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Human Molecular Genetics Pages 85-90

Biosynthesis and intracellular targeting of the CLN3 protein defective in Batten disease
Introduction
Results
   Expression of CLN3 in COS-1 cells
   Intracellular synthesis and maturation of the CLN3 polypeptide
   Subcellular distribution of CLN3 protein in transfected HeLa cells
Discussion
Materials And Methods
   Construction of expression plasmids
   In vitro transcription and translation
   Cell culture and transfection
   Antibodies
   Western analysis
   Pulse labelling of COS-1 cells and immunoprecipitation
   Confocal microscopy
Acknowledgements
References

Biosynthesis and intracellular targeting of the CLN3 protein defective in Batten disease

Biosynthesis and intracellular targeting of the CLN3 protein defective in Batten disease Irma Järvelä1,*, Markku Sainio3, Terhi Rantamäki1, Vesa M. Olkkonen2, Olli Carpén3, Leena Peltonen1 and Anu Jalanko1

National Public Health Institute, 1Department of Human Molecular Genetics, 2Department of Biochemistry, Mannerheimintie 166, 00300 Helsinki, Finland and 3The Haartman Institute, Department of Pathology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki, Finland

Received August 21, 1997; Revised and Accepted October 10, 1997

Batten disease (juvenile-onset neuronal ceroid lipofuscinosis, JNCL), the most common neurodegenerative disorder of childhood, is caused by mutations in a recently identified gene (CLN3) localized to chromosome 16p11.2-12.1. To elucidate the biosynthesis and localization of the CLN3 protein, we expressed CLN3 cDNA in COS-1 and HeLa cell lines. In vitro translation, immunoprecipitation and Western blotting analyses detected an ~43 kDa polypeptide. Pulse-chase experiments indicated that the CLN3 protein is synthesized as an N-glycosylated single-chain polypeptide, which was not detected in growth medium. Confocal immunofluorescence microscopy revealed that the CLN3 protein is localized to the lysosomal compartment. These results provide evidence that Batten disease can be classified as a member of lysosomal diseases.

INTRODUCTION

Batten disease (juvenile-onset neuronal ceroid lipofuscinosis, JNCL) is the most common neurodegenerative disorder of childhood, characterized by visual failure, epilepsy and progressive dementia, and leads to premature death at the age of ~25 years (1 ). The hallmark of central nervous system pathology is cerebral atrophy (2 ) which can be visualized by magnetic resonance imaging (MRI) from ~15 years of age on (3 ). The 15 kb CLN3 gene, which consists of 15 exons forming an open reading frame (ORF) of 1314 bp, has been shown recently to be mutated in Batten disease patients (4 ,5 ). The ORF encodes a 438 amino acid protein with a deduced mol. wt of 48 kDa. The predicted amino acid sequence of the CLN3 protein shows no homology with any known protein, but the computer-based structural analysis indicates several hydrophobic regions suggesting that CLN3 represents an integral transmembrane protein (4 ,6 ). The function of the CLN3 protein remains unknown but the high evolutionary conservation demonstrated with homologous genes in Saccharomyces cerevisiae (BNT1) (7 ), Caenorhabditis elegans (8 ), mouse (9 ) and dog (8 ) suggests an important role for this protein in eukaryotic cells.

In mutation screening of Batten patients, one founder mutation, a 1.02 kb genomic deletion, has been found in ~85% of the disease alleles worldwide (10 ). In addition, 22 other mutations have been identified so far (4 ,10 ,11 ). This autosomal recessive disorder is enriched in the genetically isolated Finnish population, with an estimated carrier frequency of 1/70. In this population, 90% of disease alleles carry the 1.02 kb deletion (12 ).

Here we characterize the biosynthesis and intracellular targeting of the CLN3 protein by expressing the CLN3 cDNA in COS-1 and HeLa cells. The ~43 kDa single-chain polypeptide was N-glyco- sylated and targeted to lysosomes. These findings indicate that Batten disease may fall into the category of lysosomal storage diseases.

