Mutation of the glycosylated asparagine residue 286 in human CLN2 protein results in loss of enzymatic activity

Kostas Tsiakas2, Robert Steinfeld2, Stephan Storch2, Junji Ezaki3, Zoltan Lukacs2, Eiki Kominami3, Alfried Kohlschütter2, Kurt Ullrich2 and Thomas Braulke1,2

2 Department of Biochemistry, Children's Hospital, University Hospital Hamburg Eppendorf, Martinistraße 52, Bldg W23, D20246 Hamburg, Germany; and 3 Department of Biochemistry, Juntendo University School of Medicine; Tokyo 113, Japan

Received on July 25, 2003; revised on December 11, 2003; accepted on January 2, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by the deficiency of the lysosomal tripeptidyl peptidase-I encoded by CLN2. We previously detected in two LINCL patients a homozygous missense mutation, p.Asn286Ser, that affects a potential N-glycosylation site. We introduced the p.Asn286Ser mutation into the wild-type CLN2 cDNA and performed transient expression analysis to determine the effect on the catalytic activity, intracellular targeting, and glycosylation of the CLN2 protein. Expression of mutant p.Asn286Ser CLN2 in HEK293 cells revealed that the mutant was enzymatically inactive. Western blot analysis demonstrated that at steady state the amounts of expressed p.Asn286Ser CLN2 were reduced compared with wild-type expressing cells. The rate of synthesis and the sorting of the newly synthesized p.Asn286Ser CLN2 in the Golgi was not affected compared with wild-type CLN2 protein. The electrophoretic mobility of the immunoprecipitated mutant p.Asn286Ser CLN2 was increased by approximately 2 kDa compared with the wild-type CLN2 protein, whereas deglycosylation led to the generation of polypeptides of the same apparent size. The data suggest that mutant p.Asn286Ser CLN2 lacks one oligosaccharide chain resulting in enzymatic inactivation.

Key words: expression analysis / glycosylation / late infantile neuronal ceroid lipofuscinosis / loss of function / tripeptidyl peptidase I


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a rare inherited neurodegenerative disease due to mutations in the CLN2 gene. Affected children show the first clinical symptoms between 1.5 and 4 years of age—characterized by seizures, ataxia, myoclonus, and vision loss with blindness by 5 or 6 years (Williams et al., 1999Go). The CLN2 gene encodes the tripeptidyl peptidase-I (TPP-I, EC 3.4.14.9), a soluble lysosomal aminopeptidase with substrate-dependent endopeptidase activity (Ezaki et al., 2000Go; Sleat et al., 1997Go; Vines and Warburton, 1999). Whereas the identity of natural substrates for TPP-I remains unclear, the loss of TPP-I activity in patients with LINCL leads to the lysosomal accumulation of subunit c of ATP synthase and the ubiquitous deposition of curvilinear storage bodies.

TPP-I is synthesized in the endoplasmic reticulum as N-glycosylated 66/67 kDa precursor protein (Ezaki et al., 1999Go; Lin et al., 2001Go). During the passage to the Golgi, the oligosaccharides are processed, which includes the formation of mannose 6-phosphate (M6P) residues on high-mannose type oligosaccharides. The M6P residues function as a high-affinity recognition signal for M6P receptors that mediate in the trans-Golgi network the segregation of TPP-I from the secretory route. The receptor–TPP-I complexes are then transported in clathrin-coated vesicles to the acidic endosomal/prelysosomal compartment followed by the dissociation of the enzymes due to the low pH (Braulke, 1996Go). The delivery of TPP-I to lysosomes is accompanied by proteolytic cleavage into the 46-kDa mature enzyme form. In cells overexpressing TPP-I variable amounts of the newly synthesized precursor form were found in the medium (Golabek et al., 2003Go; Lin and Lobel, 2001Go; Lin et al., 2001Go).

