DSpace at VNU: New cysteine protease inhibitors in physiological secretory fluids and their medical significance

Advan Enzyme Regul 43 (2003) 393–410 New cysteine protease inhibitors in physiological secretory fluids and their medical significance N Katunumaa,*, A Ohashia, E Sanoa, E Murataa, H Shiotab, K Yamamotoc, E Majimac, Q.T Led a Institute for Health Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima City, Tokushima 770-8514, Japan b School of Medicine, University of Tokushima, Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan c APRO Life Science Institute, 45-56 Kurosaki-Matsushima, Naruto City, Tokushima 772-0001, Japan d Biotechnology Center, Vietnam National University, Hanoi, 144 Xuan thuy-Cau giay, Hanoi, Viet Nam Introduction This work has helped to elucidate the biological significance of cathepsin inhibitors at the molecular level The finding of new inhibitors in biological materials and the elucidation of their properties were studied Furthermore, it is important to know the inhibitory mechanisms between cathepsins and their endogenous inhibitors This is useful for differential diagnosis of eye diseases Classification of Cysteine Proteases and their Physiological Roles in All Animals, Plants and Microorganisms Cysteine proteases, cathepsins, play an essential role to keep life in all living things Cysteine proteases are synthesized in bound ribosomes and secreted from transgolgy and then translocated in two ways, one is targeted into lysosomes and the other is secreted to outside of the cells as the secretion vesicles Cathepsins located in lysosomes play mainly a role of protein catabolism via autophagy and heterophagy On the other hand, the secreted cathepsins play a role in processing various biological proteins in the outside of the cells About 10 kinds of cathepsins have been reported and they show different properties and different biological functions They have a role not only in protein catabolism, but also in the production of biological active peptides by their limited proteolysis, such as antigen processing to make antigenic peptides and present to MHC class II (Matsunaga et al., 1993; Maekawa et al., 1998; Zhang et al., 2000; Katunuma et al., 1994), proteolytic regulation of *Corresponding author Tel.: +81-886-22-9611; fax: +81-886-22-2503 E-mail address: katunuma@tokushima.bunri-u.ac.jp (N Katunuma) 0065-2571/03/$ - see front matter r 2003 Elsevier Science Ltd All rights reserved doi:10.1016/S0065-2571(02)00041-9 394 N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 retinoic acid receptor a by cathepsin L on the modulation of thyroid hormone action (Nagaya et al., 1998) Therefore, intracellular regulation of cathepsin activities in situ by their endogenous inhibitors is very important The defect, or low function, of an individual cathepsin resulted in the specific metabolic error disease Physiological Functions of Endogenous Cysteine Protease Inhibitors, Including Cystatins Cystatin a (A) in skin and cystatin b (B) in liver was discovered by Katunuma group and Turk group, as the first endogenous proteinous inhibitor of cathepsins (Katunuma et al., 1995) The cystatin family is classified into two groups, one group having low molecular inhibitors with molecular weights of 10–15 kDa and the other high molecular weight inhibitors such as kinin in serum Various kinds of cystatins are located in various organs and they have a common sequence peptide part in their molecules as the binding site with cathepsins Intracellular cystatins are principally located in cytoplasm and also some cystatins are secreted into milk, tears, saliva or serum However, the mechanism of action of cystatins to inhibit the intralysosomal cathepsins has been unclear On the other hand, it was reported that the cystatins show strong bacteriocidal and virocidal functions due to inhibition of bacterial cathepsins Katunuma et al reported that phosphorylated cystatin a located in skin epidermis shows strong cidal action to Staphylococcus aureus V8 (Takahashi et al., 1994) and also Korant et al reported that proliferation of polio-virus is strongly inhibited (Korant et al 1985) Cysteine proteases play important roles in the metabolism and life in bacteria and viruses Recently a different type of cathepsin inhibitor from typical cystatin family was reported, by Hof et al., that is Von Ebner’s Grand (VEG) protein in human tears (Hof et al., 1997) The VEG protein inhibits cathepsins considerably; however the VEG protein contains