International Journal of Molecular Sciences

Article Extended Cleavage Specificities of Two -Related Proteases and One 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 and . Here we present the extended cleavage specificities of two such proteases from the platypus. Both of them show an extended -like specificity almost identical to other mammalian MC chymases. This suggests that MC chymotryptic 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 -inducing protease from cytotoxic T cells, . 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 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 (-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 G related in light green. B related in dark green and the 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 , 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 and and MC MC expressed expressed protease, protease, cathepsin cathepsin G; G; andand twotwo T-cellT-cell , 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 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. mammals. This branch This of the branch mammalian of the mammalian tree has been tree estimated has been to estimated have separated to have from separated the commonfrom the mammalian common mammalian branch around branch 220 around million 220 millionyears ago. years Today ago. Today the monotremes the monotremes are areonly only representedrepresented by by three three extant extant members, members, the the platypus platypus as as well asas thethe long-long- and and short-nosed short-nosed echidnas. echidnas. The Theplatypus platypus genome genome is stillis still incomplete incomplete and and currently currently we we have have found found three three genes, genes, which which cluster cluster closely closelyin a phylogeneticin a phylogenetic tree withtree with the placentalthe placental and and marsupial marsupial chymase chymase loci loci genes genes (Figure (Figure1). The1). The three threegenes genes are foundare found in two in separatetwo separate contigs contigs where wh twoere of these,two of named these, platypusnamed platypus granzyme granzyme B (GzmB) B and (GzmB)platypus and DDN1-like, platypus DDN1-like, cluster together cluster with together the chymases, with the and chymases, one gene and called one platypus gene called granzyme platypus BGH granzyme(GzmBGH), BGH clusters (GzmBGH), with clusters the granzymes. with the granzyme When lookings. When at thelooking three at major the three specificity-determining major specificity- determiningamino acids, amino which acids, sit in which the substrate sit in bindingthe substr pocketate binding (based onpocket crystallized (based 3Don structures),crystallized we 3D find structures),that the two we firstfind platypusthat the two enzymes first platypus have very enzy similarmes triplets have very as the similar previously triplets characterized as the previously classical characterizedchymases (SGN classical and chymases SVN, compared (SGN and with SVN, SGA). compared The third, with platypusSGA). The granzyme third, platypus BGH, granzyme has a triplet BGH,that has is similar a triplet to that the granzymeis similar to Bs the of several granzyme placental Bs of mammalianseveral placental species, mammalian AGR, indicating species, that AGR, it has indicatingasp-ase activitythat it has similar asp-ase to granzyme activity similar B of placental to granzyme mammals. B of placental To obtain mammals. a better view To obtain of the origina better and viewevolution of the origin of MC and chymases evolution and granzymeof MC chymases B during and vertebrate granzyme evolution, B during we vertebrate have studied evolution, the extended we havecleavage studied specificity the extended of these cleavage three platypusspecificity enzymes. of these three We can platypus show that enzymes. both platypus We can ‘granzymeshow that B’ (the chymase) and platypus DDN1-like are classical chymases with specificities very similar to human and mouse MC chymases, whereas the platypus GzmBGH is an asp-ase with very similar specificity to granzyme B of placental mammals. This shows that these enzymes have been conserved for more than Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 13 both platypus ‘granzyme B’ (the chymase) and platypus DDN1-like are classical chymases with Int. J. Mol. Sci. 2020, 21, 319 4 of 13 specificities very similar to human and mouse MC chymases, whereas the platypus GzmBGH is an asp-ase with very similar specificity to granzyme B of placental mammals. This shows that these enzymes200 million have yearsbeen ofconserved mammalian for more evolution, than 200 which million gives years strong of indicationsmammalian for evolution, their importance which gives in the strongimmune indications functions for oftheir MCs importance and cytotoxic in the T cells.immune functions of MCs and cytotoxic T cells.