To date, more than 50 mutations in the CLN2 gene have been described to be associated with LINCL (www.ucl.ac.uk./ncl/CLN2.html). Recently we found two unrelated patients of Kurdish ethnicity to be homozygous for an allele containing the novel missense mutation p.Asn286Ser affecting one of the five potential N-glycosylation sites (Steinfeld et al., 2002Go). For both patients a more protracted clinical course was observed when compared with patients with a typical progression of the disease. In the present study, the p.Asn286Ser mutation was introduced in the CLN2 cDNA and expressed in HEK-293 cells to examine enzymatic activity, intracellular transport and carbohydrate processing.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The p.Asn286Ser mutation was introduced into the CLN2 cDNA and transiently transfected HEK-293 cells were examined for expression of CLN2. Nontransfected and vector-transfected HEK-293 cells were used as controls. Western blot analysis showed in wild-type expressing cells a prominent immunoreactive band of 65 kDa, representing the precursor CLN2 form (Figure 1A), which was hardly seen in vector-transfected controls. In cells transfected with the mutant p.Asn286Ser CLN2 cDNA, the precursor form exhibiting a slightly increased mobility of ~2 kDa was detected. The steady-state concentrations of mutant CLN2 were constantly lower than that in cells expressing the wild type. Similar amounts of the endogenous LAMP-1 protein and of the coexpressed green fluorescence protein (GFP) indicate equal protein loading and transfection efficiency (Figure 1A). Incubation of transfected HEK-293 cells with a mixture of leupeptin and pepstatin A, potent inhibitors of lysosomal cysteine and aspartic proteases, failed to increase the intensity of the p.Asn286Ser CLN2. The data suggest that the lower amounts of mutant CLN2 are presumably due to increased degradation in prelysosomal compartments.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Expression and activity of CLN2 in overexpressing HEK-293 cells. (A) HEK-293 cells were transiently transfected with vector pIRES2-EGFP vector alone or wild-type or mutant p.Asn286Ser CLN2 cDNA. Twenty-four hours after transfection, cells were lysed; cell extracts (30 µg protein) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted onto nitrocellulose, probed with anti-human CLN2 IgG (0.3 µg/ml), anti-human LAMP-1 (1:1000), and anti-GFP IgG (1:500), followed by incubation with peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection. In parallel, HEK-293 cells were treated 24 h prior and after transfection with leupeptin and pepstatin A (Leu/Pep; each 100 µM). The position of the molecular mass markers (in kDa) are indicated. (B) The activities of TPP-I (white bars) and ß-galactosidase (black bars) were measured in cells transiently transfected with vector only or wild-type and mutant p.Asn286Ser CLN2 cDNA. Both enzyme activities were expressed in relation to nontransfected cells (0.11 ± 0.01 and 2.05 ± 0.26 nmoles/min/mg cell protein, respectively). The values are means ± SD from three or four independent transfection experiments carried out in triplicate.

 
In HEK-293 cells expressing the mutant p.Asn286Ser CLN2, the TPP activity was comparable with vector-only transfected and nontransfected HEK-293 cells, which were used for comparison (Figure 1B). In extracts of cells transfected with wild-type CLN2 cDNA, the TPP activity was on average ninefold higher than in vector-transfected cells. The activity of a second lysosomal enzyme, ß-galactosidase, was unchanged in all three cell types (Figure 1B).

To compare synthesis and sorting of wild-type and mutant CLN2, transfected HEK-293 cells were metabolically labeled for 1 h with [35S]-methionine and then incubated in nonradioactive medium for 24 h. CLN2 was immunoprecipitated from cell extracts and media (Figure 2). One major band of 65 and two minor bands of 52 and 47 kDa were precipitated from cells expressing wild-type CLN2. About 12% of the newly synthesized wild-type CLN2 precursor was found in the medium. From cells expressing the mutant p.Asn286Ser CLN2, only a 63 kDa [35S]-labeled precursor form was precipitated. The weakly labeled 47-kDa band presumably represents the endogenous mature form. This shift in electrophoretic mobility was also observed in the mutant p.Asn286Ser CLN2 precipitated from the medium. Densitometric evaluation of the fluorograms revealed that about fourfold more [35S]-labeled CLN2 forms were immunoprecipitated from wild-type than from mutant p.Asn286Ser CLN2-expressing cells. From extracts of vector-transfected cells but not from media weak [35S]-labeled bands of 66, 65, 64, and 47 kDa could be immunoprecipitated presenting endogenous CLN2 protein.



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 2. Biosynthesis of wild-type and mutant CLN2 protein. Twenty-four hours after transfection of HEK-293 cells with pcDNA3.1 vector only or wild-type and mutant CLN2 cDNAs, cells were labeled with [35S]-methionine for 1 h and chased for 24 h. CLN2 was immunoprecipitated from cell extracts and media followed by electrophoresis (10% acrylamide) and fluorography. The experiment was repeated twice with similar results.