only one homological sequence with a common active site of cystatin family, while the cystatin family has three common binding site sequences Therefore, lactoferrin may be said to be VEG protein type inhibitor Recently, we found a new VEG protein type inhibitor in milk and human tears and also we found completely different new type inhibitors from cystatin family in human tears in the case of some special autoimmune diseases The detection method of cysteine protease inhibitors in biological materials was used, that is a new reverse zymography technique for cysteine protease inhibitors This paper describes the finding of new cathepsin inhibitors in physiological materials and their medical significance from the aspects of pathogenesis and diagnosis Materials and methods Inhibition Analysis of Transferrin Family Against Cysteine Proteases Recombinant rat liver cathepsins B, L, and C were used for inhibitory assay Recombinant cathepsins K and S were expressed and purified according to the N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 395 methods of Katunuma (Katunuma et al., 1999), Kopitar (Kopitar et al., 1996), and Bossard (Bossard et al., 1996) The cysteine proteases were assayed using Z-Phe-ArgMCA as a substrate for cathepsins L, B, S, K and papain, following the method of Barrett (Barrett and Kirschke, 1981) Synthesized Peptide of Near C-terminus 17 Mer Peptide of Lactoferrin The near C-terminus 17 mer peptide (Y679–K695) of lactoferrin and 19 mer (L142– H160) of human b-casein were chemically synthesized by Asahi Technoglass Co (Chiba, Japan) with 95% purity The synthesized peptide sequences were YEKYLGPQYVAGITNLK (Y679–K695) and LTDVENLHLPLPLLQSWMH (L142–H160) Preparation of Intramolecular Peptides of b-casein Bovine b-casein (250 mg) in 100 mM Tris-HCl buffer pH 8.5 was digested by lysylendopeptidase at 35 C for 16 h The digested sample was applied to HPLC, TSK gel DDS-80Ts and eluted by a linear gradient using solvents of 0.1% TFA and 0.1% TFA in 90% acetonitrile The main eluted peaks were used to assay the inhibitory activities and to determine the amino acid sequences Analysis of N-terminus Amino Acid Sequence The N-terminus amino acid sequences of proteins and the isolated intramolecular peptides were determined with an HP G1005A protein sequencing system (Hewlett-Packard, Palo Alto, CA) After SDS-PAGE, the bands were transferred to a polyvinylidene difluoride membrane, and then were subjected to amino acid sequence analysis using Majima’s method (Majima et al., 2001) Negative Staining Method of SDS-PAGE Gel Negative staining of gel was performed by the method of Fernandez et al (1992) Samples of milk (10–15 ml) were mixed with the same amount of sample buffer (0.125 M Tris-HCl, 4% SDS, 20% glycerol, 0.02% bromophenol blue pH 6.8) After electrophoresis, the gels were incubated in a 0.2 M imidazole solution for 10 The incubation time could be modified depending on the acrylamide percentage Then the gels were transferred to a bath containing 0.2–0.3 M zinc sulfate for For visualization, the protein bands were cut and washed with 2% citric acid to remove the staining solution The gel pieces containing the protein bands were eluted and the eluates were used to check the inhibitory activity of the various authentic cysteine proteases 396 N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 Results and discussion Reverse Zymography for Detection of Cysteine Protease Inhibitors in Natural Materials We developed a new detection technique for cysteine protease inhibitors in crude natural materials, and named it ‘‘reverse zymography.’’ The principle of this detection method of protease inhibitors on SDS-PAGE gel is the reverse of usual zymography and the practical procedure is illustrated in Fig The inhibitor samples were applied to special SDS gels coagulated with gelatin or without gelatin as the control To digest the embedded gelatin, the gels were immersed in a papain (31 unit/ml) solution The embedded gelatin and the sample proteins were digested, thereby removing stainable proteins except where inhibitor bands were present These preserved gelatin bands, in which the inhibitors were located, were stained with Coomassie brilliant blue The SDS-PAGE was performed following the Laemmli method (Laemmli et al., 1920) SDS-polyacrylamide slab gels were cast with substrate gelatin as follows: slab gels were cast with 12.5% acrylamide, 0.3% bis-acrylamide and 0.1% gelatin, or without gelatin as the control The stacking gel