Aspartyl proteases in Caenorhabditis elegans. Isolation, identification and characterization by a combined use of affinity chromatography, two-dimensional gel electrophoresis, microsequencing and databank analysis.
Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal proteolytic activity under acidic pH conditions. About 90% of this activity is inhibited by the oligopeptide pepstatin, which specifically inhibits the activity of aspartyl proteases such as pepsin, cathepsins D and E or renin. We have purified enzymes responsible for this proteolytic activity by a single-step affinity chromatography on pepstatin-agarose. Analysis of the purified fraction by 1D SDS gel electrophoresis revealed six bands ranging from 35 to 52 kDa. After electrotransfer to poly(vinylidene difluoride) membranes, all bands were successfully subjected to N-terminal microsequencing. On 2D gels, the purified protein bands split into 19 spots which, after renewed microsequencing, were identified as isoelectric variants of the six proteins already described. The N-termini obtained for these proteins could be correlated to genomic DNA sequences determined in the course of the C. elegans genome sequencing project. All these sequences were predicted to code for expressed proteins as collected in the WORMPEP database. Five of the six coding sequences identified in this study were found to contain the typical active-site consensus sequence of aspartyl proteases and displayed an overall amino acid identity between 25 and 66% as compared to aspartyl proteases from other organisms. In addition to the five aspartyl proteases detected at the protein level, we have identified the coding sequences for seven other enzymes of this protease family by a similarity search in the genomic DNA of C. elegans which has recently been completely sequenced.
Geier
G
G
Institute of Zoology, University of Heidelberg, Germany.
Banaj
H J
HJ
Heid
H
H
Bini
L
L
Pallini
V
V
Zwilling
R
R
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D016384:Consensus Sequence; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D016282:Aspartic Acid Endopeptidases