 
To examine whether the differences in the electrophoretic mobility between the wild-type and mutant p.Asn286Ser CLN2 were due to an altered glycosylation, CLN2 immunoprecipitates from transfected HEK-293 cells labeled with [35S]-methionine for 2 h were either treated with peptide N-glycosidase F (PNGase F) or endoglucosaminidase H (endo H). The amounts of the [35S]-labeled CLN2 forms immunoprecipitated from wild-type and mutant expressing cells were similar, indicating comparable rates of synthesis of both CLN2 proteins (Figure 3). After treatment with PNGase F, the apparent molecular masses of both wild-type and the mutant p.Asn286Ser CLN2 were completely reduced to ~55 kDa. Treatment with endo H resulted also in the deglycosylation to the 55-kDa form of the majority of the wild type and mutant CLN2 precursor protein. However, a small fraction (11–15%) of both CLN2 proteins contained complex oligosaccharides resistant to endo H. These findings demonstrate that the slightly increased electrophoretic mobility of mutant p.Asn286Ser CLN2 is due to the loss of one oligosaccharide chain rather than to proteolytic modification.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3. Deglycosylation analysis of wild-type and mutant p.Asn286Ser CLN2. CLN2 was immunoprecipitated from wild-type and mutant p.Asn286Ser CLN2-expressing HEK-293 cells labeled for 2 h with [35S]-methionine (0.1 mCi/ml medium), and one-third each was incubated in the presence or absence of 10 and 1 mU of PNGase F and endo H, respectively. The samples were analyzed by electrophoresis (10% acrylamide) and fluorography. The position of the glycosylated (gly), deglycosylated (non-gly), and endo H–resistent (asterisk) CLN2 forms are indicated.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The present study shows that the asparagine residue 286 of CLN2/TPP-I expressed in HEK-293 cells is glycosylated, and its substitution by a serine residue results in a complete loss of enzymatic activity, identifying p.Asn286Ser as a pathogenic mutation in LINCL patients. Whether the mutation has a direct effect on the function of the CLN2 protein or on the stability of the mature, enzymatically active form remains to be demonstrated and requires in vitro studies with recombinant mutant p.Asn286Ser CLN2. It has been reported that the purified wild-type CLN2 precursor protein can undergo autocatalytic processing on acidification to an active protease (Lin et al., 2001Go).

Human CLN2/TPP-I has five potential N-glycosylation sites (Asn210, Asn222, Asn286, Asn313, and Asn443), which are completely conserved in all deduced CLN2 amino acid sequences published (Bos taurus, Rattus norvegicus, Canis familiaris, Mus musculus, Macaca fascicularis). All N-glycosylation sites are located in the domain forming the mature enzyme comprising the amino acid residues 196–563. Complete deglycosylation of the CLN2 protein by PNGase F resulted in an expected decrease in the apparent molecular mass deduced from the cDNA sequence by 10 kDa (Figure 3; Golabek et al., 2003Go). Considering a molecular mass of ~2 kDa per carbohydrate chain (Millat et al., 1997Go; Yamashita et al., 1993Go), each N-glycosylation site in CLN2 is used. The observed shift in electrophoretic mobility of the expressed mutant p.Asn286Ser CLN2 by 2 kDa in comparison with the wild-type CLN2 confirms the usage of asparagine residue 286 as glycosylation site.

The results presented here indicate that the amounts of [35S]-methionine incorporated during a 2-h labeling period into mutant p.Asn286Ser CLN2 and wild-type CLN2 protein were similar (Figure 3), indicating no alterations in the rate of synthesis. On the other hand, both the amounts of labeled mutant p.Asn286Ser CLN2 recovered after a 24-h chase (Figure 2), and the steady-state level of immunoreactive mutant protein (Figure 1A) were four- and twofold reduced, respectively. Because the transfection efficiency was found to be similar for the wild-type and mutant CLN2 cDNA, the mutant p.Asn286Ser CLN2 appears to exhibit a reduced stability. It is likely that mutant p.Asn286Ser CLN2 will be degraded shortly after synthesis and escape immunoprecipitation. This is supported by the failure of potent inhibitors of lysosomal proteases to increase the amounts of CLN2 proteins in contrast to the lysosomal membrane protein LAMP-1 (Figure 1A). Generally, the expression pattern of CLN2 transiently expressed in HEK-293 cells differ from CLN2 in stably transfected cells (Golabek et al., 2003Go; Lin and Lobel, 2001Go; Lin et al., 2001Go). The majority of wild-type CLN2 in HEK-293 cells is presented as 65-kDa precursor forms. However, ~10% of the newly synthesized CLN2 is secreted into the medium, indicating correct sorting in the Golgi. Whether the strong overexpression in transfected cells interfere with the folding and/or exit of newly synthesized CLN2 in the endoplasmic reticulum or with post-Golgi sorting and/or processing is not known.