2. Results2. Results

2.1.2.1. Production, Production, Activation Activation and and Purificat Purificationion of Recombinant of Recombinant Platypus Platypus Enzymes Enzymes TheThe coding coding regions regions for for the the three three separate separate chymas chymasee locus-related locus-related platypus platypus enzymes enzymes were were ordered ordered as asdesigner designer genes genes from from Genscript Genscript and and inserted inserted in inthe the mammalian mammalian expression expression vector vector pCEP-Pu2 pCEP-Pu2 [20]. [20 ]. TheseThese three three clones clones were were then then transfected transfected into into the the human human embryonic embryonic kidney cell cell (HEK) (HEK) 293-EBNA 293-EBNA for for expression.expression. After After establishing establishing confluent confluent cultures cultures of ofthe the transfected transfected cells, cells, the the medium medium was was collected collected andand the the secreted secreted protein protein was was purified purified on on Ni-chelating Ni-chelating IMAC IMAC columns. columns. After After analysis analysis of ofthe the protein protein fractionsfractions from from the the purification, purification, all allthree three clones clones gave gave sufficient sufficient amounts amounts of ofprotein protein for for further further analyses analyses (Figure(Figure 3).3 ).After After purification, purification, the the proteins proteins were were ac activatedtivated by by removing removing the the N-terminal N-terminal His-6 His-6 tag tag and and thethe enterokinase enterokinase site site by bycleavage cleavage with with enterokinase. enterokinase. The The active active protein protein (i.e., (i.e., enterokinase enterokinase cleaved) cleaved) is is approximatelyapproximately 1.5 1.5 kDa kDa smaller smaller than than the the uncleaved uncleaved protein protein (no (no enterokinase enterokinase addition) addition) (Figure (Figure 3).3 ).

FigureFigure 3. Analysis 3. Analysis of the of therecombinant recombinant platypus platypus enzymes enzymes used used in the in the determination determination of their of their extended extended cleavagecleavage specificities. specificities. Three Three different different recombinan recombinantt platypus platypus enzymes enzymes were were produced produced as proenzymes, as proenzymes, containing an N-terminal His -tag and an enterokinase site, in the HEK-293 EBNA cells with the containing an N-terminal His6-tag6 and an enterokinase site, in the HEK-293 EBNA cells with the episomalepisomal vector vector pCEP-Pu2. pCEP-Pu2. These These proenzymes proenzymes were were first first purified purified on onNi-NTA Ni-NTA beads beads (-EK) (-EK) and and then then activatedactivated by by removal removal of of thethe His-6His-6 tag tag by by enterokinase enterokinase digestion digestion (+EK). (+EK). After purificationAfter purification and activation, and activation,the three the enzymes three wereenzymes analysed were by analysed separation by on separation SDS-PAGE on and SDS-PAGE visualized withand Coomassievisualized Brilliantwith CoomassieBlue staining. Brilliant The Blue proteases staining. before The andproteases after enterokinase before and after cleavage enterokinase are marked cleavage with small are marked red arrow withheads. small The red few arrow additional heads. bandsThe few seen additional on the gel bands are originating seen on the from gel the are conditioned originating cell from medium, the conditionedand the major cell medium, band of a and size the of approximately major band of 69 a kDasize isof bovineapproximately serum albumin 69 kDa fromis bovine the serum serum that represent the major protein of the cell medium. albumin from the serum that represent the major protein of the cell medium. 2.2. Chromogenic Substrate Assay and Phage Display 2.2. Chromogenic Substrate Assay and Phage Display A panel of 11 different chromogenic substrates was used to try to determine the primary specificities of two of these enzymes, GzmB and Gzm BGH. However, no activity was detected with any of the Int. J. Mol. Sci. 2020, 21, 319 5 of 13

11 substrates with these two enzymes indicating high specificities. We also tried several times with substrate phage display without success with all three of these enzymes. A similar phenomenon has been observed for a few other hematopoietic serine proteases, which later have been found to be highly specific. This further supports the indication from the chromogenic substrate assay that all enzymes are more specific than the other mammalian enzymes previously investigated.

