HUMAN MUTATION Mutation in Brief #747 (2004) Online
MUTATION IN BRIEF
A Novel Aspartylglucosaminuria Mutation Affects Translocation of Aspartylglucosaminidase Jani Saarela, Carina von Schantz, Leena Peltonen, and Anu Jalanko* Department of Molecular Medicine, National Public Health Institute, and Department of Medical Genetics, University of Helsinki *Correspondence to: Anu Jalanko, National Public Health Institute, PO Box 104, FIN-00251 Helsinki, Finland; Tel.: +358 9 47448392; Fax: +358 9 47448480; E-mail:
[email protected] Grant sponsor: Academy of Finland, Centre of Excellence is Disease Genetics; Grant number: 44870. Communicated by Mark H. Paalman
The AGA gene is mutated in patients with aspartylglucosaminuria (AGU), a lysosomal storage disease enriched in the Finnish population. The disease mechanism of AGU and the biochemistry and cell biology of the lysosomal aspartylglucosaminidase (AGA) enzyme are well characterized. Here, we have investigated a novel AGU mutation found in a Finnish patient. The mutation was detected as a compound heterozygote with the Finnish major mutation in the other allele. The novel point mutation, c.44T>G, causes the L15R amino acid substitution in the signal sequence of the AGA enzyme. The mutated AGA enzyme was here analyzed by over expression in BHK and COS-1 cells. The L15R AGA protein was only faintly detectable by immunofluorescence analysis and observed in the endoplasmic reticulum. Metabolic labeling and immunoprecipitation revealed only a small amount of AGA polypeptides but the specific activity of the mutant enzyme was surprisingly high, 37% of the wild type. The amino acid substitution probably affects translocation of AGA polypeptides by altering a critical hydrophobic core structure of the signal sequence. It appears that the small amounts of active enzyme are not able to reach the lysosomes thus explaining the development of AGU disease in the patient. © 2004 Wiley-Liss, Inc. KEY WORDS: Aspartylglucosaminuria; AGU; aspartylglucosaminidase; AGA; signal sequence; translocation
INTRODUCTION
Aspartylglucosaminuria (AGU; MIM# 208400; GenBank: X55330) results from deficient activity of a lysosomal hydrolase, aspartylglucosaminidase (AGA, glycosylasparaginase, N4-(β-N-acetyl-glucosaminyl)-1asparaginase, EC 3.5.1.26). This recessively inherited disease is enriched in the Finnish population [Norio, 2003] and is manifested by excessive accumulation of uncleaved glycoasparagines in lysosomes and elevated metabolite levels in urine [Pollitt, et al., 1968]. The clinical phenotype of patients includes progressive mental retardation and skeletal and connective tissue abnormalities [Autio, et al., 1973]. The phenotype is rather uniform and the average life span of patients is 40 years. One founder mutation [Ikonen, et al., 1991a] represents 98% of AGU alleles in Finland [Syvänen, et al., 1992]. Prior to this study, altogether 26 different mutations had been characterized
Received 23 March 2004; accepted revised manuscript 18 June 2004.
© 2004 WILEY-LISS, INC. DOI: 10.1002/humu.9276
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[Saarela, et al., 2001]. The structure of the native human AGA enzyme has been solved by us [Oinonen, et al., 1995]. The active molecule consists of two α- and two β-subunits. The processing of human AGA in cells is relatively complex [Ikonen, et al., 1993]. The AGA gene [Ikonen, et al., 1991a] encodes an inactive 346 amino acid precursor polypeptide, which is translocated into the lumen of the endoplasmic reticulum (ER). The 23 amino acid signal sequence is cleaved co-translationally by signal peptidase [Ikonen, et al., 1993]. In the lumen of the ER, two precursor polypeptides fold and dimerize, which is a prerequisite of subsequent activation [Riikonen, et al., 1996]. The activation occurs autocatalytically resulting in the active tetrameric (αββα) enzyme [Saarela, et al., 1998; Saarela, et al., 2004]. The activated molecule is transported to the Golgi complex, where the lysosomal targeting signal, mannose-6-phosphate, is added to the four oligosaccharide chains of the molecule [Tikkanen, et al., 1997]. Sorting for lysosomal transportation by mannose-6-phosphate receptors occurs in the trans-Golgi network. In the lysosomes, both subunits of AGA are trimmed by lysosomal hydrolases, which does not affect AGA activity. In this study, a novel AGU mutation is characterized. The mutant cDNA was expressed in BHK and COS-1 cells, and the consequences of the mutation to the intracellular processing, transport, and activity of AGA were determined.