This is the first report describing the fatal effect of an elimination of a single glycosylation site (Asn286) of CLN2/TPP-I detected in two patients with LINCL. To our knowledge, there are only two studies reporting on glycosylation site mutations in patients with lysosomal nonenzymatic sphingolipid activator protein (saposin) deficiency leading to metachromatic leukodystrophy (Regis et al., 1999Go; Wrobe et al., 2000Go). However, there are several other studies investigating the role of N-glycosylation for structural integrity, intracellular transport, and/or catalytic function by site-directed mutagenesis and expression of mutant proteins. In some of the tested lysosomal enzymes—such as arylsulfatase A (Gieselmann et al., 1992Go), ß-glucuronidase (Shipley et al., 1993Go), alpha-galactosidase (Ioannou et al., 1998Go), and sulphamidase (Di Natale et al., 2001Go)—the elimination of individual occupied glycosylation sites and/or a combination of occupied sites induces loss of enzymatic activities, whereas N-glycosylation of alpha-glucosidase (Hermans et al., 1993Go) or iduronate sulfatase (Millat et al., 1997Go) are not required for catalytic activity. In a preliminary ternary structural model of human CLN2 constructed by homology modeling (Wlodawer et al., 2003Go) the Asn286 residue appears not to be located in the immediate vicinity of the substrate-binding sites. Thus the reasons for the severe effect of p.Asn286Ser CLN2 on enzymatic activity led to the variant phenotype in both LINCL patients are presently not clear and require further studies.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Fetal calf serum, Dulbecco's modified minimal essential medium, OptiMEM, and Lipofectamine were from Gibco Life Technologies (Karlsruhe, Germany); the primers for cloning of CLN2 and mutagenesis were from MWG Biotech (Munich, Germany). [35S]-Methionine and prestained rainbow molecular weight marker were from Amersham-Pharmacia-Biotech (Freiburg, Germany). Nitrocellulose was purchased from Sartorius (Göttingen, Germany), 4-methylumbelliferyl-ß-D-galactopyranoside and the protease inhibitor cocktail were from Sigma (Taufkirchen, Germany), and H-Ala-Ala-Phe-AMC was purchased from Bachem (Weil am Rhein, Germany). Rabbit IgG against human CLN2 was described previously (Ezaki et al., 1999Go). The monoclonal antibody H4A3 developed by J. T. August against human LAMP-1 was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). Monoclonal anti-GFP antibody was from BD Biosciences Clontech (Palo Alto, CA), peroxidase-conjugated anti-rabbit and anti-mouse IgG were from Dianova (Hamburg, Germany), and ECL Supersignal was from Pierce (Rockford, IL). Enzymes for molecular biology were from New England Biolabs (Frankfurt, Germany) and Stratagene (La Jolla, CA).

DNA constructs
Total RNA purified from human lymphoblasts was used to clone the CLN2 cDNA through reverse transcription polymerase chain reaction (PCR) using oligo d(T)16-primer (Applied Biosystems, Foster City, CA). PCR reactions were performed using Pfu-Turbo polymerase with the primers (cln2-for) 5'-GCT AGC AGA ATG GGA CTC CAA GCC TGC-3' and (cln2-his6-rev) 5'-GGT ACC TTA GTG ATG GTG ATG GTG ATG AGA TCT GGG GTT GAG TAG AGT CTTC AG-3'. The resulting PCR product was cloned into the pCR-blunt-TOPO vector (Invitrogen, Groningen, Netherlands). After excision the CLN2 cDNA was subcloned into NheI and Kpn I restriction sites of the expression vector pcDNA3.1.+ (Invitrogen). The construct was sequenced using the Abi prism sequenator to rule out that mutations had been introduced during PCR amplification. Mutation of asparagine residue 286 to serine (p.Asn286Ser) was performed by PCR using the Quik Change Site-Directed Mutagenesis Kit (Stratagene) and the primers (cln2/286-for) 5'-AGT GCT GGT GCC AGC ATC TCC ACC TGG-3' and (cln2/286-rev) 5'-CCA GGT GGA GAT GCT GGC ACC AGC ACT-3'. The nucleotide sequences of the plasmids were verified by sequencing. Wild-type and mutant CLN2 cDNAs were also subcloned into the pIRES2-EGFP expression vector (BD Biosciences Clontech).

