László Szilágyi

Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis.

Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene. The role of mutations has been investigated by in vitro studies using recombinant rat and human trypsinogen (TG). In this study we compare the enzymatic properties and inhibition by human pancreatic secretory trypsin inhibitor (hPSTI) of the native, postsynthetically modified and recombinant cationic trypsin, and found these values practically identical. We also determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin. Both forms were equally stable. Similarly, we found no difference in the rate of activation of the two zymogens by human cationic and anionic trypsin. Mesotrypsin did not activate either form. The rate of autocatalytic activation of Hu1Asn21 TG and Hu1Ile21 TG was also identical at pH 8 both in the presence and absence of Ca2+. At pH 5 Hu1Ile21 TG autoactivated about twice as fast as Hu1Asn21 TG. The presence of physiological amount of hPSTI completely prevented autoactivation of both zymogens at pH 8 and at pH 5 as well. Cathepsin B readily activated both zymogens although Hu1Ile21 TG was activated about 2.5–3 times as fast as Hu1Asn21 TG. The presence of hPSTI did not prevent the activation of zymogens by cathepsin B. Our results underlie the central role of cathepsin B in the development of different forms of pancreatitis.

Photoaffinity labelling with an ATP analog of the N-terminal peptide of myosin.

Photoaffinity labelling of tryptic and chymotryptic heavy meromyosin with 3′O-3-[N-(4-azido-2-nitrophenyl) amino]propionyl-adenosine 5′-triphosphate (arylazido-β-alanine ATP) resulted in incorporation of radioactivity and inhibition of the ATPase activity. ATP prevented the reaction with the photoaffinity label, as shown by the lack of incorporation of 3H and intact ATPase activity. On the tryptic digestion of either type of photoaffinity labeled HMM the label was found in a 25K peptide identifiable with the N-terminus of the myosin heavy chain (Lu et al., Fed. Proc. 37 1695 1978). The results are discussed in the light of previous localization of the reactive thiol groups, SH-1 and SH-2 (Balint et al., Arch. Biochem. Biophys. 190, 793 1978).

Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay

Stem cells reside in customized microenvironments (niches) that contribute to their unique ability to divide asymmetrically to give rise to self and to a daughter cell with distinct properties. Notch receptors and their ligands are highly conserved and have been shown to regulate cell‐fate decisions in multiple developmental systems through local cell interactions. To assess whether Notch signaling may regulate hematopoiesis to maintain cells in an immature state, we examined the functional role of the recombinant, secreted form of the Notch ligand Jagged‐1 during mouse hematopoietic stem cell (HSC) and progenitor cell proliferation and maturation. We found that ligand immobilization on stromal layer or on Sepharose‐4B beads is required for the induction of self‐renewing divisions of days 28–35 cobblestone area‐forming cell. The free, soluble Jagged‐1, however, has a dominant‐negative effect on self‐renewal in the stem‐cell compartment. In contrast, free as well as immobilized Jagged‐1 promotes growth factor‐induced colony formation of committed hematopoietic progenitor cells. Therefore, we propose that differences in Jagged‐1 presentation and developmental stage of the Notch receptor‐bearing cells influence Notch ligand‐binding results toward activation or inhibition of downstream signaling. Moreover, these results suggest potential clinical use of recombinant Notch ligands for expanding human HSC populations in vitro.

Attempts to convert chymotrypsin to trypsin.

Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the PI site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to Typsin‐like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin‐like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly Typsin‐like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin → trypsin specificity conversion.

Myelin basic protein, an autoantigen in multiple sclerosis, is selectively processed by human trypsin 4

Demyelination, the proteolytic degradation of the major membrane protein in central nervous system, myelin, is involved in many neurodegenerative diseases. In the present in vitro study the proteolytic actions of calpain, human trypsin 1 and human trypsin 4 were compared on lipid bound and free human myelin basic proteins as substrates. The fragments formed were identified by using N‐terminal amino acid sequencing and mass spectrometry. The analysis of the degradation products showed that of these three proteases human trypsin 4 cleaved myelin basic protein most specifically. It selectively cleaves the Arg79‐Thr80 and Arg97‐Thr98 peptide bonds in the lipid bound form of human myelin basic protein. Based on this information we synthesized peptide IVTPRTPPPSQ that corresponds to sequence region 93–103 of myelin basic protein and contains one of its two trypsin 4 cleavage sites, Arg97‐Thr98. Studies on the hydrolysis of this synthetic peptide by trypsin 4 have confirmed that the Arg97‐Thr98 peptide bond is highly susceptible to trypsin 4. What may lend biological interest to this finding is that the major autoantibodies found in patients with multiple sclerosis recognize sequence 85–96 of the protein. Our results suggest that human trypsin 4 may be one of the candidate proteases involved in the pathomechanism of multiple sclerosis.

