- Email: [email protected]
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[50] Craik, C.S., Choo, Q.L., Swift, G.H., Quinto, C., MacDonald, R.J., Rutter, W.J. (1984). Structure of two related rat pancreatic trypsin genes. J. Biol. Chem. 259, 1425514264. [51] MacDonald, R., Stary, S.J., Swift, G.H. (1982). Two similar but nonallelic rat pancreatic trypsinogens. Nucleotide sequences of the cloned cDNAs. J. Biol. Chem. 257, 97249732. [52] Rowen, L., Koop, B.F., Hood, L. (1996). The complete 685-kilobase DNA sequence of the human beta T cell receptor locus. Science 272, 17551762. [53] Honey, N.K., Sakaguchi, A.Y., Quinto, C., MacDonald, R.J., Bell, G.I., Craik, C., Rutter, W.J., Naylor, S.L. (1984). Chromosomal assignments of human genes for serine proteases trypsin, chymotrypsin B, and elastase. Somat. Cell Mol. Genet. 10, 369376. [54] Whitcomb, D.C., Gorry, M.C., Preston, R.A., Furey, W., Sossenheimer, M.J., Ulrich, C.D., Martin, S.P., Gates, L.K. Jr., Amann, S.T., Toskes, P.P., Liddle, R., McGrath, K., Uomo, G., Post, J.C., Ehrlich, G.D. (1996). Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nature Genet. 14, 141145. [55] Honey, N.K., Sakaguchi, A.Y., Lalley, P.A., Quinto, C., MacDonald, R.J., Craik, C., Bell, G.I., Rutter, W.J., Naylor, S.L. (1984). Chromosomal assignments of genes for trypsin, chymotrypsin B, and elastase in mouse. Somat. Cell Mol. Genet. 10, 377383. [56] Wang, K., Gan, L., Lee, I., Hood, L. (1995). Isolation and characterization of the chicken trypsinogen gene family. Biochem. J. 307, 471479. [57] Craik, C.S., Rutter, W.J., Fletterick, R. (1983). Splice junctions: association with variation in protein structure. Science 220, 11251129.
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[58] Craik, C., Sprang, S., Fletterick, R., Rutter, W.J. (1982). Intronexon splice junctions map at protein surfaces. Nature 299, 180182. [59] Wiegand, U., Corbach, S., Minn, A., Kang, J., Mu¨ller-Hill, B. (1993). Cloning of the cDNA encoding human brain trypsinogen and characterization of its product. Gene 136, 167175. [60] Read, R.J., James, M.N. (1988). Refined crystal structure of ˚ resolution. J. Mol. Biol. 200, Streptomyces griseus trypsin at 1.7 A 523551. [61] James, M.N.G. (1976). Relationship between the structures and activities of some microbial serine proteases. II. Comparison of the tertiary structures of microbial and pancreatic serine proteases, in: Proteolysis and Physiological Regulation, Ribbons, D.W., Brew, J., eds., New York: Academic Press, pp. 125142. [62] Sakanari, J.A., Staunton, C.E., Eakin, A.E., Craik, C.S., McKerrow, J.H. (1989). Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes. P. Natl. Acad. Sci. USA 86, 48634867. [63] Hewett-Emmett, D., Czelusniak, J., Goodman, M. (1981). The evolutionary relationships of the enzymes involved in blood coagulation and hemostasis. Ann. N.Y. Acad. Sci. 370, 511527. [64] Rypniewski, W.R., Hastrup, S., Betzel, C., Dauter, M., Dauter, Z., Papendorf, G., Branner, S., Wilson, K.S. (1993). The sequence and X-ray structure of the trypsin from Fusarium oxysporum. Protein Eng. 6, 341348. [65] Hedstrom, L. (1996). Trypsin: a case study in the structural determinants of enzyme specificity. Biol. Chem. 377, 465470. [66] Hedstrom, L. (2002). Serine protease mechanism and specificity. Chem. Rev. 102, 45014524. [67] The PyMOL Molecular Graphics System, Version 1.4, Schro¨dinger, LLC.
Teaster T. Baird, Jr. Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132, USA. Email: [email protected]
Charles S. Craik Department of Pharmaceutical Chemistry, University of California, San Francisco, 600 16th Street, S512C, Box 2280, San Francisco, CA 94158, USA. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00575-5
Chapter 576
Human Trypsins DATABANKS MEROPS name: cationic trypsin (Homo sapiens-type)
MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.127 Tertiary structure: Available
Clan PA S1 | 576. Human Trypsins
Species distribution: subphylum Vertebrata Reference sequence from: Homo sapiens (UniProt: P07477) MEROPS name: trypsin-2 type A MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.258 Species distribution: superfamily Hominoidea Reference sequence from: Homo sapiens (UniProt: P07478) MEROPS name: mesotrypsin MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.174 Tertiary structure: Available Species distribution: known only from Homo sapiens Reference sequence from: Homo sapiens (UniProt: P35030) MEROPS name: neurotrypsin MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.237 Species distribution: subphylum Vertebrata Reference sequence from: Mus musculus (UniProt: O08762) MEROPS name: trypsin C MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.298 Species distribution: superfamily Hominoidea Reference sequence from: Homo sapiens
Name and History Proteins Haverback et al. [1] reported the presence of trypsinogen in human pancreatic juice in 1960. Buck et al. [2] described the properties of partially purified trypsin from extracts of human pancreas in 1962. Keller & Allan [3] identified two anionic ‘trypsins’ in human pancreatic juice in 1967, and Figarella et al. [4] purified the precursor forms of the two anionic trypsins, trypsinogen 1 and trypsinogen 2 in 1969. The two isoforms are present in a 2:1 ratio, together accounting for B19% of proteins in human pancreatic juice [5]. A third minor isoform, characterized by its intermediate electrophoretic mobility and isoelectric point, was discovered by Rinderknecht et al. in 1979 [6]; it occurs in very low concentrations and probably accounts for ,0.5% of proteins in human pancreatic juice [7]. All human trypsinogens are anionic, but to avoid confusion, the least anionic isoform, trypsinogen 1, was termed ‘cationic’ and the most anionic form, trypsinogen 2, ‘anionic’ [6]. The third isoform was initially named zymogen-X [6] and later renamed mesotrypsinogen [7].
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However, these three trypsinogen isoforms have also been designated as trypsinogens 3, 1 and 2, numbered consecutively from anode to cathode in accordance with the IUPAC-IUB Commission on the biochemical nomenclature of multiple forms of enzymes [8]. The comparative nomenclature of Rinderknecht et al. [7] is used throughout this chapter in accordance with previous recommendation [9]. The molecular mass and pI of the three human trypsinogen isoforms are summarized in Table 576.1.
cDNAs cDNAs encoding for cationic and anionic trypsinogens, known as TRYI and TRYII respectively, were cloned by Emi et al. in 1986 [10]. The subsequently reported TRYIII [11] would have represented the cDNA for mesotrypsinogen, if it had not unfortunately contained sequencing errors [9]. The correct cDNA sequence for mesotrypsinogen was published by Nyaruhucha et al. in 1997 [12].
Genes The human genome contains six highly homologous trypsinogen genes, exhibiting a nucleotide sequence similarity to each other of 91%. Genes T4-T8 2 five tandemly arranged 10-kb repeats 2 are located toward the 30 end of the T-cell receptor (TCR) β locus (TRB@) on chromosome 7q35 [13]. The sixth 10-kb repeat, T9, became translocated from chromosome 7q35 to chromosome 9p13 some 1520 million years ago [1314]. T5 and T7 are obvious pseudogenes [13] whilst T6 is likely an expressed pseudogene [15] (see Distinguishing Features Related Genes later). T4 (PRSS1), T8 (PRSS2) and T9 (PRSS3), each of which is composed of five exons, encode the aforementioned three trypsinogen isoforms, respectively [13] (Table 576.2). The divergently evolved T1, T2 and T3 genes will be discussed in Distinguishing Features Related Genes. TABLE 576.1 Physical properties of the three human trypsinogen isoforms Physical properties
Cationic trypsinogen
Mesotrypsinogen
Anionic trypsinogen
Molecular mass (kDa)
26 700
26 000
28 000
pIa
6.4/6.2
5.5/5.7
4.4/4.9
a
The pI values represent isoelectric points of undenatured (native) proteins/isoelectric points of proteins denatured in 8 M urea. Data from Scheele et al. [8].
Clan PA S1 | 576. Human Trypsins
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TABLE 576.2 Correlation of the nomenclature of proteins, cDNAs, genes and official gene symbols of the human trypsinogen family Proteina Figarella et al. [4]
Rinderknecht et al. [67]
Scheele et al. [8]
Trypsinogen 1
Cationic trypsinogen
Trypsinogen 3
Trypsinogen 2
cDNA
Gene [13]
Gene symbolb
Chromosome location
TRYI [10]
T4
PRSS1
7q35
T5 (pseudogene)
7q35
T6 (expressed pseudogene [15])
7q35
T7 (pseudogene)
7q35
Anionic trypsinogen
Trypsinogen 1
TRYII [10]
T8
PRSS2
7q35
Mesotrypsinogen
Trypsinogen 2
Mesotrypsinogen [12]
T9
PRSS3
9p13
a
The comparative nomenclature of Rinderknecht et al. [6,7] is now the most commonly used. Approved by the HUGO Gene Nomenclature Committee. PRSS stands for protease, serine.
b
Numbering of Trypsinogen Amino Acid Residues The chymotrypsinogen numbering [16] provides an easy comparison of the primary structural features, common disulphide bridges, and similar tertiary structures between the peptidases of family S1 of clan PA. However, this numbering system was extremely confusing to geneticists and clinicians not directly involved in protein study: not only does the numbering not start from the translation initiator methionine but also the family members vary in lengths [17]. The new standard system with the translation initiator Met numbered as 1 has been widely used since 2000. A parallel comparison of the two numbering systems is provided in Figure 576.1. The new standard system is used throughout the article unless specified otherwise.
