α and β Changed Rapidly during Primate Speciation and Evolved from γ-Like Transmembrane Peptidases in Ancestral Vertebrates This information is current as of September 25, 2021. Neil N. Trivedi, Qiao Tong, Kavita Raman, Vikash J. Bhagwandin and George H. Caughey J Immunol 2007; 179:6072-6079; ; doi: 10.4049/jimmunol.179.9.6072 http://www.jimmunol.org/content/179/9/6072 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Mast Cell ␣ and ␤ Tryptases Changed Rapidly during Primate Speciation and Evolved from ␥-Like Transmembrane Peptidases in Ancestral Vertebrates1

Neil N. Trivedi, Qiao Tong, Kavita Raman, Vikash J. Bhagwandin, and George H. Caughey2

Human mast cell tryptases vary strikingly in secretion, catalytic competence, and inheritance. To explore the basis of variation, we compared from a range of primates, including humans, great apes (chimpanzee, gorilla, orangutan), Old- and New- World monkeys (macaque and marmoset), and a prosimian (galago), tracking key changes. Our analysis reveals that extant soluble -like proteins, including ␣- and ␤-like tryptases, mastins, and implantation serine proteases, likely evolved from membrane-anchored ancestors because their more deeply rooted relatives (␥ tryptases, pancreasins, prostasins) are type I trans- membrane peptidases. Function-altering mutations appeared at widely separated times during primate speciation, with tryptases Downloaded from evolving by duplication, conversion, and point mutation. The ␣-tryptase Gly216Asp catalytic domain mutation, which di- minishes activity, is present in macaque tryptases, and thus arose before great apes and Old World monkeys shared an ancestor, ,and before the ␣␤ split. However, the Arg؊3Gln processing mutation appeared recently, affecting only human ␣. By comparison the transmembrane ␥-tryptase gene, which anchors the telomeric end of the multigene tryptase locus, changed little during primate evolution. Related transmembrane peptidase genes were found in reptiles, amphibians, and fish. We identified soluble

tryptase-like genes in the full spectrum of mammals, including marsupial (opossum) and monotreme (platypus), but not in http://www.jimmunol.org/ nonmammalian vertebrates. Overall, our analysis suggests that soluble tryptases evolved rapidly from membrane-anchored, two-chain peptidases in ancestral vertebrates into soluble, single-chain, self-compartmentalizing, inhibitor-resistant oligomers expressed primarily by mast cells, and that much of present numerical, behavioral, and genetic diversity of ␣- and ␤-like tryptases was acquired during primate evolution. The Journal of Immunology, 2007, 179: 6072–6079.

uman mast cell tryptases exhibit impressively diverse apparent failure of autocatalytic maturation (9). Also, ␣ appears to processing, secretion, solubility, catalytic activity, and have a critical catalytic domain mutation, which greatly reduces inheritance. As a group, tryptases are implicated in al- activity toward substrates readily cleaved by ␤ tryptases. This mu-

H by guest on September 25, 2021 lergic and other types of inflammation. All mast cell tryptase genes tation perturbs the in a manner that is unproductive for cluster tightly on 16p13.3 (1). Products of these substrate binding (10–12). In contrast to ␤ tryptase, ␣ fails to genes fall into two general categories: membrane-anchored and evoke neutrophilic inflammation (13) and most or all of it appears soluble. The ␥ gene, TPSG1, encodes a type I transmembrane pep- to be secreted (probably as a proenzyme) and not stored in mast tidase anchored to the cell surface after secretion (2, 3). Behavior cell granules (14). ␣ Genes are absent in a substantial portion of of peptidase chimeras containing part of mouse ␥ tryptase suggests most humans (6). This appears to be because they are alleles at a that the anchor is a C-terminal hydrophobic peptide, which is not locus that also accepts ␤I genes (1). Humans without ␣ genes have exchanged for a lipid anchor, as occurs in some related , diminished circulating levels of immunoreactive tryptase com- including prostasin (4). In humans, ␥ is the sole membrane-an- pared with those with ␣ genes (15). Thus, ␣ and ␤ tryptases differ chored tryptase and is hypothesized to be proinflammatory (5). The in important ways. other three transcribed tryptase genes encode soluble enzymes ␤ Tryptases are products of two adjacent loci. The major alleles lacking an anchor. These are TPSAB1 (encoding ␣ and ␤I trypta- (I–III) are highly similar (16, 17). ␤ Tryptases encode soluble, ses), TPSB2 (encoding ␤II and ␤III), and TPSD1 (encoding ␦ active enzymes that are stored in secretory granules and released in tryptases) (1, 6, 7). response to allergen-bound IgE and other stimuli (18). They self- ␣ Tryptase, the first human tryptase to have its full primary assemble into tetramers, which shield the active site from inacti- structure determined (8), features a propeptide mutation causing vation by circulating inhibitors (19). ␤ Tryptases are the dominant forms isolated from tissue extracts and are targets for therapeutic inhibition because of postulated importance in diseases such as Cardiovascular Research Institute and Department of Medicine, University of Cali- , anaphylaxis, and inflammatory bowel disease (7, 20, 21). fornia, San Francisco, CA 94143; Northern California Institute for Research and ␦ Education, San Francisco, CA 94121; and Veterans Affairs Medical Center, San Fran- Human tryptases differ in that they are C-terminally truncated cisco, CA 94121 (1). They also feature the propeptide mutation that blocks process- Received for publication June 27, 2007. Accepted for publication August 10, 2007. ing in ␣ tryptases. Consequently, ␦ tryptase has little catalytic ac- The costs of publication of this article were defrayed in part by the payment of page tivity (22) and may have defective propeptide processing. Al- charges. This article must therefore be hereby marked advertisement in accordance though the ␦ gene TPSD1 was first hypothesized to be a with 18 U.S.C. Section 1734 solely to indicate this fact. (1, 23), transcripts and immunoreactivity were later 1 This work was supported by National Institutes of Health Grant HL024136 and the detected in multiple organs (22). However, the targets and roles, if Northern California Institute for Research and Education. any, of ␦ tryptases remain to be determined. 2 Address correspondence and reprint requests to Dr. George H. Caughey, Veterans Affairs Medical Center, Mailstop 111D, 4150 Clement Street, San Francisco, CA The origins of human tryptase isoforms and of mammalian 94121. E-mail address: [email protected] tryptases in general are obscure. Unlike many other mammalian www.jimmunol.org The Journal of Immunology 6073