RESULTS

Expression of CLN3 in COS-1 cells

The cDNA coding for CLN3 polypeptide was, based on the reported sequence (4 ), obtained by RT-PCR amplification from total human brain RNA and cloned into the pGEM4 and pCMV5 expression vectors. In vitro translation of the CLN3-pGEM4 construct revealed an ~43 kDa polypeptide. In the presence of microsomal membranes, the major polypeptide detected was of the same size. However, an additional form of ~45 kDa appeared, indicating translocation to the microsomal membranes accompanied by a post-translational modification (Fig. 1 A). For the immunological detection of the CLN3 polypeptide, a polyclonal rabbit antibody 385/CLN3 was raised against a synthetic peptide corresponding to amino acid residues 242-258, predicted to be localized in a hydrophilic region of the CLN3 protein. The 385/CLN3 antibody reacted with a doublet of a major ~43 kDa band and a weaker ~45 kDa band in immunoblots of COS-1 cells transfected with CLN3-pCMV5 (Fig. 1 B).


Figure 1.(A) In vitro translation analysis of the CLN3 cDNA subcloned in the pGEM-4Z vector in the presence (+M) or absence (-M) of microsomal membranes. The pGEM-4Z vector was used as control (C). The molecular weights of the low range marker (BioRad) are indicated on the left. (B) Western blot analysis of the CLN3 polypeptide using the polyclonal 385/CLN3 antibody. Polypeptides of COS-1 cells transfected with CLN3-pCMV5 DNA (CLN3) and untransfected COS-1 cells as a control (C) were resolved on 11% SDS-PAGE. The molecular weights of the low range marker (BioRad) are indicated on the right.

Intracellular synthesis and maturation of the CLN3 polypeptide

To monitor the biosynthesis and intracellular processing of the CLN3 protein, metabolic labelling was performed and CLN3 polypeptides were immunoprecipitated from transfected COS-1 cells and media. A single intracellular polypeptide of ~43 kDa was detectable after 1 h labelling. During chase time up to 6 h, a characteristic doublet of ~43 kDa and ~45 bands appeared (Fig. 2 A). After 24 h of chase, the relative intensity of the labelled intracellular polypeptides was found to be reduced, most probably due to intracellular turnover of the protein. The CLN3 polypeptide could not be detected in the growth medium even after extended chase times (Fig. 2 B).


Figure 2. Pulse-chase analysis of the CLN3 polypeptide expressed in COS-1 cells. Cells were transiently transfected with the CLN3-pCMV5 construct, pulse labelled with [35S]cysteine for 1 h and chased for 0, 1, 3, 6 or 24 h. The polypeptides were immunoprecipitated from both cells (A) and media (B), resolved on 11% SDS-PAGE and visualized by fluorography. C is the control sample. The molecular weights of the low range marker (Biorad) are indicated on the left. Digestion of CLN3 polypeptide with endoglycosidase H (Endo-H) (E) and protein:N-glycosidaseF (P) under conditions described in Materials and Methods (C). Lane (-) represents the undigested CLN3 polypeptide.

The apparent maturation of the polypeptide into a doublet of the ~43 and ~45 kDa forms was suggestive of a post-translational modification taking place. To test whether the protein is N-glycosylated, endoglycosidase H (EndoH) and protein:N-glycosidase F (PNGaseF) treatments of the CLN3 protein expressed in COS-1 cells were performed after the immunoprecipitation. Both treatments yielded a single ~43 kDa polypeptide band (Fig. 2 C), demonstrating that the CLN3 polypeptide is N-glycosylated but, as shown by the EndoH sensitivity, does not attain complex type sugars.