Structural Chemistry The primary translated polypeptides of PRSS1, PRSS2 and PRSS3 share substantial sequence similarity (Figure 576.2). Each of them contains an N-terminal signal peptide (15 amino acids); a short activation peptide (eight amino acids); the catalytic triad His, Asp and Ser that are found in all members of subclan PA(S); the three key pocket specificity residues, Asp194, Gly217 and Gly227 [18]; and the six absolutely conserved cysteine residues necessary to build the three disulphide bridges observed in all vertebrate trypsins, 4864, 171185, and 196220 [19].
Human cationic trypsin has a similar tertiary structure to bovine, rat, and porcine trypsins, with root-mean˚ for all 223 Cα positions square differences of 0.4 to 0.6 A [20]. Human mesotrypsin (described as ‘human brain trypsin’ in Katona et al. [21]) has a similar crystal structure to human cationic trypsin, with a root-mean-square ˚ for all Cα positions [21]. deviation of only 0.5 A
Activity and Specificity Trypsin belongs to the chymotrypsin superfamily of serine endopeptidases that are characterized by the catalytic triad His57, Asp102 and Ser195 (chymotrypsinogen numbering) [22]. Human trypsins hydrolyze peptide bonds after arginine or lysine residues, their activity being optimal between pH 7.5 and 8.5 and in the presence of Ca21 [7]. The substrate specificity of trypsins is conferred by the substrate-binding pocket composed of Asp194, Gly217 and Gly227. Whereas Asp194 forms a strong electrostatic band with arginine or lysine residues of the substrate, Gly217 and Gly227 permit entry of large amino acid side chains into the hydrophobic pocket [18]. Most synthetic inhibitors inhibit all three trypsins to about the same extent. For example, under standard conditions, p-aminobenzamidine and DFP, EDTA, and HgCl2 inhibit about 100, 50, and 11% of the activity, respectively. The specific inhibitor Tos-Lys-CH2Cl inhibits cationic and anionic trypsins completely, but reacts only slowly and incompletely with mesotrypsin (99 versus 65% inhibition) [7]. In sharp contrast to cationic and anionic trypsins, mesotrypsin shows almost total resistance to most protein
Clan PA S1 | 576. Human Trypsins
2603
T 8
F 9
V 10
A 11
A A 12 13
L 14
A 15
8 A 16
9 P 17
10 F 18
18 19 G G 26 27
20 Y 28
21 22 N C 29 30
23 E 31
24 25 E N 32 33
26 S 34
27 28 V P 35 36
29 Y 37
30 31 Q V 38 39
32 S 40
39 Y 45
40 41 H F 46 47
42 C 48
43 44 G G 49 50
45 S 51
46 47 L I 52 53
48 N 54
49 50 E Q 55 56
51 W 57
52 53 V V 58 59
54 S 60
57 58 H C 63 64
59 Y 65
60 61 K S 66 67
62 R 68
63 64 I Q 69 70
65 V 71
65 66 R L 72 73
69 G 74
70 71 72 E H N 75 76 77
73 74 75 I E V 78 79 80
78 79 G N 83 84
80 E 85
81 82 Q F 86 87
83 I 88
84 85 N A 89 90
86 A 91
87 88 K I 92 93
89 I 94
90 91 R H 95 96
93 94 Q Y 98 99
M 1
N 2
P 3
13 D 21
14 D 22
33 L 41
L 4
L 5
I 6
15 16 K I 23 24
17 V 25
34 N 42
37 38 S G 43 44
55 A 61
56 G 62
76 L 81
77 E 82
L 7
92 P 97
11 12 D D 19 20
95 D 100
96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 R K T L N N D I M L I K L S S R A V I N 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 116 117 118 119 120 121 122 123 124 125 127 128 129 130 132 133 134 135 136 137 A R V S T I S L P T A P P A T G T K C L 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 I S G W G N T A S S G A D Y P D E L Q C 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 L D A P V L S Q A K C E A S Y P G K I T 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 178 179 180 181 182 183 184 184 185 186 187 187 188 189 190 191 192 193 194 195 S N M F C V G F L E G G K D S C Q G D S 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 196 197 198 199 200 201 202 203 204 209 210 211 212 213 214 215 216 217 219 220 G G P V V C N G Q L Q G V V S W G D G C 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 A Q K N K P G V Y T K V Y N Y V K W I K 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 240 241 242 243 244 245 245 N T I A A N S 241 242 243 244 245 246 247 FIGURE 576.1 Amino acid sequence of the human cationic trypsinogen. Numbers below the sequences are in accordance with the new standard system in which the ATG translation initiator codon is numbered as 1. Numbers above the sequences correspond to those of the bovine chymotrypsinogen A [16]. Note the artificial deletions (downward arrows) and insertions (stars) introduced by the chymotrypsinogen numbering system. Signal peptide is in italics, activation peptide is shaded in gray, and the catalytic triad (His63, Asp107 and Ser200) are in bold and red. The numbering also applies to human anionic trypsinogen and mesotrypsinogen because all the three human trypsinogen isoforms have the same number of amino acids (see Figure 576.2). Amino acid sequence is from the NCBI Gene Database.
inhibitors of trypsin including pancreatic trypsin inhibitor, soybean trypsin inhibitor, lima bean trypsin inhibitor, ovomucoid and α1-antitrypsin inhibitor [7]; although it is readily inhibited by serpin-type inhibitors with Arg in the reactive loop including the α1-antitrypsin Pittsburgh variant [23] and protease nexin-1 [24]. The resistance of mesotrypsin to protein inhibitors was suggested by
Nyaruhucha et al. [12] to be due to the presence at residue 198 of an Arg in mesotrypsin, but a Gly in human cationic and anionic trypsins and in other vertebrate trypsins. Indeed, in the crystal structure, the side chain of Arg198 is in an extended conformation and occupies the S20 subsite [21], which in the absence of conformational change would result in a steric clash with the P20 residue
Clan PA S1 | 576. Human Trypsins
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48
64
PRSS1 PRSS2 PRSS3
MNPLLILTFVAAALAAPFDDDDKIVGGYNCEENSVPYQVSLNSGYHFCGGSLINEQWVVSAGHCY 65 --L----------V--------------I------------------------S----------- 65 ---F---A--G--V-V------------T-----L---------S--------S-------A--- 65 Trypsin Trypsinogen Pretrypsinogen
PRSS1 PRSS2 PRSS3
KSRIQVRLGEHNIEVLEGNEQFINAAKIIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPT 130 --------------------------------K-NSR--D---L------P----S---A----- 130 -T-----------K------------------K-N-D--D----------P-------------- 130
PRSS1 PRSS2 PRSS3
APPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDS 195 ----A--ES--------L---------------------E----------N-------------- 195 ----A--E---------L-F--------K-------T--E-K--------NS------------- 195
107
171
196
PRSS1 PRSS2 PRSS3
200
220
185
194
227
CQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIKNTIAANS 247 ----------S--E---I----Y------R-----------D---D------ 247 --R-------------------H---W--R-----------D---D------ 247
PRSS2
% 86
89 %
PRSS1
88%
PRSS3
FIGURE 576.2 Amino acid sequences of human cationic trypsinogen (PRSS1), anionic trypsinogen (PRSS2) and mesotrypsinogen (PRSS3). Note that trypsinogen is synthesized in the form of a prezymogen (i.e. pretrypsinogen). Removal of the signal peptide (italics) results in the formation of trypsinogen. Further removal of the activation peptide (shaded in gray) by either the physiological activator enteropeptidase or trypsin(ogen) itself leads to the formation of trypsin. Some of the residues that are critical for trypsin structure and function and that are consequently absolutely conserved throughout vertebrate evolution are highlighted in color: the catalytic triad residues (His63, Asp107 and Ser200) are in red; the three residues determining trypsin specificity (Asp194, Gly217 and Gly227) are in blue; and the three common disulfide bridges (4864, 171185 and 196220) are in green. Dashes indicate identity with the PRSS1 sequence. Amino acid sequence similarity between the three human trypsinogen isoforms is shown below the aligned sequences. Amino acid sequences are from the NCBI Gene Database.
of a protein inhibitor [25]. In addition, the side chain of Arg198 contributes to an unusually strong clustering of positive charges around the primary specificity pocket of mesotrypsin [21]. Most importantly, changing Arg198 back to Gly made the human mesotrypsin fully sensitive to protein trypsin inhibitors [26]. The human trypsins differ as well in selectivity toward protein substrates. While cationic and anionic trypsins are believed to be fairly promiscuous enzymes capable of cleaving nearly any accessible peptide bond that follows Lys or Arg, mesotrypsin shows evidence of greater specificity. Mesotrypsin does not activate other pancreatic zymogens, as do cationic and anionic trypsins [26], nor is it very efficient in cleaving protease activated receptors (PARs) [27,28]. However, it displays strikingly enhanced catalytic activity relative to other trypsins for cleavage of specific protein substrates including human pancreatic secretory trypsin inhibitor (SPINK1), soybean trypsin inhibitor (SBTI), amyloid precursor protein Kunitz protease inhibitor domain (APPI), and bovine pancreatic trypsin inhibitor (BPTI) [25,26,29]. The more restricted specificity of mesotrypsin appears to derive both from a preference for substrate cleavage sites stabilized in a three-dimensional canonical conformation [30], and from distinct specificity at individual subsites, including a
stronger preference for Arg at the S1 subsite [30] and a preference for Ser or Thr at the S10 subsite [23].
Biological Aspects Autoactivation and Autolysis Trypsinogen is generally regarded as the inactive precursor of trypsin, its physiological activator being enteropeptidase that is located on the brush border membrane of enterocytes in the duodenum. However, it has been firmly established that trypsinogen possesses proteolytic activity to activate itself [31]. This process of trypsinogen activation, catalyzed first slowly by trypsinogen and then accelerated by the resulting trypsin, is termed here trypsinogen autoactivation (Figure 576.3). (Trypsinogen activation by enteropeptidase-activated trypsin in the duodenum is also known as trypsinogen autoactivation.) Activation of human trypsinogens by enteropeptidase, trypsinogen or trypsin occurs through hydrolyzing the peptide bond after Lys23 (Figure 576.2). Trypsin samples also autolyze in the absence of inhibitor as the result of self-digestion (i.e. one trypsin molecule acts upon another; termed autolysis). Arg122 has been identified to be the primary autolysis site of trypsins
Clan PA S1 | 576. Human Trypsins
2605
(A)
Initial activation by trypsinogen
(B) Trypsinogen
Trypsinogen Autoactivation (within pancreas)
Enteropeptidase (within duodenum)
Trypsin
Trypsin
Subsequent activation by trypsin
FIGURE 576.3 Schematic representation of trypsinogen autoactivation. Top panel indicates the initial activation, in which one trypsinogen molecule acts upon another. Lower panel indicates the subsequent activation, in which a newly formed trypsin molecule acts upon a trypsinogen molecule. Trypsinogen activation catalyzed by trypsinogen is much slower than that catalyzed by trypsin (indicated by the different sizes of the lightning symbols). Box, activation peptide. Bar, trypsin.