2.3. Analysis of the Extended Cleavage Specificity by the Use of Recombinant Protein Substrates As neither chromogenic substrates nor phage display resulted in any information concerning the extended specificities of these three platypus enzymes, we instead tried a third alternative; to use a new type of recombinant substrate. A panel of such substrates has previously been designed and produced for the verification of the extended cleavage specificity following phage display analysis for a number of previously analysed mammalian hematopoietic and serine proteases [17,21–30]. In this type of substrate, the consensus sequence obtained from the phage display analysis is generally used, often a region of approximately nine amino acids. This region is inserted into a linker region between two E. coli thioredoxin (Trx) molecules by ligating a double stranded oligonucleotide encoding the actual sequence into a BamHI and a SalI site of the vector construct (Figure4A). For purification purposes we also inserted a His-6 tag to the C-terminal of the second Trx molecule of this protein (Figure4A). A number of related and unrelated substrate sequences were also produced with this system, by ligating the corresponding oligonucleotides into the BamHI/SalI sites of the vector. All of these substrates were expressed as soluble proteins in a bacterial host, E. coli rosetta gami, and purified on IMAC columns to obtain a protein with a purity of 90–95%. We have produced more than 270 such substrates for the analysis of other serine proteases and selected a few such with different types of amino acids, including aromatic, aliphatic, negatively or positively charged amino acids in the central position of the substrate for an initial screening of the three platypus serine proteases. We started the analysis with the two enzymes expected to have chymotryptic activity: the platypus GzmB and platypus DDN-1-like. For these two enzymes, we selected a panel of different chymase substrates as well as an elastase and a substrate as negative controls. Both of these two enzymes showed a relatively strict chymotryptic activity. The best activity for both of these very closely related enzymes was seen with the substrate VVLF SGVL (arrow indicating the potential cleavage site) ↓ (Figures4 and5). A slight drop in cleavage activity was seen when a negative charge was introduced in the P1´or P2´positions. In contrast, by introducing a positive charge in the P2 position, we observed an almost complete block in cleavage (Figures4 and5). Furthermore, almost no cleavage was seen for substrates with a Trp or a Leu in the P1 position (Figures4 and5). These two enzymes did not display any tryptase activity as substrates with an Arg in the P1 position were not cleaved (Figures4 and5). Changing the P1’position from a Ser to a Leu also resulted in a dramatic reduction in cleavage, indicating the importance of the in this position. An Arg in the P1´position was more favoured but still considerably less than Ser in this position (Figures4 and5). These two enzymes showed very similar cleavage patterns, which was expected due to their very high similarity in primary sequence. However, a few minor differences could be observed. One such difference was that platypus DDN-1-like tolerated a negatively charged residue in the P1’position better than granzyme B (Figures4 and5). Int. J. Mol. Sci. 2020, 21, 319 6 of 13 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 13

FigureFigure 4. 4.AnalysisAnalysis of of the cleavagecleavage specificity specificity of platypusof platypus GzmB GzmB (the chymase)(the chymase) by the useby ofthe recombinant use of recombinantprotein substrates. protein Panelsubstrates. (A) shows Panel the (A overall) shows structure the overall of the structure recombinant of the protein recombinant substrates protein used for substratesanalysis used of the for effi analysisciency in of cleavage the efficiency by the in three cleavage different by the enzymes three diff (analysederent enzymes in Figures (analysed4–6). In these in figuressubstrates, 4, 5 and two 6). thioredoxinIn these substrates, molecules two are thiore positioneddoxin molecules in tandem are and positioned the proteins in tandem have aand His-6 the tag proteinspositioned have in a theirHis-6 C-termini tag positioned of the second in their thioredoxin. C-termini Theof the diff erentsecond cleavable thioredoxin. sequences The aredifferent inserted cleavablein a linker sequences region are between inserted the in two a linker thioredoxin region between molecules the by two the thioredoxin use of two molecules unique restriction by the use sites, of onetwo Bamunique HI andrestriction one SalI sites,site, one which Bam are HI indicated and one in SalI the site, bottom which of panel are indicated (A). In panel in the (B ),bottom an example of panelcleavage (A). In is panel shown (B to), highlightan example possible cleavage cleavage is shown patterns. to highlight Panels possible (C–E) show cleavage the cleavage patterns. of aPanels number (C–ofE) substratesshow the cleavage by the platypus of a number granzyme of substrates B, the chymase. by the platypus The sequences granzyme of theB, the diff chymase.erent substrates The sequencesare indicated of the above different the imagessubstrates of theare gels. indicate Thed red above residues the images highlight of thethe majorgels. The diff erencered residues between highlightthis substrate the major and difference the reference between substrate this substrat positionede and at the beginningreference substrate (Left side) positioned of this panel. at the The beginningtime of cleavage,(Left side) in of minutes, this pane is alsol. The indicated time of above cleavage, the corresponding in minutes, is lanes also of indicated the different above gels. the The correspondingun-cleaved substrateslanes of the have different a molecular gels. weightThe un-cleaved of approximately substrates 25 have kDa anda molecular the cleaved weight substrates of approximatelyappear as two 25 closely-located kDa and the cleaved bands withsubstrates a size appe of 12–13ar as kDa. two closely-located bands with a size of 12–13 kDa.

