CATHEPSIN, A MAJOR PROTEASE OF THE MARINE SPONGE GEODIA-CYDONIUM - PURIFICATION OF THE ENZYME AND MOLECULAR-CLONING OF CDNA

Sponges are suspension-feeders that are devoid of body cavities. Phago cytosis is the major route of nutrition in these animals. In an attemp t to understand protein digestion, cathepsin was identified in crude e xtracts hum the sponge Geodia cydonium. This enzyme was purified from lysosomes by a two-step procedure-pH precipitation and FPLC separation -to apparent homogeneity; it showed an M-r of 26,000. Inhibitor as wel l as substrate studies showed that the sponge cathepsin belongs to the subfamily L of these cysteine proteases. The complete cDNA coding for cathepsin L was isolated and characterized. The deduced aa sequence c ontains 322 residues, has an M-r of 36,085, and shows the characterist ic signatures known from other cathepsins of the L subfamily: e.g., cl eavage site fur the proregion, the ERFNIN motif, and the conserved reg ions forming the catalytic triad of cysteine proteases. Phylogenetic a nalyses revealed that the sponge sequence groups with the cathepsin L subfamily and branches off first from the other metazoan members. The sponge sequence shows high homology to that isolated from Dictyosteliu m discoideum and only low similarity to the protozoan cathepsins L fro m Paramecium tetraurelia and Tetrahymena thermophila. From the data pr esented it is concluded that cathepsin L is the major digestive protea se in sponges.