Zeeuwen 2004-evidence that unrest

Human Molecular Genetics, 2004, Vol. 13, No. 10 1069–1079
DOI: 10.1093/hmg/ddh115
Advance Access published on March 25, 2004
Evidence that unrestricted legumain activity is
involved in disturbed epidermal cornification in
cystatin M/E deficient mice
Patrick L.J.M. Zeeuwen1,*, Ivonne M.J.J. van Vlijmen-Willems1, Diana Olthuis1,
Harald T. Johansen2, Kiyotaka Hitomi3, Ikuko Hara-Nishimura4, James C. Powers5,
Karen E. James5, Huub J. op den Camp6, Rob Lemmens1 and Joost Schalkwijk1
1Department of Dermatology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen,
PO Box 9101, 6500 HB Nijmegen, The Netherlands, 2Department of Pharmacology, School of Pharmacy, Oslo,
Norway, 3Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya, Japan, 4Department of Botany, Graduate School of Science, Kyoto University,
Kyoto, Japan, 5School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia,
USA and 6Department of Microbiology, Faculty of Science, University of Nijmegen, The Netherlands
Received January 28, 2004; Revised and Accepted March 11, 2004
Homozygosity for Cst6 null alleles causes the phenotype of the ichq mouse, which is a model for human
harlequin ichthyosis (OMIM 242500), a genetically heterogeneous group of keratinization disorders. Here we
report evidence for the mechanism by which deficiency of the cysteine protease inhibitor cystatin M/E (the Cst6
gene product) leads to disturbed cornification, impaired barrier function and dehydration. Absence of cystatin
M/E causes unrestricted activity of its target protease legumain in hair follicles and epidermis, which is the
exact location where cystatin M/E is normally expressed. Analysis of stratum corneum proteins revealed a
strong decrease of soluble loricrin monomers in skin extracts of ichq mice, although normal levels of loricrin
were present in the stratum granulosum and stratum corneum of ichq mice, as shown by immunohisto-chemistry.
This suggested a premature or enhanced crosslinking of loricrin monomers in ichq mice by
transglutaminase 3 (TGase 3). In these mice, we indeed found strongly increased levels of TGase 3 that was
processed into its activated 30 and 47 kDa subunits, compared to wild-type mice. This study shows that cystatin
M/E and legumain form a functional dyad in epidermis in vivo. Disturbance of this protease–antiprotease
balance causes increased enzyme activity of TGase 3 that could explain the observed abnormal cornification.
INTRODUCTION
Over the past decade, significant progress has been made in our
understanding of the molecular basis of disorders of epidermal
differentiation (1– 4). A variety of genes have been identified
that underlie inherited forms of ichthyosis, keratoderma and
skin fragility. The encoded proteins have diverse cellular
functions, ranging from structural properties (loricrin, keratins),
cell–cell adhesion (desmoplakin, desmoglein-1, plakophilin,
plakoglobin), lipid metabolism and/or trafficking (steroid
sulfatase, fatty aldehyde dehydrogenase, lipoxygenases,
ABCA12), regulation of post-translational protein proces-sing
(transglutaminase 1, cathepsin C, LEKT1), intercellular
communication (connexins) and calcium signaling (ATP2C1,
ATP2A2). Despite these advances, a number of genodermatoses
characterized by disturbed cornification remain unexplained at
the molecular level. These include, for example, forms of
lamellar ichthyosis that are not caused by known mutations,
ichthyosis vulgaris (OMIM 146700) and harlequin ichthyosis
(OMIM 242500). Harlequin ichthyosis (HI) is a severe
congenital skin disorder usually leading to a stillborn fetus or
early neonatal death. Its clinical features at birth include
ectropion, eclabium, ear dysmorphology and a thickened
fissured epidermis (5). We have recently demonstrated that
the phenotype of the ichq mouse, a model for human HI, is
caused by a null mutation in the Cst6 gene leading to
deficiency for the epidermal cysteine protease inhibitor cystatin
M/E (6). The ichq mice phenotype has morphological and
*To whom correspondence should be addressed. Tel: þ31 243617245; Fax: þ31 243541184; Email: p.zeeuwen@derma.umcn.nl
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biochemical similarities to human HI, which includes excessive
epidermal and follicular hyperkeratosis, abnormal large
mitochondria, and a characteristic keratin expression pattern
in the interfollicular epidermis. Although we have so far
excluded CST6 mutations in humans as a major cause of
harlequin ichthyosis type 2, CST6 and genes that have a role in
biological pathways that are controlled by cystatin M/E, should
be considered as candidate genes for human disorders of
cornification of unknown etiology (7). The function of cystatin
M/E in normal epidermal homeostasis is unknown and the
mechanism that leads to the observed pathology in ichq mice
remains unresolved. Clearly, the identification of its target
protease(s) in vivo would be an essential step in answering
these questions.
A biochemical study on the novel asparaginyl endopeptidase
legumain has indicated that cystatin M/E in vitro binds to this
protease with high affinity and could possibly represent a
physiological target enzyme, although no biological evidence
has been provided so far (8). Legumain or asparaginyl
endopeptidase (AEP, EC 3.4.22.34) belongs to clan CD of
the cysteine proteases and has a strict specificity for hydrolysis
of asparaginyl bonds. Initially, the enzyme was purified as a
cysteine protease responsible for the maturation of seed storage
proteins and designated vacuolar processing enzyme (VPE) (9).