TABLE 576.3 Autoactivation and autolysis properties of the three human trypsin(ogen) isoforms Isoform
Autoactivation (ref.) Autolysis (ref.)
Cationic trypsin(ogen) 11 [38,41]
1 [38,42]
Anionic trypsin(ogen)
1 [38,41]
11 [38,42]
Mesotrypsin(ogen)
2 [26]
2 [26]
in cows [32], pigs [33], humans [20] and rats [34,35]. Human cationic trypsin was shown to undergo autolysis very slowly in vitro and to be stabilized against autolysis by calcium [3638]; furthermore, in vitro autolysis of the Arg122-Val123 bond did not proceed to completion [36,39]. By contrast, rapid and complete autolysis was achieved following the selective cleavage of the Leu81Glu82 peptide bond by chymotrypsin C (CTRC) [40]. In vitro autoactivation and autolysis properties of the three human trypsin(ogen)s [26,38,41,42] are compared in Table 576.3. As indicated in the table, mesotrypsinogen is deficient in autoactivation and is significantly impaired in cleavage of anionic and cationic trypsinogens at the autolysis site; the deficiency in autoactivation is rescued by mutation of Arg198 to Gly [26].
Trypsinogens in the Pancreas The three pretrypsinogen isoforms are synthesized in pancreatic acinar cells by ribosomes attached to the rough
Chymotrypsinogen
Chymotrypsin
Proelastase
Elastase
Kallikreinogen
Kallikrein
Procarboxypeptidase A
Carboxypeptidase A
Procarboxypeptidase A
Carboxypeptidase B
Prophospholipase A2
Phospholipase A2
Procolipase
Colipase
Physiology (within intestine)
Food digestion
Pathology (within pancreas)
Pancreatic autodigestion (Pancreatitis)
FIGURE 576.4 Trypsinogen, a double-edged sword. (A) Illustration of the physiological role of trypsinogen in digestion; (B) Illustration of the pathogenic role of prematurely activated trypsinogen within the pancreas. Normally, prematurely activated trypsin within the pancreas can be inhibited by the human pancreatic secretory trypsin inhibitor and/or degraded by chymotrypsinogen C and trypsin itself. A defect in one or a combination of these defense mechanisms may trigger the zymogen activation cascade leading to pancreatic autodigestion. Adapted from Chen & Fe´rec [66].
endoplasmic reticulum, where the signal peptides are removed co-translationally. The resulting trypsinogens are secreted into the pancreatic duct and finally discharged into the duodenal lumen where they are activated by enteropeptidase. The newly formed trypsins play a central role in digestion by acting as the trigger enzymes which activate all other pancreatic digestive pro-enzymes as well as the trypsinogens themselves (Figure 576.4a). Intuitively, prematurely activated trypsin within the pancreas, if not inhibited or/and inactivated, can trigger the zymogen activation cascade leading to pancreatic autodigestion (Figure 576.4b). Indeed, both gain-of-function missense mutations (through enhancing trypsinogen autoactivation or/and reducing trypsin autolysis) and copy number mutations in the PRSS1 gene (see Chapter 577) as well as loss-of-function mutations in the SPINK1 (encoding trypsin’s physiological inhibitor [43]) and CTRC genes [44,45] predispose to chronic pancreatitis. In an established model of acute pancreatitis, activation of trypsinogen was shown to occur in large endocytic vacuoles of pancreatic acinar cells [46]. Another mechanism potentially leading to pancreatic autodigestion is the ‘co-localization hypothesis’, in which digestive zymogens are thought to be activated by
Clan PA S1 | 576. Human Trypsins
2606
lysosomal hydrolases when the two types of enzymes become co-localized within the zymogen-containing secretory compartment [47]. Supporting evidences include that: (1) cathepsin B (CTSB), a lysosomal protease, has long been shown to be a potent activator of trypsinogen in vitro [4849]; (2) ctsb/ mice have significantly reduced intrapancreatic trypsin activity and severity of pancreatitis as compared with the wild-type mice [50]; and (3) CTSB is abundantly present in the secretory compartment of the human exocrine pancreas [51]. These notwithstanding, chronic pancreatitiscausing PRSS1 mutations p.Asn29Ile, p.Asn29Thr and p.Arg122His resulted in neither increase nor decrease of trypsinogen activation by CTSB in vitro [51]. Moreover, there exist controversial findings regarding association of CTSB polymorphisms with chronic pancreatitis [5254].
Extrapancreatic Expression of Trypsinogens It has long been thought that trypsinogens are specifically expressed in the pancreas and perform no function other than alimentary digestion. However, since the report of Bohe et al. in 1986 [55], mRNA or/and protein expression of human trypsinogens has been detected in a variety of tumors and cancer cell lines as well as in endothelial and epithelial cells of various normal tissues. Additionally, expression of a splice isoform of mesotrypsinogen lacking a typical signal sequence has been identified in human brain at both the transcript level [56] and the protein level [57] (see later T9-derived Chimeric Genes). These findings suggest that extrapancreatic trypsins may be involved in tissue remodeling and in tumor invasion. Moreover, there is increasing evidence that trypsin is a signaling molecule that may regulate multiple cellular functions by activating proteinase-activated receptors (for reviews, see Paju & Stenman [58] and Itkonen [59]).
Preparation The three human trypsinogen isoforms were analyzed simultaneously by polyacrylamide gel electrophoresis [6,7] or two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis [8]. Cationic trypsin can be purified rapidly from pancreatic juice: the first step is to isolate trypsinogen by fast protein liquid chromatography on a MonoS column at pH 4.5; trypsinogen is then activated by enteropeptidase, and finally isolated by affinity chromatography on STI-Sepharose column [37]. Cationic and anionic trypsinogens have also been purified from cyst fluid of ovarian cancer patients [60] and from human seminal fluid [61] by immunoaffinity chromatography and anion exchange chromatography. (The slight differences in substrate specificities between extra-pancreatic and pancreatic preparations [60] are likely to be caused by
sulfation of Tyr154 in pancreatic trypsinogens [62,63].) Cloned trypsins are usually produced by recombinant expression in Escherichia coli. In some expression systems, the soluble and correctly folded zymogens are isolated from the periplasm, while in others the zymogens are isolated in inclusion bodies and oxidatively refolded in vitro prior to chromatographic purification. Proteolytically activated trypsins are obtained by adding enteropeptidase to the zymogen preparation [12,37,39].
Distinguishing Features - Related Genes Divergently Evolved T1, T2 and T3 Genes Besides the T4T9 genes, three other genes (i.e. T1T3) located toward the 50 end of TRB@ were also identified as trypsinogen genes [13]. However, the T1T3 gene cluster and the T4T8 gene cluster have evolved largely as separate gene families after duplication, evidenced by the lack of significant nucleotide sequence similarity between these two clusters. Whereas T2 and T3 are clearly non-functional genes, T1 (known as trypsin X3 (TRYX3) in the NCBI Gene Database) has an inframe coding sequence and appears to have counterparts in other species including chimpanzee, Rhesus monkey, cattle, rat and mouse in the NCBI Gene Database. In addition, search of the NCBI EST database revealed transcripts that correspond to the putative TRYX3 gene in humans and some other species. Nonetheless, these genes would no longer encode a functional trypsinogen protein owing to their highly divergent evolution. As opined by Chen & Fe´rec [64], TRYX3 presumably represents a new gene, possibly with a new function, that has evolved from an ancient trypsinogen gene duplicate.
T6 Rowen et al. [13] annotated T6 (known as TRY6 or trypsinogen C in the NCBI Gene Database) as an apparently functional trypsinogen gene but noted the following three points. First, reverse transcriptase PCR analysis of pancreas, thymus and liver suggested that T6 may be expressed in minute amounts in the thymus. Second, the surface charge and shape of the predicted T6 protein would differ significantly from those of the cationic and anionic trypsinogens. Third, T6 is deleted in a common insertion/deletion polymorphism. Chen & Fe´rec [15] further suggested that the presence of His122 instead of Arg in the predicted T6 protein would indicate a relaxation of the selection pressure acting on the evolutionarily conserved Arg122 autolysis site. Taken together, T6 most probably represents a transition state between a functional duplicated gene and a nonfunctional pseudogene, that is, an expressed pseudogene [15].
Clan PA S1 | 576. Human Trypsins
T9-derived Chimeric Genes The T9 gene was translocated from chromosome 7q35 to chromosome 9p13 some 1520 million years ago [14]. This event has certainly liberated the T9 gene from shared functional or regulatory constraints exerted at the original locus, reflected by the dramatic differences between mesotrypsinogen and the other two trypsinogen isoforms in terms of expression level in pancreas and biochemical properties. More importantly, the splicing donor site of intron 1 of the T9 gene is changed from the consensus ‘GT’ to the less commonly used GC. Evidently, this latter change has contributed directly to the generation of two chimeric genes, both retaining T9’s exons 25 but differing in exon 1 due to alternative splicing [56,65]. Were these new chimeric genes to be really functional, their biological roles would be different from that performed by the pancreatic mesotrypsin since: (1) the alternative exon 1 sequences do not encode a typical signal peptide; and (2) both chimeric genes are expressed predominantly in non-pancreatic tissues. From an evolutionary viewpoint, it is possible that the T9 gene is gradually losing its function as a trypsinogen gene but acquiring new (not yet characterized) function(s) through the formation of new chimeric genes [64].
