International Journal of Molecular Sciences Article Extended Cleavage Specificities of Two Mast Cell Chymase-Related Proteases and One Granzyme B-Like Protease from the Platypus, a Monotreme Zhirong Fu, Srinivas Akula , Michael Thorpe and Lars Hellman * Department of Cell and Molecular Biology, Uppsala University, Uppsala, The Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden; [email protected] (Z.F.); [email protected] (S.A.); [email protected] (M.T.) * Correspondence: [email protected]; Tel.: +46-(0)18-471-4532; Fax: +46-(0)18-471-4862 Received: 20 November 2019; Accepted: 31 December 2019; Published: 2 January 2020 Abstract: Mast cells (MCs) are inflammatory cells primarily found in tissues in close contact with the external environment, such as the skin and the intestinal mucosa. They store large amounts of active components in cytoplasmic granules, ready for rapid release. The major protein content of these granules is proteases, which can account for up to 35 % of the total cellular protein. Depending on their primary cleavage specificity, they can generally be subdivided into chymases and tryptases. Here we present the extended cleavage specificities of two such proteases from the platypus. Both of them show an extended chymotrypsin-like specificity almost identical to other mammalian MC chymases. This suggests that MC chymotryptic enzymes have been conserved, both in structure and extended cleavage specificity, for more than 200 million years, indicating major functions in MC-dependent physiological processes. We have also studied a third closely related protease, originating from the same chymase locus whose cleavage specificity is closely related to the apoptosis-inducing protease from cytotoxic T cells, granzyme B. The presence of both a chymase and granzyme B in all studied mammals indicates that these two proteases bordering the locus are the founding members of this locus. Keywords: platypus; monotremes; mast cell; chymase; human chymase; cleavage specificity; animal model 1. Introduction Mast cells (MC) are hematopoietic cells distributed along both external and internal surfaces of the body where they most likely act as a first line of defence [1–3]. They are tissue resident cells that are frequently found in connective tissue of the skin and around blood vessels and nerves as well as in the mucosa of the airways and intestines. MCs pre-store a number of inflammatory mediators in cytoplasmic granules. These mediators are rapidly exocytosed from the cell following activation triggered by various stimulators, including cross-linking of receptor-bound IgE, anaphylatoxins (C3a, C4a and C5a) and substance P. The mediators released from MCs include histamine, heparin, various proteases, prostaglandins and leukotrienes. Histamine, heparin and proteases are granule-stored whereas leukotrienes and prostaglandins are produced from arachidonic acid upon cell stimulation and are not granule-stored. The majority of proteins found in the MC granules are serine proteases [4–6]. These proteases can generally be sub-divided into chymases and tryptases [7,8]. Chymases are chymotrypsin-like and cleave substrates after aromatic amino acids. Phylogenetic analyses of the chymases have led to the identification of two distinct subfamilies, the α-chymases and the β-chymases (Figure1)[ 9–12]. The α-chymases are found as a single gene in all species investigated, except for ruminants, where two very similar α-chymase genes have been identified [12,13]. β-chymases have Int. J. Mol. Sci. 2020, 21, 319; doi:10.3390/ijms21010319 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 319 2 of 13 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 13 onlyand been cats identified [12]. Interestingly, in rodents, the and rodent as singleα-chymasesβ-chymase-like mouse MC genesprotease in (mMCP)-5, dogs andcats rat MC [12 ].protease Interestingly, the rodent(rMCP)-5α-chymases and hamster mouse chymase MC II protease have changed (mMCP)-5, their primary rat MC proteasecleavage (rMCP)-5specificities and from hamster aromatic chymase II haveamino changed acids (chymotrysin-like) their primary cleavage to alipha specificitiestic amino acids from (elastase-like) aromatic amino[14–17]. acids (chymotrysin-like) to aliphatic amino acids (elastase-like) [14–17]. FigureFigure 1. A1. phylogeneticA phylogenetic tree tree of of chymasechymase loci encoded encoded se serinerine proteases. proteases. The The amino amino acidacid sequences sequences of of a panela panel of chymase of chymase loci loci encoded encoded proteases proteases were an analysedalysed for for sequence sequence relatedness relatedness with withthe program the program MrBase.MrBase. A bootstrapA bootstrap tree tree based based on 1000on 1000 replicates replicates was was generated generated and and the the bootstrap bootstrap values values are are depicted at eachdepicted branch at each of the branch tree. of The the tree. different The different subfamilies subfamilies of chymase of chymase loci genesloci genes were were colour-coded colour-coded for easy for easy