Molecular cloning of the cDNA which encodes beta-N-acetylhexosaminidase A from Dictyostelium discoideum. Complete amino acid sequence and homology with the human enzyme.
beta-N-Acetylhexosaminidase A (EC 3.2.1.52), the product of the nag A gene, is a lysosomal enzyme which is developmentally regulated in Dictyostelium discoideum. The enzyme plays a role during the slug stage of development in the maintenance of pseudoplasmodia of normal size. We used a homogeneous preparation of deglycosylated enzyme subunits to generate antibody. The antibody was suitable for screening a lambda gt11 cDNA expression library derived from the mRNA of late log stage axenic cells. We isolated seven positive clones. One of these contains the complete coding sequence of the protein. We also isolated a genomic clone which contains 800 base pairs of 5'-flanking sequence and 728 base pairs of coding sequence. Analysis of the sequences and of primer extension studies indicates an inferred transcript size of 1665 bases which closely matches the 1.8-kilobase mRNA size estimated by Northern blot analysis of poly(A+) mRNA from the organism. The sequence contains an open reading frame which encodes a protein of 59,787 kDa. This equals the apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gels of in vitro translated enzyme. The amino terminus of the purified enzyme appeared to be blocked, but internal peptide sequences were obtained by automated Edman degradation of gel-purified peptides generated by treatment of protein subunits with staphylococcal V-8 protease. These sequences are included in the inferred sequence. In addition to a typical signal sequence, the open reading frame encodes a second candidate transmembrane region, a serine-rich region, and four potential N-glycosylation sites. These are discussed with regard to the localization and processing of the enzyme during its biogenesis. Beginning at amino acid 100 of the Dictyostelium enzyme sequence, 36% of its amino acids are identical to the corresponding sequence of the beta chain, and 33% are identical with those of the alpha chain of human beta-N-acetylhexosaminidase. This is strong evidence that the Dictyostelium enzyme is homologous to the alpha and beta chains of the human enzyme.
Graham
T R
TR
Department of Microbiology, St. Louis University School of Medicine, Missouri 63104-1028.
Zassenhaus
H P
HP
Kaplan
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004023:Dictyostelium / Q000201:enzymology*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics*
D004247:DNA; D001619:beta-N-Acetylhexosaminidases