Subcellular distribution of CLN3 protein in transfected HeLa cells

To analyse the subcellular distribution of the CLN3 protein, we carried out confocal microscopic analysis of transfected HeLa cells stained for CLN3 and several markers of subcellular compartments (Fig. 3 ). The CLN3 protein was distributed throughout the cytoplasm in a punctate vesicular fashion. A highly overlapping staining pattern was detected for the CLN3 protein and the lysosomal marker Lamp1; only a minority of juxtanuclear lysosomal vesicles were devoid of CLN3. The CLN3 protein did not demonstrate a cell surface staining pattern when cell contours were visualized by differential interference (DIC) microscopy or cell membrane staining [fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA)], and no CLN3 immunoreactivity was seen in unpermeabilized cells (not shown). Double staining for the CLN3 protein and endoplasmic reticulum (ER) proteins revealed only a minor co-distribution at juxtanuclear areas. No significant co-localization of CLN3 and mitochondrial or early endosomal markers was detected.

DISCUSSION

The current data demonstrate that the CLN3 protein defective in Batten disease is a lysosomal protein. The analyses of the intracellular synthesis and maturation of the CLN3 polypeptide demonstrate that it is synthesized as a single ~43 kDa precursor which is translocated into the ER membrane, attains heterogeneous N-glycosylation and is targeted to the lysosomes. The protein is not secreted at a detectable level even in the overexpression situation in transfected COS-1 cells.


Figure 3. Distribution of the CLN3 protein in transfected HeLa cells. The cells were transfected with wild-type CLN3-pCMV5 DNA and grown on glass coverslips. Double immunofluorescence staining of CLN3 protein (A, D, G, J and C, F, I, L) and lysosomes (B and green in C), endoplasmic reticulum (E and green in F), mitochondria (H and green in I) and early endosomes (K and green in L) was carried out with anti-385/CLN3 for CLN3 protein (cln3), mAb G1/139 for the lysosomal membrane protein Lamp1 (lys), mAb 1D3 for endoplasmic reticulum proteins (er), mAb M113-1 for a 60 kDa antigen of human mitochondria (mito) and mAb AT for the transferrin receptor in early endosomes (ee). Confocal microscope sections at 1.5 µm above the growth substratum are shown. In colour overlays, the staining pattern of CLN3 protein (red) and subcellular compartment markers (green) is shown (overlapping distribution in yellow). Bars (A-C) 25 µm, (D-L) 10 µm.

Evidence for the lysosomal localization of CLN3 was obtained by transient expression followed by double immunofluorescence studies of the cDNA in HeLa cells, where the transfection does not result in a vast overexpression which might disturb the intracellular transport of the protein. Double immunofluorescence studies using confocal microscopy showed a clear co-localization of CLN3 with the endogenous lysosomal membrane protein Lamp1, both displaying a punctate vesicular staining throughout the cytoplasm, typical of lysosomes. Interestingly, the highest CLN3-specific signal as well as the best co-localization with Lamp1 were observed at the edges of the cells, which is suggestive of polarization, characteristic of many membrane-associated proteins. Furthermore, CLN3 could not be seen associated with the plasma membrane, indicating that CLN3 is a lysosomal protein that does not become transported to its destination via the endocytic process. The CLN3 polypeptide showed partial co-localization also with the markers of the ER, suggesting that the intracellular transport of CLN3 proceeds through the ER-Golgi compartments.

Since the structural predictions of CLN3 suggest that it is an integral transmembrane protein with 5-6 membrane spans (4 ,6 ), this polypeptide could be an integral component of the lysosomal membrane. The lysosomal membrane is composed of highly glycosylated proteins, termed LGPs or lysosome-associated membrane proteins. The CLN3 protein is shown here to be glycosylated and not secreted out of the cell, both features typical of lysosomal membrane proteins. The overall structure of lysosomal integral membrane glycoproteins is very conserved and consists of a large lumenal domain containing 16-20 N-glycosylation sites, a single transmembrane domain and a short cytoplasmic tail, which contains the lysosomal targeting signal (13 -15 ). The hydrophilicity plot as well as the relatively low number of predicted glycosylation sites clearly place CLN3 protein into a distinct group. Some lysosomal membrane proteins that are targeted to lysosomes via the intracellular Golgi-endosome route contain a di-leucine motif at their C-terminal cytoplasmic tail (16 ,17 ). A putative di-leucine motif can be found in the C-terminal tail of the CLN3 polypeptide (Leu425Leu426). However, it remains to be studied whether this motif truly is involved in the lysosomal targeting of the CLN3 protein.