Table I. Primate tryptase sequences: nomenclature, sources, and identifiers

Primate Protein GenBank Identifier

Homo sapiens (human) Tryptase, ␣I, ␣II M30038; AF098328 Tryptase, ␤I M33494 Tryptase, ␤II M33492 and AF099145 Tryptase, ␤III AF099143 Tryptase, ␥I, ␥II NM_012467; AAF76458 Tryptase, ␦I NM_012217 ISP2 AF529082a Pan troglodytes (chimpanzee) Tryptase, ␣ XM_001158624 Tryptase, ␤ EF206351a Gorilla gorilla (gorilla) Tryptase, ␤1 EF208020a Tryptase, ␤2 EF208021a Tryptase, ␤3 EF208022a Pongo pygmaeus abelii (Sumatran orangutan) Tryptase, ␤1 EF452227a Tryptase, ␤2 EF452228a Tryptase, ␤3 EF452229a Tryptase, ␤4 EF452230a Macaca fascicularis (crab-eating macaque) Tryptase, ␣␤1 EF212445a Tryptase, ␣␤2 EF212444a Macaca mulatta (rhesus macaque) Tryptase, ␣␤ XM_001088289 Downloaded from Tryptase, ␥ AANU01106415b ISP2 XP_001118570 Callithrix jacchus (common marmoset) Tryptase Contig12371.9c Tryptase, ␥ Contig12371.8c Otolemur garnettii (small-eared galago) Tryptase, ␥ AAQR01303204b

