in contrast to the EBP widespread distribution on cell surface expressed in normal controls (Figure 6C)
.
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Because qualitative estimate by immunohistochemistry in the fibroblasts from the patient with the juvenile form showed abnormal accumulation of keratan sulfate, quantitative estimate of this mucopolysaccharide has been performed in the 48-hour-old cultured fibroblasts of normal (2 ng/ml ± SD 0.66) and juvenile patient (4 ng/ml ± SD 0.7) and in the 10-day culture medium of normal (24 ng/ml ± SD 0.71) and juvenile patient (28 ng/ml ± SD 1.19).
Tropoelastin Production and Assembly
To further endorse the nature of the elastic fiber defect detected in the juvenile patient’s fibroblasts, Western blot analysis with antibody against tropoelastin, and insoluble elastin estimates were also performed. Western blot showed that the ratio of soluble undegraded tropoelastin/degraded tropoelastin products is greater in the normal control compared with the juvenile patient’s cell lysate (Figure 7)
. On the other hand insoluble elastin, the major component of elastic fibers, was significantly reduced in the juvenile patient’s fibroblasts compared with normal controls (Figure 8)
.
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Discussion |
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Expression studies and enzyme assays were performed with the aim of assigning a putative role of these mutations in the GLB1 stability or catalytic activity. Because S54N and R59C, identified in the infantile patient, and C230Y and T329A, identified in the juvenile and in the adult patients, respectively, abolished the enzyme activity, they can be considered as severe mutations.
The T329 amino acid is found in a conserved cluster region of five amino acids including the catalytic D332.27 At present, only E268, D332, and W273 are predicted to be involved in the catalytic site, even if many other residues are likely to play a role in the enzyme activity.27 It has been suggested that some enzyme defects may be due to the alteration of binding interaction of the catalytic residues or other residues in the active site.28 Thus, a comprehensive correlation between the mutations and the enzyme defects will be available only when GLB1 is completely characterized by identification of its crystallographic structure. Nevertheless, our expression studies showed that the T329A mutation abolishes the GLB1 activity but does not alter the synthesis of the precursor that was still detected on Western blots. This finding confirms the importance of the conserved T329 residue in the formation of the catalytic site. On the other hand, the GLB1 enzyme carrying the S54N mutation is predicted to be a misfolded and rapidly degraded protein because its presence was not detected.
The juvenile and adult patients also carry GLB1 mutations associated with residual enzyme activity, which is in agreement with their clinical phenotype. It has been suggested that the R201C mutation affects the enzyme folding process.27 In vitro expression studies and enzyme assay confirmed that the R201H mutation does not greatly alter the GLB1 catalytic activity. These data are in keeping with Western blot analysis that revealed a reduced amount of the enzyme carrying the R201H mutation. The relatively high activity of the R201H GLB1 variant may arise from the high affinity of this mutated protein for the synthetic fluorogenic substrate, in agreement with previous expression studies on that mutation.29,30
Defects in the GLB1 gene are very rare, and most of the genetic lesions reported up to now affect both GLB1 enzyme and EBP. The detailed clinical evaluation of those GM1-gangliosidosis patients with mutations affecting either the GLB1 coding region alone or both GLB1 and EBP, could identify specific clinical manifestations associated with defects on either of the two proteins or both. Our juvenile patient showed very peculiar mutations (R201H and C230Y) located in a region of pre-mRNA encoding only the lysosomal enzyme. Thus, molecular and cellular studies of this patient became extremely useful in shedding light on the pathogenesis of GM1-gangliosidosis.
Some GLB1 mutations, giving rise to a deficiency in the degradation of keratan sulfate, have been previously hypothesized to cause lesions in connective tissue and bones.25 It has been also predicted that EBP mutations are involved in the extracellular deposition of keratan sulfate, stored in Morquio type B disease.27 Thus, the disruption of both GLB1 and EBP functions may give rise to clinical features typical of both GM1-gangliosidosis and Morquio type B disease.27
Decreased elastin deposition has been previously linked to the lack of EBP in infantile GM1-gangliosidosis patients, who carried nonsense mutations, and in Morquio type B patients, due to mutations that cause deficiency in both GLB1 and EBP.9 Interestingly, the EBP amount of the fibroblasts from the juvenile patient here reported is reduced even thought the mutational analysis has excluded its involvement. However, it has been demonstrated that the abnormal accumulation of some galactosugar-bearing moieties such as chondroitin sulfate and dermatan sulfate causes EBP functional inactivation in Hurler disease and Costello syndrome, respectively.10,11 Our juvenile GM1-gangliosidosis patient was initially diagnosed as suffering from Morquio type B disease, because of an elevated urine excretion of keratan sulfate and his skeletal abnormalities. Keratan sulfate accumulation was also detected in his cultured fibroblasts by immunohistochemistry. Based on these data and taking into consideration that even keratan sulfate has galactosugar-bearing moieties, the relationship between keratan sulfate and EBP reduction in this patient was investigated. Severely impaired elastogenesis of the patient’s fibroblasts was detected by immunostaining, but this was restored by incubation of the juvenile patient’s fibroblasts with keratanase. On the other hand, normal control fibroblasts did not assemble any elastic fibers after the addition of keratan sulfate in the culture medium.