MATERIALS AND METHODS DNA Sequencing
DNA samples of a Finnish AGU patient and the father were obtained. The mother’s DNA was unavailable for research. The patient’s and the father’s chromosomal DNA was amplified using intron-specific primers [Isoniemi, et al., 1995] and sequencing of both strands of the AGA gene (GenBank: X55330) was performed with an ABIPrism 377 DNA sequencer (Perkin Elmer). Sequencer software (Gene Codes) was used to analyze the data. The DNA mutation numbering is based on cDNA sequence and is marked with a “c” before the number. The number +1 corresponds to the A of the ATG translation initiation codon in the reference sequence. In vitro mutagenesis
Mutagenesis was performed with QuikChange site-directed mutagenesis kit (Stratagene) on an AGA cDNA template [Ikonen, et al., 1991b]. In the template, the gene had been cloned into the BamHI site of the mammalian expression vector SVpoly [Stacey and Schnieke, 1990]. After mutagenesis, the AGA gene was sequenced to exclude the possibility of unwanted mutations. Transfection, metabolic labeling and immunoprecipitation
The methods used have been described earlier [Saarela, et al., 2001]. In short, COS-1 cells (ATCC: CRL-1650) were transfected and, after 48 h, the cells were labeled for 1 h with [35S]Cys. A 3 h chase was followed by immunoprecipitation using AGA-specific polyclonal antibodies against the native enzyme [Halila, et al., 1991]. The labeled polypeptides were separated by 14% SDS-PAGE under reducing conditions and visualized by autoradiography. Assay for AGA activity
The modified AGA activity assay [Peltola, et al., 1996] was performed after metabolic labeling. The assay is based on colorimetric measurement of liberated N-acetylglucosamine [Reissig, et al., 1955]. Immunofluorescence analysis and confocal microscopy
The intracellular localization of AGA polypeptides was studied by staining of the AGA transfected BHK-21 cells (ATCC: CCL-10). Following 48 h incubation after transfection, the cells were treated with 50 µg/ml cycloheximide (Sigma) for 3 h to stop protein synthesis and subsequently fixed with 4% paraformaldehyde. After permeabilization, the cells were stained with AGA-specific antibodies (against the native enzyme and against the denatured subunits of AGA) and fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories) as described earlier [Saarela, et al., 2001]. Specimens were viewed with a 63x objective on a Leica DMR confocal microscope (Leica) with Leica NT software.
Novel AGU Mutation Affects Translocation 3
RESULTS Identification of a novel AGU mutation
A novel mutation, c.44T>G, was found in the first exon in one chromosome of the patient and the father. The novel mutation has not been found from other DNA samples. In the other chromosome, the patient, but not the father, had the major Finnish founder mutation (c482G>A + c488G>C). The novel and the Finnish major mutation were thus found as compound heterozygotes in the patient, who presumably had inherited the Finnish major mutation from the mother. At the protein level the novel mutation results in the amino acid substitution L15R. The mutation is located in the signal sequence of the AGA polypeptide. Intracellular targeting, processing, and activity of mutant AGA polypeptides
The intracellular localization of AGA polypeptides was studied using immunofluorescence analysis of transfected BHK cells. The L15R mutant AGA polypeptides were observed only in the ER, by co-localization studies using ER-specific markers, and the AGA staining was very faint (data not shown). In immunoprecipitation analysis, small amounts of L15R AGA polypeptides were observed (Figure 1). The sizes of L15R polypeptides corresponded with the WT AGA polypeptides. However, the AGA activity of L15R polypeptides was surprisingly high considering the small amount of AGA polypeptides observed by immunofluorescence and immunoprecipitation analyses. Compared to WT AGA, 37% of AGA activity was detected in L15R transfected cells and 7% in mock-transfected cells.
Figure 1. Intracellular processing of AGA polypeptides. AGA polypeptides from COS-1 cells transfected with wild-type (WT) or mutant (L15R) AGA-SVpoly were labelled and subsequently immunoprecipitated after a 3 h chase using antibodies produced against the native enzyme. The endogenous AGA background of COS-1 cells is also shown (COS). The polypeptides were resolved on 14% SDS-PAGE and observed with autoradiography.
DISCUSSION
Signal sequences control the entry of virtually all proteins to the secretory pathway [Hartmann, et al., 1992]. The common structure of signal sequences from various proteins is commonly described as a positively charged nregion, followed by a hydrophobic h-region and a neutral but polar c-region. The residues at positions -3 and -1 (relative to the cleavage site) must be small and neutral for cleavage to occur [von Heijne, 1983; von Heijne, 1985]. Analysis of signal sequence mutants have shown that the middle hydrophobic core region is the most essential part required for targeting and membrane insertion [von Heijne, 1985]. The hydrophobicity of signal sequences has been suggested to correlate with preference for translocation pathway. Signal sequences for proteins
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that follow the signal recognition particle (SRP) –independent pathway were found to be relatively less hydrophobic than those from proteins targeted by SRP or those utilizing both pathways [Ng, et al., 1996]. Posttranslational protein translocation across the membrane of the ER is mediated by the Sec complex. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the transmembrane channel formed by multiple copies of the Sec61 protein [Van den Berg, et al., 2004]. While bound, the signal sequence is in contact with both protein components of the channel and lipid of the membrane.
Figure 2. The signal sequence of WT and L15R AGA. The colouring of amino acid residues follows the classification used by Branden and Tooze [Branden and Tooze, 1999] e.g. hydrophobic-green, charged-red, polar uncharged-white.
The AGA signal sequence contains 23 amino acids (Figure 2). The WT signal sequence contains two charged residues at the N-terminal part of the signal sequence (residues 3 and 4), a 10-amino acid hydrophobic middle part, and at positions -3 and -1 small and neutral residues. The mutant structure is identical, except that at position 15 a hydrophobic residue has been substituted by a charged residue. Thus, the most essential component of the signal sequence normally required for ER translocation is abnormal in the L15R polypeptides. A charged residue could prevent or weaken the binding of the signal sequence with the protein and lipid components in the translocon complex. This in turn could well affect the translocation of the L15R polypeptides into the ER. Translocation and subsequent folding and dimerization are required for AGA activation. The L15R polypeptides could be mostly misfolded due to lack of transport into the lumen of the ER where AGA acquires its’ correct three-dimensional structure and enzyme activity. Therefore, only trace amounts of AGA polypeptides would be expected to be visible in immunofluorescence analysis. The relatively high degree of AGA activity and normal processing pattern of a small amount of AGA polypeptides observed here would indicate that to some extent the L15R polypeptides are translocated to the ER, and autocatalycally activated. However, we were not able to detect traces of over expressed enzyme in the lysosomes, and in the patient’s cells, the amounts of produced mutant enzyme are evidently below detection levels and lysosomal accumulation of glycoasparagines occurs, thus leading to the development of the AGU disease.
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