a Cloned and sequenced, this work. b Deduced from unannotated whole genome shotgun sequence. http://www.jimmunol.org/ c Deduced from unannotated sequences from http://blast.wustl.edu. peptidases, tryptases lack obvious orthologs in nonmammalian genomic DNA from species-specific cultured fibroblasts. DNA encoding vertebrates. To understand origins and consequences of the diver- primate ␣- and ␤-like genes was amplified by PCR using Advantage 2 (BD Clontech). For ␣- and ␤-like genes, primers bracketed full protein-coding sity of expressed human tryptases, the present study probes the Ј Ј ␣ ␤ ␥ sequence and were based on highly conserved portions of the 3 and 5 evolution of human , , and tryptases. The data acquired for flanks of human ␣ and ␤ tryptases. Individual amplimers were ligated into this study suggest that tryptases proliferated and changed rapidly pCR2.1-TOPO (Invitrogen Life Technologies). Resulting recombinant during mammalian evolution, arising from ancestral membrane- plasmid DNA was purified and the inserted portion was sequenced. by guest on September 25, 2021 anchored peptidases, which are present in a variety of vertebrate Sequencing of the human implantation (ISP) gene genomes. The pace of change accelerated during evolution of pri- mates. This work’s comparison of primate tryptases suggests that We obtained full sequence of the human ISP2 gene from a fragment of several idiosyncratic features of human enzymes are recent devel- human chromosome 16p13.3 in bacterial artificial chromosome (BAC) 48D21 (Invitrogen Life Technologies). A fragment generated by digestion opments. The resulting analysis reveals human tryptase origins and of the pBeloBAC11 clone with HindIII was subcloned as described (3). highlights peptidases of likely functional importance. This ϳ8-kb subclone (48K), which contains the 5Ј half and flank of the ␦II-tryptase gene, was further sequenced by gene walking to obtain the Materials and Methods adjacent ISP2 gene, which is oriented tail-to-tail with respect to the ␦II gene (24). Data mining Full primary sequences of mammalian tryptases not already published or Phylogenetic comparisons annotated were obtained by data mining, including basic local alignment 3 Preprotryptase amino acid sequences were compared using Geneious soft- search tool (BLAST) and BLAST-like alignment tool searches of high ware (Biomatters). Aligned sequences were subjected to phylogenetic anal- throughput genome sequence and whole genome shotgun databases at the ysis, including tree preparation, using unweighted pair group method with National Center for Biotechnology Information using mammalian tryptase arithmetic mean (UPGMA) and neighbor-joining techniques with bootstrap genes and cDNAs as query sequences. Marmoset sequences were obtained resampling. from BLAST searches of genomic sequence archived at the Washington University School of Medicine Genome Sequencing Center (http:// genome.wustl.edu). Amino acid sequences of previously unreported trypta- Results ses were extracted from genomic DNA using existing tryptase gene struc- Nucleotide sequence, size, and intron/exon structure of primate tures as a guide, following standard rules for placement of intron-exon ␣-like/␤-like genes boundaries. To avoid connecting two closely related but separate genes, only genomic fragments containing complete coding sequence of the cat- Genomic sequence bracketing the full protein coding sequence of alytic domain were subjected to this analysis. Most tryptase genes are small primate tryptases was obtained by sequencing ϳ10 cloned am- compared with other serine protease genes, so that shotgun-derived frag- plimers each from gorilla, chimpanzee, orangutan, and crab-eating ments can be long enough to contain a complete gene. macaque (cynomolgus monkey) genomic DNA. Nucleotide se- Sequencing of primate tryptase genes quence of genomic amplimers yielding unique tryptase sequences was deposited into GenBank (see accession numbers in Table I). Primate genomic DNA was obtained from the Primate Cell Repositories of the Coriell Institute for Medical Research (Camden, NJ), which provides The length of sequenced genes was similar to that of related human genes. Exonic sequence was predicted by aligning human tryptase cDNAs. In all cases, introns began with dinucleotide GT and 3 Abbreviations used in this paper: BLAST, basic local alignment search tool; ISP, implantation serine protease; BAC, bacterial artificial chromosome; UPGMA, un- ended with AG. Intron phase (0, 1, or 2) was identical with that of weighted pair group method with arithmetic mean. homologous introns in other soluble mammalian tryptases. 6074 HYPEREVOLUTION OF SOLUBLE TRYPTASES

Primary structure of primate ␣-like/␤-like tryptases: general features The preprotryptase amino acid sequence predicted from each of the cloned genes is 275 aa, comprised of a Ϫ30 residue leader and a ϩ245 residue catalytic portion (Fig. 1). Each possesses “” residues His44, Asp91, and Ser195 (corresponding to His57, Asp102, and Ser195 of chymotrypsinogen) common to active pep- tidases of the family. Each has the Gly-1 character- istic of mast cell tryptases at the site of propeptide removal as well as most of the residues forming the hydrophobic interfaces be- tween subunits in crystallized human ␤II tryptase tetramer (19). These interface-forming residues include Pro48, Tyr66, Tyr67, Tyr84, Pro140, Pro141, and His163, which are absolutely conserved in known soluble primate and murine tryptases (Fig. 1). Of these residues, only Pro141 (Pro138 in ␥ tryptase) is conserved in nono- ligomerizing ␥ tryptases. The six surface loops involved in making intersubunit contacts are underlined in Fig. 1. None of the deduced tryptases, including the chimpanzee otherwise similar to ␣, has the GlnϪ3 found in human ␣ and ␦ tryptases (1, 8). Con- Downloaded from sensus glycosylation sites at Asn102 and Asn203 are conserved in this group, although human ␤II, which lacks the Asn102 consensus site, is aberrant in this regard. Two of the residues forming the “specificity triad,” which shapes the pocket accommodating the substrate P1 side chain at the site of hydrolysis, are absolutely conserved. One of these is Asp188 (189 in chymotrypsinogen), as http://www.jimmunol.org/ expected of enzymes that are primarily tryptic in specificity. The other entirely conserved residue is Gly225 (226 in chymotrypsin- ogen). However, residue 215 (216 in chymotrypsinogen) varies, being Asp in human and chimpanzee ␣ and in all macaque ␣␤ tryptases, but Gly in all others, including human ␤ tryptases and classic chymotrypsin-family peptidases of tryptic specificity. This is somewhat surprising because this residue in human ␣ distorts the substrate and limits peptidase activity (11, 12). by guest on September 25, 2021 Although rhesus and crab-eating macaque tryptases feature this ␣-like mutation, overall they are nearly as similar to human ␤ as to ␣, as revealed by Fig. 2, therefore, we refer to them here as “␣␤” tryptases. Marmoset tryptase, in contrast, possesses the classic specificity triad but is nearly as similar to ␣ as to ␤ (Table II), hence, we refer to this enzyme simply as tryptase. The tryptases most similar to human are from gorilla (Table II, Fig. 2). All three gorilla tryptases are more nearly identical with human than to chimpanzee ␤. Although orangutan tryptases share some residues with human and chimpanzee ␣ and others with human and gorilla ␤; overall, they are more ␤-like, especially in regard to function- ally important residues like Gly225. Therefore, we apply the ␤ label to these orangutan tryptases.

