Krabbe disease: isolation and characterization of a full-length cDNA for human galactocerebrosidase.
Human galactocerebrosidase, the enzyme deficient in Krabbe disease, was purified, through several hydrophobic column steps and gel filtration, 22,650-fold from human lymphocytes. Using information on its N-terminal and internal amino acid sequences, and the polymerase chain reaction method, we cloned a full-length cDNA for the enzyme. The deduced amino acid sequence matched all amino acid sequences determined. The 3780 nucleotide sequence included 2007 nucleotides which encoded a single chain peptide of 669 amino acid residues with a 26 amino acid N-terminal signal peptide and six potential asparagine-linked glycosylation sites. The galactocerebrosidase cDNA detected an about 4 kb mRNA band material in human cultured skin fibroblasts. A nonsense mutation was found at codon 369 (GAA-->TAA) in the coding sequence of cDNA amplified from cultured skin fibroblast mRNA from a patient with typical Krabbe disease.
Sakai
N
N
Department of Pediatrics, Osaka University School of Medicine, Japan.
Inui
K
K
Fujii
N
N
Fukushima
H
H
Nishimoto
J
J
Yanagihara
I
I
Isegawa
Y
Y
Iwamatsu
A
A
Okada
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts / Q000737:chemistry; D005698:Galactosylceramidase / Q000172:deficiency / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007965:Leukodystrophy, Globoid Cell / Q000201:enzymology / Q000235:genetics*; D008214:Lymphocytes / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D017422:Sequence Analysis, DNA; D012867:Skin / Q000737:chemistry / Q000166:cytology
D018076:DNA, Complementary; D011498:Protein Precursors; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D005698:Galactosylceramidase