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

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc, E.C. 3.2.1.49), the lysosomal glycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties from glycoconjugates, is encoded by a gene localized to chromosome 22q13----qter. The deficient activity of alpha-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 lambda gt11 cDNA library, had a 2158-base pair (bp) insert with an open reading frame which predicted an amino acid sequence that was colinear 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 alpha-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 alpha-GalNAc cDNA had remarkable amino acid homology with the human alpha-galactosidase A (alpha-Gal A) cDNA suggesting the evolutionary relatedness of these genes. The alpha-GalNAc cDNA had 46.9-64.7% amino acid identity in sequences (codons 1-319) corresponding to alpha-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.(ABSTRACT TRUNCATED AT 400 WORDS)