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 13 Int. J. Mol. Sci. 2020, 21, 319 7 of 13

FigureFigure 5. Analysis 5. Analysis of the of the cleavage cleavage specificity specificity of platypus of platypus DDN1-like DDN1-like (also (also a chymase) a chymase) by bythe the use use of of recombinantrecombinant protein protein substrates. substrates. The The different different panels panels show show the the cleavage cleavage of a number ofof substratessubstrates byby the theplatypus platypus DDN1-like. DDN1-like. The The sequences sequences of of the the di differefferentnt substrates substrates are are indicated indicated above above the the pictures pictures of of the thegels. gels. The The red red residues residues highlight highlight the the major major diff erencedifference between between this substratethis substrate and the and reference the reference substrate substratepositioned positioned at the beginning at the beginning (Left side) (Left of side) this panel.of this Thepanel. time The of time cleavage, of cleavage, in minutes, in minutes, is also indicatedis also indicatedabove the above corresponding the corresponding lanes of the lanes diff erentof the gels. different The un-cleaved gels. The substrates un-cleaved have substrates a molecular have weight a molecularof approximately weight of 25approximately kDa and the cleaved25 kDa substratesand the cleaved appear substrates as two closely-located appear as two bands closely-located with a size of bands12–13 with kDa. a size of 12–13 kDa.

TheThe names names of ofthe the platypus platypus proteases proteases do do not not reflect reflect other other close close mammalian mammalian homologs homologs and and have have beenbeen generated generated by byearly early sequence sequence similarity similarity analyses analyses of few of few members members of this of this subfamily subfamily of proteases. of proteases. TheThe platypus platypus GzmB GzmB is not is not the the classical classical granzyme granzyme B homolog B homolog but but instead instead the the platypus platypus MC MC chymase. chymase. Currently,Currently, this this is issomewhat somewhat confusing confusing but but it it may may ta takeke some some time time before before the name will changechange basedbased on onthe the availableavailable evidence.evidence. AsAs a anext next step, step, we we analysed thethe extended extended cleavage cleavage specificity specificity of theof platypusthe platypus GzmBGH, GzmBGH, the enzyme the enzymewith a with granzyme B-like substrateB-like substrate pocket. pocket. The substrates The substrates with a negativelywith a negatively charged charged amino acid amino in the acidP1 in position the P1 position were the were only the ones only that ones showed that anyshowed cleavage any cleavage activity (Figure activity6 ).(Figure This enzyme 6). This was enzyme found wasto found prefer to sequences prefer sequences with a Phe with preceding a Phe preceding the P1 Asp the and P1 dislikedAsp and positively disliked positively charged amino charged acids aminolike Argacids in like this Arg position, in this the position, P2 position the P2 (Figure position6). (Figure An Arg 6). in An the Arg P2’ wasin the also P2’ negatively was also negatively influencing influencingcleavage (Figurecleavage6). The(Figure most 6). optimal The most sequence optimal in thissequence analysis in was this LIE analysis↓FD↓VFVQ was LIE (arrows↓FD↓VFVQ indicate (arrowspotential indicate cleavage potential sites), acleavage sequence sites), with a two sequen negativelyce with charged two negatively residues, charged one in the residues, P1 and oneone in in the theP3 P1 position and one and in the an aromaticP3 position amino and acidan aromatic in the P2 amino position acid (Figure in the 6P2). position (Figure 6).

Int. J. Mol. Sci. 2020, 21, 319 8 of 13 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 13

