- Email: [email protected]
Trypsin: is there anything new under the Sun?
Journal of Molecular Structure (Theochem) 666–667 (2003) 481–485 www.elsevier.com/locate/theochem
Trypsin: is there anything new under the Sun? La´szlo´ Gra´f*, La´szlo´ Szila´gyi Department of Biochemistry, Eo¨tvo¨s Lora´nd University, Puskin u. 3, Budapest H-1088, Hungary
Abstract Though trypsin is the first discovered and probably the best characterized enzyme, recent studies have led to the discovery of new properties and even a new form of this enzyme. The molecular mechanisms of autolysis of both trypsin and chymotrypsin have recently been explored and it has been proposed that the elimination of the major autolytic site by mutation in human cationic trypsin might cause pancreatitis. Other highlights of trypsin research are the discovery, X-ray crystallography and immunohistochemical localization of human brain trypsin. q 2003 Elsevier B.V. All rights reserved. Keywords: Trypsin; Autoactivation; Autolysis; Pancreatitis; Human brain trypsin
1. Trypsin is the first discovered and probably the best characterized enzyme It has been known for more than 130 years that pancreatic juice is able to digest proteins [4]. Ku¨hne suggested that the this property of the juice was due to an ‘unorganized ferment’ or enzyme that he named ‘trypsin’. He also showed that the extracts of fresh pancreas or freshly secreted pancreatic juice had no proteolytic activity, but the activity appeared and were increasing when the pancreas was allowed to stand. After a longer period of standing the proteolytic activity of the pancreatic juice started to decrease (Fig. 1). This was the first description, in 1867, of autoactivation of the inactive (zymogen) form of trypsin and the autolytic inactivation of active trypsin. Since then autoactivation and autolyis of trypsinogen/ trypsin have been favourite topics of biochemical and physiological studies on trypsin. * Corresponding author. Tel.: þ 36-1-2667858; fax: þ 36-12667830. E-mail address: [email protected] (L. Gra´f). 0166-1280/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2003.08.072
2. Pancreatitis-associated mutations in trypsin While the molecular mechanism and biological function of trypsinogen autoactivation are not clear yet, the mechanisms of autolysis of trypsin [6] and chymotrypsin [1] have recently been reported from our laboratory. According to these studies the interdomain loops of both enzymes contain the major autolytic sites, Arg117– Vall118 in trypsin and Phe114 – Ser 115 in chymotrypsin. The cleavages of these peptide bonds lead to the inactivation of the proteinases (Fig. 2). What makes these results particulary interesting is the discovery that an Arg117 to His mutation in human cationic trypsin is associated with hereditary pancreatitis [7]. Since this mutation clearly abolishes the major autolytic site of trypsin, one can speculate that autolyis may play a physiological role in the pancreas also. Autolysis might function as a safety mechanism to eliminate prematurely activated (autoactivated?) trypsin in the pancreas [6,8] (Figs. 3 and 4). The presence and accumulation of an autolysis-resistant trypsin in
482
L. Gra´f, L. Szila´gyi / Journal of Molecular Structure (Theochem) 666–667 (2003) 481–485
mutant 2-3 times as fast as the wild-type zymogen, and the presence of hPSTI did not prevent the activation of the zymogens by cathepsin B.
3. Brain Associated Trypsin (BAT): will exploration of its biochemical properties give a clue to its biological function?
Fig. 1. The time-course of proteolytic activity in extracts from fresh pancreas according to Ref. [4].