Other methods
Transient transfection of HEK-293 cells with wild type, mutant p.Asn286Ser CLN2, or vector alone using Lipofectamine 2000 and the immunoblot expression analysis were carried out as described previously (Storch et al., 2003Go). Protein concentrations were determined using the Bradford protein assay (Bio-Rad, Munich, Germany). Cells were metabolically labeled with [35S]-methionine followed by immunoprecipitation of CLN2 according to a standard protocol (Partanen et al., 2003Go). Immune complexes were solubilized and incubated in the absence or presence of 1 mU endoglucosaminidase H (Roche, Mannheim, Germany) or 10 mU PNGase F (Roche) as described (Braulke et al., 1988Go; Partanen et al., 2003Go). TPP-I and ß-galactosidase activities were determined as described previously (Kleijer et al., 1976Go; Lukacs et al., 2003Go)


    Acknowledgements
 
This work was supported by the Deutsche Forschungsgemeinschaft (BR 990/10-2).


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: braulke{at}uke.uni-hamburg.de


    Abbreviations
 
Endo H, endoglucosaminidase H; GFP, green fluorescence protein; LINCL, late infantile neuronal ceroid lipofuscinosis; PNGase F, peptide N-glycosidase F; TPP, tripeptidyl peptidase


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Braulke T. (1996) Origin of lysosomal proteins in subcellular biochemistry. In Lloyd, J.B. and Mason, R.W. (eds.), Biology of the lysosome, vol 27. Plenum Press, New York, pp. 15–49.

Braulke, T., Hasilik, A., and von Figura, K. (1988) Low temperature blocks transport and sorting of cathepsin D in fibroblasts. Biol. Chem. Hoppe-Seyler, 369, 441–449.[ISI][Medline]

Di Natale, P., Vanacore, B., Daniele, A., and Esposito, S. (2001) Heparan N-sulfatase: in vitro mutagenesis of potential N-glycosylation sites. Biochem. Biophys. Res. Commun., 280, 1251–1257.[ISI][Medline]

Ezaki, J., Tanida, I., Kanehagi, N., and Kominami, E. (1999) A lysosomal proteinase, the late infantile neuronal ceroid lipofuscinosis gene (CLN2) product, is essential for degradation of a hydrophobic protein, the subunit c of ATP synthase. J. Neurochem., 72, 2573–2582.[CrossRef][ISI][Medline]

Ezaki, J., Takeda-Ezaki, M., Oda, K., and Kominami, E. (2000) Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis. Biochem. Biophys. Res. Commun., 268, 904–908.[CrossRef][ISI][Medline]

Giesclmann, V., Schmidt, O. and von Figura, K. (1992) In vitro mutagenesis of potential N-glycosylation sites of arulsulphatase. Biochem. J., 267, 13262–13266.

Golabek, A.A., Kida, E., Walus, M., Wujek, P., Mehta, P., and Wisniewski, K.E. (2003) Biosynthesis, glycosylation, and enzymatic processing in vivo of human tripeptidyl-peptidase I. J. Biol. Chem., 278, 7135–7145.[Abstract/Free Full Text]

Hermans, M.M., de Graaff, E., Kroos, M.A., Wisselaar, H.A., Willemsen, R., Oostra, B.A., and Reuser, A.J. (1993) The conservative substitution Asp-645->Glu in lysosomal alpha glucosidase affects transport and phosphorylation of the enzyme in an adult patient with glycogen-storage disease type II. Biochem. J., 289, 687–693.[ISI][Medline]

Ioannou, Y.A., Zeidner, K.M., Grace, M.E., and Desnick, R.J. (1998) Human {alpha}-galactosidase A: glycosylation site 3 is essential for enzyme solubility. Biochem. J., 332, 789–797.[ISI][Medline]

Kleijer, W.J., van der Veer, E., and Niermeijer, M.F. (1976) Rapid prenatal diagnosis of GM1-gangliosidosis using microchemical methods. Hum. Genet., 33, 299–305.[ISI][Medline]

Lin, L. and Lobel, P. (2001) Production and characterization of recombinant human CLN2 protein for enzyme-replacement therapy in late infantile neuronal ceroid lipofuscinosis. Biol. J., 357, 49–55.

