Molecular cloning and characterization of the structural gene coding for the developmentally regulated lysosomal...

The gene coding for the Dictyostelium discoideum lysosomal enzyme, alpha-mannosidase, has been cloned and sequenced. To accomplish this, the mature 60- and 58-kDa subunits of the enzyme were purified and subjected to liquid-phase N-terminal amino acid sequencing. Sequence information was obtained for both of the mature subunits, and a 48-mer oligonucleotide was synthesized based on the determined amino acid sequence of the 58-kDa subunit. Using this oligonucleotide as a probe, an 8-kilobase HindIII fragment of genomic DNA was isolated and subjected to Sanger dideoxy DNA sequencing. The first 4400 nucleotides contained the complete alpha-mannosidase gene and 1100 nucleotides of 5'-flanking DNA. Primer extension analysis indicated that transcription begins at multiple sites -48 to -64 nucleotides upstream of the first nucleotide of the predicted translation initiation codon. A single open reading frame (ORF) of 3015 nucleotides was found that was interrupted by a single intron and that contained the amino acid sequences of the N termini of the two mature alpha-mannosidase subunits; a polyadenylation signal was also found just downstream of the termination codon. A potential cleavable signal sequence was identified in the first 22 amino acids of the predicted precursor protein, and two propeptide regions (Pro I and II) were identified that were immediately upstream of the N termini of the 60- and 58-kDa mature subunits, respectively. These propeptide regions are not present in the mature protein and are therefore predicted to be proteolytically removed as the membrane associated 140-kDa precursor is transported to lysosomes and processed to the soluble 60- and 58-kDa mature forms of the enzyme. In fact, potential proteolytic cleavage sites were identified flanking the Pro I and Pro II regions. Pro I, which immediately follows the signal sequence, consists of 18 amino acids, most of which are highly charged and hydrophilic residues, while Pro II, found in the central portion of the precursor, is very hydrophobic. While no obvious transmembrane regions were identified, several short hydrophobic amino acid stretches were found to be localized in and around the Pro II region, and these may be responsible for attachment of precursors to membranes.