Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes [Protein Structure and Folding]

Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases which digest dietary proteins. CELAs are typically expressed in multiple isoforms which can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reason for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible, high-affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage-display selected inhibitors, we recombinantly expressed and purified twelve SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino-acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception which was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable while CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.

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