identification and the genes of primary interest, monotreme and marsupial enzymes, are identification and the genes of primary interest, monotreme and marsupial enzymes, are marked by red marked by red arrows and the related alligator and Xenopus proteases are marked with green arrows. arrows and the related alligator and Xenopus proteases are marked with green arrows. The granzyme The granzyme B related in dark green and the cathepsin G related in light green. B related in dark green and the cathepsin G related in light green. In mammals, the mast cell chymotryptic enzymes are found in one chromosomal locus, the chymase locus. In humans, this locus encodes four genes: one MC expressed enzyme, the α-chymase; one Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 13 marked by red arrows and the related alligator and Xenopus proteases are marked with green arrows. The granzyme B related in dark green and the cathepsin G related in light green. Int. J. Mol. Sci. 2020, 21, 319 3 of 13 In mammals, the mast cell chymotryptic enzymes are found in one chromosomal locus, the chymase locus. In humans, this locus encodes four genes: one MC expressed enzyme, the α-chymase; oneneutrophil neutrophil and and MC MC expressed expressed protease, protease, cathepsin cathepsin G; G; andand twotwo T-cellT-cell granzymes, granzymes (gzm) H H and B (Figure2 2))[ [12].12]. ThisThis locuslocus isis presentpresent in in all all studied studied mammals mammals and and related related enzymes enzymes have have also also been beenidentified identified in thein the American American alligator alligator and and in the in clawedthe clawed frog, frog,Xenopus Xenopus laevis laevis[12]. [12]. However, However, no closely no closelyrelated related members members of this of locusthis locus have have been been found fo inund fishes in fishes or birds or birds [12]. The[12]. chymaseThe chymase locus locus also hasalso the hassame the same bordering bordering genes genes in all in mammals all mammals studied studied from from opossums opossums to humans; to humans; at one at one end end by by the the mast mastcell cellα-chymase α-chymase and and at the at otherthe other end end by granzyme by granzyme B (Figure B (Figure2). However, 2). However, there havethere been have massive been massivechanges changes in gene in numbers gene numbers in some in placental some placental mammals mammals within these within borders, these primarilyborders, primarily in rodents in but rodentsalso in but ruminants. also in ruminants. As previously As previously described, descri thebed, human the human locus contains locus contains four active four serineactive serine protease proteasegenes: genes: the chymase, the chymase, cathepsin cathepsin G and twoG and granzymes, two granzymes, B and H. B Both and miceH. Both and mice rats haveand experiencedrats have experiencedlarge increases large in increases gene numbers in gene in this numbers locus, most in likelythis locus, by successive most likely gene duplications.by successive Mice gene have duplications.15 active serine Mice proteasehave 15 active genes andserine rats protease have 28 ge suchnes and genes rats [12 have]. All 28 of such the studiedgenes [12]. mammals, All of the from studiedmarsupials mammals, to placental from marsupials mammals, to have placental a classical mammals, chymotryptic have a classical enzyme chymotryptic expressed by enzyme mast cells, expressedexcept for by the mast rabbit cells, and except the guinea for the pig, rabbit where and the the chymases guinea pig, have where become the restrictedchymases in have their become substrate restrictedselectivity in their to become substrate strict selectivity Leu-ases [to18 ,become19]. All ofstrict these Leu-ases chymotryptic [18,19]. enzymes, All of these and chymotryptic the Leu-ases are enzymes,encoded and from the the Leu-ases chymase are locus encoded of the from respective the chymase species. locus of the respective species. FigureFigure 2. The 2. chymaseThe chymase locus. The locus. chymase The chymaselocus encodes locus a encodesnumber of a hematopoietic number of hematopoietic serine proteases, serine includingproteases, theincluding α-chymases, the αβ-chymases,-chymases, βcathepsin-chymases, G, cathepsin and several G, andgranzymes several granzymes[12]. Genes [ 12are]. colour- Genes are coded:colour-coded: the α-chymase-related the α-chymase-related genes are genesmarked are in marked light blue, in light the blue,β-chymases the β-chymases in slightly in darker slightly blue, darker cathepsinblue, cathepsin G in green, G in the green, M8 family the M8 in family light green, in light the green, granzymes the granzymes in dark blue. in dark blue. ToTo look look deeper deeper into into the theconservation conservation of the of ch theymase chymase locus locus and andthe presence the presence and conservation and conservation of classicalof classical MC chymases, MC chymases, here our here interest our interest lies with lies the with monotremes, the monotremes, which are which an early are branch an early of branchegg- layingof egg-laying mammals.