Figure 6. Analysis of the cleavage specificity of platypus GzmBGH (the granzyme B equivalent) by Figurethe use6. Analysis of recombinant of the cleavage protein specificity substrates. of The platypus different GzmBGH panels show(the granzyme the cleavage B equivalent) of a number by of thesubstrates use of recombinant by the platypus protein GzmBGH. substrates. The The sequences different of thepanels different show substrates the cleavage are indicated of a number above of the substratespictures by of the gels.platypus The GzmBGH. red residues The highlight sequences the of major the different difference substrates between are this indicated substrate above and the thereference pictures substrateof the gels. positioned The red residues at the beginning highlight (Left the side)major of differe this panel.nce between The time this of cleavage,substrate inand minutes, the referenceis also indicatedsubstrate abovepositioned the corresponding at the beginning lanes (Lef of thet side) different of this gels. panel. The un-cleavedThe time of substrates cleavage, have in a minutes,molecular is also weight indicated of approximately above the 25correspondi kDa and theng cleavedlanes of substrates the different appear gels. as twoThe closely-locatedun-cleaved substratesbands with have a sizea molecular of 12–13 weight kDa. of approximately 25 kDa and the cleaved substrates appear as two closely-located bands with a size of 12–13 kDa. 3. Discussion 3. DiscussionThe chymase loci of all previously analysed mammals, including both placental mammals and marsupials,The chymase are borderedloci of all at previously one end by analysed an α-chymase mammals, and atincluding the other both end placental by granzyme mammals B [12]. and Based marsupials,on the analyses are bordered of two at genomic one end contigs by an α in-chymase the platypus, and at which the other encode end by enzymes granzyme that B cluster [12]. Based closely onwith the analyses other mammalian of two genomic chymase contigs loci genes,in the weplatypus, can now which conclude encode that enzymes these platypus that cluster enzymes closely also withmost other likely mammalian represent chymase these bordering loci genes, genes. we can On now one conclude of these that contigs, these there platypus are two enzymes very closelyalso mostrelated likely chymases, represent which these both bordering appear togenes. be α -chymases,On one of indicatingthese contigs, a relatively there are recent two gene very duplication, closely relatedand onchymases, the second which contig both there appear is an asp-ase to be closelyα-chymases, related indicating to the other a mammalianrelatively recent granzyme gene Bs. duplication,Therefore, and all of on the the major second extant contig mammalian there is lineagesan asp-ase have closely the same related general to the pattern other withmammalian a classical granzymeMC chymase Bs. Therefore, and a granzyme all of the B [major12]. The extant platypus mammalian is so far lineages the only have species the analysed same general where pattern the locus withhas a beenclassical separated MC chymase into two and regions a granzyme which are B [12]. located The on platypus two diff erentis so far chromosomes the only species (Figure analysed2). In the whereplatypus the locus to chromosomes has been separated 13 and into 14, two where region onlys which the chymases are located on chromosomeon two different 13 chromosomes have previously (Figure 2). In the platypus to chromosomes 13 and 14, where only the chymases on chromosome 13 have previously identified bordering genes related to the chymase locus of other mammals (Figure