contained 4% acrylamide and 0.14% bis-acrylamide Milk (10–15 ml) or tears was diluted with the same amount of a solution of 4.0% SDS, 20% glycerol, 0.25 Tris-Cl pH 6.8 (0.02% bromophenol) After electrophoresis was completed, the gel was removed, washed and transferred to a tray of 100 ml of acetate buffer at pH 5.5 containing mg papain (31 unit/ml) and incubated at 37 C for 10 h to digest the gelatin The gel was washed with distilled water and then stained with 0.025% Coomassie brilliant blue R250 The gels were then washed with destaining solution (40% methanol, 10% acetic acid, 50% distilled water) Putative protease inhibitors were detected as blue bands on a clear background The reverse zymography was compared with and without gelatin plates Basic Demonstrations for Detection of Authentic Protease Inhibitors using this Reverse Zymography Method To demonstrate specific detection of the correspondent inhibitors for the various target proteases using our reverse zymography, well-established authentic protease inhibitors and the pure correspondent proteases were employed For example, the pairs of cystatin C for papain, lactoferrin for papain and soybean trypsin inhibitor for trypsin, were demonstrated in Fig After electrophoresis of the gelatin gel applied cystatin C or lactoferrin, the gel was incubated with papain to hydrolyze the background gelatin for 10 h at 37 C The washed gel was stained with Coomassie blue Only the bands in which cystatin C and lactoferrin were located was stained in the position of 15 kDa and 78 kDa, respectively, because the embedded gelatin remained only in the inhibitor bands, as shown in Fig 2a and c, respectively Using the same method, soybean trypsin inhibitor was detected in the 25 kDa area using trypsin as the corresponding protease trypsin to remove the embedded gelatin as Fig 2a shows The without-gelatin gels were used as their controls (the N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 397 Fig Schematic illustration of reverse zymography procedure for specific detection of cysteine protease inhibitors correspondent proteases were treated) We could demonstrate the selective detection of correspondent inhibitors to various target proteases by choosing target proteases as digesting proteases of embedded gelatin in the gel Without-gelatin gels were used 398 N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 Fig Specific detection of authentic protease inhibitors by reverse zymography; soybean trypsin inhibitor for trypsin and cystatin C or lactoferrin for papain Lanes and are molecular markers (a) Soybean trypsin inhibitor was applied in lanes and 3, and embedded gelatin was digested out by trypsin; Lane is with gelatin gel and lane is without-gelatin gel as the control The remaining gelatin band in the soybean trypsin inhibitor band of 25 kDa is stained by Coomassie blue in lane (b) Cystatin C was applied in lanes and 5, and the embedded gelatin was digested out by papain; lane is with gelatin gel and lane is without-gelatin gel as the control The remaining gelatin band in the cystatin C band of 15 kDa is stained in lane (c) Lactoferrin was applied in lanes and 7, and the embedded gelatin was digested out by papain; lane is with gelatin gel and lane is without gelatin gel as the control The remaining gelatin in the lactoferrin band of 78 kDa is stained in lane The without gelatin control gels in lanes 3, and are not stained as the correspondent controls as shown in lanes 3, and of Fig The contaminated proteins in the natural materials were also digested out to produce a clean background Disease-specific Expression of New Inhibitors in Human Tears Inhibitory proteins of cysteine proteases in normal human tears More than 10 kinds of major protein components in normal human tears were detected using Coomassie-blue staining and also the negative staining of the SDSPAGE, as shown in Fig To detect the bands of cysteine protease inhibitors in N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 399 Fig Reverse zymography staining of human tears Lane is a molecular marker The same amount of human normal tears were applied in lanes and Lane was stained by the reverse zymography method Lane was stained all proteins in tears by Coomassie blue Lane was stained by reverse zymography with gelatin gel and three main papain inhibitors were stained in 72, 20 and 15 kDa human tears, our reverse zymography of gelatinolysis inhibition to papain was employed As shown in Fig 3, at least two different kinds of