References [1] Haverback, B.J., Dyce, B., Bundy, H., Edmondson, H.A. (1960). Trypsin, trypsinogen and trypsin inhibitor in human pancreatic juice: Mechanism for pancreatitis associated with hyperparathyroidism. Am. J. Med. 29(3), 424433. [2] Buck, F.F., Bier, M., Nord, F.F. (1962). Some properties of human trypsin. Arch. Biochem. Biophys. 98, 528530. [3] Keller, P.J., Allan, B.J. (1967). The protein composition of human pancreatic juice. J. Biol. Chem. 242(2), 281287. [4] Figarella, C., Clemente, F., Guy, O. (1969). On zymogens of human pancreatic juice. FEBS Lett. 3(5), 351353. [5] Guy, O., Lombardo, D., Bartelt, D.C., Amic, J., Figarella, C. (1978). Two human trypsinogens. Purification, molecular properties, and N-terminal sequences. Biochemistry 17(9), 16691675. [6] Rinderknecht, H., Renner, I.G., Carmack, C. (1979). Trypsinogen variants in pancreatic juice of healthy volunteers, chronic alcoholics, and patients with pancreatitis and cancer of the pancreas. Gut 20(10), 886891. [7] Rinderknecht, H., Renner, I.G., Abramson, S.B., Carmack, C. (1984). Mesotrypsin: a new inhibitor-resistant protease from a zymogen in human pancreatic tissue and fluid. Gastroenterology 86(4), 681692. [8] Scheele, G., Bartelt, D., Bieger, W. (1981). Characterization of human exocrine pancreatic proteins by two-dimensional isoelectric focusing/ sodium dodecyl sulfate gel electrophoresis. Gastroenterology 80(3), 461473. [9] Chen, J.M., Fe´rec, C. (2000). Genes, cloned cDNAs, and proteins of human trypsinogens and pancreatitis-associated cationic trypsinogen mutations. Pancreas 21(1), 5762.
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[10] Emi, M., Nakamura, Y., Ogawa, M., Yamamoto, T., Nishide, T., Mori, T., Matsubara, K. (1986). Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens. Gene 41(23), 305310. [11] Tani, T., Kawashima, I., Mita, K., Takiguchi, Y. (1990). Nucleotide sequence of the human pancreatic trypsinogen III cDNA. Nucleic Acids Res. 18(6), 1631. [12] Nyaruhucha, C.N., Kito, M., Fukuoka, S.I. (1997). Identification and expression of the cDNA-encoding human mesotrypsin(ogen), an isoform of trypsin with inhibitor resistance. J. Biol. Chem. 272(16), 1057310578. [13] Rowen, L., Koop, B.F., Hood, L. (1996). The complete 685-kilobase DNA sequence of the human beta T cell receptor locus. Science 272(5269), 17551762. [14] Rowen, L., Williams, E., Glusman, G., Linardopoulou, E., Friedman, C., Ahearn, M.E., Seto, J., Boysen, C., Qin, S., Wang, K., Kaur, A., Bloom, S., Hood, L., Trask, B.J. (2005). Interchromosomal segmental duplications explain the unusual structure of PRSS3, the gene for an inhibitor-resistant trypsinogen. Mol. Biol. Evol. 22(8), 17121720. [15] Chen, J.M., Montier, T., Fe´rec, C. (2001). Molecular pathology and evolutionary and physiological implications of pancreatitisassociated cationic trypsinogen mutations. Hum. Genet. 109(3), 245252. [16] Hartley, B.S. (1970). Homologies in serine proteinases. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 257(813), 7787. [17] Chen, J.M., Fe´rec, C. (2000). Wanted: a consensus nomenclature for cationic trypsinogen mutations. Gastroenterology 119(1), 277278. [18] Craik, C.S., Largman, C., Fletcher, T., Roczniak, S., Barr, P.J., Fletterick, R., Rutter, W.J. (1985). Redesigning trypsin: alteration of substrate specificity. Science 228(4697), 291297. [19] Roach, J.C., Wang, K., Gan, L., Hood, L. (1997). The molecular evolution of the vertebrate trypsinogens. J. Mol. Evol. 45(6), 640652. [20] Gaboriaud, C., Serre, L., Guy-Crotte, O., Forest, E., FontecillaCamps, J.C. (1996). Crystal structure of human trypsin 1: unexpected phosphorylation of Tyr151. J. Mol. Biol. 259(5), 9951010. [21] Katona, G., Berglund, G.I., Hajdu, J., Gra´f, L., Szila´gyi, L. (2002). Crystal structure reveals basis for the inhibitor resistance of human brain trypsin. J. Mol. Biol. 315(5), 12091218. [22] Higaki, J.N., Gibson, B.W., Craik, C.S. (1987). Evolution of catalysis in the serine proteases. Cold Spring Harb. Symp. Quant. Biol. 52, 615621. [23] Szepessy, E., Sahin-Toth, M. (2006). Human mesotrypsin exhibits restricted S10 subsite specificity with a strong preference for small polar side chains. FEBS J. 273(13), 29422954. [24] Koistinen, H., Koistinen, R., Zhang, W.M., Valmu, L., Stenman, U.H. (2009). Nexin-1 inhibits the activity of human brain trypsin. Neuroscience 160(1), 97102. [25] Salameh, M.A., Soares, A.S., Hockla, A., Radisky, E.S. (2008). Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin. J. Biol. Chem. 283(7), 41154123. [26] Szmola, R., Kukor, Z., Sahin-Toth, M. (2003). Human mesotrypsin is a unique digestive protease specialized for the degradation of trypsin inhibitors. J. Biol. Chem. 278(49), 4858048589. [27] Grishina, Z., Ostrowska, E., Halangk, W., Sahin-Toth, M., Reiser, G. (2005). Activity of recombinant trypsin isoforms on
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human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1. Br. J. Pharmacol. 146(7), 990999. Knecht, W., Cottrell, G.S., Amadesi, S., Mohlin, J., Skaregarde, A., Gedda, K., Peterson, A., Chapman, K., Hollenberg, M.D., Vergnolle, N., Bunnett, N.W. (2007). Trypsin IV or mesotrypsin and p23 cleave protease-activated receptors 1 and 2 to induce inflammation and hyperalgesia. J. Biol. Chem. 282(36), 2608926100. Salameh, M.A., Robinson, J.L., Navaneetham, D., Sinha, D., Madden, B.J., Walsh, P.N., Radisky, E.S. (2010). The amyloid precursor protein/protease nexin 2 Kunitz inhibitor domain is a highly specific substrate of mesotrypsin. J. Biol. Chem. 285(3), 19391949. Salameh, M.A., Soares, A.S., Navaneetham, D., Sinha, D., Walsh, P.N., Radisky, E.S. (2010). Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin. J. Biol. Chem. 285(47), 3688436896. Kassell, B., Kay, J. (1973). Zymogens of proteolytic enzymes. Science 180(90), 10221027. Maroux, S., Rovery, M., Desnuelle, P. (1967). An autolyzed and still active form of bovine trypsin. Biochim. Biophys. Acta 140(2), 377380. Ru, B.G., Du, J.Z., Zeng, Y.H., Chen, L.S., Ni, Y.S., Tan, G.H., Zhang, L.X. (1980). Active products of porcine trypsin after autolysis. Sci. Sin. 23(11), 14531460. Li, X.F., Nie, X., Tang, J.G. (1998). Anti-autolysis of trypsin by modification of autolytic site Arg117. Biochem. Biophys. Res. Commun. 250(2), 235239. Varallyay, E., Pal, G., Patthy, A., Szila´gyi, L., Gra´f, L. (1998). Two mutations in rat trypsin confer resistance against autolysis. Biochem. Biophys. Res. Commun. 243(1), 5660. Sahin-Toth, M., Toth, M. (2000). Gain-of-function mutations associated with hereditary pancreatitis enhance autoactivation of human cationic trypsnogen. Biochem. Biophys. Res. Commun. 278(2), 286289. Szila´gyi, L., Kenesi, E., Katona, G., Kaslik, G., Juhasz, G., Gra´f, L. (2001). Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis. J. Biol. Chem. 276(27), 2457424580. Kukor, Z., Toth, M., Sahin-Toth, M. (2003). Human anionic trypsinogen: properties of autocatalytic activation and degradation and implications in pancreatic diseases. Eur. J. Biochem. 270(9), 20472058. Kukor, Z., Toth, M., Pal, G., Sahin-Toth, M. (2002). Human cationic trypsinogen. Arg(117) is the reactive site of an inhibitory surface loop that controls spontaneous zymogen activation. J. Biol. Chem. 277(8), 61116117. Szmola, R., Sahin-Toth, M. (2007). Chymotrypsin C (caldecrin) promotes degradation of human cationic trypsin: identity with Rinderknecht’s enzyme Y. Proc. Natl. Acad. Sci. USA 104(27), 1122711232. Colomb, E., Figarella, C., Guy, O. (1979). The two human trypsinogens. Evidence of complex formation with basic pancreatic trypsin inhibitor-proteolytic activity. Biochim. Biophys. Acta 570(2), 397405. Colomb, E., Guy, O., Deprez, P., Michel, R., Figarella, C. (1978). The two human trypsinogens: catalytic properties of the corresponding trypsins. Biochim. Biophys. Acta 525(1), 186193.