The Role of the Individual Domains in the Structure and Function of the Catalytic Region of a Modular Serine Protease, C1r

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal α region interacting with other subcomponents and C-terminal γB region mediating proteolytic activity. The γB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the γB region we produced the CCP1-CCP2-SP (γB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.

Probing conformational plasticity of the activation domain of trypsin: The role of glycine hinges

Trypsin-like serine proteases play essential roles in diverse physiological processes such as hemostasis, apoptosis, signal transduction, reproduction, immune response, matrix remodeling, development, and differentiation. All of these proteases share an intriguing activation mechanism that involves the transition of an unfolded domain (activation domain) of the zymogen to a folded one in the active enzyme. During this conformational change, activation domain segments move around highly conserved glycine hinges. In the present study, hinge glycines were replaced by alanine residues via site directed mutagenesis. The effects of these mutations on the interconversion of the zymogen-like and active conformations as well as on catalytic activity were studied. Mutant trypsins showed zymogen-like structures to varying extents characterized by increased flexibility of some activation domain segments, a more accessible N-terminus and a deformed substrate binding site. Our results suggest that the trypsinogen to trypsin transition is hindered by the mutations, which results in a shift of the equilibrium between the inactive zymogen-like and active enzyme conformations toward the inactive state. Our data also showed, however, that the inactive conformations of the various mutants differ from each other. Binding of substrate analogues shifted the conformational equilibrium toward the active enzyme since inhibited forms of the trypsin mutants showed similar structural features as the wild-type enzyme. The catalytic activity of the mutants correlated with the proper conformation of the active site, which could be supported by varying conformations of the N-terminus and the autolysis loop. Transient kinetic measurements confirmed the existence of an inactive to active conformational transition occurring prior to substrate binding.

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine A possible means to regulate gene expression

Summary Chromosomal rearrangements apparently account for the presence of a primate‐specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72‐residue leader peptide on Isoform A, and a CUG site that codes for a 28‐residue leader peptide on Isoform B. We report studies that provide evidence for the N‐terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28‐amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.

Regional Distribution of Human Trypsinogen 4 in Human Brain at mRNA and Protein Level

Gene PRSS3 on chromosome 9 of the human genome encodes, due to alternative splicing, both mesotrypsinogen and trypsinogen 4. Mesotrypsinogen has long been known as a minor component of trypsinogens expressed in human pancreas, while the mRNA for trypsinogen 4 has recently been identified in brain and other human tissues. We measured the amount of trypsinogen 4 mRNA and the quantity of the protein as well in 17 selected areas of the human brain. Our data suggest that human trypsinogen 4 is widely but unevenly distributed in the human brain. By immunohistochemistry, here we show that this protease is localized in neurons and glial cells, predominantly in astrocytes. In addition to cellular immunoreactivity, human trypsinogen 4 immunopositive dots were detected in the extracellular matrix, supporting the view that human trypsinogen 4 might be released from the cells under special conditions.

Stable monomeric form of an originally dimeric serine proteinase inhibitor, ecotin, was constructed via site directed mutagenesis

Ecotin, a homodimer protein of E. coli, is a unique member of canonical serine proteinase inhibitors, since it is a potent agent against a variety of serine proteinases having different substrate specificity. Monomers of ecotin are held together mostly by their long C‐terminal strands that are arranged as a two‐stranded antiparallel β‐sheet in the functional dimer. One ecotin dimer can chelate two proteinase molecules, each of them bound to both subunits of ecotin at two different sites, namely the specific primary and the non‐specific secondary binding sites. In this study the genes of wild type ecotin and its Met84 Arg P1 site mutant were truncated resulting in new forms of ecotin that lack 10 amino acid residues at their C‐terminus. These mutants do not dimerize spontaneously, though in combination with trypsin they assemble into the familiar heterotetramer. Our data suggest that this heterotetramer exists even in extremely diluted solutions, and the interaction, which is responsible for the dimerization of ecotin, contributes to the stability of the heterotetrameric complex.