strong staining bands of 78 kDa and 15 kDa and very weak staining bands of 65 kDa and 20 kDa in normal tears were detected by reverse zymography and the reverse zymography patterns of eight normal individuals are demonstrated, although the comparative strengths showed some individual differences The 20 kDa band was stained using Western blot with polyclonal anti-VEG protein antibody, but not with anti-cystatin S antibody, and the 15 kDa band was stained specifically with anti-cystatin S antibody (data not shown) (Isemura et al., 1984) The nature of 72, 65, 20 and 15 kDa bands was finally determined as lactoferrin, Ig heavy chain-V-III region, Von Ebner’s Gland (VEG) protein and cystatin S, respectively, based on their amino acid sequences Very weak 65 kDa band was detected in rare case of normal tears 400 N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 Identification of inhibitors in normal tears based on sequence analysis The structures of the three bands, showing strong inhibitory activity, were identified using amino acid sequence analysis of their N-terminus area and/or their sequences of the intramolecular peptides The 20 kDa inhibitor was identical to VEG protein based on the amino acid sequence analysis, as shown in Fig (Korant et al., 1985), and the band was cross-reacted with polyclonal antibody of anti-19 mer of Nterminus peptide, L21–A39 (chemically synthesized), of VEG protein molecule The VEG protein was reported by Hof et al., to be a member of the cystatin super-family (Hof et al 1997) The 15 kDa inhibitor was estimated to be cystatin S, which is known as a member of cystatin family in saliva based on the molecular weight, the inhibitory profiles and the cross-reactivity with anti-cystatin S antibody (Isemura et al., 1984) The 78 kDa band inhibitor was determined as being a lactoferrin from the N-terminus sequence as Fig shows As shown in Fig 6, the near C-terminus peptide Y679–K695 of lactoferrin molecule showed a strong homologous sequence with a common active site (binding site) of the cystatin family Practically, this domain peptide synthesized showed considerable inhibition to various cysteine proteases as Table shows Fig Identification of 20 kDa inhibitor in reverse zymography with Von Ebner’s Grand protein based on N-terminus sequence Fig Identification of 78 kDa inhibitor in reverse zymography of human normal tears with lactoferrin based on N-terminus sequence The N-terminus 10 amino acid sequence of the 72 kDa inhibitor in reverse zymography of human tears is completely identical with N-terminus sequence of lactoferrin precursor protein N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 401 Fig Homology of lactoferrin sequence near the C-terminus with available high frequency amino acids of a common active site of the cystatin family Lactoferrin sequence of Y679–K695 showed 89% homology and 61% identity with that of active site of cystatin family and transferrin sequence of the correspondent part was 78% homology and 28% identity with that of cystatin family Table Inhibition specificities of lactoferrin and the synthetic peptide of an active site of lactoferrin to cysteine Inhibitors Target proteases Inhibition % at 10À7 M 10À6 M 10À5 M 10À4 M 40 100 100 0 80 90 100 100 0 0 50 10 70 50 10À3 M Lactoferrin Cathepsin Papain Cathepsin Cathepsin Cathepsin Trypsin Synthetic Y679–K695 L B S C Cathepsin L Papain Cathepsin B 100 100 50 Since the inhibition kinetics of lactoferrin to papain shows non-competitive type, it is suggested that the lactoferrins not compete with the synthetic substrate of papain Practically, recombinant lactoferrin and b-casein were not degraded after incubation with papain using the SDS-PAGE, as shown in Fig Lactoferrin has been known to possess bacterio-static action (Bhimani et al., 1999; Shimizu et al., 2002), but the mechanism was unknown We clarified that the inhibition of cysteine proteases by lactoferrin must play a major role in exhibiting bacteriocidal function, due to the strong cysteine protease inhibition of bacterias and viruses (Takahashi et al., 1994; Korant et al., 1985) Characteristic changes of the inhibitor profiles in pathological tears and detection of new disease-specific inhibitors in special autoimmune diseases The cysteine protease inhibitors in tears of special diseases showed characteristic changes from those of normal tears, and, furthermore, novel disease-specific 402 N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 Fig Lactoferrin and b-casein are