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Technical Briefs |
-N-Acetylgalactosaminidase Deficiency
Departments of
1
Biochemistry and
2 Dermatology, Faculty of Medicine, Kagoshima University, Kagoshima 890-8520, Japan
aauthor for correspondence: fax 81-99-264-6274, e-mail masakun{at}m.kufm.kagoshima-u.ac.jp
Thin-layer chromatography (TLC) has been widely applied to the diagnosis of lysosomal storage diseases by analyzing abnormally excreted oligosaccharides in urine. However, TLC does not necessarily identify the excreted compounds accurately. Labeling the oligosaccharides with a tag, such as 8-aminonaphthalene-1,3,6-trisulfonic acid, allows easy identification of the excreted compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (1). However, this labeling is based on reductive amination of the reductive end of the oligosaccharides with the labeling compound, so abnormal amino acid O-glycosides with no reducing end cannot be identified. Indeed, patients with Kanzaki/Schindler disease or aspartylglucosaminuria, who mainly excrete abnormal amino acid O-glycosides, can be identified only with difficulty by this method.
In the present study, we successfully identified abnormal amino acid O-glycosides in urine from Kanzaki/Schindler disease [
-N-acetylgalactosaminidase (-NAGA) deficiency] patients (2), using partition chromatography and MALDI-TOF MS. The method involves three steps: purification on a solid-phase extraction column filled with cellulose;removal of sialic acids; and mass spectrometric analysis. This simple procedure reduces both the amount of urine and analysis time and precisely identifies excreted abnormal substances.
For MS, 5- to 15-µL urine samples (equivalent to 5 µg of creatinine) were mixed with 250 pmol of raffinose as an internal standard. Urinary creatinine was measured by colorimetric analysis (3). The samples were then mixed with an ethanol–1-butanol solution (1:4 by volume) and applied to a column filled with cellulose (Cellulose Cartridge Glycan preparation reagent set; Takara Co.). These solid-phase extraction cartridges retain all the glycoconjuguates possessing an oligosaccharide containing at least three sugar residues. The eluted samples were dried on a centrifugal vacuum evaporator, suspended in 50 µL of 50 mmol/L HCl, and incubated at 80 °C for 1 h to remove sialic acids from compounds. They were then dried and dissolved with 20 µL of 1 mL/L trifluoroacetic acid in acetonitrile–H2O (50:50 by volume). Finally, each sample was mixed with 5 µL of 10 g/L 2,5-dihydroxybenzoic acid (DHB) as a matrix. From each sample, 1 µL was analyzed by TOF MS (VoyagerTM Pro; Applied Biosystems) in positive linear mode at an accelerating voltage of 20 kV. The resolution calculator from the GRAMS/32 software was supplied with the instrument. We calibrated the ions with the mass position of the [M +Na]+ form of raffinose [monoisotopic mass (M), 527.2; Wako Pure Chemical Co.] as an internal or external standard.
We first checked the masses of the ions from the compounds (GP-D2) that are abnormally excreted in the urine of patients with -NAGA deficiency (4)(5)(6). The purified compounds from a patient with -NAGA deficiency (4), kindly provided by Dr Hirabayashi (Riken, Tsukuba, Japan), were NeuAc2-3Galß1-3[NeuAc2-6]GalNAc1-Ser/Thr, where NeuAc is acetylneuraminic acid and GalNAc is N-acetylgalactosamine. The sample was composed mainly of the threonine type of GP-D2 (monoisotopic mass, 1066.4) and <5% of the serine type (monoisotopic mass, 1052.4). After 500 pmol of the GP-D2 was dried, it was treated with 20 µL of 1 mL/L trifluoroacetic acid in acetonitrile–H2O (50:50 by volume), mixed with DHB as a matrix, and analyzed by TOF MS. Mass spectrometric analysis showed several ions around m/z 1150 (m/z 1090.9, 1135.9, 1157.3, and 1172.6; data not shown). No chemical composition could be deduced from these ions, and the spectra were unsuitable for disease diagnosis. Because of difficulties of ionizing GP-D2, we treated it to remove the two sialic acids from the compounds described above. As shown in Fig. 1A
, GP-D2 gave clear signals at m/z 507.2, 523.2, 529.2, and 545.2, which were exactly the ones expected for [M +Na]+, [M +K]+, [M + 2 Na -H]+, and [M + Na + K -H]+, respectively. Several ions around m/z 1150 in the untreated sample disappeared in the treated sample. This suggested that sialic acid moieties were completely removed by the treatment. In addition, the signal at m/z 493.1 was in accordance with a pseudomolecular ion, indicating a Na+ ion adduct of the serine type of GP-D2 (Fig. 1A
, 
). The ions in Fig. 1
labeled m1–m4 (m/z 473.2, 489.0, 495.2, and 511.1) were from the matrix (DHB) because they appeared even without the standard material (data not shown).
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We next analyzed urine samples with the addition of raffinose as an internal standard. In general, ionization with TOF MS is problematic with samples containing detergent and high concentrations of salts. We therefore partially purified urine (volume equivalent to 5 µg of creatinine) with a partition column (Takara Shuzo Co.) filled with cellulose. After desalting and partially purifying the urine with partition chromatography, we treated it with an acidic solution to remove sialic acids from the compounds as well as the standard. Through this simple procedure, we identified the abnormal amino acid O-glycosides in the urine of two patients with -NAGA as the asialo compounds on the basis of their molecular masses, which corresponded to the ions produced by GP-D2 (Fig. 1C
; ions 1–4); total preparation and analysis time was <9 h. The TOF MS profiles of the two patients were very similar. Representative data from six healthy volunteers and the two patients are presented in Fig. 1
, B and C. The samples from healthy volunteers showed only the ions produced by the matrix and the internal standard within this range. These results were in accordance with the reports that patients with -NAGA deficiency in adult form excrete mainly compounds composed of one or two sialic acid moieties and Galß1-3GalNAc1-Thr as a core structure (4)(5).
-NAGA deficiency is one of the rarest and probably the most heterogeneous of lysosomal storage disorders (3)(7). At present, only nine patients are known from six families. Of these, infants (four patients) and adults (five patients) display obviously different phenotypic expression. Adults commonly have angiokeratoma, loss of hearing, dizziness, and cutaneous sensory disturbance (numbness). To date, we have hesitated to diagnose the disease through the somewhat intricate procedure of TLC and electron microscopy, but the newly established method will make it easier to survey patients showing the above signs. In proposing this new method for identifying abnormal amino acid O-glycosides in urine from patients with -NAGA deficiency, we are also suggesting a way to identify compounds containing no reductive moiety excreted in the urine of patients with other lysosomal storage diseases.
Acknowledgments
We thank Dr. G. Bierwirth and M. Gore for editorial assistance and Dr. Y. Hirabayashi for the kind gift of GP-D2. This research was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Kodama Foundation for Research in Medical Science.
References
-N-Acetylgalactosaminidase deficiency: Schindler disease. Scriver CR Beaudet AL Sly WS Valle D eds. The metabolic and molecular bases of inherited disease, 8th ed 2001:3483-3505 McGraw-Hill New York. .
-N-acetylgalactosaminidase activity. Biol Chem Hoppe-Seyler 1989;370:661-672.[Medline]
[Order article via Infotrieve]
-N-acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum, with Meniere’s syndrome and without mental retardation. Br J Dermatol 2001;144:363-368.[Medline]
[Order article via Infotrieve]
-N-acetylgalactosaminidase (-NAGA) deficiency: new mutations and the paradox between genotype and phenotype. J Med Genet 1996;33:458-464.[Abstract]
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