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: (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 min. 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 1 min. 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. 1. 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 1 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. 2. 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. 1. 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. 2. Specific detection of authentic protease inhibitors by reverse zymography; soybean trypsin
inhibitor for trypsin and cystatin C or lactoferrin for papain. Lanes 1 and 8 are molecular markers. (a)
Soybean trypsin inhibitor was applied in lanes 2 and 3, and embedded gelatin was digested out by trypsin;
Lane 2 is with gelatin gel and lane 3 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 2. (b) Cystatin C was
applied in lanes 4 and 5, and the embedded gelatin was digested out by papain; lane 4 is with gelatin gel
and lane 5 is without-gelatin gel as the control. The remaining gelatin band in the cystatin C band of
15 kDa is stained in lane 4. (c) Lactoferrin was applied in lanes 6 and 7, and the embedded gelatin was
digested out by papain; lane 6 is with gelatin gel and lane 7 is without gelatin gel as the control. The
remaining gelatin in the lactoferrin band of 78 kDa is stained in lane 6. The without gelatin control gels in
lanes 3, 5 and 7 are not stained.

as the correspondent controls as shown in lanes 3, 5 and 7 of Fig. 2. 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. 3. To detect the bands of cysteine protease inhibitors in

N. Katunuma et al. / Advan. Enzyme Regul. 43 (2003) 393–410

399

Fig. 3. Reverse zymography staining of human tears. Lane 1 is a molecular marker. The same amount of
human normal tears were applied in lanes 2 and 3. Lane 2 was stained by the reverse zymography method.
Lane 3 was stained all proteins in tears by Coomassie blue. Lane 2 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. 4 (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. 5 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 1 shows.

Fig. 4. Identification of 20 kDa inhibitor in reverse zymography with Von Ebner’s Grand protein based
on N-terminus sequence.

Fig. 5. 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. 6. 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 1
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
0

100
100
0
0

80
90

100

100
0
0

0
0

50
10

70
50
0

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 do 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. 7. 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. 7. Lactoferrin and b-casein are not degraded by papain. 10À5 M of lactoferrin or b-casein was
incubated at 37 C for 30 min. These samples were applied to SDS-PAGE. Lane 2 was lactoferrin alone
and Lane 3 was incubated with papain. Lane 3 was b-casein alone and Lane 4 was incubated with papain.

No degradation patterns were observed, while papain activities in lane 3 and 5 were completely inhibited
for 30 min 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. 8 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. 9. 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. 8. 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. 8.

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
2 shows all the protein in milk using normal SDS-PAGE staining with Coomassie
brilliant blue. Lane 3 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 4
shows the control without gelatin plate. Lanes 5 and 6 show reverse zymography of
recombinant lactoferrin and lane 6 shows the control without the gelatin plate.
Reverse zymography of recombinant human b-casein is shown in lanes 7 and 8, and
lane 8 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. 5 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. 9. 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 2 was lactoferrin alone and Lane 3 was
incubated with papain. Lane 3 was b-casein alone and Lane 4 was incubated with
papain. No degradation patterns were observed, while papain activities in lanes 3
and 5 were completely inhibited for 30 min 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. 8. Disease-specific inhibitor patterns in typical cases of special eye diseases were compared
quantitatively using the gel scanning method of reverse zymography. No. 1 band of the 72 kDa is

lactoferrin. No. 2 band of the 65 kDa is Ig heavy chain V–III region. No. 4 reddish band of the 31 kDa is
lachrymal acidic proline-rich protein of Behcet’s disease-specific inhibitor. No. 5 band of the 20 kDa is
VEG protein. No. 6 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 1 and 9 show molecular weight markers. Lane 2 shows all the proteins in cow milk stained by
Coomassie brilliant blue. Lanes 3 and 4 show reverse zymography of cow milk and lane 4 shows it without
the gelatin plate as the control. Lanes 5 and 6 show reverse zymography of authentic lactoferrin and lane 6
shows it without the gelatin plate as the control. Lanes 7 and 8 show reverse zymography of authentic bcasein and lane 8 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 logðv=Vm À vÞ ¼ nlog½SŠ À log Km
(Vmax ¼ 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 2.