not degraded by papain 10À5 M of lactoferrin or b-casein was incubated at 37 C for 30 These samples were applied to SDS-PAGE Lane was lactoferrin alone and Lane was incubated with papain Lane was b-casein alone and Lane was incubated with papain No degradation patterns were observed, while papain activities in lane and were completely inhibited for 30 by 10À5 M of these inhibitors inhibitors were found in some autoimmune diseases Characteristic reddish bands of 31 kDa stained strongly with Coomassie blue (the other normal bands were stained blue) were detected specifically in all eight cases of Behcet’s disease as Fig shows, which is an autoimmune disease, and also the lactoferrin content in these cases was relatively high The N-terminus sequence of the 31 kDa reddish band specifically detected in the cases of Behcet’s disease was 100% identical with that of human lachrymal acidic proline-rich protein as shown in Fig Furthermore, the prolinerich proteins are known to be stained a reddish color by Coomassie-blue staining, in N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 403 general Furthermore, the eluates of 31 kDa band area of SDS-PAGE (not stained) in the tears of Behcet’s disease showed about 50% inhibition of papain at 10À6 M protein concentration The cysteine protease inhibition of this kind of acidic prolinerich protein family has not been reported before In the tears of Harada’s disease (four cases), which is a typical autoimmune disease, the 65 kDa inhibitors were strong and the lactoferrin content was relatively weak compared with those of normal tears as shown in Fig The 65 kDa band inhibitor which was expressed strongly in the cases of Harada’s disease tears was determined as being a human Ig heavy chain V–III region based on the N-terminus sequence analysis as shown in Fig 10 The N-terminus 10 mer sequence of the 65 kDa band inhibitor was 100% identical to the human Ig heavy chain V–III region sequence These inhibitors may have a relation with pathogenesis of these autoimmune diseases The Ig heavy chain of variable III region was secreted extensively in Harada’s autoimmune-disease tears Quantitative changes of typical patterns of these inhibitors in special eye diseases are compared with a scanning densitometry method in Fig The characteristic changes in these inhibitor contents and the expression of disease-specific inhibitors were found in Behcet’s disease and Harada’s disease These unique changes of cysteine protease inhibitors in tears of special autoimmunediseases may not only lead to the elucidation of their pathogenesis, but also be useful for diagnosis The autoantigen of Behcet’s diseases is still unknown It may be speculated that the proline-rich proteins are possible candidates of specific autoantigen to induce Behcet’s diseases Lactoferrin and b-Casein in Milk as a New Cysteine Protease Inhibitor Detection of lactoferrin and b-casein as cysteine protease inhibitors in human and cow milk Human and cow milk was found to contain two cysteine protease inhibitors, lactoferrin and b-casein, using our reverse zymography for papain inhibition The main inhibition bands in cow and human milk were found with apparent molecular weights of 78 kDa and 35 kDa, which showed the same migration with recombinant lactoferrin and b-casein on their SDS-PAGE, respectively, as shown in Fig 11 Lane shows all the protein in milk using normal SDS-PAGE staining with Coomassie brilliant blue Lane shows the papain inhibition bands due to 78 kDa of lactoferrin and 35 kDa of b-casein in human milk (cow) using reverse zymography, and lane shows the control without gelatin plate Lanes and show reverse zymography of recombinant lactoferrin and lane shows the control without the gelatin plate Reverse zymography of recombinant human b-casein is shown in lanes and 8, and lane is the control without the gelatin plate Lactoferrin and b-casein are the major inhibitors of cysteine proteases in mammalian milk Identification of 78 and 35 kDa bands with lactoferrin and b-casein The 78 and 35 kDa staining bands of human milk were identified as lactoferrin and b-casein based on the analysis of their N-terminus sequences, as shown in Figs and 12, respectively The N-terminus 10 mer sequence of the 78 kDa band was completely 404 N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 405 Fig Identification of 31 kDa inhibitor in Behcet’s disease tears with lachrymal proline-rich protein Fig 10 Identification of 65 kDa inhibitor in Harada’s disease