Translation initiation in the HEXB gene encoding the beta-subunit of human beta-hexosaminidase.

摘要: The human lysosomal enzyme beta-hexosaminidase (EC 3.2.1.52) is a glycoprotein composed of dimers alpha- and/or beta-subunits. subunits the enzymes are synthesized in rough endoplasmic reticulum and transported through Golgi apparatus to lysosome. As such, each subunit contains an amino-terminal signal peptide that directs nascent polypeptide into lumen reticulum. cleavage site beta-polypeptide known, but its NH2 terminus has not been determined due presence three candidate initiation codons upstream site. In this study, we identified mRNA cap site, confirming all AUGs majority HEXB mRNA. To identify translation initiation, mutated ATGs by deletion site-directed mutagenesis showed AUG can be used for after expression COS cells. Furthermore, case, fully processed, i.e. mature lysosomal, enzymatically active was produced indicating functional synthesized. However, frameshift mutation normal construct, created insertion single nucleotide between first second ATG, resulted no significant activity or beta-subunit protein. We conclude, therefore, in-frame ATG exclusively vivo, keeping with scanning model eukaryotic initiation. Interestingly, substitution CTG amount beta-hexosaminidase, showing under these conditions, could occur from non-AUG codons. Translation gives prepro-beta-polypeptide 42 amino acids unusually long hydrophobic core more typical membrane spanning domains. Such large found other cleavable peptides.