[43] Witt, H., Luck, W., Hennies, H.C., Classen, M., Kage, A., Lass, U., Landt, O., Becker, M. (2000). Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis. Nat. Genet. 25(2), 213216. [44] Rosendahl, J., Witt, H., Szmola, R., Bhatia, E., Ozsvari, B., Landt, O., Schulz, H.U., Gress, T.M., Pfutzer, R., Lohr, M., Kovacs, P., Bluher, M., Stumvoll, M., Choudhuri, G., Hegyi, P., te Morsche, R.H., Drenth, J.P., Truninger, K., Macek, M. Jr., Puhl, G., Witt, U., Schmidt, H., Buning, C., Ockenga, J., Kage, A., Groneberg, D.A., Nickel, R., Berg, T., Wiedenmann, B., Bodeker, H., Keim, V., Mossner, J., Teich, N., Sahin-Toth, M. (2008). Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis. Nat. Genet. 40(1), 7882. [45] Masson, E., Chen, J.M., Scotet, V., Le Mare´chal, C., Fe´rec, C. (2008). Association of rare chymotrypsinogen C (CTRC) gene variations in patients with idiopathic chronic pancreatitis. Hum. Genet. 123(1), 8391. [46] Sherwood, M.W., Prior, I.A., Voronina, S.G., Barrow, S.L., Woodsmith, J.D., Gerasimenko, O.V., Petersen, O.H., Tepikin, A.V. (2007). Activation of trypsinogen in large endocytic vacuoles of pancreatic acinar cells. Proc. Natl. Acad. Sci. USA 104(13), 56745679. [47] van Acker, G.J., Perides, G., Steer, M.L. (2006). Co-localization hypothesis: a mechanism for the intrapancreatic activation of digestive enzymes during the early phases of acute pancreatitis. World J. Gastroenterol. 12(13), 19851990. [48] Greenbaum, L.M., Hirshkowitz, A., Shoichet, I. (1959). The activation of trypsinogen by cathepsin B. J. Biol. Chem. 234, 28852890. [49] Figarella, C., Miszczuk-Jamska, B., Barrett, A.J. (1988). Possible lysosomal activation of pancreatic zymogens. Activation of both human trypsinogens by cathepsin B and spontaneous acid. Activation of human trypsinogen 1. Biol. Chem. Hoppe Seyler 369(Suppl), 293298. [50] Halangk, W., Lerch, M.M., Brandt-Nedelev, B., Roth, W., Ruthenbuerger, M., Reinheckel, T., Domschke, W., Lippert, H., Peters, C., Deussing, J. (2000). Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis. J. Clin. Invest. 106(6), 773781. [51] Kukor, Z., Mayerle, J., Kruger, B., Toth, M., Steed, P.M., Halangk, W., Lerch, M.M., Sahin-Toth, M. (2002). Presence of cathepsin B in the human pancreatic secretory pathway and its role in trypsinogen activation during hereditary pancreatitis. J. Biol. Chem. 277(24), 2138921396. [52] Mahurkar, S., Idris, M.M., Reddy, D.N., Bhaskar, S., Rao, G.V., Thomas, V., Singh, L., Chandak, G.R. (2006). Association of cathepsin B gene polymorphisms with tropical calcific pancreatitis. Gut 55(9), 12701275. [53] Weiss, F.U., Behn, C.O., Simon, P., Ruthenburger, M., Halangk, W., Lerch, M.M. (2007). Cathepsin B gene polymorphism Val26 is not associated with idiopathic chronic pancreatitis in European patients. Gut 56(9), 13221323. [54] Witt, H., Bhatia, E. (2008). Genetic aspects of tropical calcific pancreatitis. Rev. Endocr. Metab. Disord. 9(3), 213226. [55] Bohe, M., Borgstrom, A., Lindstrom, C., Ohlsson, K. (1986). Pancreatic endoproteases and pancreatic secretory trypsin inhibitor
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[56]
[57]
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immunoreactivity in human Paneth cells. J. Clin. Pathol. 39(7), 786793. Wiegand, U., Corbach, S., Minn, A., Kang, J., Muller-Hill, B. (1993). Cloning of the cDNA encoding human brain trypsinogen and characterization of its product. Gene 136(12), 167175. Nemeth, A.L., Medveczky, P., Toth, J., Siklodi, E., Schlett, K., Patthy, A., Palkovits, M., Ovadi, J., Tokesi, N., Nemeth, P., Szila´gyi, L., Gra´f, L. (2007). Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine. A possible means to regulate gene expression. FEBS J. 274(6), 16101620. Paju, A., Stenman, U.H. (2006). Biochemistry and clinical role of trypsinogens and pancreatic secretory trypsin inhibitor. Crit. Rev. Clin. Lab. Sci. 43(2), 103142. Itkonen, O. (2010). Human trypsinogens in the pancreas and in cancer. Scand. J. Clin. Lab. Invest. 70(2), 136143. Koivunen, E., Huhtala, M.L., Stenman, U.H. (1989). Human ovarian tumor-associated trypsin. Its purification and characterization from mucinous cyst fluid and identification as an activator of prourokinase. J. Biol. Chem. 264(24), 1409514099.
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[61] Paju, A., Bjartell, A., Zhang, W.M., Nordling, S., Borgstrom, A., Hansson, J., Stenman, U.H. (2000). Expression and characterization of trypsinogen produced in the human male genital tract. Am. J. Pathol. 157(6), 20112021. [62] Sahin-Toth, M., Kukor, Z., Nemoda, Z. (2006). Human cationic trypsinogen is sulfated on Tyr154. FEBS J. 273(22), 50445050. [63] Itkonen, O., Helin, J., Saarinen, J., Kalkkinen, N., Ivanov, K.I., Stenman, U.H., Valmu, L. (2008). Mass spectrometric detection of tyrosine sulfation in human pancreatic trypsinogens, but not in tumor-associated trypsinogen. FEBS J. 275(2), 289301. [64] Chen, J.M., Fe´rec, C. (2003). Trypsinogen genes: evolution, in: Nature Encyclopedia of the Human Genome, vol. 5, Cooper, D.N., ed., London: Macmillan Publishers Ltd., Nature Publishing Group, pp. 645650. [65] Nakanishi, J., Yamamoto, M., Koyama, J., Sato, J., Hibino, T. (2010). Keratinocytes synthesize enteropeptidase and multiple forms of trypsinogen during terminal differentiation. J. Invest. Dermatol. 130(4), 944952. [66] Chen, J.M., Fe´rec, C. (2009). Chronic pancreatitis: genetics and pathogenesis. Annu. Rev. Genomics Hum. Genet. 10, 6387.
Jian-Min Chen Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), U1078 and Etablissement Franc¸ais du Sang Bretagne, 46 rue Fe´lix Le Dantec, 29218 Brest, France. Email: [email protected]
Evette S. Radisky Department of Cancer Biology, Mayo Clinic Cancer Center, 4500 San Pablo Road, Jacksonville, FL 32224, USA. Email: [email protected]
Claude Fe´rec Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), U1078 and Etablissement Franc¸ais du Sang Bretagne, 46 rue Fe´lix Le Dantec, 29218 Brest, France. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00576-7
[50] Craik, C.S., Choo, Q.L., Swift, G.H., Quinto, C., MacDonald, R.J., Rutter, W.J. (1984). Structure of two related rat pancreatic trypsin genes. J. Biol. Chem. 259, 1425514264. [51] MacDonald, R., Stary, S.J., Swift, G.H. (1982). Two similar but nonallelic rat pancreatic trypsinogens. Nucleotide sequences of the cloned cDNAs. J. Biol. Chem. 257, 97249732. [52] Rowen, L., Koop, B.F., Hood, L. (1996). The complete 685-kilobase DNA sequence of the human beta T cell receptor locus. Science 272, 17551762. [53] Honey, N.K., Sakaguchi, A.Y., Quinto, C., MacDonald, R.J., Bell, G.I., Craik, C., Rutter, W.J., Naylor, S.L. (1984). Chromosomal assignments of human genes for serine proteases trypsin, chymotrypsin B, and elastase. Somat. Cell Mol. Genet. 10, 369376. [54] Whitcomb, D.C., Gorry, M.C., Preston, R.A., Furey, W., Sossenheimer, M.J., Ulrich, C.D., Martin, S.P., Gates, L.K. Jr., Amann, S.T., Toskes, P.P., Liddle, R., McGrath, K., Uomo, G., Post, J.C., Ehrlich, G.D. (1996). Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nature Genet. 14, 141145. [55] Honey, N.K., Sakaguchi, A.Y., Lalley, P.A., Quinto, C., MacDonald, R.J., Craik, C., Bell, G.I., Rutter, W.J., Naylor, S.L. (1984). Chromosomal assignments of genes for trypsin, chymotrypsin B, and elastase in mouse. Somat. Cell Mol. Genet. 10, 377383. [56] Wang, K., Gan, L., Lee, I., Hood, L. (1995). Isolation and characterization of the chicken trypsinogen gene family. Biochem. J. 307, 471479. [57] Craik, C.S., Rutter, W.J., Fletterick, R. (1983). Splice junctions: association with variation in protein structure. Science 220, 11251129.
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[58] Craik, C., Sprang, S., Fletterick, R., Rutter, W.J. (1982). Intronexon splice junctions map at protein surfaces. Nature 299, 180182. [59] Wiegand, U., Corbach, S., Minn, A., Kang, J., Mu¨ller-Hill, B. (1993). Cloning of the cDNA encoding human brain trypsinogen and characterization of its product. Gene 136, 167175. [60] Read, R.J., James, M.N. (1988). Refined crystal structure of ˚ resolution. J. Mol. Biol. 200, Streptomyces griseus trypsin at 1.7 A 523551. [61] James, M.N.G. (1976). Relationship between the structures and activities of some microbial serine proteases. II. Comparison of the tertiary structures of microbial and pancreatic serine proteases, in: Proteolysis and Physiological Regulation, Ribbons, D.W., Brew, J., eds., New York: Academic Press, pp. 125142. [62] Sakanari, J.A., Staunton, C.E., Eakin, A.E., Craik, C.S., McKerrow, J.H. (1989). Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes. P. Natl. Acad. Sci. USA 86, 48634867. [63] Hewett-Emmett, D., Czelusniak, J., Goodman, M. (1981). The evolutionary relationships of the enzymes involved in blood coagulation and hemostasis. Ann. N.Y. Acad. Sci. 370, 511527. [64] Rypniewski, W.R., Hastrup, S., Betzel, C., Dauter, M., Dauter, Z., Papendorf, G., Branner, S., Wilson, K.S. (1993). The sequence and X-ray structure of the trypsin from Fusarium oxysporum. Protein Eng. 6, 341348. [65] Hedstrom, L. (1996). Trypsin: a case study in the structural determinants of enzyme specificity. Biol. Chem. 377, 465470. [66] Hedstrom, L. (2002). Serine protease mechanism and specificity. Chem. Rev. 102, 45014524. [67] The PyMOL Molecular Graphics System, Version 1.4, Schro¨dinger, LLC.