Genomic and amino acid sequence of primate ␥ tryptases As shown in Fig. 3, we deduced ␥-tryptase sequence from three nonhuman primates, including a prosimian (the small-eared ga- lago, Otolemur garnettii), an Old World monkey (rhesus ma- caque), a New World monkey (common marmoset, Callithrix jac- chus), and a great ape (orangutan). Table III demonstrates the percentage identity of pairs of primate and rodent ␥ tryptases. We predict that mature versions of these tryptases are two-chain, type I transmembrane, tryptic peptidases, like human ␥. Hydropathy FIGURE 1. Alignment of primate ␣-like/␤-like/␦-like tryptases. Exam- analysis (http://gcat.davidson.edu/rakarnik/kyte-doolittle.htm) us- ples of tryptases sequenced or deduced for this work are aligned as indi- ing a 19-residue window reveals C termini typical of membrane- cated and compared with human ␤I, ␣II, and ␦ tryptases and mouse mast spanning regions (data not shown). Inspection of these sequences cell protease (MMCP) 6 and 7 tryptases. See Table I for GenBank iden- tifiers. Hyphens (-) indicate amino acid identity compared with correspond- -identify gaps in the align- serine peptidases of tryptic specificity; ϩ, the predicted sites of N-glyco (ء) ing residues in human ␤I tryptase. Asterisks ment. Symbol meanings: #, “catalytic triad” residues common to all active sylation conserved in all of these enzymes; underlined residues indicate serine peptidases; %, conserved “specificity triad” residues typical of most loops involved in subunit contacts. The Journal of Immunology 6075

FIGURE 2. Tree of ␣-like/␤-like tryptases. Potential phylogenetic relationships between classic murine and primate soluble mast cell tryptases, including proteins newly sequenced or deduced for this study, are probed with this rooted dendrogram generated by UPGMA with 500 iterations of bootstrap resampling. The tree also is a template for tracking other tryptases with the propeptide processing mutation of human ␣ (R-3Q, heavy solid line) as well as the catalytic domain muta- tion (G215D, hatched line). Proposed branch points for ␣ and ␤ clades are identified by the appropriate symbol. Sources of primate tryptase sequences are in Table I. Nonprimate sources are as follows: opossum (M. do- mestica, deduced from whole genome shotgun sequence AAFR03046243), rat Mcpt6 and 7 (R. norvegicus, AAB48262 and AAB48263), mouse Mcpt6 and 7 (M. musculus, P21845 and Q02844). Note that the ␣␤ pri- mate tryptases share ancestry more recently with rodent