the pancreas would lead to the clinical symptoms of pancreatitis (Fig. 4). One weakness of this explanation is that the physiological concentration of human pancreatic secretory trypsin inhibitor (hPSTI) in the pancreas should completely prevent both autoactivation and autolyis of trypsinogen/trypsin. Therefore, in a more recent study of us on another pancreatitisassociated human cationic trypsin mutant, the Asn21Ile one, we proposed that cathepsin B, rather than trypsin, might be the pathological activator of trypsinogen in pancreatitis [5] (Fig. 5). In in vitro experiments, cathepsin B activated the Asn21Ile
While the involvement of pancreatitis-associated trypsin mutants in the pathomechanism of pancreatitis is evident, the physiological function and possible pathological role of human trypsinogen 4 has not been understood yet. The common gene encoding pancreatic mesotrypsinogen and human trypsinogen 4 (PRSS3) is located on chromosome 9, in contrast to the genes for pancreatic trypsinogens 1 and 2 that are located on chromosome 7. As a result of alternative splicing mesotrypsinogen and human trypsinogen 4 differ (and only differ) in there N-terminal amino sequences: while the former one has a typical signal sequence, human trypsinogen 4, dependent on the translation initiation site, has a 72 or 28 amino acid Nterminal leader sequence (Fig. 6). To our knowledge the mRNA for human trypsinogen 4 was found in
Fig. 2. Superimposed structures of trypsin and chymotrypsin with the major autolytic cleavage sites, Arg117–Val118 for trypsin and Phe114– Ser115 for chymotrypsin [1,6], respectively.
L. Gra´f, L. Szila´gyi / Journal of Molecular Structure (Theochem) 666–667 (2003) 481–485
483
Fig. 3. Schematic representation of the production of trypsinogen, chymotrypsinogen and their inhibitors in the pancreas, their transport through the ductus and their activation/autolysis in the duodenum.
human brain only [9], and for the first time by using two specific monoclonal antibodies raised against the recombinant enzyme and a synthetic fragment of the N-terminal ‘leader’ peptide we were able to localize human trypsinogen 4-like immunoreactivity in glia
cells of human cerebral cortex and spinal cord [2] (Fig. 7). Initiated by these new findings we named human trypsin 4 as Brain Associated Trypsin (BAT). Our preliminary studies on the interaction of some artificial membranes with a synthetic peptide
Fig. 4. Schematic representation of the possible consequence of trypsinogen mutation Arg117 to His that prevents autoactivation of trypsin [6].
484
L. Gra´f, L. Szila´gyi / Journal of Molecular Structure (Theochem) 666–667 (2003) 481–485
Fig. 5. Cathepsin B might be the pathological activator of trypsinogen in hereditary pancreatitis [5].
fragment of the ‘leader’ sequence of BAT suggest that this region might serve as an anchor to attach the inactive proteinase to the cell membrane. Mesotrypsin ¼ BAT is a unique isoform of trypsin in which an arginine replaces the conserved glycine at position 193. It has long been thought that this
Fig. 6. The N-terminal sequences of human mesotrypsinogen and two possible isoforms of human trypsinogen 4.
Fig. 7. Immunohistochemical localization of BAT (human trypsin 4) in cortical white matter by staining with monoclonal antibodies raised against the protease domain (A) and the 28-residue synthetic ‘anchor’ peptide (B).
Fig. 8. The effect of mutation Arg193 to Gly in human trypsin 4 (T4). The log Ki inhibitory contants ðMÞ for benzamidine, soy-bean trypsin inhibitor (STI), bovine pancreatic trypsin inhibitor (BPTI), human pancreatic secretory trypsin inhibitor (hPSTI) and Alzheimer precursor protein inhibitor (APPI) are shown on the ordinate.
L. Gra´f, L. Szila´gyi / Journal of Molecular Structure (Theochem) 666–667 (2003) 481–485
485
Arg193 on the enzymatic properties of BAT by changing Arg193 to glycine (Arg193Gly). The results of a comparison of the inhibitor sensitivities of the arginine and glycine containing forms are shown in Fig. 8. The Fig. clearly shows that a single arginine to glycine substitution at position 193 is sufficient to restore the trypsin-like inhibitor sensitivity of BAT towards natural canonical trypsin inhibitors. These data are in agreement with the structural studies that show that in BAT Arg193 occupies the P20 subsite of the substrate and inhibitor binding surface [3]. Localization of BAT in the glia cells of human brain, the likely association of the zymogen with the cell membrane, the resistance of the active proteinase towards canonical trypsin inhibitors and its limited substrate specificity are properties that make it a unique enzyme of the human brain. Its function, however, does not follow from these properties, and is a mistery at this stage. Our best guess is that the membrane bound BAT gets activated and released from the membrane (Fig. 9) under certain physiological and/or pathological conditions and cleaves specific, so far unknown neuropeptide or neuroprotein substrates in the brain One explanation for its resistance towards the naturally occurring proteinase inhibitors may be that its action is controlled by its release and unique substrate specificity rather than by specific inhibitors.
