1)Tokushima Bunri University, Institute for Health
Sciences, Tokushima, Japan ; 2)Department of
Opthalmology and Visual Neuroscience, The University of
Tokushima School of Medicine, Tokushima, Japan; and 3)Biotechnology
Center, Vietnam National University, Hanoi, Vietnam
Abstract: New cysteine protease inhibitors in human tears
and milk and their medical significance are reviewed in
this paper. As protective components against bacterial infection
in the eyes, we detected four kinds of anti-bacterial proteins
in normal human tears including lysozyme and three kinds
of cysteine protease inhibitors. Using our reverse zymography
of normal tears, three kinds of cysteine protease inhibitors
were found to be 78kDa, 20kDa and 15kDa and were determined
to be lactoferrin, Von Ebner's Gland (VEG) protein and cystatin
S, respectively. All of them belong to the cystatin super
family and VEG protein and cystatin S are well known cysteine
protease inhibitors. The C-terminus area 17mer peptide,
Y679-K695, of lactoferrin showed strong homology with a
common active domain of the cystatin family and the synthesized
peptide showed inhibition of cysteine proteases. Not only
were disease-specific changes found in these inhibitor profiles,
but also disease-specific new inhibitors in patients tears
with certain autoimmune diseases. A 35kDa inhibitor, which
was detected specifically in tears with Behcet's disease,
an typical autoimmune disease, was determined to be a lacrimal
acidic proline-rich protein based on the N-terminus sequence
analysis. A65kDa inhibitor of tears with Harada's autoimmune
disease was determined to be an Ig heavy chain V-III region.
In addition, lactoferrin content in Harada's disease was
very low. We found two cathepsin inhibitors in bovine milk
using reverse zymography, namely lactoferrin andβ-casein.
The L133-Q151, in the humanβ-casein molecule is
the active inhibitory domain. They may play an important
role in antiseptic and anti-infectious functions. J. Med.
Invest. 50:154-161, 2003
Keywords:cystein protease inhibitor, human tears, reverse
zymography, Behcet's disease, lactoferrin, milk, autoimmune
disease
INTRODUCTION
[I] Physiological roles of cysteine proteases.
Cysteine proteases, cathepsins, play an essential role in
maintaining life in all living organisms. Cysteine proteases
are synthesized in bound ribosomes and secreted from the
trans-golgi, and then translocated into two ways, one is
targeted into lysosomes and the other is secreted to the
outside of cells through the secretion vesicles. Cathepsins
located in lysosomes mainly play a role in protein catabolism
via autophagy and heterophagy. On the other hand, the secreted
cathepsins play a role in the processing of various biological
active proteins, such as the processing of cytokines, hormones
and inflammatory peptides. About 10 kinds of cathepsins
have been reported and they have different functions and
different catalytic properties. They play roles not only
in protein catabolism, but also in the production of biologically
active peptides by their limited proteolysis, such as antigen
processing to present to MHC class ?(1 - 4), and also in
the processing of various precursor proproteins (5). The
hyper-function or low function of certain cathepsins results
in specific metabolic error diseases. Therefore, intracellular
regulation of cathepsin activities by their endogenous inhibitors
in situ is very important.
[II] Physiological functions of endogenous cysteine protease
inhibitors.
Cystatin α (A) in the skin and cystatin β
(B) in the liver have been discovered by Katunuma's group
and Turk's group, as the first endogenous proteinous inhibitors
of cathepsins in mammals (6, 22). The cystatin family is
classified into two groups, one group having low molecular
weight with molecular weights of 10 -20 kDa, the others
being high molecular weight inhibitors having a repeated
peptide domain, such as kinin in the serum. Various kinds
of cystatins are located in various organs and secretory
fluids and they have three common binding domains with cathepsins
in their molecules. Intracellular cystatins are principally
located in the cytoplasm and various cystatins are also
secreted into physiological secretory fluids such as tears,
saliva or serum. However, the mechanism of action of cystatins
in regulating the intralysosomal cathepsins has been relatively
unclear. On the other hand, it was reported that the cystatins
show strong bactericidal and virucidal functions due to
the inhibition of cathepsins in bacteria. Katunuma et al
reported that phosphorylated cystatin α located
in the skin epidermis shows strong bactericidal action to
Staphylococcus aureus V8 (7) and also Korant et al. reported
that proliferation of poliovirus is strongly inhibited (8).
Because, cysteine proteases in bacteria and viruses play
important roles in maintaining their metabolism and life.
Therefore, cysteine protease inhibitor play important roles
in anti-septic and anti-infectious functions.