The major protein component of the abnormal storage material in Batten disease is the mitochondrial ATP synthase subunit c, which has been localized in the inner mitochondrial membrane (18 ,19 ). A vast majority of mitochondrial proteins coded by nuclear genes are synthesized as precursor proteins on cytosolic ribosomes from where they are targeted directly to mitochondria (20 ). The CLN3-specific antibody and the human mitochondrial antibody M113-1 showed no co-localization in the confocal immunofluorescence microscopy. Interestingly, recent studies of BTN1, a yeast gene corresponding to the CLN3 gene, show that deletion in BTN1 is not essential for viability, mitochondrial function or degradation of mitochondrial ATP synthase in yeast (7 ).

The palmitoyl protein thioesterase (PPT) gene was shown recently to be responsible for the infantile form of neuronal ceroid lipofuscinosis (INCL) (21 ). Furthermore, PPT was confirmed to be a soluble lysosomal protein (22 -25 ) which uses the traditional mannose-6-phosphate receptor pathway for its lysosomal targeting. PPT has been shown to remove palmitate groups from its in vitro substrate, Ras. The natural substrates of PPT, probably localizing within the lysosomal membrane, have not yet been identified. Therefore, the question of the possible interaction of CLN3 and PPT arises, especially due to the related nature of the diseases. Indicatively, the C-terminal tail of CLN3 contains a myristoylation signal and a neighbouring cysteine residue, characteristic of proteins that are both myristoylated and palmitoylated (26 ).

Electron microscopic studies, which before the DNA era were the cornerstone in Batten disease diagnosis, have shown patient cells to contain cytosomes with lipopigments encircled by trilaminar unit membrane, indicating a lysosomal origin. These lipopigment granules are present in both neural and non-neural cells. Also, an intense acid phosphatase activity of storage material suggests association with lysosomes (2 ,27 ,28 ). In this study, we present, for the first time, evidence that the CLN3 protein is a lysosomal membrane protein: confocal immunofluorescence microscopy shows co-localization of the CLN3 protein and the lysosomal membrane protein Lamp1. In agreement with sequence-based predictions revealing membrane-spanning domains, the radiolabelled CLN3 polypeptide was retained in cells, suggesting the character of an integral membrane protein. Although these data allow the classification of Batten's disease as a lysosomal storage disorder, the actual disease mechanism awaits to be resolved.

MATERIALS AND METHODS

Construction of expression plasmids

The CLN3 cDNA was obtained by reverse transcription and PCR amplification of the CLN3-coding region from human brain RNA followed by ligation into pCMV5 expression vector (29 ). The cloned cDNA was analysed by dideoxynucleotide sequencing (30 ). For in vitro translation experiments, CLN3 cDNA was ligated to the pGEM4 vector under the SP6 promoter.

In vitro transcription and translation

CLN3 cDNA cloned into pGEM4 under the SP6 promoter was first transcribed to mRNA using the CapScribe transcription kit (Boehringer-Mannheim) and then in vitro translated to protein using an in vitro translation kit (Boehringer-Mannheim) with and without microsomal membranes.

Cell culture and transfection

COS-1 and HeLa cells were obtained from the American Type Culture Collection, cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics. One day prior to transfection, the cells were seeded on 3-cm cell culture dishes at 4×105 cells per dish. The cells were transfected with 5 µg of CLN3 in the plasmid pCMV5 using the Lipofectamine (Gibco/BRL) transfection method (31 ). COS-1 cells were prepared for analysis 48 h after transfection. HeLa cells were plated on coverslips on the day following the transfection.

Antibodies

For the preparation of the 385/CLN3antibody, a synthetic peptide corresponding to amino acid residues 242-258 of the full-length CLN3 cDNA, with one cysteine residue added to the N-terminus of the peptide, was coupled to keyhole limpet hemocyanin (KLH) using 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) as a coupling reagent. For rabbit immunizations, 500 µg of peptide-KLH conjugate were emulsified in complete Freund's adjuvant and injected intradermally at multiple sites. Injections were performed at 3-week intervals for a total of 9 weeks. The antiserum was purified by an affinity chromatography with a Sepharose CL4B column in which the synthetic peptide had been cross-linked.