Int. J. Mol. Sci. 2020, 21, 319 9 of 13 identified bordering genes related to the chymase locus of other mammals (Figure2). Interestingly, in the majority of rodents the α-chymases have changed primary specificities and become . However, their lack of a chymotryptic α-chymase seems to have been compensated for by the presence of one or several chymotryptic β-chymases. All the rodents that have elastolytic α-chymases, including mice, rats and golden hamsters, have mainly chymotryptic β-chymases [14–17]. In this locus, all the analysed mammals also have granzyme B or a granzyme B-like protease with asp-ase activity, which indicates a strong conservation of both a MC chymotryptic enzyme and of an apoptosis-inducing granzyme B in cytotoxic T cells [12,28]. Currently, the only two species which differ from this pattern are the rabbit and guinea pig, where the β-chymase or α-chymase, respectively, have become strict leu-ases. Furthermore, they both appear to lack a compensating chymotryptic enzyme encoded from the chymase locus [19]. The follow-up question is what central targets these enzymes have and how the rabbit and guinea pig can compensate for this loss of a chymase in MCs. One possibility is that another locus is compensating for this loss possibly by gene duplication and a change in tissue specificity. A similar situation has occurred in pigs, sheep and cows where genes within the chymase locus have duplicated, most likely a granzyme or a cathepsin G gene, and that these new copies have changed tissue specificities, from expression in hematopoietic cells to primarily expression in the duodenum of the intestinal region; therefore, they are named duodenases [12,31,32]. However, no analyses of the protease content of rabbit or guinea pig mast cells have yet been performed to screen for chymotryptic enzymes originating from other protease loci, which could substantiate or disprove such a possibility. A number of potential targets for the MC chymotryptic enzymes have been identified, including angiotensin I cleavage in the context of blood pressure regulation, cleavage of snake or scorpion toxins in the protection against their toxic effects, cleavage of fibronectin and the activation of pro-collagenases in connective tissue turnover, cleavage of to regulate coagulation and several additional potential targets (reviewed in [11,33]). One question is how the rabbit and guinea pig have solved these functions if they lack a MC chymotryptic enzyme? One interesting finding, which touches on one of these problems, is the major chymotryptic enzyme of rat connective tissue MCs, rMCP-1. This enzyme has been shown to primarily activate angiotensin I, by cleavage at Phe-8 to form angiotensin II but also has the ability to cleave at Tyr-4, which leads to inactivation of angiotensin I. The rat has an extra β-chymase gene that is a potent angiotensin I converter, which has changed tissue specificity and is now primarily expressed in vascular smooth muscular cells, where it can activate angiotensin independently of mast cells. This enzyme has therefore been named vascular chymase [34,35]. In our minds, this supports the role of MC chymotryptic enzymes in blood pressure regulation. When this function has been lost from MCs, it has to be compensated for by another tissue, and in this case also another enzyme. Another serine protease locus may have duplicated copies of a chymotryptic enzyme that is either expressed in rabbit and/or guinea pig MCs, or that such enzymes are expressed in other cell types in tissues where MCs normally reside. Another striking observation in this study was the lack of cleavage of the chromogenic substrates and also the difficulty in obtaining good amplification during phage display pannings of the platypus enzymes. We have observed this phenomenon with several related enzymes from non-mammalian species including enzymes from both Xenopus laevis, an amphibian, and the Chinese alligator, a reptile. Two of these enzymes have a specificity determining triplet in their predicted substrate binding pocket of the enzyme, which strongly indicates asp-ase specificity, similar to the granzyme Bs of mammals, and the third, an alligator enzyme, has a triplet that is identical to primate cathepsin G (Figure1). However, when using a panel of chromogenic substrates and phage display, we also saw no cleavage and no increase in titers of phages after the pannings, similar to what we observed with the platypus enzymes (unpublished data). However, in contrast to the platypus where the recombinant substrates were cleaved, we have not had any success with the reptile and amphibian supposedly chymase locus genes. This is true of all the recombinant substrates currently analysed, which includes a large selection of over 270 different possible substrates. We have also analysed a few hematopoietic fish proteases and generally it seems that the mammalian enzymes have broader specificities and thereby can be Int. J. Mol. Sci. 2020, 21, 319 10 of 13 more easily studied with chromogenic substrates and phage display in comparison to their reptile, amphibian and fish homologues [27]. Currently, we have no good explanation to this phenomenon except perhaps the extended specificities are so specific that they are not picked up against classical granzyme B substrates. Even minor differences in substrate sequences compared to the proteases’ consensus may result in a complete loss of cleavage. Similarly, the phage library used may also have very low numbers of clones with such specifically preferred sequences and therefore the phage pannings do not result in amplification of any/enough clones compared to the background. In summary, this study shows that the platypus has both a MC chymotryptic enzyme and a granzyme B homolog. This reveals that all three mammalian lineages, the (egg-laying) monotremes, the marsupials and the placental mammals, have these two enzymes in their immune repertoire, which is a very strong indication that they play essential functions in the of mammals. Interestingly, there is no clear candidate for a chymase in the few chymase locus-related enzymes identified in reptiles and amphibians. However, we have identified enzymes that look like granzymes Bs in their potential substrate pockets, indicating that the apoptosis-inducing function was the first function of an enzyme within this locus and the chymase followed later. An alternative scenario is that the chymases have been lost in the two lineages and been replaced by an enzyme from another locus. This is possibly valid for the leu-ase expressing rabbit and the guinea pig; questions that we hope can be addressed in the near future.

4. Materials and Methods

4.1. Production and Purification of Recombinant Platypus Enzymes The sequences of the three platypus enzymes were retrieved from the NCBI database. cDNA copies of the mRNA were designed, based on the gene sequences, and ordered as designer genes from GenScript (Piscataway, NJ, USA). The individual designer gene was inserted into the mammalian expression vector pCEP-Pu2 and sequence verified. The construct contains a signal sequence, an N-terminal His-6 tag and an enterokinase site for purification and later activation by enterokinase cleavage. The vector was transfected into the human embryonic kidney (HEK) 293-EBNA cell line for expression of the recombinant enzyme. After purification on IMAC Ni 2+ agarose, the enzyme was activated by the addition of 1 µL enterokinase into 90 µL column eluate containing the recombinant protein. The sample was mixed, followed by a 5 h incubation at 37 ◦C to activate the protease. The purity and activation of the enzyme was determined by separation on 4–12% pre-cast SDS-PAGE gels (Invitrogen, Carlsbad, CA, USA). An amount of 2.5 µL sample buffer, containing sodium dodecyl sulfate (SDS), and 0.5 µL β-mercapto-ethanol was added to the 10 µL of protein sample followed by 85 ◦C heating, before separation on the SDS-PAGE gel. The gels were stained overnight in colloidal Coomassie staining solution followed by de-staining [36].