Mcpt6 tryptases than with Mcpt7, that key ␣ mutations Downloaded from appeared at times widely separated in primate evolution, and that the ␣␤ dichotomy is recent and only evident among great apes. http://www.jimmunol.org/ for glycosylphosphatidylinositol anchor attachment sites using the Also included in the analysis are tryptases already deduced and Big-PI algorithm (http://mendel.imp.ac.at/sat/gpi/gpi_server.html) searchable in GenBank as follows: mouse (Mus musculus), P21845 reveals no consensus sites. Therefore, these primate ␥ tryptases, and Q02844; rat (Rattus norvegicus), U67909 and U67910; dog like mouse (as revealed by ␥/prostasin chimeras; Ref. 4), are not (Canis familiaris), M24664; horse (Equus caballus), AJ515902; likely to have C-terminal peptide anchors swapped for lipids. gerbil (Meriones unguiculatus), D31789; sheep (Ovis aries), Y18223 and Y18224; cattle (Bos taurus), NP_776627; and pig Primate ISP2 “implantation tryptase” genes (Sus scrofa), NP_999356. The full sequence of human ISP2 was obtained from subclone 48K ␥ of a human BAC (as noted in Materials and Methods), which also Nonprimate tryptase-like mastins, ISPs, -like prostasins, and by guest on September 25, 2021 contains tryptase genes (3). The sequence of the human ISP2 gene related genes (which appears to encode a nonfunctional protein because the cod- See Fig. 4 legend for sources of newly or previously deduced ing sequence goes out of frame compared with the mouse ISP2 full-length protein sequences used to construct a master tree, in- sequence) was deposited in GenBank (Table I). Its presence on cluding tryptase-related type I transmembrane peptidases in non- the same subclone as ␦ tryptase confirms that it lies close to mammalian vertebrates. classic tryptase genes and belongs to the tryptase locus. Be- cause it appears to be encoded by a pseudogene, human ISP2 is Discussion not included in the tree in Fig. 4. Also, mining of available This work illuminates origins of human ␣, ␤, and ␥ tryptases. In chimpanzee and orangutan genome sequence yielded only particular, it reveals that much of the diversity of form and func- flawed ISP2 genes, which therefore are excluded from the tree. tion among expressed human tryptases was generated during pri- However, amino acid sequence representing apparently intact mate evolution. The data further suggest that the ISP2 was deduced from whole genome shotgun sequence from harbors tryptase-like , some of which are expressed rhesus macaque and is included in the phylogenetic analysis. and active in other mammals, including nonhuman primates. Sev- Thus, it appears that the ISP2 gene may have remained func- eral genetic mechanisms contributed to creation, alteration, and tional in primates at least until macaques and great apes shared inactivation of primate tryptase-like genes. These include segmen- a common ancestor. tal duplication, gene conversion, chimera formation, and point mu- tation. Overall, our analysis charts an evolutionary path from Nonprimate classical soluble tryptase sequences ␥-like, membrane-anchored, two-chain, inhibitor-sensitive pepti- The cDNA and corresponding amino acid sequence were deduced dases in ancestral vertebrates to soluble, single-chain, self-com- for this work from GenBank-deposited, unannotated whole ge- partmentalizing, inhibitor-resistant oligomers. nome shotgun sequences from the following nonprimate mam- Compared with opossum and dog tryptase loci, the murine mals: little brown bat (Myotis lucifugus), AAPE01472347; Euro- Mcpt6/Mcpt7 tryptase locus is duplicated and the human ␣/hu- pean hedgehog (Erinaceus europaeus), AANN01723492; short man ␤/human ␦ (TPSAB1/TPSB2/TPSD1) locus is triplicated. gray-tailed opossum (Monodelphis domestica), AAFR03046243; As shown in prior work from this laboratory, the ␦ (TPSD1) and duck-billed platypus (Ornithorhynchus anatinus), gene product is a truncated tryptase chimera created by gene AAPN01127233, AAPN01196394, AAPN01292231. The platy- conversion events (1). The present analysis, focusing on ␣- and pus sequence provides evidence of a complete tryptase sequence in ␤-like tryptases, indicates that ␣␤ dichotomization and key mu- a monotreme, which is believed to be a survivor of an early branch tations affecting activation, storage, secretion, and activity of ␣ in the mammalian tree. Similarly, the marsupial opossum sequence tryptase occurred during evolution of primates. As indicated by is an example of a tryptase sequence from a nonplacental mammal. branching in Figs. 2 and 4, the ␣␤ dichotomy is unrelated to the 6076 HYPEREVOLUTION OF SOLUBLE TRYPTASES

Table II. Pairwise comparisons of percentage identity of mouse and primate soluble tryptase catalytic domainsa

Go Ch Or Hu Ch Rh Mf Ma MP MP

␤1 ␤␤4 ␣II ␣␣␤␣␤167

Hu ␤I99979794949190867877 Go ␤1989694949190867877 Ch ␤ 96 95 95 90 88 84 77 77 Or ␤493939391877877 Hu ␣II 98 91 90 85 76 77 Ch ␣ 91 91 84 76 76 Rh ␣␤ 98 86 76 76 Mf ␣␤1857675 Ma 73 76 MP 6 71

a Hu, Human; Go, gorilla; Ch, chimpanzee; Or, orangutan; Rh, rhesus; Mf, M. fascicularis macaque; Ma, marmoset; MP, mouse mast cell protease. Downloaded from Mcpt6/Mcpt7 split in rodents. Indeed, human and chimpanzee TPSAB1 (␣ and ␤⌱) and TPSB2 (␤II/␤III) genes are duplicated in relation to the corresponding mouse locus (24). This dupli- cation likely arose from an ancestor shared with Mcpt6, given that the mouse MCP-6 sequences aligns more closely than

