Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C)

Abstract

Mucopolysaccharidosis IIIC (MPS IIIC), or Sanfilippo C, represents the only MPS disorder in which the responsible gene has not been identified; however, the gene has been localized to the pericentromeric region of chromosome 8. In an ongoing proteomics study of mouse lysosomal membrane proteins, we identified an unknown protein whose human homolog, TMEM76, was encoded by a gene that maps to 8p11.1. A full-length mouse expressed sequence tag was expressed in human MPS IIIC fibroblasts, and its protein product localized to the lysosome and corrected the enzymatic defect. The mouse sequence was used to identify the full-length human homolog (HGSNAT), which encodes a protein with no homology to other proteins of known function but is highly conserved among plants and bacteria. Mutational analyses of two MPS IIIC cell lines identified a splice-junction mutation that accounted for three mutant alleles, and a single base-pair insertion accounted for the fourth.

Figure 1.

Identification of D8Ertd354e by MS/MS. The lysosomal membrane proteins from mutant (Beige) and normal mice were labeled with either isotopically heavy (¹³C) or light (¹²C) cICAT reagent, were digested with trypsin, and were affinity purified. The upper right inset is the MS pattern of a pair of these labeled peptides. The main spectrogram shows the MS/MS fragmentation pattern for the Cys-ICAT light–labeled peptide. The obtained peptide sequence is shown at the upper left. *C indicates the labeled Cys residue.

Figure 2.

The epitopically expressed mouse D8Ertd354e (N-acetyltransferase) localizes to lysosomes in HeLa cells. C-terminal V5-epitope–tagged mouse N-acetyltransferase cDNA was transiently expressed in HeLa cells. The fixed cells were processed for indirect immunofluorescence confocal microscopy. N-acetyltransferase was detected with V5-antibody (green), whereas lysosomes were detected with LAMP-1 (H4A3) antibody (red). The merged images show substantial overlap between the signals (yellow), which indicates colocalization. Bar=10 μm.

Figure 3.

Chromosomal localization and structure of the human HGSNAT gene and the predicted topology of the enzyme it encodes, N-acetyltransferase, with its 11 TMDs (A–K) after the cleavage of the signal peptide. The exons and their encoded protein domain, shown in red, are unique to higher metazoans. Red circles on the protein diagram represent the positions of putative Asn-linked oligosaccharide sites, and the green triangles delineate the sections of the protein encoded by each of the 18 exons in HGSNAT.

Figure 4.

Aligned primary sequences of N-acetyltransferase from higher metazoans. The predicted human 30-residue, cleavable signal peptide is shown in a cyan-filled rectangle. Red circles and green triangles represent putative Asn-linked oligosaccharide sites and delineate the portions of the protein encoded by each of the 18 exons of the gene, respectively. Black bars indicate the 11 predicted TMDs (A–K), and the yellow underlined sequence in the mouse is the tryptic peptide we initially identified by MS/MS (fig. 1).

Figure 5.

Northern-blot analysis of the HGSNAT transcripts in various human tissues. PBL = peripheral-blood leukocytes.