Murine monoclonal antibody mAb 1D3 against the KDEL retrieval motif of soluble ER proteins was a gift of Stephen Fuller (32 ), mAb G1/139 against the lysosomal membrane protein Lamp1 was a gift from Hans-Peter Hauri (Biocenter, Basel, Swizerland), monoclonal antibody M113-1 against 60 kDa antigen of human mitochondria was purchased from BioGenex, and monoclonal antibody AT against human transferrin receptor from Boehringer-Mannheim. The secondary antibodies used in immunofluorescence microscopy were tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Dako) and FITC-conjugated goat anti-mouse IgG (Dako).

Western analysis

For Western blot analysis, transfected COS-1 cells were trypsinized and the pellets were solubilized in 100 µl of Laemmli buffer (33 ). Samples were run on 11% SDS-PAGE and the proteins were transferred to a nitrocellulose membrane (Amersham, Hybond-C Extra) by electroblotting at 400 mA for 1 h. Immunostaining was performed with the polyclonal 385/CLN3-peptide antibody followed by anti-rabbit IgG-AP (Promega). The bands were visualized by ProtoBlot® NBT and BCIP Color Development System (Promega).

Pulse labelling of COS-1 cells and immunoprecipitation

For metabolic labelling, transfected cells were starved in cysteine-free medium for 1 h and thereafter labelled with 100 µCi/ml of [35S]cysteine (3000 Ci/mmol; Amersham) in cysteine-free medium. After a 1 h pulse, the cells and media were either harvested immediately or the cells were subjected to a 3-24 h chase in DMEM without FCS. The cells were harvested by trypsinization and lysed by freeze-thawing in 100 µl of 1% Triton X-100 in phosphate-buffered saline (PBS) with protease inhibitors (pepstatin and leupeptin). Proteins from the collected media were concentrated by Centricon 10 (Amicon) prior to immunoprecipitation. Immunoprecipitations were carried out with the 385/CLN3 antibody as previously described (34 ). The polypeptides were separated by 11% SDS-PAGE (33 ) and visualized by fluorography using the Amplify reagent (Amersham). One µl (200 mU) of EndoH (Boehringer-Mannheim) and 2 µl (1000 U) of PNGaseF (New England Biolabs) were added to an immunoprecipitate dissolved in 67 mM potassium phosphate, pH 6.7 and the samples were incubated for 20 h at 37°C.

Confocal microscopy

For indirect immunofluorescence, HeLa cells were seeded on 12-mm coverslips and grown overnight to 30-50% confluency. Transfected cells were fixed in 4% paraformaldehyde (PFA), pH 7.4, and permeabilized with 0.05% saponin (Sigma). For double staining of the CLN3 protein with the compartmental markers, fixed cells were incubated simultaneously with anti-385/CLN3 and different mAbs, followed by detection of the bound antibodies using TRITC-conjugated goat anti-rabbit IgG (Dako) and FITC-conjugated goat anti-mouse IgG (Dako). Specimens were viewed with a confocal 410 Invert Laser Scan Microscope (Carl Zeiss, Oberkochen, Germany). In some experiments, cell contours were visualized with FITC-WGA (Sigma) and using the DIC option of the confocal microscope.

ACKNOWLEDGEMENTS

We thank Auli Haikka, Tuula Manninen and Tuula Halmesvaara for excellent technical assistance. We are grateful for Camilla Cantor and Jouni Vesa for preparing the CLN3 antibody and Hans-Peter Hauri (Biocenter, The University of Basel, Swizerland) for Lamp1 antibody. This work was supported by The Academy of Finland and The Paulo Foundation.

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*To whom correspondence should be addressed. Tel: +358 9 4744394; Fax: +358 9 4744480; Email irma.jarvela@ktl.fi
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