The relationship between keratan sulfate accumulation and impaired elastic fiber assembly was further investigated by testing elastin and microfibrillar synthesis and deposition. The normal amount of EBPand elastin mRNA in all our patients, as detected by RT-PCR analysis, excludes a defect in mRNA synthesis. It has been previously shown that tropoelastin content is reduced in fibroblasts from Costello syndrome due to accumulation of chondroitin sulfate that causes the degradation of tropoelastin products.10 A reduction of tropoelastin in the juvenile patient’s fibroblasts, detected by Western blot analysis, suggested a partial decrease of this protein. Also, insoluble elastin assay, that estimates the only integral elastin content present in the mature elastic fibers, was reduced in the patient’s cell-layer extracts. These data confirmed that tropoelastin did not properly aggregate on the cell-surface of the juvenile patient’s fibroblasts and that tropoelastin, even if normally synthesized, was likely to be quickly shipped to degradation.
Together with tropoelastin, a key molecule in the process of elastic fibers formation is fibrillin-1.31 The first step in elastogenesis is marked by the deposition of the microfibrillar components into the extracellular space. The amount and distribution of fibrillin-1 in the juvenile patient’s fibroblasts revealed by immunohistochemistry did not differ from these detected in normal controls. Thus, the elastic fibers assembly defect observed in the juvenile GM1 gangliosidosis patient is likely to be independent from microfibril secretion. Rather, an EBP depletion of that patient’s cell-layer extracts was visualized by Western blot analysis and immunostaining. Keratan sulfate was abnormally elevated in the patient’s urine, in his fibroblasts, and culture medium. Monoclonal antibody also revealed a remarkable focal and punctate cytoplasmic amount of keratan sulfate in 48-hour cell cultures of that patient compared with normal control.
In summary, taking into account that the fibroblasts from the GM1-gangliosidosis juvenile patient showed an EBP protein reduction, impaired elastic fibers assembly, and large keratan sulfate accumulation, we hypothesized that his fibroblasts’ EBP depletion is of secondary origin caused by keratan sulfate. Additional studies of the role of the single amino acid substitutions on the GLB1 crystallographic structure, would probably clarify the nature of the GLB1 active site involved in the degradation of keratan sulfate.
Lack of EBP of primary origin, has been linked to impaired elastogenesis in infantile GM1-gangliosidosis patients bearing nonsense mutations of the GLB1 gene.9 Interestingly, decreased elastin assembly may also result from a primary defect of EBP function in which a normal amount of the protein is present, as demonstrated in our infantile patient’s fibroblasts.
In summary, GLB1 and EBP proteins, altered in function and/or distribution, contribute differently to the specific clinical manifestations of patients with mutations in the GLB1 gene. The missense mutations detected in these patients are responsible for the characteristic clinical manifestations and they confirm the genetic heterogeneity of GM1-gangliosidosis. In addition, impaired elastogenesis, due to a secondary deficiency of EBP and to keratan sulfate storage, has been shown in the fibroblasts from the juvenile GM1-gangliosidosis patient here reported. Understanding the role of EBP is crucial for the diagnosis and the pathogenesis of GM1-gangliosidosis and Morquio type B, and should be investigated in all diseases in which the EBP-protein and receptor are involved.
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Acknowledgements |
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Footnotes |
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Supported in part by the Fondi Ateneo (MURST ex 60%), MIUR-PRIN 2004, Associazione Malattie Metaboliche Congenite ereditarie, and Associazione Italiana Mucopolisaccaridosi e Malattie Affini.
Accepted for publication August 12, 2005.
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