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
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Abstract |
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Key words: expression analysis / glycosylation / late infantile neuronal ceroid lipofuscinosis / loss of function / tripeptidyl peptidase I
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Introduction |
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TPP-I is synthesized in the endoplasmic reticulum as N-glycosylated 66/67 kDa precursor protein (Ezaki et al., 1999; Lin et al., 2001
). 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 receptorTPP-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, 1996
). 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., 2003
; Lin and Lobel, 2001
; Lin et al., 2001
).
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., 2002). 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.
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Results |
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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.
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Discussion |
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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 196563. 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., 2003). Considering a molecular mass of
2 kDa per carbohydrate chain (Millat et al., 1997
; Yamashita et al., 1993
), 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., 2003; Lin and Lobel, 2001
; Lin et al., 2001
). 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., 1999; Wrobe et al., 2000
). 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 enzymessuch as arylsulfatase A (Gieselmann et al., 1992
), ß-glucuronidase (Shipley et al., 1993
), alpha-galactosidase (Ioannou et al., 1998
), and sulphamidase (Di Natale et al., 2001
)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., 1993
) or iduronate sulfatase (Millat et al., 1997
) are not required for catalytic activity. In a preliminary ternary structural model of human CLN2 constructed by homology modeling (Wlodawer et al., 2003
) 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.
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Materials and methods |
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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., 2003). 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., 2003
). 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., 1988
; Partanen et al., 2003
). TPP-I and ß-galactosidase activities were determined as described previously (Kleijer et al., 1976
; Lukacs et al., 2003
)
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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