Wang A M, Bishop D F, Desnick R J
Division of Medical and Molecular Genetics, Mount Sinai School of Medicine, New York, New York 10029.
J Biol Chem. 1990 Dec 15;265(35):21859-66.
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)
人α-N-乙酰半乳糖胺酶（α-GalNAc，E.C. 3.2.1.49）是一种溶酶体糖水解酶，可从糖缀合物中切割α-N-乙酰半乳糖胺部分，由定位于22q13----qter染色体的一个基因编码。α-GalNAc活性缺乏是辛德勒病（一种遗传性神经轴索性营养不良）的酶缺陷。为了分离全长cDNA，将人肺中的该酶纯化至同质，通过对N端和七个胰蛋白酶肽段进行微量测序确定了129个不重叠的氨基酸，并使用四种合成寡核苷酸混合物筛选人成纤维细胞cDNA文库。从胎盘λgt11 cDNA文库中分离出的全长cDNA pAGB-3，其插入片段为2158个碱基对（bp），有一个开放阅读框，预测的氨基酸序列与纯化酶的所有129个微量测序残基共线。pAGB-3插入片段有一个344-bp的5'-非翻译区、一个编码411个氨基酸的1236-bp开放阅读框、一个514-bp的3'-非翻译区和一个64-bp的聚腺苷酸尾。预测有一个17个氨基酸的信号肽序列以及六个N-糖基化位点。将pAGB-3 cDNA亚克隆到p91023(B)哺乳动物表达载体中，人α-GalNAc活性在COS-1细胞中瞬时表达，证明了全长cDNA的功能完整性。mRNA的Northern杂交分析显示有两个约3.6和2.2千碱基（kb）的转录本，引物延伸研究表明2.2-kb转录本的帽位点在核苷酸-347处。分离出了3.6-kb的cDNA（pAGB-35）；3598-bp的pAGB-35插入片段与2.2-kb插入片段相同，但有额外的5'-和3'-非翻译序列，包括在核苷酸3100 - 3105处的第二个下游聚腺苷酸化信号。分离出一个基因组克隆gAGB-1，并对包括pAGB-3在内的2048-bp区域进行测序，发现在密码子319和320之间有一个1754-bp的内含子，这也是其他cDNA克隆中一个70-bp插入和一个45-bp缺失的位点。值得注意的是，α-GalNAc cDNA与人类α-半乳糖苷酶A（α-Gal A）cDNA有显著的氨基酸同源性，表明这些基因在进化上的相关性。α-GalNAc cDNA在对应于α-Gal A外显子1至6的序列（密码子1 - 319）中有46.9% - 64.7%的氨基酸同一性，而可比的外显子7序列（pAGB-3密码子320 - 411）与众多缺口的同源性仅为15.8%。（摘要截短于400字）