Teaster T. Baird, Jr. Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132, USA. Email: [email protected]
Charles S. Craik Department of Pharmaceutical Chemistry, University of California, San Francisco, 600 16th Street, S512C, Box 2280, San Francisco, CA 94158, USA. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00575-5
Chapter 576
Human Trypsins DATABANKS MEROPS name: cationic trypsin (Homo sapiens-type)
MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.127 Tertiary structure: Available
Clan PA S1 | 576. Human Trypsins
Species distribution: subphylum Vertebrata Reference sequence from: Homo sapiens (UniProt: P07477) MEROPS name: trypsin-2 type A MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.258 Species distribution: superfamily Hominoidea Reference sequence from: Homo sapiens (UniProt: P07478) MEROPS name: mesotrypsin MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.174 Tertiary structure: Available Species distribution: known only from Homo sapiens Reference sequence from: Homo sapiens (UniProt: P35030) MEROPS name: neurotrypsin MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.237 Species distribution: subphylum Vertebrata Reference sequence from: Mus musculus (UniProt: O08762) MEROPS name: trypsin C MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase S01.298 Species distribution: superfamily Hominoidea Reference sequence from: Homo sapiens
Name and History Proteins Haverback et al. [1] reported the presence of trypsinogen in human pancreatic juice in 1960. Buck et al. [2] described the properties of partially purified trypsin from extracts of human pancreas in 1962. Keller & Allan [3] identified two anionic ‘trypsins’ in human pancreatic juice in 1967, and Figarella et al. [4] purified the precursor forms of the two anionic trypsins, trypsinogen 1 and trypsinogen 2 in 1969. The two isoforms are present in a 2:1 ratio, together accounting for B19% of proteins in human pancreatic juice [5]. A third minor isoform, characterized by its intermediate electrophoretic mobility and isoelectric point, was discovered by Rinderknecht et al. in 1979 [6]; it occurs in very low concentrations and probably accounts for ,0.5% of proteins in human pancreatic juice [7]. All human trypsinogens are anionic, but to avoid confusion, the least anionic isoform, trypsinogen 1, was termed ‘cationic’ and the most anionic form, trypsinogen 2, ‘anionic’ [6]. The third isoform was initially named zymogen-X [6] and later renamed mesotrypsinogen [7].
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However, these three trypsinogen isoforms have also been designated as trypsinogens 3, 1 and 2, numbered consecutively from anode to cathode in accordance with the IUPAC-IUB Commission on the biochemical nomenclature of multiple forms of enzymes [8]. The comparative nomenclature of Rinderknecht et al. [7] is used throughout this chapter in accordance with previous recommendation [9]. The molecular mass and pI of the three human trypsinogen isoforms are summarized in Table 576.1.
cDNAs cDNAs encoding for cationic and anionic trypsinogens, known as TRYI and TRYII respectively, were cloned by Emi et al. in 1986 [10]. The subsequently reported TRYIII [11] would have represented the cDNA for mesotrypsinogen, if it had not unfortunately contained sequencing errors [9]. The correct cDNA sequence for mesotrypsinogen was published by Nyaruhucha et al. in 1997 [12].
Genes The human genome contains six highly homologous trypsinogen genes, exhibiting a nucleotide sequence similarity to each other of 91%. Genes T4-T8 2 five tandemly arranged 10-kb repeats 2 are located toward the 30 end of the T-cell receptor (TCR) β locus (TRB@) on chromosome 7q35 [13]. The sixth 10-kb repeat, T9, became translocated from chromosome 7q35 to chromosome 9p13 some 1520 million years ago [1314]. T5 and T7 are obvious pseudogenes [13] whilst T6 is likely an expressed pseudogene [15] (see Distinguishing Features Related Genes later). T4 (PRSS1), T8 (PRSS2) and T9 (PRSS3), each of which is composed of five exons, encode the aforementioned three trypsinogen isoforms, respectively [13] (Table 576.2). The divergently evolved T1, T2 and T3 genes will be discussed in Distinguishing Features Related Genes. TABLE 576.1 Physical properties of the three human trypsinogen isoforms Physical properties
Cationic trypsinogen
Mesotrypsinogen
Anionic trypsinogen
Molecular mass (kDa)
26 700
26 000
28 000
pIa
6.4/6.2
5.5/5.7
4.4/4.9
a
The pI values represent isoelectric points of undenatured (native) proteins/isoelectric points of proteins denatured in 8 M urea. Data from Scheele et al. [8].
Clan PA S1 | 576. Human Trypsins
2602
TABLE 576.2 Correlation of the nomenclature of proteins, cDNAs, genes and official gene symbols of the human trypsinogen family Proteina Figarella et al. [4]
Rinderknecht et al. [67]
Scheele et al. [8]
Trypsinogen 1
Cationic trypsinogen
Trypsinogen 3
Trypsinogen 2
cDNA
Gene [13]
Gene symbolb
Chromosome location
TRYI [10]
T4
PRSS1
7q35
T5 (pseudogene)
7q35
T6 (expressed pseudogene [15])
7q35
T7 (pseudogene)
7q35
Anionic trypsinogen
Trypsinogen 1
TRYII [10]
T8
PRSS2
7q35
Mesotrypsinogen
Trypsinogen 2
Mesotrypsinogen [12]
T9
PRSS3
9p13
a
The comparative nomenclature of Rinderknecht et al. [6,7] is now the most commonly used. Approved by the HUGO Gene Nomenclature Committee. PRSS stands for protease, serine.
b
Numbering of Trypsinogen Amino Acid Residues The chymotrypsinogen numbering [16] provides an easy comparison of the primary structural features, common disulphide bridges, and similar tertiary structures between the peptidases of family S1 of clan PA. However, this numbering system was extremely confusing to geneticists and clinicians not directly involved in protein study: not only does the numbering not start from the translation initiator methionine but also the family members vary in lengths [17]. The new standard system with the translation initiator Met numbered as 1 has been widely used since 2000. A parallel comparison of the two numbering systems is provided in Figure 576.1. The new standard system is used throughout the article unless specified otherwise.
Structural Chemistry The primary translated polypeptides of PRSS1, PRSS2 and PRSS3 share substantial sequence similarity (Figure 576.2). Each of them contains an N-terminal signal peptide (15 amino acids); a short activation peptide (eight amino acids); the catalytic triad His, Asp and Ser that are found in all members of subclan PA(S); the three key pocket specificity residues, Asp194, Gly217 and Gly227 [18]; and the six absolutely conserved cysteine residues necessary to build the three disulphide bridges observed in all vertebrate trypsins, 4864, 171185, and 196220 [19].
Human cationic trypsin has a similar tertiary structure to bovine, rat, and porcine trypsins, with root-mean˚ for all 223 Cα positions square differences of 0.4 to 0.6 A [20]. Human mesotrypsin (described as ‘human brain trypsin’ in Katona et al. [21]) has a similar crystal structure to human cationic trypsin, with a root-mean-square ˚ for all Cα positions [21]. deviation of only 0.5 A
Activity and Specificity Trypsin belongs to the chymotrypsin superfamily of serine endopeptidases that are characterized by the catalytic triad His57, Asp102 and Ser195 (chymotrypsinogen numbering) [22]. Human trypsins hydrolyze peptide bonds after arginine or lysine residues, their activity being optimal between pH 7.5 and 8.5 and in the presence of Ca21 [7]. The substrate specificity of trypsins is conferred by the substrate-binding pocket composed of Asp194, Gly217 and Gly227. Whereas Asp194 forms a strong electrostatic band with arginine or lysine residues of the substrate, Gly217 and Gly227 permit entry of large amino acid side chains into the hydrophobic pocket [18]. Most synthetic inhibitors inhibit all three trypsins to about the same extent. For example, under standard conditions, p-aminobenzamidine and DFP, EDTA, and HgCl2 inhibit about 100, 50, and 11% of the activity, respectively. The specific inhibitor Tos-Lys-CH2Cl inhibits cationic and anionic trypsins completely, but reacts only slowly and incompletely with mesotrypsin (99 versus 65% inhibition) [7]. In sharp contrast to cationic and anionic trypsins, mesotrypsin shows almost total resistance to most protein
Clan PA S1 | 576. Human Trypsins
2603
T 8
F 9
V 10
A 11
A A 12 13
L 14
A 15
8 A 16
9 P 17
10 F 18
18 19 G G 26 27
20 Y 28
21 22 N C 29 30
23 E 31
24 25 E N 32 33
26 S 34
27 28 V P 35 36
29 Y 37
30 31 Q V 38 39
32 S 40
39 Y 45
40 41 H F 46 47
42 C 48
43 44 G G 49 50
45 S 51
46 47 L I 52 53
48 N 54
49 50 E Q 55 56
51 W 57
52 53 V V 58 59
54 S 60
57 58 H C 63 64
59 Y 65
60 61 K S 66 67
62 R 68
63 64 I Q 69 70
65 V 71
65 66 R L 72 73
69 G 74
70 71 72 E H N 75 76 77
73 74 75 I E V 78 79 80
78 79 G N 83 84
80 E 85
81 82 Q F 86 87
83 I 88
84 85 N A 89 90
86 A 91
87 88 K I 92 93
89 I 94
90 91 R H 95 96
93 94 Q Y 98 99
M 1
N 2
P 3
13 D 21
14 D 22
33 L 41
L 4
L 5
I 6
15 16 K I 23 24
17 V 25
34 N 42
37 38 S G 43 44
55 A 61
56 G 62
76 L 81
77 E 82
L 7
92 P 97
11 12 D D 19 20
95 D 100
96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 R K T L N N D I M L I K L S S R A V I N 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 116 117 118 119 120 121 122 123 124 125 127 128 129 130 132 133 134 135 136 137 A R V S T I S L P T A P P A T G T K C L 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 I S G W G N T A S S G A D Y P D E L Q C 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 L D A P V L S Q A K C E A S Y P G K I T 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 178 179 180 181 182 183 184 184 185 186 187 187 188 189 190 191 192 193 194 195 S N M F C V G F L E G G K D S C Q G D S 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 196 197 198 199 200 201 202 203 204 209 210 211 212 213 214 215 216 217 219 220 G G P V V C N G Q L Q G V V S W G D G C 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 A Q K N K P G V Y T K V Y N Y V K W I K 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 240 241 242 243 244 245 245 N T I A A N S 241 242 243 244 245 246 247 FIGURE 576.1 Amino acid sequence of the human cationic trypsinogen. Numbers below the sequences are in accordance with the new standard system in which the ATG translation initiator codon is numbered as 1. Numbers above the sequences correspond to those of the bovine chymotrypsinogen A [16]. Note the artificial deletions (downward arrows) and insertions (stars) introduced by the chymotrypsinogen numbering system. Signal peptide is in italics, activation peptide is shaded in gray, and the catalytic triad (His63, Asp107 and Ser200) are in bold and red. The numbering also applies to human anionic trypsinogen and mesotrypsinogen because all the three human trypsinogen isoforms have the same number of amino acids (see Figure 576.2). Amino acid sequence is from the NCBI Gene Database.