Recently, a different type of cathepsin inhibitor from the
typical cystatin family was reported, by Hof et al., that
is, Von Ebner's Gland (VEG) protein in human tears(9). The
VEG protein inhibits cathepsins considerably, however the
VEG protein contains only one homologous active domain sequence
with a common active site with the cystatin family, while
the cystatin family possesses is three common binding sites.
Since lactoferrin possesses only one binding domain, it
may be considered a VEG protein-type inhibitor, although
it belongs to the cystatin super family.
Furthermore, we found some novel types of new inhibitors
from the cystatin family in human tears with certain autoimmune
diseases.
A new method for detecting cysteine protease inhibitors
in biological materials, named “reverse zymography",
was used in this study.
MATERIALS AND METHODS
Inhibition analysis of the 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 methods of Katunuma (10),
Kopitar (11), and Bossard(12). The cysteine proteases were
assayed using Z-Phe-Arg-MCA as a substrate for cathepsins
L, B, S, K and papain, following the method of Barrett (13).
Synthesized peptide of near C-terminus 17 mer peptide of
lactoferrin
The near C-terminus17mer peptide (Y679-K695)of lactoferrin
and 19mer (L142-H160) of human β-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 β-casein
Bovine β-casein (250µg) in 100mM Tris-HCl
buffer pH8.5 was digested by lysylendopeptidase at 35°C
for 16 hours. The digested sample was applied to HPLC, TSK
gel DDS-80 Ts 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 the 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 subjected
to amino acid sequence analysis using Majima's method (14).
Negative staining method of the SDS-PAGE gel
Negative staining of the gel was performed by the method
of Fernandez, C. et al. (15). Samples of milk(10 -15µl)
were mixed with the same amount of sample buffer (0.125M
Tris-HCl 4% SDS, 20% glycerol, 0.02%bromophenol blue pH
6.8). After the electrophoresis, the gels were incubated
in a 0.2M imidazole solution for 10 minutes. The incubation
time could be modified depending on the acrylamide percentage.
Then, the gels were transferred to a bath containing 0.2-0.3M
zinc sulfate for1minute. 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.
RESULTS AND DISCUSSION
[1] Reverse zymography for the 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"as shown in Fig.1. The principal of this
detection method of protease inhibitors on SDS -PAGE is
the reverse of that in usual zymography. The inhibitor samples
were applied to special SDS gels co-coagulated with gelatin
or without gelatin as the control. To digest the embedded
gelatin, the gels were incubated with papain solution. The
embedded gelatin and the coexistent proteins in the sample
were digested. Gelatin preserved in the bands, in which
the inhibitors were located, was stained with Coomassie
brilliant blue. The SDS-PAGE was performed following the
Laemmli method (16). Milk or tears were diluted with the
same amount of a solution. After electrophoresis, the gel
was removed, washed and transferred to a tray of 100 ml
of acetate buffer containing papain and incubated at 37
°C for 10hours to digest the gelatin. The gel was
washed with distilled water and then stained with Coomassie
brilliant blue. The gels were then washed with destaining
solution. Putative protease inhibitors were detected as
blue bands on a clear background. The reverse zymography
was compared with and without gelatin plates.
[2] Basic demonstrations for detection of authentic protease
inhibitors using the reverse zymography method
To demonstrate the corresponding inhibitors against the
various target proteases using our reverse zymography, well-established
authentic protease inhibitors and the pure corresponding
proteases were employed. For example, the pairs for reverse
zymography, cystatin C for papain, lactoferrin for papain
and soybean trypsin inhibitor for trypsin, are demonstrated
in Fig.2. After electrophoresis of the gelatin gel to which
cystatin C or lactoferrin was applied, the gel was incubated
with papain to hydrolyze the background gelatin. The washed
gel was stained with Coomassie brilliant blue. Only the
bands in which cystatin C or lactoferrin was located were
stained at positions corresponding to15kDa or 78kDa, respectively,
because the embedded gelatin in the inhibitor bands remained,
as shown in Fig.2-B or Fig.2-C, respectively. Using the
same method, soybean trypsin inhibitor was detected in the
25 kDa area using trypsin as the corresponding digestive
protease to remove the embedded gelatin as Fig.2-A shows.
The without-gelatin gels were used as their controls. We
could selectively detected corresponding inhibitors to various
target proteases by choosing various target proteases as
digesting proteases of the embedded gelatin in the gel.
Without-gelatin gels were used as the corresponding controls
as shown in lanes 3, 5 or7 in Fig.2. The contaminated proteins
in the natural materials were also digested out to produce
a clean background. The remaining gelatin in the inhibitor
band was stained blue on a white background.
