title: Human arylsulfatase B: MOPAC cloning, nucleotide sequence of a full-length cDNA, and regions of amino acid identity with arylsulfatases A and C.
abstract: cDNAs encoding the human lysosomal hydrolase, arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase, EC 3.1.6.1), were isolated from a hepatoma cell cDNA library using an ASB-specific oligonucleotide generated by the MOPAC (mixed oligonucleotide primed amplification of cDNA) technique. To facilitate cDNA cloning, human ASB was purified to apparent homogeneity and a total of 112 amino acid residues were microsequenced from the N-terminus and four internal tryptic peptides of the 47-kDa subunit. Based on the ASB N-terminal amino acid sequence, two oligonucleotide mixtures containing inosines to reduce the mixture complexity were constructed and used as primers to amplify an ASB-specific product from human placental cDNA by the polymerase chain reaction. DNA sequencing of this MOPAC product demonstrated colinearity with 21 N-terminal ASB amino acids. Based on this sequence and on codon usage for the adjacent conserved amino acids in human arylsulfatases A and C, a unique 66-mer was synthesized and used to screen a human hepatoma cell cDNA library. Four putative positive cDNA clones were isolated, and the largest insert (pASB-1) was sequenced in both orientations. The 1834-bp pASB-1 insert had a 1278-bp open reading frame encoding 425 amino acids that was colinear with 85 microsequenced amino acids of the purified enzyme, demonstrating its authenticity. Using the pASB-1 cDNA as a probe, a full-length cDNA clone, pASB-4, was isolated from a human testes library and sequenced in both orientations. pASB-4 had a 2811-bp insert containing a 559-bp 5' untranslated sequence, a 1602-bp open reading frame encoding 533 amino acids (six potential N-glycosylation sites), a 641-bp 3' untranslated sequence, and a 9-bp poly(A) tract. Comparison of the predicted amino acid sequences of arylsulfatases A, B, and C revealed regions of identity, particularly in their N-termini.
authors.affiliation: Division of Medical and Molecular Genetics, Mount Sinai School of Medicine, New York, New York 10029.
authors.forename: E H
authors.initials: EH
authors.lastname: Schuchman
authors.forename: C E
authors.initials: CE
authors.lastname: Jackson
authors.forename: R J
authors.initials: RJ
authors.lastname: Desnick
mesh_terms: D000595:Amino Acid Sequence; D001192:Arylsulfatases / Q000235:genetics*; D001483:Base Sequence; D002553:Cerebroside-Sulfatase / Q000235:genetics*; D002803:Chondro-4-Sulfatase / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D043266:Steryl-Sulfatase; D013429:Sulfatases / Q000235:genetics*
chemical_list: D004247:DNA; D013429:Sulfatases; D001192:Arylsulfatases; D043266:Steryl-Sulfatase; D002553:Cerebroside-Sulfatase; D002803:Chondro-4-Sulfatase