tears with the N-terminus of human Ig heavy chain V–III region identical with that of lactoferrin and also the N-terminus 15 mer sequence of the 35 kDa band was completely identical with that of human b-casein Furthermore, the eluates from the 78 kDa band and the 35 kDa band of negative staining SDS-PAGE gel of both milks showed the same inhibitory profiles to various cysteine proteases as those of recombinant lactoferrin and b-casein, respectively (data not shown) These samples were applied to SDS-PAGE Lane was lactoferrin alone and Lane was incubated with papain Lane was b-casein alone and Lane was incubated with papain No degradation patterns were observed, while papain activities in lanes and were completely inhibited for 30 by 10À5 M of these inhibitors Inhibition characteristics of human b-casein to cysteine proteases b-Casein inhibited papain completely at 10À6 M The inhibitory specificities of bcasein to various cysteine proteases are shown in Fig 13 b-Casein inhibited papain strongly and inhibited cathepsin L weakly at 10À5 M, but cathepsin B was not inhibited at 10À5 M However, we could not find a homological domain in b-casein molecule with an common active site sequence of cystatin family Therefore, the inhibition mechanisms must be different from that of cystatin The inhibition mode Fig Disease-specific inhibitor patterns in typical cases of special eye diseases were compared quantitatively using the gel scanning method of reverse zymography No band of the 72 kDa is lactoferrin No band of the 65 kDa is Ig heavy chain V–III region No reddish band of the 31 kDa is lachrymal acidic proline-rich protein of Behcet’s disease-specific inhibitor No band of the 20 kDa is VEG protein No band of the 15 kDa is cystatin S 406 N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 Fig 11 Detection of papain inhibitors in cow milk using reverse zymography of gelatinolysis inhibition Lanes and show molecular weight markers Lane shows all the proteins in cow milk stained by Coomassie brilliant blue Lanes and show reverse zymography of cow milk and lane shows it without the gelatin plate as the control Lanes and show reverse zymography of authentic lactoferrin and lane shows it without the gelatin plate as the control Lanes and show reverse zymography of authentic bcasein and lane shows it without the gelatin plate as the control of human b-casein to papain showed sigmoidal allosteric inhibition kinetics as shown in Fig 14(a) and (b) The inhibition kinetics of human b-casein showed a second order sigmoidal curve to the substrate and the reciprocal plot between 1=v and 1=½SŠ2 gave a straight-line as shown in Fig 14(b) A Hill constant was calculated as n ¼ 2:4 using the Hill equation of logv=Vm vị ẳ nlogẵS log Km (Vmax ẳ 9000 U and Km ¼ 0:0079) The hydrolyzed products of bovine b-casein by lysylendopeptidase showed about the same inhibition as that of intact b-casein The digested product peptides were separated using reverse-phase HPLC and the papain inhibitions of these main peptides were assayed and the inhibitory peptide sequences N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 407 Fig 12 Identity of N-terminus 15 mer sequence of 35 kDa inhibitor in human milk with b-casein The Nterminus 15 mer sequence of the 35 kDa inhibitor in human milk was completely identical with that of human b-casein and showed strong homology with that of bovine b-casein The active inhibition domains in human and bovine b-casein molecules are indicated as underlined sequences Determination of inhibitory domain in b-casein molecule b-Casein was digested by lysilendopeptidase and the digested product mixture showed strong inhibitory activity Therefore, the product was separated by HPLC, TSK gel DDS-80Ts and eluted by a linear gradient using solvents of 0.1% TFA and 0.1% TFA in 90% acetonitril The main eluted peaks were used to assay the inhibitory activities of papain and to determine the amino acid sequences To confirm the inhibitory activities, chemical synthesized peptides were used as shown in Table Fig 13 Inhibitory specificity of b-casein to various cysteine proteases were determined as LTDVENLHLPLPLLQSWMH (L142–H160) in bovine b-casein and LTDLENLHLPLPPLPLLQPLMH (L133–Q151) in human b-casein Both peptide sequences showed 79% identity and 84% homology with each other The 408 N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 Fig 14 Inhibition kinetics of b-casein to papain Panel (a): Substrate–activity relationship Without inhibitor: – and with