[3] Disease-specific expression of new inhibitors in human
tears
(1) Inhibitory proteins of cysteine proteases in normal
human tears
More than10kinds of major protein components in normal human
tears were detected using Coomassie brilliant blue staining
of SDS -PAGE and as well as by the negative staining of
the SDS -PAGE, as shown in Fig.3. To detect the bands of
cysteine protease inhibitors in 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 78kDa and 15kDa and very weak staining
bands of 65kDa and 20kDa in normal tears were detected by
the reverse zymography, although the comparative strengths
showed some individual differences. And very weak 65kDa
band was detected in a rare case of normal tears.
(2) Identification and properties of normal tears inhibitors
The structures of the three bands, showing strong inhibitory
activity, were identified using amino acid sequence analysis
of their N-terminus area and/or the sequences of their intramolecular
peptides. The20kDa inhibitor was identical to VEG protein
based on the amino acid sequence analysis (8), and the band
was cross-reacted with polyclonal antibody against anti-19mer
of N-terminus peptide, L21-A39(chemically synthesized),
of the VEG protein molecule. The VEG protein was reported
by Hof et al. to be a member of the cystatin super-family
(8). The 15kDa inhibitor was estimated to be cystatin S,
which is known to be a member of the cystatin family in
saliva based on the molecular weight, the inhibitory profiles
and the cross-reactivity with anti-cystatin S antibody (17).
The 78 kDa band inhibitor was determined as being a lactoferrin
from the N-terminus sequence (20). The natures of the78kDa,
65kDa, 20kDa and 15kDa bands were finally determined as
lactoferrin, Ig heavy chain-V-? region, VEG protein and
cystatin S, respectively, based on their amino acid sequence
analysis. As shown in Fig.4, the near C-terminus peptide
Y679-K695, of the lactoferrin molecule showed a strong homologous
sequence with a common active site (binding site) of the
cystatin family. In practice, this domain peptide synthesized
chemically showed considerable inhibition to various cysteine
proteases. Since the inhibition kinetics of lactoferrin
to papain is of a non-competitive type, it is suggested
that the lactoferrin does not compete with the synthetic
substrate of papain. Authentic lactoferrin and β-casein
were not degraded after incubation with papain using SDS-PAGE.
Lactoferrin has been known to possess bacteriostatic action(18,
19), but the mechanism has not been determined. We clarified
that the inhibition of cysteine proteases by lactoferrin
must play a major role in exhibiting bactericidal and anti-septic
functions, due to the strong cysteine protease inhibition
of bacteria and viruses (7, 8).
(3) Characteristic changes in the inhibitor profiles in
pathological tears and detection of new disease-specific
inhibitors in tears with specific autoimmune diseases
The cysteine protease inhibitor profiles in tears of certain
diseases showed characteristic changes from those of normal
tears, and furthermore, novel disease-specific inhibitors
were found in some autoimmune diseases (21). Characteristic
reddish bands of 31kDa stained strongly with Coomassie brilliant
blue were detected specifically in all eight cases of Behcet's
disease which is an autoimmune disease, as Fig.5 (b) shows,
and the lactoferrin contents in these cases were relatively
high. The N-terminus sequence of the 31kDa reddish band
specifically detected in the cases of Behcet's disease was100%
identical with that of human lacrimal acidic proline-rich
protein. Furthermore, the eluates of the31kDa band area
of SDS-PAGE (not stained) in the tears with Behcet's disease
showed about 50% inhibition of papain at the protein concentration
of 10-6 M. The cysteine protease inhibition of this kind
of acidic proline-rich protein family has not been reported
before. In the tears from four cases with Harada's disease,
which is a typical autoimmune disease, the 65kDa inhibitors
were strong and the lactoferrin content was relatively weak
compared with those of normal tears as shown in Fig.5 (c).
The65kDa band inhibitor expressed strongly in the cases
of Harada's disease tears was determined as being a human
Ig heavy chain V-? region based on the N-terminus sequence
analysis. The N-terminus 10 mer sequence of the 65kDa band
inhibitor was 100% identical with the human Ig heavy chain
V-? region sequence. These inhibitors may have a relationship
with the pathogenesis of these autoimmune diseases. The
Ig heavy chain of variable ? region was secreted extensively
in Harada's autoimmune disease tears. Quantitative changes
in the typical patterns of these inhibitors in specific
eye diseases are compared by a scanning densitometry method
in Fig.5.
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
in cysteine protease inhibitors in tears with specific autoimmune
diseases may not only lead to the elucidation of their pathogenesis,
but also be useful for differential diagnosis. The autoantigen
of Behcet's diseases is still unknown. It could be speculated
that the proline-rich proteins are possible candidate of
specific autoantigens that induce Behcet's disease, because
these proteins belong to the collagen family.