4.2. Generation of Recombinant Substrates for the Analysis of the Cleavage Specificity A new type of substrate was originally developed to verify the results obtained from the phage display analysis. Two copies of the E. coli thioredoxin (Trx) gene were inserted in tandem into the pET21 vector for bacterial expression (Figure4A). In the C-terminal end a His-6 tag was inserted for purification on Ni2+ IMAC columns. In a linker region, between the two thioredoxin molecules, the different substrate sequences were inserted by ligating double-stranded oligonucleotides into two unique restriction sites, one BamHI and one SalI site (Figure4A). This linker region contains a kinker region of repeating Ser-Gly residues which forms a flexible region open for access by a protease to cleave. The sequences of the individual clones were verified after cloning by sequencing of both DNA strains. The plasmids were then transformed into the E. coli Rosetta gami strain for protein expression (Novagen, Merck, Darmstadt, Germany). A 10 mL overnight culture of the bacteria harbouring the plasmid was diluted 10 times in LB + Amp and grown at 37 ◦C for 1–2 h until the OD (600 nm) reached 0.5. IPTG was then added to a final concentration of 1 mM. The culture was Int. J. Mol. Sci. 2020, 21, 319 11 of 13

grown at 37 ◦C for an additional 3 hours under vigorous shaking, after which the bacteria were pelleted by centrifugation at 3500 rpm for 12 minutes. The pellet was washed once with 25 ml PBS + 0.05 % Tween 20. The pellet was then dissolved in 2 mL PBS and sonicated 6 30 s to open the × cells. The lysate was centrifuged at 13,000 rpm for 10 min and the supernatant was transferred to a new tube. Five hundred microliters of Ni-NTA slurry (50:50) (Qiagen, Hilden, Germany) was added and the sample was slowly rotated for 45 min at room temperature. The sample was subsequently transferred to a 2 mL column and the supernatant was allowed to slowly pass through the filter leaving the Ni-NTA beads with the bound protein in the column. The column was then washed four times with 1 ml washing buffer (PBS + 0.05 % Tween + 10 mM Imidazole + 1 M NaCl). Elution of the protein was performed by adding 150 µL elution buffer followed by five 300 µL fractions of elution buffer (PBS + 0.05 % Tween 20 + 100 mM imidazole). Each fraction was collected individually. Ten microliters from each of the eluted fractions was then mixed with 1 volume of 2 sample buffer and 1 × µL β-mercapto-ethanol and then heated for 3 min at 80 ◦C. The samples were analysed on an SDS bis tris 4–12% PAGE gel and the second and third fractions that contained the most protein were pooled. The protein concentration of the combined fractions was determined by Bio-Rad DC Protein assay (Bio-Rad Laboratories Hercules, CA USA). Approximately 60 µg of recombinant protein was added to each 120 µL cleavage reaction (in PBS). Twenty microliters from this tube was removed before adding the enzyme, the 0 minute time point. The active enzyme was then added and the reaction was kept at room temperature during the entire experiment. Twenty microliter samples were removed at the indicated time points (15, 45 and 150 min) and stopped by addition of one volume of 2 sample buffer. × One microliter β-mercapto-ethanol was added to each sample followed by heating for 3 minutes at 80 ◦C. Twenty microliters from each of these samples was then analysed on 4–12% pre-cast SDS-PAGE gels (Invitrogen, Carlsbad, CA, USA). The gels were stained overnight in colloidal Coomassie staining solution and de-stained for several hours according to previously described procedures [36].

Author Contributions: Conceptualization, L.H.; validation, Z.F., S.A., and L.H.; formal analysis, Z.F., S.A. and L.H.; investigation Z.F. and S.A.; writing original draft preparation, L.H. writing review and editing, M.T.; visualization Z.F., S.A., and L.H.; supervision, L.H.; project administration, L.H.; funding acquisition, L.H. All authors have read and agreed to the published version of the manuscript. Funding: The study was supported by grants from Knut and Alice Wallenberg Foundation (KAW 2017-0029). Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

MC mast cell aa amino acid(s) mMCP mouse mast cell protease LD linear dichroism rMCP rat mast cell protease HC human chymase Ang angiotensin Ni-NTA nickel-nitrilotriacetic acid EK enterokinase

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