References
Fig. 9. Brain Associated Trypsinogen (BAT, human trypsinogen 4) might be a membrane associated zymogen. Upon activation with enterokinase or another activating protease the active BAT might be released.
substitution is responsible for the resistance of mesotrypsin towards naturally occurring protein inhibitors of trypsin. The crystal structure of recombinant mesotrypsin/BAT confirmed this notion revealing the orientation of the side-chain of Arg193. It assumes an extended conformation and fills the S20 substrate binding subsite of the enzyme [3]. By sitedirected mutagenesis also we studied the effect of
[1] A. Bo´di, G. Kaslik, I. Venekei, L. Gra´f, Eur. J. Biochem. 268 (2001) 6238. [2] K. Gallatz, P. Medveczky, P. Ne´meth, L. Szila´gyi, L. Gra´f, M. Palkovits, 2003. in preparation. [3] G. Katona, G.I. Berglund, J. Hajdu, L. Gra´f, L. Szila´gyi, J. Mol. Biol. 315 (2002) 1209. [4] W. Ku¨hne, Virchows. Arch. 39 (1867) 130. [5] L. Szila´gyi, E. Ke´nesi, G. Katona, G. Kaslik, G. Juha´sz, L. Gra´f, J. Biol. Chem. 276 (2001) 24574. [6] E. Va´rallay, G. Pa´l, A. Patthy, L. Szila´gyi, L. Gra´f, Biochem. Biophys. Res. Commun. 243 (1998) 56. [7] D.C. Whitcomb, M.C. Gorry, R.A. Preston, W. Furey, M.J. Sossenheimer, C.D. Ulrich, S.P. Martin, L.K. Gates, S.T. Amman, P.P. Toskes, R. Liddle, K. McGrath, G. Uomo, J.C. Post, G.D. Ehrlich, Nat. Genet. 259 (1996) 995. [8] D.C. Whitcomb, GUT 45 (1999) 317. [9] U. Wiegand, S. Corbach, A. Minn, J. Kang, B. Muller-Hill, Gene 136 (1993) 167.
Trypsin: is there anything new under the Sun? La´szlo´ Gra´f*, La´szlo´ Szila´gyi Department of Biochemistry, Eo¨tvo¨s Lora´nd University, Puskin u. 3, Budapest H-1088, Hungary
Abstract Though trypsin is the first discovered and probably the best characterized enzyme, recent studies have led to the discovery of new properties and even a new form of this enzyme. The molecular mechanisms of autolysis of both trypsin and chymotrypsin have recently been explored and it has been proposed that the elimination of the major autolytic site by mutation in human cationic trypsin might cause pancreatitis. Other highlights of trypsin research are the discovery, X-ray crystallography and immunohistochemical localization of human brain trypsin. q 2003 Elsevier B.V. All rights reserved. Keywords: Trypsin; Autoactivation; Autolysis; Pancreatitis; Human brain trypsin
1. Trypsin is the first discovered and probably the best characterized enzyme It has been known for more than 130 years that pancreatic juice is able to digest proteins [4]. Ku¨hne suggested that the this property of the juice was due to an ‘unorganized ferment’ or enzyme that he named ‘trypsin’. He also showed that the extracts of fresh pancreas or freshly secreted pancreatic juice had no proteolytic activity, but the activity appeared and were increasing when the pancreas was allowed to stand. After a longer period of standing the proteolytic activity of the pancreatic juice started to decrease (Fig. 1). This was the first description, in 1867, of autoactivation of the inactive (zymogen) form of trypsin and the autolytic inactivation of active trypsin. Since then autoactivation and autolyis of trypsinogen/ trypsin have been favourite topics of biochemical and physiological studies on trypsin. * Corresponding author. Tel.: þ 36-1-2667858; fax: þ 36-12667830. E-mail address: [email protected] (L. Gra´f). 0166-1280/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2003.08.072
2. Pancreatitis-associated mutations in trypsin While the molecular mechanism and biological function of trypsinogen autoactivation are not clear yet, the mechanisms of autolysis of trypsin [6] and chymotrypsin [1] have recently been reported from our laboratory. According to these studies the interdomain loops of both enzymes contain the major autolytic sites, Arg117– Vall118 in trypsin and Phe114 – Ser 115 in chymotrypsin. The cleavages of these peptide bonds lead to the inactivation of the proteinases (Fig. 2). What makes these results particulary interesting is the discovery that an Arg117 to His mutation in human cationic trypsin is associated with hereditary pancreatitis [7]. Since this mutation clearly abolishes the major autolytic site of trypsin, one can speculate that autolyis may play a physiological role in the pancreas also. Autolysis might function as a safety mechanism to eliminate prematurely activated (autoactivated?) trypsin in the pancreas [6,8] (Figs. 3 and 4). The presence and accumulation of an autolysis-resistant trypsin in
482
L. Gra´f, L. Szila´gyi / Journal of Molecular Structure (Theochem) 666–667 (2003) 481–485
mutant 2-3 times as fast as the wild-type zymogen, and the presence of hPSTI did not prevent the activation of the zymogens by cathepsin B.