[4] Lactoferrin and β-casein in mammalian milk
as cysteine protease inhibitors
(1) Detection of lactoferrin and β-casein as cysteine
protease inhibitors in human and cow milk
Human and cow milk were found to contain two cysteine protease
inhibitors, namely lactoferrin and β-casein, using
our reverse zymography for papain inhibition. The main inhibition
bands in cow and human milk were found with apparent molecular
weights of78kDa and 35kDa, which showed the same migration
with recombinant lactoferrin and β-casein on their
SDS-PAGE, respectively, as shown in Fig.6. Lane1shows all
the proteins in milk using normal SDS-PAGE staining with
Coomassie brilliant blue. Lane 2 shows the papain inhibition
bands due to 78kDa of lactoferrin and 35kDa of β-casein
in milk using reverse zymography, and lane3shows the control
without the gelatin plate. Lanes4and lane 5 show reverse
zymography of recombinant lactoferrin and lanes 5shows the
control without the gelatin plate. Reverse zymography of
recombinant human β-casein is shown in lanes 6
and 7, and lane 8 is the control without the gelatin plate.
Lactoferrin and β-casein are the major inhibitors
of cysteine proteases in mammalian milk.
The78kDa and35kDa staining bands of human milk were identified
as lactoferrin and β-casein based on analysis of
their N-terminus sequences. The N-terminus10mer sequence
of the 78kDa band was completely identical to that of lactoferrin
and the N-terminus 15mer sequence of the 35kDa band was
completely identical with that of human β-casein.
Furthermore, the eluates from the 78kDa band and the 35kDa
band of the negative staining SDS -PAGE gel showed the inhibition
of various cysteine proteases.
(2) Inhibition characteristics of human β-casein
to cysteine proteases
β-Casein inhibited papain completely at 10-6 M.
The inhibitory specificities of β-casein to various
cysteine proteases were tested. β-Casein inhibited
papain strongly and inhibited cathepsin L weakly at 10-5
M, but cathepsin B was not inhibited at10-5 M. However,
we could not find homologous domain in the β-casein
molecule with a common active site sequence of the cystatin
family. Therefore, the inhibition mechanism must be different
from that of cystatin. The inhibition mode of human β-casein
to papain showed sigmoidal allosteric inhibition kinetics.
The inhibition kinetics of human β-casein showed
a second order sigmoidal curve to the substrate and the
reciprocal plot between1/v and1/[S]2gave a straight-line.
A Hill constant was calculated as n=2.4using the Hill equation
of log (v/Vm-v)= n log [S]-log Km (Vmax=9,000U and Km=0.0079).
The hydrolyzed products of bovine β-casein by lysylendopeptidase
showed about the same inhibition as that of intact β-casein.
The product peptides by the digestion were separated using
reverse-phase HPLC, the papain inhibitions of these main
peptides were assayed and the inhibitory peptide sequences
were determined as LTDVENLHLPLPLLQSWMH (L142-H160)in bovine
β-casein and LTDLENLHLPLPPLPLLQPLMH (L133-Q151)
in human β-casein. Both peptide sequences showed
79% identity and 84% homology to each other. The synthesized
peptide of L133-Q151in human β-casein showed significant
inhibition to papain, and the peptide showed 68% inhibition
at10-5M and 100% inhibition at 10-4M, but the other parts
of the separated peptides showed no inhibition. β-Casein
is not only a nutritional protein source, but also plays
a role as a cysteine protease inhibitor. The biological
roles of cysteine protease inhibitors of lactoferrin and
β-casein in mammalian milk are important from medical
aspects. One of the important functions of lactoferrin and?-casein
in milk may be to exert inhibitory effects to cysteine proteases
of bacteria and viruses to play a role in anti-infectious
and anti-septic functions (7, 8).
These contents were published in the following original
papers.
1)A. Ohashi, E. Murata, K. Yamamoto, E. Majima, E. Sano,
Q. T. Le and N. Katunuma: New functions of lactoferrin and
β-casein in mammalian milk as cysteine protease
inhibitors., BBRC 306, 98-103 (2003). 2)Q. T. Le, E. Sano,
A. Ohashi, E. Murata, H. Shiota, M. Ishimaru, K. Yamamoto,
E. Majima, S. Isemura, and N. Katunuma : Disease-specific
cysteine protease inhibitors in tears with autoimmune-diseases.
(2003)in preparation of Archives of Ophthalmology. 3) N.
Katunuma, A. Ohashi, E. Sano, E. Murata, H. Shiota, K. Yamamoto,
E. Majima and Q. T. Le : New cysteine protease inhibitors
in physiological secretory fluids and their medical significances.,
Advances in Enzyme Regulation 43, 393-410 (2003).
ACKNOWLEDGEMENTS
Many thanks to Miss Rika Takahashi for her valuable help
in preparing this manuscript.
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