inhibitors of trypsin including pancreatic trypsin inhibitor, soybean trypsin inhibitor, lima bean trypsin inhibitor, ovomucoid and α1-antitrypsin inhibitor [7]; although it is readily inhibited by serpin-type inhibitors with Arg in the reactive loop including the α1-antitrypsin Pittsburgh variant [23] and protease nexin-1 [24]. The resistance of mesotrypsin to protein inhibitors was suggested by
Nyaruhucha et al. [12] to be due to the presence at residue 198 of an Arg in mesotrypsin, but a Gly in human cationic and anionic trypsins and in other vertebrate trypsins. Indeed, in the crystal structure, the side chain of Arg198 is in an extended conformation and occupies the S20 subsite [21], which in the absence of conformational change would result in a steric clash with the P20 residue
Clan PA S1 | 576. Human Trypsins
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48
64
PRSS1 PRSS2 PRSS3
MNPLLILTFVAAALAAPFDDDDKIVGGYNCEENSVPYQVSLNSGYHFCGGSLINEQWVVSAGHCY 65 --L----------V--------------I------------------------S----------- 65 ---F---A--G--V-V------------T-----L---------S--------S-------A--- 65 Trypsin Trypsinogen Pretrypsinogen
PRSS1 PRSS2 PRSS3
KSRIQVRLGEHNIEVLEGNEQFINAAKIIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPT 130 --------------------------------K-NSR--D---L------P----S---A----- 130 -T-----------K------------------K-N-D--D----------P-------------- 130
PRSS1 PRSS2 PRSS3
APPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDS 195 ----A--ES--------L---------------------E----------N-------------- 195 ----A--E---------L-F--------K-------T--E-K--------NS------------- 195
107
171
196
PRSS1 PRSS2 PRSS3
200
220
185
194
227
CQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIKNTIAANS 247 ----------S--E---I----Y------R-----------D---D------ 247 --R-------------------H---W--R-----------D---D------ 247
PRSS2
% 86
89 %
PRSS1
88%
PRSS3
FIGURE 576.2 Amino acid sequences of human cationic trypsinogen (PRSS1), anionic trypsinogen (PRSS2) and mesotrypsinogen (PRSS3). Note that trypsinogen is synthesized in the form of a prezymogen (i.e. pretrypsinogen). Removal of the signal peptide (italics) results in the formation of trypsinogen. Further removal of the activation peptide (shaded in gray) by either the physiological activator enteropeptidase or trypsin(ogen) itself leads to the formation of trypsin. Some of the residues that are critical for trypsin structure and function and that are consequently absolutely conserved throughout vertebrate evolution are highlighted in color: the catalytic triad residues (His63, Asp107 and Ser200) are in red; the three residues determining trypsin specificity (Asp194, Gly217 and Gly227) are in blue; and the three common disulfide bridges (4864, 171185 and 196220) are in green. Dashes indicate identity with the PRSS1 sequence. Amino acid sequence similarity between the three human trypsinogen isoforms is shown below the aligned sequences. Amino acid sequences are from the NCBI Gene Database.
of a protein inhibitor [25]. In addition, the side chain of Arg198 contributes to an unusually strong clustering of positive charges around the primary specificity pocket of mesotrypsin [21]. Most importantly, changing Arg198 back to Gly made the human mesotrypsin fully sensitive to protein trypsin inhibitors [26]. The human trypsins differ as well in selectivity toward protein substrates. While cationic and anionic trypsins are believed to be fairly promiscuous enzymes capable of cleaving nearly any accessible peptide bond that follows Lys or Arg, mesotrypsin shows evidence of greater specificity. Mesotrypsin does not activate other pancreatic zymogens, as do cationic and anionic trypsins [26], nor is it very efficient in cleaving protease activated receptors (PARs) [27,28]. However, it displays strikingly enhanced catalytic activity relative to other trypsins for cleavage of specific protein substrates including human pancreatic secretory trypsin inhibitor (SPINK1), soybean trypsin inhibitor (SBTI), amyloid precursor protein Kunitz protease inhibitor domain (APPI), and bovine pancreatic trypsin inhibitor (BPTI) [25,26,29]. The more restricted specificity of mesotrypsin appears to derive both from a preference for substrate cleavage sites stabilized in a three-dimensional canonical conformation [30], and from distinct specificity at individual subsites, including a
stronger preference for Arg at the S1 subsite [30] and a preference for Ser or Thr at the S10 subsite [23].
Biological Aspects Autoactivation and Autolysis Trypsinogen is generally regarded as the inactive precursor of trypsin, its physiological activator being enteropeptidase that is located on the brush border membrane of enterocytes in the duodenum. However, it has been firmly established that trypsinogen possesses proteolytic activity to activate itself [31]. This process of trypsinogen activation, catalyzed first slowly by trypsinogen and then accelerated by the resulting trypsin, is termed here trypsinogen autoactivation (Figure 576.3). (Trypsinogen activation by enteropeptidase-activated trypsin in the duodenum is also known as trypsinogen autoactivation.) Activation of human trypsinogens by enteropeptidase, trypsinogen or trypsin occurs through hydrolyzing the peptide bond after Lys23 (Figure 576.2). Trypsin samples also autolyze in the absence of inhibitor as the result of self-digestion (i.e. one trypsin molecule acts upon another; termed autolysis). Arg122 has been identified to be the primary autolysis site of trypsins
Clan PA S1 | 576. Human Trypsins
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(A)
Initial activation by trypsinogen
(B) Trypsinogen
Trypsinogen Autoactivation (within pancreas)
Enteropeptidase (within duodenum)
Trypsin
Trypsin
Subsequent activation by trypsin
FIGURE 576.3 Schematic representation of trypsinogen autoactivation. Top panel indicates the initial activation, in which one trypsinogen molecule acts upon another. Lower panel indicates the subsequent activation, in which a newly formed trypsin molecule acts upon a trypsinogen molecule. Trypsinogen activation catalyzed by trypsinogen is much slower than that catalyzed by trypsin (indicated by the different sizes of the lightning symbols). Box, activation peptide. Bar, trypsin.
TABLE 576.3 Autoactivation and autolysis properties of the three human trypsin(ogen) isoforms Isoform
Autoactivation (ref.) Autolysis (ref.)
Cationic trypsin(ogen) 11 [38,41]
1 [38,42]
Anionic trypsin(ogen)
1 [38,41]
11 [38,42]
Mesotrypsin(ogen)
2 [26]
2 [26]
in cows [32], pigs [33], humans [20] and rats [34,35]. Human cationic trypsin was shown to undergo autolysis very slowly in vitro and to be stabilized against autolysis by calcium [3638]; furthermore, in vitro autolysis of the Arg122-Val123 bond did not proceed to completion [36,39]. By contrast, rapid and complete autolysis was achieved following the selective cleavage of the Leu81Glu82 peptide bond by chymotrypsin C (CTRC) [40]. In vitro autoactivation and autolysis properties of the three human trypsin(ogen)s [26,38,41,42] are compared in Table 576.3. As indicated in the table, mesotrypsinogen is deficient in autoactivation and is significantly impaired in cleavage of anionic and cationic trypsinogens at the autolysis site; the deficiency in autoactivation is rescued by mutation of Arg198 to Gly [26].
Trypsinogens in the Pancreas The three pretrypsinogen isoforms are synthesized in pancreatic acinar cells by ribosomes attached to the rough
Chymotrypsinogen
Chymotrypsin
Proelastase
Elastase
Kallikreinogen
Kallikrein
Procarboxypeptidase A
Carboxypeptidase A
Procarboxypeptidase A
Carboxypeptidase B
Prophospholipase A2
Phospholipase A2
Procolipase
Colipase
Physiology (within intestine)
Food digestion
Pathology (within pancreas)
Pancreatic autodigestion (Pancreatitis)
FIGURE 576.4 Trypsinogen, a double-edged sword. (A) Illustration of the physiological role of trypsinogen in digestion; (B) Illustration of the pathogenic role of prematurely activated trypsinogen within the pancreas. Normally, prematurely activated trypsin within the pancreas can be inhibited by the human pancreatic secretory trypsin inhibitor and/or degraded by chymotrypsinogen C and trypsin itself. A defect in one or a combination of these defense mechanisms may trigger the zymogen activation cascade leading to pancreatic autodigestion. Adapted from Chen & Fe´rec [66].
endoplasmic reticulum, where the signal peptides are removed co-translationally. The resulting trypsinogens are secreted into the pancreatic duct and finally discharged into the duodenal lumen where they are activated by enteropeptidase. The newly formed trypsins play a central role in digestion by acting as the trigger enzymes which activate all other pancreatic digestive pro-enzymes as well as the trypsinogens themselves (Figure 576.4a). Intuitively, prematurely activated trypsin within the pancreas, if not inhibited or/and inactivated, can trigger the zymogen activation cascade leading to pancreatic autodigestion (Figure 576.4b). Indeed, both gain-of-function missense mutations (through enhancing trypsinogen autoactivation or/and reducing trypsin autolysis) and copy number mutations in the PRSS1 gene (see Chapter 577) as well as loss-of-function mutations in the SPINK1 (encoding trypsin’s physiological inhibitor [43]) and CTRC genes [44,45] predispose to chronic pancreatitis. In an established model of acute pancreatitis, activation of trypsinogen was shown to occur in large endocytic vacuoles of pancreatic acinar cells [46]. Another mechanism potentially leading to pancreatic autodigestion is the ‘co-localization hypothesis’, in which digestive zymogens are thought to be activated by
Clan PA S1 | 576. Human Trypsins
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lysosomal hydrolases when the two types of enzymes become co-localized within the zymogen-containing secretory compartment [47]. Supporting evidences include that: (1) cathepsin B (CTSB), a lysosomal protease, has long been shown to be a potent activator of trypsinogen in vitro [4849]; (2) ctsb/ mice have significantly reduced intrapancreatic trypsin activity and severity of pancreatitis as compared with the wild-type mice [50]; and (3) CTSB is abundantly present in the secretory compartment of the human exocrine pancreas [51]. These notwithstanding, chronic pancreatitiscausing PRSS1 mutations p.Asn29Ile, p.Asn29Thr and p.Arg122His resulted in neither increase nor decrease of trypsinogen activation by CTSB in vitro [51]. Moreover, there exist controversial findings regarding association of CTSB polymorphisms with chronic pancreatitis [5254].
