Translation initiation in the HEXB gene encoding the beta-subunit of human beta-hexosaminidase.
The human lysosomal enzyme beta-hexosaminidase (EC 3.2.1.52) is a glycoprotein composed of dimers of alpha- and/or beta-subunits. The subunits of the enzymes are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the lysosome. As such, each subunit contains an amino-terminal signal peptide that directs the nascent polypeptide into the lumen of the endoplasmic reticulum. The signal peptide cleavage site of the beta-polypeptide is known, but its NH2 terminus has not been determined due to the presence of three candidate initiation codons upstream of the cleavage site. In this study, we identified the mRNA cap site, confirming the presence of all three AUGs in the majority of HEXB mRNA. To identify the site of translation initiation, we mutated the three ATGs by deletion and site-directed mutagenesis and showed that all three AUG codons can be used for translation initiation after expression in COS cells. Furthermore, in each case, a fully processed, i.e. mature lysosomal, and enzymatically active beta-hexosaminidase was produced indicating that a functional signal peptide was synthesized. However, expression of a frameshift mutation in the normal construct, created by insertion of a single nucleotide between the first and second ATG, resulted in no significant enzyme activity or beta-subunit protein. We conclude, therefore, that the first in-frame ATG is used exclusively in vivo, in keeping with the scanning model of eukaryotic translation initiation. Interestingly, substitution of all three ATGs with CTG resulted in a significant amount of mature beta-hexosaminidase, showing that under these conditions, initiation could occur from non-AUG codons. Translation initiation from the first AUG gives the prepro-beta-polypeptide a signal peptide of 42 amino acids that has an unusually long hydrophobic core more typical of membrane spanning domains. Such a large hydrophobic core has not been found in other cleavable signal peptides.
Neote
K
K
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Brown
C A
CA
Mahuran
D J
DJ
Gravel
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003062:Codon / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D054819:Hexosaminidase B; D006801:Humans; D008247:Lysosomes / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010442:Peptide Chain Initiation, Translational; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D014162:Transfection; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics*
D003062:Codon; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases