Aspartic Proteases from the Nematode Caenorhabditis elegans: STRUCTURAL ORGANIZATION AND DEVELOPMENTAL AND CELL-SPECIFIC EXPRESSION OF asp-1

Irina Tcherepanova,Lokesh Bhattacharyya,Charles S Rubin,Jonathan H Freedman

doi:10.1074/jbc.m000956200

Abstract

A Caenorhabditis elegans gene (asp-1) and cDNA that encode a homologue of cathepsin D aspartic protease were cloned and characterized. The asp-1 mRNA is transcribed from a single exon, and it begins with the SL1 trans-splice leader sequence. The protein (ASP-1) is expressed as a 396-amino acid, 42.7-kDa pre-pro-peptide that is post-translationally processed into a approximately 40-kDa lysosomal protein. ASP-1 shares approximately 60% sequence identity with the aspartic protease precursor from the nematode Strongyloides stercoralis. The amino acid sequences adjacent to the two active site aspartic acid residues in ASP-1 are 100% identical to those in other eukaryotic aspartic proteases. In addition, ASP-1 contains conserved, potential disulfide bond-forming cysteine residues and N-glycosylation sites. The asp-1 gene is exclusively transcribed in the intestinal cells, with the highest levels of expression observed at late embryonic and early larval stages of development. asp-1 transcription is not observed in adult nematodes or mature larvae. Furthermore, transcription predominantly occurs in eight anterior cells of the intestine (int6-int8). Analyses of ASP-1 nucleotide and amino acid sequences revealed the presence of five additional C. elegans aspartic proteases.

Highlights

Aspartic proteases are a class of proteolytic enzymes that include pepsins, renins, cathepsin D and E, and chymosins [1]
Cloning and Sequencing Analysis of cDNAs that Encode a C. elegans Aspartic Protease - A cDNA library was prepared in the bacteriophage λZAPII, using template mRNA isolated from nematodes that were exposed to 2 mM cadmium [31,48]
The sequence of the initial 22 bp of the ASP-1 cDNA was identical with the splice leader 1 (SL1) RNA sequence described by Krause and Hirsh [49] (Fig. 1)

Summary

Introduction

Aspartic proteases are a class of proteolytic enzymes that include pepsins, renins, cathepsin D and E, and chymosins [1]. Expression of Recombinant ASP-1 Protein in Insect Cells - An ASP-1 cDNA fragment that contains the entire coding region was prepared with 5’-PstI and 3’-blunt termini. Analysis of the ASP-1 Protein Sequence – The derived amino acid sequence of ASP-1 reveals the presence of two potential active site aspartic acid residues (amino acids 86 and 278) contained within the aspartic proteases consensus sequences, (F/I/L)-D-T-G-S and D-(T/S)-G-(S-T), respectively (Fig. 1).

Results

Conclusion