b-casein E–E Panel (b): Reciprocal plotting of substrate–activity relationship using a Lineweaver–Burk plot Line – is without inhibitor Line ’–’ and m–mline are with b-casein and line ’–’ is reciprocal plotting of 1=v to 1=½SŠ: The symbols are the same as in Panel (a) Reciprocal plotting between 1=v and 1=½SŠ2 gave almost a straight line; m–m Second order sigmoidal curve by the Lineweaver–Burk equation was obtained Panel (c): Hill plot of papain inhibition with b-casein The equation of log v=Vm2v ẳ n logẵS À log Km was used for Vm ¼ 9000 U, Km ¼ 0:0079: Hill constant was calculated as n ¼ 2:4: synthesized peptide of L133–Q151 in human b-casein showed significant inhibition to papain, with 68% inhibition at 10À5 M and 100% inhibition at 10À4 M, and the other parts of the separated peptides showed no inhibition as shown in Table b-Casein is not only a nutritional protein, but also a cysteine protease inhibitor The biological role of cysteine protease inhibitors of transferrin family and b-casein in mammalian milk are important from the medical aspects One of the important functions of lactoferrin and b-casein in milk may be to exert inhibitory effects to cysteine proteases of bacteria and viruses to protect from infection (Takahashi et al., 1994; Korant et al., 1985) N Katunuma et al / Advan Enzyme Regul 43 (2003) 393–410 409 Table Inhibition percent of papain by various synthesized peptides in b-casein Peptides b-casein (Human) L133–Q151 (Human) V176–Q182 (Human) I64–Y175 (Bovine) Concentrations (M) 10À6 10À5 73% 0% 100% 68% 10À4 100% 0% 0% Summary New cysteine protease inhibitors in human tears and milk were found and their medical significance was studied As the protective components against bacterial infection in eyes, we detected four kinds of biologically active proteins in normal human tears including three kinds of cysteine protease inhibitors Using our reverse zymography of normal tears, the three kinds of cysteine protease inhibitors were found to be 78, 20 and 15 kDa and were determined to be lactoferrin, VEG protein and cystatin S, respectively The C-terminus area 17 mer peptide, Y679–K695 of lactoferrin molecule showed strong homology with a common active domain of cystatin family and the synthesized peptide itself showed considerable inhibition of cysteine proteases Not only disease-specific changes of these inhibitor contents, but disease-specific new inhibitors were also found in patient tears in special autoimmune diseases The characteristic 35 kDa inhibitor band which was detected specifically in the cases of Behcet’s disease tears, an autoimmune disease, was determined to be a lachrymal acidic proline-rich protein family based on the N-terminus sequence analysis The 65 kDa inhibitor of tears in Harada’s autoimmune-disease was determined to be a human Ig heavy chain V–III region Also lactoferrin content in Harada’s disease was very low compared with that of normal tears Also we found two cathepsin inhibitors, lactoferrin and b-casein, in milk of human and bovine using reverse zymography They may also play a role in bacterio-cidal and viro-cidal functions in milk The L133–Q151 in human b-casein molecule is the active inhibitory domain It is most important to know from biological aspects that the concentration of these inhibitors in natural milk can inhibit cysteine proteases of bacteria Surprisingly, the 50 times diluted milk inhibited papain completely, because lactoferrin and casein contents in milk are very high We want to emphasize that these inhibitors in milk play a sufficient role in the protection of bacteria References Barrett AJ, Kirschke H Cathepsin B, cathepsin H, cathepsin L Methods Enzymol 1981;80:535–61 Bhimani RS, Vendrov Y, Furmanski P Influence of lactoferrin feeding and injection against systemic staphylococcal infections in mice J Appl Microbiol 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Endogenous Cysteine Protease Inhibitors, Including Cystatins Cystatin a (A) in skin and cystatin b (B) in liver was discovered by Katunuma group and Turk group, as the first endogenous proteinous inhibitor... Lactoferrin Cathepsin Papain Cathepsin Cathepsin Cathepsin Trypsin Synthetic Y679–K695 L B S C Cathepsin L Papain Cathepsin B 100 100 50 Since the inhibition kinetics of lactoferrin to papain shows... of cysteine protease inhibitors in biological materials was used, that is a new reverse zymography technique for cysteine protease inhibitors This paper describes the finding of new cathepsin inhibitors