Lin, L., Sohar, I., Lackland, H., and Lobel, P. (2001) The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH. J. Biol. Chem., 276, 2249–2255.[Abstract/Free Full Text]

Lukacs, Z., Santavuori, P., Keil, A., Steinfeld, R., and Kohlschütter, A. (2003) Rapid and simple assay for the determination of tripeptidyl peptidase and palmitoyl protein thioesterase activities in dried blood spots. Clin Chem., 49, 509–511.[Free Full Text]

Millat, G., Froissart, R., Maire, I., and Bozon, D. (1997) Characterization of iduronate sulphatase mutants affecting N-glycosylation sites and the cysteine-84 residue. Biochem. J., 326, 243–247.[ISI][Medline]

Partanen, S., Storch, S., Löffler, H.-G., Hasilik, A., Tyynelä, J., and Braulke, T. (2003) A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells. Biochem. J., 369, 55–62.[CrossRef][ISI][Medline]

Regis, S., Filocamo, M., Corsolini, F., Caroli, F., Keulemans, J.L., van Diggelen, O.P., and Gatti, R. (1999) An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity. Eur. J. Hum. Genet., 7, 125–130.[ISI][Medline]

Shipley, M., Grubb, J.H., and Sly, S.W. (1993) The role of glycosylation and phosphorylation in the expression of active human ß-glucuronidase. J. Biol. Chem., 268, 12193–12198.[Abstract/Free Full Text]

Sleat, D.E., Donelly, R.J., Lackland, H., Liu, C.-G., Sohar, I., Pullarkat, R.K., and Lobel, P. (1997) Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis. Science, 277, 1802–1804.[Abstract/Free Full Text]

Steinfeld, R., Heim, P., von Gregory, H., Meyer, K., Ullrich, K., Goebel, H.H., and Kohlschütter, A. (2002) Late infantile neuronal ceroid lipofuscinosis: quantitative description of the clinical course in patients with CLN2 mutations. Am. J. Med. Genet., 112, 347–354.[CrossRef][ISI][Medline]

Storch, S., Wittenstein, B., Islam, R., Ullrich, K., Sly, W.S., and Braulke, T. (2003) Mutational analysis in longest known survivor of mucopolysaccharidosis type VII. Hum. Genet., 112, 190–194.[ISI][Medline]

Vines, D.J. and Warburton, M.J. (1998) Purification and characterization of a tripeptidyl aminopeptidase I from rat spleen. Biochim. Biophys. Acta, 1384, 233–242.[ISI][Medline]

Williams, R.E., Gottlob, I., Lake, B.D., Goebel, H.H., Winchester, B.G., and Wheeler, R.B. (1999) CLN2. Classic late infantile NCL. In Goebel, H.H., Mole, S.E., and Lake, B.D. (eds.), The neuronal ceroid lipofuscinoses (Batten disease). Biomedical Health Research, vol 33. IOS Press, Amsterdam, pp. 37–54.

Wlodawer, A., Durell, S.R., Li, M., Oyama, H., Oda, K., and Dunn, B.M. (2003) A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases. BMC Struct. Biol., 3, 8.[CrossRef][Medline]

Wrobe, D., Henseler, M., Huettler, S., Pascual, S.I., Chabas, A., and Sandhoff, K. (2000) A non-glycosylated and functionally deficient mutant (N215H) of the sphingolipid activator protein B (SAP-B) in a novel case of metachromatic leukodystrophy (MLD). J. Inherit. Metab. Dis., 23, 63–76.[CrossRef][ISI][Medline]

Yamashita, K., Ohkura, T., Ideo, H., Ohno, K., and Kanai, M. (1993) Electrospray ionization-mass spectrometric analysis of serum transferrin isoforms in patients with carbohydrate-deficient glycoprotein syndrome. J. Biochem., 114, 766–769.[Abstract]