MCP-7 with the ␣␤ clade, as shown in Fig. 2. The duplication http://www.jimmunol.org/ probably occurred during primate evolution, because compara- ble duplications have not been noted in nonprimates. The rodent Mcpt7 gene shares mixed ancestry with the chimeric human TPSD1 (␦) gene, which appears to have been created by a con- version event involving a recent ancestor of a primate ␣␤-like gene and a more distantly related tryptase similar to rodent Mcpt7 (1). The exact origin of the Mcpt6/Mcpt7 duplication extant in rodents and further multiplied and modified in pri- mates is unclear. However, some mammals, including dogs, by guest on September 25, 2021 possess only one orthologous tryptase gene, suggesting that the duplication of the putative ancestral soluble tryptase gene oc- curred after dogs, rodents, and primates shared a common an- cestor, but before ancestors of rodents and primates split from the tree. As demonstrated by Fig. 1 and Table II, there are numerous ␣ ␤ differences between and tryptases (e.g., 19 mismatches be- ␥ ␣ ␤ ␣ ␤ FIGURE 3. Alignment of tryptases. Hyphens (-) indicate amino tween human II and I and 15 between chimpanzee and ). acid identity compared with the corresponding residues in human ␥I Of the variant residues, two in particular cause unconventional tryptase. See Table I for identifiers of primate GenBank files, which Ϫ3 behavior in human ␣ vs ␤:1)Gln substituting for Arg in the contain full genomic sequence. Mouse (M. musculus) and rat (R. nor- ␣ propeptide apparently precludes autolytic processing, activa- vegicus) sequences are from NP_036164 and NM_175593, respectively. identify gaps in the alignment, the largest of which is in (ء) tion, oligomerization, and storage in secretory granules (9, 14), Asterisks and 2) Asp215 substituting for Gly in the catalytic domain dis- mouse and rat prosequence, corresponding to an exon that is transcribed orders the active site and reduces activity of any ␣ that manages in human and, by homology, other primate ␥ tryptases but not in murine to be correctly processed and activated from its zymogen form ␥ genes. Symbol meanings: @, an absolutely conserved cysteine via (10–12). Our analysis reveals that origins of these mutations which the propeptide remains linked to the catalytic domain after cleav- Ϫ age-activation between Arg-1 and Ile ϩ1 to yield two-chain mature en- differ. The Arg 3Gln processing mutation is nearly new, being ␣ zymes; underlined residues comprise a predicted transmembrane segment present in human but not chimpanzee . We did not detect this present in ␥ but not soluble tryptases; the meanings of #, %, and ϩ symbols mutation in other primate tryptases, with the exception of hu- are as in Fig. 1. man ␦, where its presence may be due to a recent partial con- ␣ ␦ version involving and genes, whose tandem orientation can Table III. Pairwise comparisons of percentage identity of rodent and facilitate such an event, much as ␦ itself is a chimera generated primate ␥-tryptase catalytic domains by more remote conversion events (1). Although the Ϫ3 residue is Arg in most nonprimate tryptase propeptides, this is not uni- Orangutan Rhesus Marmoset Galago Mouse Rat versal. In a gerbil tryptase, for example, the corresponding res- Human 92 93 86 79 77 77 idue is Glu (25). Whether activation of this tryptase is impaired Orangutan 93 88 80 77 78 is not known. It is worth noting that mastins possess propep- Rhesus 87 79 76 77 tides very similar to those of tryptases; they, too, lack a basic Marmoset 82 78 78 amino acid in the Ϫ3 position, yet are processed to active, oli- Galago 80 79 gomeric, -sequestered forms (26–28). Thus, for some Mouse 90 The Journal of Immunology 6077 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Tree of vertebrate tryptase-like proteases. This rooted dendrogram was prepared by UPGMA with 500 iterations of bootstrap resampling. Refer to Table I for GenBank sources of galago, rhesus, and human ␥ tryptases and rhesus ISP2, to Fig. 3 for GenBank sources of mouse and rat ␥ tryptase, and to Fig. 2 for GenBank sources of opossum tryptase and rat and mouse Mcpt7 and Mcpt6. Other sequences were obtained as follows: frog channel- activating peptidase (CAP) 1 (Xenopus laevis, AAB96905), lizard, opossum, dog, cat, mouse, rat, rhesus, orangutan, chimpanzee, and human prostasin (Anolis carolinensis DS229345; M. domestica XP_001372224; C. familiaris XP_848861; Felis catus AANG01151030; M. musculus NM_133351; R. norvegicus AAG32641; Macaca mulatta XP_001112376; Pongo pygmaeus abelii CAH90878; Pan troglodytes XM_001157456; Homo sapiens NM_002773), lizard, mouse, rat, horse, dog, marmoset, rhesus, chimpanzee, and human pancreasin (A. carolinensis AAWZ01023110; M. musculus NM_175440; R. norvegicus NM_182949; and E. caballus AAWR01029521; C. familiaris AAEX02025250; C. jacchus contig 2675.45 (http://genome.wustl.edu); M. mulatta XM_001086389; Pan troglodytes XP_510751, Homo sapiens NM_031948), horse ␥ tryptase (E. caballus AAWR01029469), platypus tryptase-like protein 1and2(O. anatinus AAPN01127233 and AAPN01196394), mouse, rat, and squirrel ISP2 (M. musculus AAK15264, R. norvegicus XP_220240, and Spermophilus tridecemlineatus AAQQ01728300), mouse, dog, cattle, and pig mastin (M. musculus AAS21652; C. familiaris P19236; B. taurus XM_869964; S. scrofa NP_998959), bat, hedgehog, horse, dog, pig, gerbil, sheep 1, sheep 2, cattle 1, and cattle 2 tryptase (M. lucifugus AAPE01472347; E. europaeus AANN01723492; E. caballus AJ515902; C. familiaris M24664; S. scrofa NP_999356; M. unguiculatus D31789; O. aries Y18223 and Y18224; B. taurus NP_776627 and AAFC03119556).