3. Brain Associated Trypsin (BAT): will exploration of its biochemical properties give a clue to its biological function?
Fig. 1. The time-course of proteolytic activity in extracts from fresh pancreas according to Ref. [4].
the pancreas would lead to the clinical symptoms of pancreatitis (Fig. 4). One weakness of this explanation is that the physiological concentration of human pancreatic secretory trypsin inhibitor (hPSTI) in the pancreas should completely prevent both autoactivation and autolyis of trypsinogen/trypsin. Therefore, in a more recent study of us on another pancreatitisassociated human cationic trypsin mutant, the Asn21Ile one, we proposed that cathepsin B, rather than trypsin, might be the pathological activator of trypsinogen in pancreatitis [5] (Fig. 5). In in vitro experiments, cathepsin B activated the Asn21Ile
While the involvement of pancreatitis-associated trypsin mutants in the pathomechanism of pancreatitis is evident, the physiological function and possible pathological role of human trypsinogen 4 has not been understood yet. The common gene encoding pancreatic mesotrypsinogen and human trypsinogen 4 (PRSS3) is located on chromosome 9, in contrast to the genes for pancreatic trypsinogens 1 and 2 that are located on chromosome 7. As a result of alternative splicing mesotrypsinogen and human trypsinogen 4 differ (and only differ) in there N-terminal amino sequences: while the former one has a typical signal sequence, human trypsinogen 4, dependent on the translation initiation site, has a 72 or 28 amino acid Nterminal leader sequence (Fig. 6). To our knowledge the mRNA for human trypsinogen 4 was found in
Fig. 2. Superimposed structures of trypsin and chymotrypsin with the major autolytic cleavage sites, Arg117–Val118 for trypsin and Phe114– Ser115 for chymotrypsin [1,6], respectively.
L. Gra´f, L. Szila´gyi / Journal of Molecular Structure (Theochem) 666–667 (2003) 481–485
483
Fig. 3. Schematic representation of the production of trypsinogen, chymotrypsinogen and their inhibitors in the pancreas, their transport through the ductus and their activation/autolysis in the duodenum.
human brain only [9], and for the first time by using two specific monoclonal antibodies raised against the recombinant enzyme and a synthetic fragment of the N-terminal ‘leader’ peptide we were able to localize human trypsinogen 4-like immunoreactivity in glia
cells of human cerebral cortex and spinal cord [2] (Fig. 7). Initiated by these new findings we named human trypsin 4 as Brain Associated Trypsin (BAT). Our preliminary studies on the interaction of some artificial membranes with a synthetic peptide
Fig. 4. Schematic representation of the possible consequence of trypsinogen mutation Arg117 to His that prevents autoactivation of trypsin [6].
484
L. Gra´f, L. Szila´gyi / Journal of Molecular Structure (Theochem) 666–667 (2003) 481–485
Fig. 5. Cathepsin B might be the pathological activator of trypsinogen in hereditary pancreatitis [5].
fragment of the ‘leader’ sequence of BAT suggest that this region might serve as an anchor to attach the inactive proteinase to the cell membrane. Mesotrypsin ¼ BAT is a unique isoform of trypsin in which an arginine replaces the conserved glycine at position 193. It has long been thought that this
Fig. 6. The N-terminal sequences of human mesotrypsinogen and two possible isoforms of human trypsinogen 4.
