Human α-N-acetylgalactosaminidase-molecular cloning, nucleotide sequence, and expression of a full-length cDNA: Homology with human α-galactosidase a suggests evolution from a common ancestral gene

Human α-N-acetylgalactosaminidase (α-GalNAc, B.C. 3.2.1.49), the lysosomal glycohydrolase that cleaves α-N-acetylgalactosaminyl moieties from glycoconjugates, is encoded by a gene localized to chromosome 22q13→qter. The deficient activity of α-GalNAc is the enzymatic defect in Schindler disease, an inherited neuroaxonal dystrophy. To isolate a full-length cDNA, the enzyme from human lung was purified to homogeneity, 129 non-overlapping amino acids were determined by microsequencing the N terminus and seven tryptic peptides, and four synthetic oligonucleotide mixtures were used to screen a human fibroblast cDNA library. A full-length cDNA, pAGB-3, isolated from a placental λgt11 cDNA library, had a 2158-base pair (bp) insert with an open reading frame which predicted an amino acid sequence that was co-linear with all 129 microsequenced residues of the purified enzyme. The pAGB-3 insert had a 344-bp 5′-untranslated region, a 1236-bp open reading frame encoding 411 amino acids, a 514-bp 3′-untranslated region, and a 64-bp poly(A) tract. A signal peptide sequence of 17 amino acids as well as six N-glycosylation sites were predicted. The pAGB-3 cDNA was subcloned into the p91023(B) mammalian expression vector and human α-GalNAc activity was transiently expressed in COS-1 cells, demonstrating the functional integrity of the full-length cDNA. Northern hybridization analysis of mRNA revealed two transcripts of about 3.6 and 2.2 kilobases (kb), and primer extension studies indicated a cap site at nucleotide -347 for the 2.2-kb transcript. The 3.6-kb cDNA (pAGB-35) was isolated; the 3598-bp pAGB-35 insert was identical to that of the 2.2-kb insert but had additional 5′- and 3′-untranslated sequences including a second downstream polyadenylation signal at nucleotide 3100-3105. Isolation of a genomic clone, gAGB-1, and sequencing the 2048-bp region including pAGB-3 revealed a 1754-bp intron between codons 319 and 320, which also was the site of a 70-bp insertion and a45-bp deletion in other cDNA clones. Notably, the α-GalNAc cDNA had remarkable amino acid homology with the human α-galactosidase A (α-Gal A) cDNA suggesting the evolutionary relatedness of these genes. The α-GalNAc cDNA had 46.9-64.7% amino acid identity in sequences (codons 1-319) corresponding to α-Gal A exons 1 through 6, while the comparable exon 7 sequence (pAGB-3 codons 320-411) had only 15.8% homology with numerous gaps. These findings implicate the genomic region at and surrounding codon 319 as a potential site for the abnormal processing of α-GalNAc transcripts as well as for a recombinational event in the evolution and divergence of α-Gal A and α-GalNAc. The availability of the full-length cDNA for human α-GalNAc will permit studies of the genomic organization and evolution of this lysosomal gene, as well as the characterization of the molecular lesions causing Schindler disease.