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 ¼ n log½SŠ 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 2.
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 2
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%

4. 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 1999;86(1):135–44.


410

N. Katunuma et al. / Advan. Enzyme Regul. 43 (2003) 393–410

Bossard MJ, Tomaszek TA, Thompson SK, Amegadzie BY, Hanning CR, Jones C, Kurdyla JT, McNulty
DE, Drake FH, Gowen M, Levy MA. Proteolytic activity of human osteoclast cathepsin K.
Expression, purification, activation, and substrate identification. J Biol Chem 1996;271:12517–24.
Fernandez CP, Castellanos S, Rodriguez P. Reverse staining of SDS-PAGE gels by imidazole-zinc salt.
Sensitive detection of unmodified protein. Biotechniques 1992;12:564–73.
Hof WV, Blankenvoorde MF, Veerman EC, Amerongen AV. The salivary lipocalin von Ebner’s gland
protein is a cysteine proteinase inhibitor. J Biol Chem 1997;272(3):1837–41.
Isemura S, Saitoh E, Ito S, Isemura M, Sanada K, Cystatin S. A cysteine proteinase inhibitor of human
saliva. J Biochem 1984;96:1311–4.
Katunuma N, Kominami E. Structure, properties, mechanisms, and assays of cysteine protease inhibitors:
cystatins and E-64 derivatives. Methods Enzymol 1995;251:382–97.
Katunuma N, Matsunaga Y, Saibara T. Mechanism and regulation of antigen processing by cathepsin B.
Adv Enzyme Regul 1994;34:145–58.

Katunuma N, Murata E, Kakegawa H, Matsui A, Tsuzuki H, Tsuge H, Turk D, Turk V, Fukushima M,
Tada Y, Asao T. Structure based development of novel specific inhibitors for cathepsin L and
cathepsin S in vitro and in vivo. FEBS Lett 1999;458:6–10.
Kopitar G, Dolinar M, Strukelj B, Pungercar J, Turk V. Folding and activation of human procathepsin S
from inclusion bodies produced in Escherichia coli. Eur J Biochem 1996;236:558–62.
Korant BD, Brzin J, Turk V. Cystatin, a protein inhibitor of cysteine proteases alters viral protein
cleavages in infected human cells. Biochem Biophys Res Commun 1985;127:1072–6.
Laemmli KU. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature
1920;227:680–5.
Maekawa Y, Himeno K, Ishikawa H, Hisaeda H, Sakai T, Dainichi T, Asao T, Good RA, Katunuma N.
Switch of CD4+ T Cell differentiation from Th2 to Th1 by treatment with cathepsin B inhibitor in
experimental Leishmaniasis. J Immunol 1998;161:2120–7.
Majima E, Ishida M, Miki S, Shimohara Y, Tada H. Specific labeling of the bovine mitochondrial
phosphate carrier with fluorescein 5-isothiocyanate. J Biol Chem 2001;276(13):9792–9.
Matsunaga Y, Saibara T, Kido H, Katunuma N. Participation of cathepsin B in processing of antigen
presentation to MHC class II. FEBS Lett 1993;324(3):325–30.
Nagaya T, Murata Y, Yamaguchi S, Nomura Y, Ohmori S, Fujieda M, Katunuma N, Yen PM, Chin
WW, Seo H. Intracellular proteolytic cleavage of 9-cis-retinoic acid receptora by cathepsin L-type
protease is a potential mechanism for modulating thyroid hormone action. J Biol Chem 1998;
273(50):33166–73.
Shimizu N, Ikehara Y, Tatematsu M. Effect of lactoferrin in H. Pylori infection animal model.
Lactoferrin. Amsterdam: Elsevier, 2002. p. 209–51.
Takahashi M, Tezuka T, Katunuma N. Inhibition of growth and cysteine proteinase activity of
Staphylococcus aureus V8 by phosphorylated cystatin a in skin cornified envelop. FEBS Lett 1994;
355:275–8.
Zhang T, Maekawa Y, Hanba J, Dainichi T, Nashed BF, Hisaeda H, Sakai T, Asao T, Himeno K, Good
RA, Katunuma N. Lysosomal cathepsin B plays an important role in antigen processing, while
cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice.
Immunology 2000;100:13–20.