Fig. 7. Immunohistochemical localization of BAT (human trypsin 4) in cortical white matter by staining with monoclonal antibodies raised against the protease domain (A) and the 28-residue synthetic ‘anchor’ peptide (B).
Fig. 8. The effect of mutation Arg193 to Gly in human trypsin 4 (T4). The log Ki inhibitory contants ðMÞ for benzamidine, soy-bean trypsin inhibitor (STI), bovine pancreatic trypsin inhibitor (BPTI), human pancreatic secretory trypsin inhibitor (hPSTI) and Alzheimer precursor protein inhibitor (APPI) are shown on the ordinate.
L. Gra´f, L. Szila´gyi / Journal of Molecular Structure (Theochem) 666–667 (2003) 481–485
485
Arg193 on the enzymatic properties of BAT by changing Arg193 to glycine (Arg193Gly). The results of a comparison of the inhibitor sensitivities of the arginine and glycine containing forms are shown in Fig. 8. The Fig. clearly shows that a single arginine to glycine substitution at position 193 is sufficient to restore the trypsin-like inhibitor sensitivity of BAT towards natural canonical trypsin inhibitors. These data are in agreement with the structural studies that show that in BAT Arg193 occupies the P20 subsite of the substrate and inhibitor binding surface [3]. Localization of BAT in the glia cells of human brain, the likely association of the zymogen with the cell membrane, the resistance of the active proteinase towards canonical trypsin inhibitors and its limited substrate specificity are properties that make it a unique enzyme of the human brain. Its function, however, does not follow from these properties, and is a mistery at this stage. Our best guess is that the membrane bound BAT gets activated and released from the membrane (Fig. 9) under certain physiological and/or pathological conditions and cleaves specific, so far unknown neuropeptide or neuroprotein substrates in the brain One explanation for its resistance towards the naturally occurring proteinase inhibitors may be that its action is controlled by its release and unique substrate specificity rather than by specific inhibitors.
References
Fig. 9. Brain Associated Trypsinogen (BAT, human trypsinogen 4) might be a membrane associated zymogen. Upon activation with enterokinase or another activating protease the active BAT might be released.
substitution is responsible for the resistance of mesotrypsin towards naturally occurring protein inhibitors of trypsin. The crystal structure of recombinant mesotrypsin/BAT confirmed this notion revealing the orientation of the side-chain of Arg193. It assumes an extended conformation and fills the S20 substrate binding subsite of the enzyme [3]. By sitedirected mutagenesis also we studied the effect of
[1] A. Bo´di, G. Kaslik, I. Venekei, L. Gra´f, Eur. J. Biochem. 268 (2001) 6238. [2] K. Gallatz, P. Medveczky, P. Ne´meth, L. Szila´gyi, L. Gra´f, M. Palkovits, 2003. in preparation. [3] G. Katona, G.I. Berglund, J. Hajdu, L. Gra´f, L. Szila´gyi, J. Mol. Biol. 315 (2002) 1209. [4] W. Ku¨hne, Virchows. Arch. 39 (1867) 130. [5] L. Szila´gyi, E. Ke´nesi, G. Katona, G. Kaslik, G. Juha´sz, L. Gra´f, J. Biol. Chem. 276 (2001) 24574. [6] E. Va´rallay, G. Pa´l, A. Patthy, L. Szila´gyi, L. Gra´f, Biochem. Biophys. Res. Commun. 243 (1998) 56. [7] D.C. Whitcomb, M.C. Gorry, R.A. Preston, W. Furey, M.J. Sossenheimer, C.D. Ulrich, S.P. Martin, L.K. Gates, S.T. Amman, P.P. Toskes, R. Liddle, K. McGrath, G. Uomo, J.C. Post, G.D. Ehrlich, Nat. Genet. 259 (1996) 995. [8] D.C. Whitcomb, GUT 45 (1999) 317. [9] U. Wiegand, S. Corbach, A. Minn, J. Kang, B. Muller-Hill, Gene 136 (1993) 167.