tryptase-like enzymes, autolytic processing at Arg-3 is not es- tryptases are no more closely related to human/chimpanzee ␣ than sential for maturation. to ␤ in overall structure, as revealed by Figs. 2 and 4. Thus, these Surprisingly, we found that the Asp215Gly catalytic domain mu- data suggest that the catalytic domain mutation appeared after New tation is present in several tryptases in Old World monkeys (i.e., and Old World monkeys diverged from the tree, but well before macaques) but we found no evidence of this mutation in tryptase the split into ␣ and ␤, which occurred after Old World monkeys from a New World monkey (i.e., marmoset) (Figs. 1 and 2). In- and great apes shared a common ancestor. Although it is possible deed, all ␣-like/␤-like sequences identified in rhesus and crab- that the Asp215Gly mutation occurred once in ancestors of ma- eating macaques contain ␣-like Asp215, although marmoset con- caque ␣␤ tryptases and a second time in ancestral chimpanzee/ tains conventional Gly215 tryptase. The macaque and marmoset human ␣, it is more likely that the Asp215Gly mutation arose once 6078 HYPEREVOLUTION OF SOLUBLE TRYPTASES

Table IV. Features of tryptases and closest relatives

Transmembrane No. of Activation No. of Mast Cell 5Ј UTR Inhibitor Protease Specificity Anchor Chains Site Prepro Exons Expression Introna Resistance 216 aa

Testisins Tryptic ϩ 2 Arg 3 ϪϪϪGly Prostasins Tryptic ϩ 2 Arg 3 ϪϪϪGly Pancreasins Tryptic ϩ/Ϫ 2 Arg 3 ϪϪϪGly ␥ Tryptases Tryptic ϩ 2 Arg 2–3 ϩϪϪGly ISP2s Tryptic Ϫ 1 Gly 2 ? ϩϩ/Ϫ Gly Mastins Tryptic Ϫ 1 Gly 2 ϩϪϩGly ␤ Tryptases Tryptic Ϫ 1 Gly 2 ϩϩϩGly ␦ Tryptases Tryptic Ϫ 1 Gly 2 ϩϩ?X ␣ Tryptases Tryptic Ϫ 1 Gly 2 ϩϩϩAsp

a UTR, Untranslated region. in an ancestor shared by macaque ␣␤ and chimpanzee/human ␣ the small second exon featured in primate ␥ tryptases (see Table IV), tryptases. The fact that we have not encountered this mutation in any which accounts for the propeptide gap in the murine ␥ propeptide tryptase-related protease in a nonprimate further supports the conclu- sequences aligned in Fig. 3. Inasmuch as this small exon is a feature sion that it arose during evolution of primates. It remains to be seen of primate ␥ tryptases as well as of related type I transmembrane Downloaded from whether macaque Asp215 tryptases are catalytically impaired, like peptidases, like prostasin (3), rodent ␥ tryptases can be seen as tran- human ␣. sitional. In light of these considerations, and because the ␥ stem is The trees in Figs. 2 and 4 suggest that classic soluble mast cell basal to that of all known soluble tryptase-like enzymes, extant ␥ tryptases were present early in mammalian evolution, existing now tryptases may be similar to tryptases in their ancestral form. in all major groups of mammals, including monotreme (platypus), Like soluble tryptase-like enzymes, ␥ tryptases have no clear