Extrapancreatic Expression of Trypsinogens It has long been thought that trypsinogens are specifically expressed in the pancreas and perform no function other than alimentary digestion. However, since the report of Bohe et al. in 1986 [55], mRNA or/and protein expression of human trypsinogens has been detected in a variety of tumors and cancer cell lines as well as in endothelial and epithelial cells of various normal tissues. Additionally, expression of a splice isoform of mesotrypsinogen lacking a typical signal sequence has been identified in human brain at both the transcript level [56] and the protein level [57] (see later T9-derived Chimeric Genes). These findings suggest that extrapancreatic trypsins may be involved in tissue remodeling and in tumor invasion. Moreover, there is increasing evidence that trypsin is a signaling molecule that may regulate multiple cellular functions by activating proteinase-activated receptors (for reviews, see Paju & Stenman [58] and Itkonen [59]).
Preparation The three human trypsinogen isoforms were analyzed simultaneously by polyacrylamide gel electrophoresis [6,7] or two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis [8]. Cationic trypsin can be purified rapidly from pancreatic juice: the first step is to isolate trypsinogen by fast protein liquid chromatography on a MonoS column at pH 4.5; trypsinogen is then activated by enteropeptidase, and finally isolated by affinity chromatography on STI-Sepharose column [37]. Cationic and anionic trypsinogens have also been purified from cyst fluid of ovarian cancer patients [60] and from human seminal fluid [61] by immunoaffinity chromatography and anion exchange chromatography. (The slight differences in substrate specificities between extra-pancreatic and pancreatic preparations [60] are likely to be caused by
sulfation of Tyr154 in pancreatic trypsinogens [62,63].) Cloned trypsins are usually produced by recombinant expression in Escherichia coli. In some expression systems, the soluble and correctly folded zymogens are isolated from the periplasm, while in others the zymogens are isolated in inclusion bodies and oxidatively refolded in vitro prior to chromatographic purification. Proteolytically activated trypsins are obtained by adding enteropeptidase to the zymogen preparation [12,37,39].
Distinguishing Features - Related Genes Divergently Evolved T1, T2 and T3 Genes Besides the T4T9 genes, three other genes (i.e. T1T3) located toward the 50 end of TRB@ were also identified as trypsinogen genes [13]. However, the T1T3 gene cluster and the T4T8 gene cluster have evolved largely as separate gene families after duplication, evidenced by the lack of significant nucleotide sequence similarity between these two clusters. Whereas T2 and T3 are clearly non-functional genes, T1 (known as trypsin X3 (TRYX3) in the NCBI Gene Database) has an inframe coding sequence and appears to have counterparts in other species including chimpanzee, Rhesus monkey, cattle, rat and mouse in the NCBI Gene Database. In addition, search of the NCBI EST database revealed transcripts that correspond to the putative TRYX3 gene in humans and some other species. Nonetheless, these genes would no longer encode a functional trypsinogen protein owing to their highly divergent evolution. As opined by Chen & Fe´rec [64], TRYX3 presumably represents a new gene, possibly with a new function, that has evolved from an ancient trypsinogen gene duplicate.
T6 Rowen et al. [13] annotated T6 (known as TRY6 or trypsinogen C in the NCBI Gene Database) as an apparently functional trypsinogen gene but noted the following three points. First, reverse transcriptase PCR analysis of pancreas, thymus and liver suggested that T6 may be expressed in minute amounts in the thymus. Second, the surface charge and shape of the predicted T6 protein would differ significantly from those of the cationic and anionic trypsinogens. Third, T6 is deleted in a common insertion/deletion polymorphism. Chen & Fe´rec [15] further suggested that the presence of His122 instead of Arg in the predicted T6 protein would indicate a relaxation of the selection pressure acting on the evolutionarily conserved Arg122 autolysis site. Taken together, T6 most probably represents a transition state between a functional duplicated gene and a nonfunctional pseudogene, that is, an expressed pseudogene [15].
Clan PA S1 | 576. Human Trypsins
T9-derived Chimeric Genes The T9 gene was translocated from chromosome 7q35 to chromosome 9p13 some 1520 million years ago [14]. This event has certainly liberated the T9 gene from shared functional or regulatory constraints exerted at the original locus, reflected by the dramatic differences between mesotrypsinogen and the other two trypsinogen isoforms in terms of expression level in pancreas and biochemical properties. More importantly, the splicing donor site of intron 1 of the T9 gene is changed from the consensus ‘GT’ to the less commonly used GC. Evidently, this latter change has contributed directly to the generation of two chimeric genes, both retaining T9’s exons 25 but differing in exon 1 due to alternative splicing [56,65]. Were these new chimeric genes to be really functional, their biological roles would be different from that performed by the pancreatic mesotrypsin since: (1) the alternative exon 1 sequences do not encode a typical signal peptide; and (2) both chimeric genes are expressed predominantly in non-pancreatic tissues. From an evolutionary viewpoint, it is possible that the T9 gene is gradually losing its function as a trypsinogen gene but acquiring new (not yet characterized) function(s) through the formation of new chimeric genes [64].
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[10] Emi, M., Nakamura, Y., Ogawa, M., Yamamoto, T., Nishide, T., Mori, T., Matsubara, K. (1986). Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens. Gene 41(23), 305310. [11] Tani, T., Kawashima, I., Mita, K., Takiguchi, Y. (1990). Nucleotide sequence of the human pancreatic trypsinogen III cDNA. Nucleic Acids Res. 18(6), 1631. [12] Nyaruhucha, C.N., Kito, M., Fukuoka, S.I. (1997). Identification and expression of the cDNA-encoding human mesotrypsin(ogen), an isoform of trypsin with inhibitor resistance. J. Biol. Chem. 272(16), 1057310578. [13] Rowen, L., Koop, B.F., Hood, L. (1996). The complete 685-kilobase DNA sequence of the human beta T cell receptor locus. Science 272(5269), 17551762. [14] Rowen, L., Williams, E., Glusman, G., Linardopoulou, E., Friedman, C., Ahearn, M.E., Seto, J., Boysen, C., Qin, S., Wang, K., Kaur, A., Bloom, S., Hood, L., Trask, B.J. (2005). Interchromosomal segmental duplications explain the unusual structure of PRSS3, the gene for an inhibitor-resistant trypsinogen. Mol. Biol. Evol. 22(8), 17121720. [15] Chen, J.M., Montier, T., Fe´rec, C. (2001). Molecular pathology and evolutionary and physiological implications of pancreatitisassociated cationic trypsinogen mutations. Hum. Genet. 109(3), 245252. [16] Hartley, B.S. (1970). Homologies in serine proteinases. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 257(813), 7787. [17] Chen, J.M., Fe´rec, C. (2000). Wanted: a consensus nomenclature for cationic trypsinogen mutations. Gastroenterology 119(1), 277278. [18] Craik, C.S., Largman, C., Fletcher, T., Roczniak, S., Barr, P.J., Fletterick, R., Rutter, W.J. (1985). Redesigning trypsin: alteration of substrate specificity. Science 228(4697), 291297. [19] Roach, J.C., Wang, K., Gan, L., Hood, L. (1997). The molecular evolution of the vertebrate trypsinogens. J. Mol. Evol. 45(6), 640652. [20] Gaboriaud, C., Serre, L., Guy-Crotte, O., Forest, E., FontecillaCamps, J.C. (1996). Crystal structure of human trypsin 1: unexpected phosphorylation of Tyr151. J. Mol. Biol. 259(5), 9951010. [21] Katona, G., Berglund, G.I., Hajdu, J., Gra´f, L., Szila´gyi, L. (2002). Crystal structure reveals basis for the inhibitor resistance of human brain trypsin. J. Mol. Biol. 315(5), 12091218. [22] Higaki, J.N., Gibson, B.W., Craik, C.S. (1987). Evolution of catalysis in the serine proteases. Cold Spring Harb. Symp. Quant. Biol. 52, 615621. [23] Szepessy, E., Sahin-Toth, M. (2006). Human mesotrypsin exhibits restricted S10 subsite specificity with a strong preference for small polar side chains. FEBS J. 273(13), 29422954. [24] Koistinen, H., Koistinen, R., Zhang, W.M., Valmu, L., Stenman, U.H. (2009). Nexin-1 inhibits the activity of human brain trypsin. Neuroscience 160(1), 97102. [25] Salameh, M.A., Soares, A.S., Hockla, A., Radisky, E.S. (2008). Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin. J. Biol. Chem. 283(7), 41154123. [26] Szmola, R., Kukor, Z., Sahin-Toth, M. (2003). Human mesotrypsin is a unique digestive protease specialized for the degradation of trypsin inhibitors. J. Biol. Chem. 278(49), 4858048589. [27] Grishina, Z., Ostrowska, E., Halangk, W., Sahin-Toth, M., Reiser, G. (2005). Activity of recombinant trypsin isoforms on
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[28]
[29]
[30]
[31] [32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
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Jian-Min Chen Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), U1078 and Etablissement Franc¸ais du Sang Bretagne, 46 rue Fe´lix Le Dantec, 29218 Brest, France. Email: [email protected]
Evette S. Radisky Department of Cancer Biology, Mayo Clinic Cancer Center, 4500 San Pablo Road, Jacksonville, FL 32224, USA. Email: [email protected]
Claude Fe´rec Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), U1078 and Etablissement Franc¸ais du Sang Bretagne, 46 rue Fe´lix Le Dantec, 29218 Brest, France. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00576-7