Asp-ase Activity of the Opossum Granzyme B Supports the Role of Granzyme B as Part of Anti-Viral Immunity Already during Early Mammalian Evolution.

Zhirong Fu,Srinivas Akula,Michael Thorpe,Lars Hellman,Adam Lesner

doi:10.1371/journal.pone.0154886

Abstract

Granzyme B is one of the key effector molecules in our defense against viruses and intracellular bacteria. This serine protease together with the pore forming protein perforin, induces caspase or Bid-dependent apoptosis in target cells. Here we present the first characterization of a granzyme B homolog, the grathepsodenase, in a non-placental mammal, the American opossum (Monodelphis domestica). The recombinant enzyme was produced in a human cell line and used to study its primary and extended cleavage specificity using a panel of chromogenic substrates and recombinant protein substrates. The opossum granzyme B was found to have a specificity similar to human granzyme B, although slightly less restrictive in its extended specificity. The identification of a granzyme B homolog with asp-ase (cleaving after aspartic acid) specificity in a non-placental mammal provides strong indications that caspase or Bid-dependent apoptosis by a serine protease with a conserved primary specificity has been part of anti-viral immunity since early mammalian evolution. This finding also indicates that an asp-ase together with a chymase were the first two serine protease genes to appear in the mammalian chymase locus.

Highlights

Secretory granules filled with inflammatory mediators are found in several of the major hematopoietic cell types
We can show that an enzyme with a primary specificity for negatively charged amino acids was present before the separation of marsupials and placental mammals, at least 180 million years ago
This indicates that the function is old, with the question remaining, how old? To trace the enzymes further back in vertebrate evolution we have recently initiated a bioinformatic analysis of related genes in early mammals and non-mammalian vertebrates

Summary

Introduction

Secretory granules filled with inflammatory mediators are found in several of the major hematopoietic cell types. NK cells, cytotoxic T cells and neutrophils, serine proteases make up the majority of the protein content of these granules [1]. Mast cells contain chymases and tryptases with chymotrypsin- and trypsin-related primary cleavage specificities [1,2,3]. NK cells and cytotoxic T cells contain a number of granzymes (Gzms) with varying specificities. Human NK cells and cytotoxic T cells express five different Gzms; -A, -K, -M, -H and–B [1]. Granzymes A and K are tryptases, GzmM a met-ase, GzmH a chymase and GzmB an asp-ase, the later cleaving after the negatively charged amino acids, aspartic and glutamic acid. Granzymes have a number of different functions, including

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