marsupial (opossum), and placental mammals (many examples). counterparts in nonmammalian vertebrates. In mammals, some of http://www.jimmunol.org/ The Fig. 4 dendrogram reveals clearly that soluble tryptases form their closest relatives are pancreasins (also known as marapsins) a clade separate from tryptase-like mastins and ISPs. At present, (31), which are tryptic serine peptidases of uncertain function, not there is no evidence that mastin and ISP are functional in humans. known to be expressed in mast cells. Except in humans and chim- However, mastins are expressed and active in several nonprimates, panzees, pancreasins (like ␥ tryptases) are predicted to be type I including dog (28, 29), pig, and mouse (in which mastin is also transmembrane peptidases (Ref. 31 and data not shown). The clos- known as TC30 tryptase and MCP-11/Prss34, respectively (27, est relatives of pancreasins are other transmembrane peptidases, 30). GenBank-deposited mastin expressed sequence tag including prostasins, testisins, and various nonmammalian, verte- EC335262, which encodes most of the protein from an opossum brate transmembrane peptidases, some of which are shown in the (silver-gray brushtail, Trichosurus vulpecula), is evidence that tree in Fig. 4. Table IV summarizes attributes of these enzymes by guest on September 25, 2021 mastins were present in early mammals—at least back to the time and supports an evolutionary path to soluble tryptases. Losing the that placental and marsupial mammals shared a common ancestor. transmembrane portion of a type I peptidase is relatively simple as Another expressed sequence tag originates from pig-tailed ma- exemplified by pancreasin, which is soluble in humans and mem- caque (DY760362), in which it may be a transcribed pseudogene, brane-anchored in mice (31). A change of just two nucleotides com- as also predicted of rhesus mastin. There is no evidence of tran- pared with mouse pancreasin caused tail loss in the human enzyme. scription of the human mastin gene. These sequences are not in- These considerations lead us to propose that premammalian vertebrate cluded in the Fig. 4 dendrogram because they are incomplete or ancestors of extant mammalian soluble mast cell tryptases were type contain early stop codons or other major flaws. I transmembrane peptidases, which lost their tails. Although the sequence deduced from the human ISP2 gene sug- Loss of the transmembrane segment in ancestral tryptases ap- gests that it is likely to be a pseudogene (even if transcribed), our parently triggered a quite dramatic increase in the pace of evolu- database screens suggest that it is potentially functional in rhesus tionary change. In the Fig. 4 tree, this is evident by comparing the macaques. This is worth noting, because ISPs (and mastins) are the proteins most closely related to classic soluble mast cell tryptases. Curiously, we do not find clearly identifiable soluble tryptase or tryptase-like proteases (including mastins and ISP2) in nonmam- malian vertebrates (or for that matter, in invertebrates). As dem- onstrated by Fig. 4, the ␣ and ␤ tryptases, mastins, and ISPs are related to ␥ tryptases, which differ in key ways summarized in Table IV. The differences include 1) an extra exon encoding the propeptide, 2) Arg rather than Gly at the site of zymogen activa- tion, 3) a two-chain mature form, and 4) a C-terminal transmem- brane anchor. Nonetheless, several features link ␥ to soluble tryptases. These include overall structural/phylogenetic similarity to soluble tryptases and tryptase-like enzymes (Fig. 4), conserva- FIGURE 5. Proposed origins of human tryptases. Pseudogenes or cat- tion of structural motifs (such as the polyproline tract LPPPF/Y, ␣␤ ␥ alytically flawed tryptases are shown in gray. Numbers 1–5 identify an 139–143 in and 136–140 in ; see Figs. 1 and 3) shared only ordered sequence of duplications, starting from an ancestral ␥-like gene in ␥ ␥ by and soluble tryptases; the position of in the tryptase gene early mammals, and giving rise to the current diversity of soluble human cluster as the nearest neighbor of soluble tryptase genes TPSB2 tryptase and tryptase-like genes. Upward arrows indicate proposed gene and Mcpt6 in human and murine genomes, respectively, tryptic conversion events leading to the creation of the ␦ chimera and to allelic specificity, and expression in mast cells. Rat and mouse ␥ genes lack disparity of ␣ and ␤I genes at the TPSAB1 locus. The Journal of Immunology 6079 extent of sequence conservation in membrane-anchored, tryptase- 9. Sakai, K., S. Ren, and L. B. Schwartz. 1996. A novel -dependent pro- related serine peptidases (␥ tryptases, pancreasins, and prostasins) cessing pathway for human tryptase: autocatalysis followed by activation with dipeptidyl peptidase I. J. Clin. Invest. 97: 988–995. with the level of conservation among soluble, tryptase-related pep- 10. Huang, C., L. Li, S. A. Krilis, K. Chanasyk, Y. Tang, Z. Li, J. E. Hunt, and tidases (ISPs, mastins, and tryptases themselves). As a group, sol- R. L. Stevens. 1999. Human tryptases ␣ and ␤II are functionally distinct due, in part, to a single amino acid difference in one of the surface loops that forms the uble tryptase-like enzymes diverge much more dramatically than substrate-binding cleft. J. Biol. Chem. 274: 19670–19676. their transmembrane relatives. For example, aligned human and 11. Marquardt, U., F. Zettl, R. Huber, W. Bode, and C. Sommerhoff. 2002. The opossum tryptase catalytic domains have more than twice as many crystal structure of human ␣1-tryptase reveals a blocked substrate-binding region. J. Mol. Biol. 321: 491–502. mismatches as the same pairing of prostasins, and pairings within 12. Selwood, T., Z. M. Wang, D. R. McCaslin, and N. M. Schechter. 2002. Diverse the ISP2 or mastin groups vary to an even greater extent. The stability and catalytic properties of human tryptase ␣ and ␤ isoforms are mediated soluble tryptase-like genes evolved not only by accumulating mu- by residue differences at the S1 pocket. Biochemistry 41: 3329–3340. 13. Huang, C., G. T. De Sanctis, P. J. O’Brien, J. P. Mizgerd, D. S. Friend, tations but by duplication and reduplication, so that some now J. M. Drazen, L. F. Brass, and R. L. Stevens. 2001. Evaluation of the substrate appear to be redundant in some mammalian genomes, including specificity of human mast cell tryptase ␤I and demonstration of its importance in human. 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