Subsequently, legumain has been described in mammals (10); it
is found at low levels in many tissues, but is particularly
abundant in kidney. Very recently, the generation of legumain
deficient mice has been reported (11). Limited functional
studies have shown disturbed biosynthetic processing of
cathepsins and accumulation of macromolecules in the
lysosomes in the proximal tubule cells of the kidney.
In the present study, we have investigated the mechanism by
which absence of cystatin M/E causes epidermal abnormalities
in ichq mice. We report that legumain is a physiologically
relevant target protease of cystatin M/E in vivo. Cystatin M/E
deficiency in ichq mice leads to free cutaneous legumain
activity at exactly the location where cystatin M/E is normally
present in wild-type mice. These mice show increased
transepidermal water loss and neonatal death, probably due to
dehydration. We provide evidence that disturbed cornification
is caused by abnormalities in loricrin processing, caused by
uncontrolled legumain activity.
RESULTS
Skin barrier function is severely disturbed in cystatin
M/E deficient mice
Cystatin M/E deficient mice have defects in epidermal
cornification and die between 5 and 12 days of age (6,12).
Autopsy on neonatal ichq mice strongly suggested that these
mice died of dehydration. This observation and the gross
abnormalities in the stratum corneum prompted us to measure
the rate of transepidermal water loss (TEWL) of ichq mice,
compared to phenotypically normal littermates. As shown in
Figure 1 (left hand y-axis), we found increased TEWL levels (up
to 3-fold) from day 9 onwards. Figure 1 (right hand y-axis)
also shows progressive weight loss in the cystatin M/E deficient
mice starting at the time point that transepidermalwater loss was
apparent. At day 11, body weights were 50% compared to
normal littermates and most mice died shortly thereafter.
Legumain is expressed in epidermis and hair follicles
The antiprotease activity of cystatin M/E has been investigated
previously (8,13–15). Cystatin M/E inhibits the asparaginyl
endoprotease legumain with a high affinity (Fig. 2A). In
addition to cathepsin B tested by others, we examined the
inhibition of cathepsins C, H, L and S. None of these proteases
was appreciably inhibited by cystatin M/E (Fig. 2B). These
data indicate that legumain is a likely physiological target of
cystatin M/E in skin in vivo, although nothing is known so far
on the presence of legumain in cutaneous tissues. Using anti-legumain
antibodies for immunohistochemical analysis of
mouse tissues we found legumain expression in the entire
epidermis of adult, wild-type mice (Fig. 3A) and in the inner
root sheet of the hair follicle (Fig. 3B), most abundantly at the
junction of dermis and subcutaneous fat (Fig. 3C). In addition
to the known presence in kidney, legumain expression was
further detected in the ciliated epithelium of the trachea and in
bronchial epithelium (Fig. 3D and E). Immunohistochemical
staining of dorsal skin in cystatin M/E deficient ichq mice
revealed normal epidermal legumain expression (Fig. 3F).
These results demonstrate that legumain is indeed expressed in
skin and hair follicles, as well as in other mouse tissues, some
of which were previously shown to express cystatin M/E (6).
These findings were confirmed at the mRNA level in mouse
and human skin by reverse transcriptase PCR analysis, and
subsequent sequencing of the PCR product (data not shown).
Free in situ legumain activity in skin of cystatin M/E
deficient mice but not in wild-type mice
Immunohistochemical localization of legumain cannot be
equated with legumain activity because legumain can be
present in an inactive zymogen form or complexed with
endogenous inhibitors. In order to demonstrate legumain enzy-matic
activity in tissue sections, we developed a cytochemical
assay, based on hydrolysis of a specific fluorescent substrate
that could be precipitated by nitrosalicylaldehyde in situ. As
it was known from literature that kidney is a rich source of
free legumain (as measured by biochemical assays) we used
this tissue as a positive control. Enzymatic legumain activity
is detected in situ as a green fluorescent precipitate in the
proximal tubulus epithelial cells of pig kidney (Fig. 4A).
Specificity of this reaction was confirmed using Cbz-Ala-Ala-
Aasn-EPCOOEt, which completely blocks proteolysis of the
substrate (Fig. 4B). This synthetic inhibitor is highly specific
for legumain (16) and shows similar affinity and specificity as
the natural inhibitor cystatin M/E. For comparison, immuno-histochemical
staining of pig kidney for legumain shows the
presence of legumain in epithelial cells of the proximal
tubuli, whereas staining of the glomeruli was weak to absent
(Fig. 4C); this is in accordance with the immunohistochemical
localization of legumain in rat kidney reported by others (17),
and it shows colocalization with enzymatically active legumain
as demonstrated in Figure 4A. We subsequently used the
cytochemical legumain assay to examine free legumain activity
in the skin of wild-type and cystatin M/E deficient mice.
1070 Human Molecular Genetics, 2004, Vol. 13, No. 10

Human Molecular Genetics, 2004, Vol. 13, No. 10 1071
Figure 1. Disturbed barrier function in cystatin M/E deficient mice. TEWL of the ichq mutant mice is increased compared to phenotypically normal mice.
Concomitantly with increased TEWL, body weights are decreasing until these mice die around day 11. Left hand y-axis: TEWL in g/m2/h. Right hand y-axis:
decrease in body weight as a percentage of the control mice.
Figure 4D visualizes free legumain activity in the stratum
granulosum and in the hair follicles at the level of the infundi-bulum
in the cystatin M/E deficient mice. No legumain activity
could be demonstrated in the skin of wild type littermates
(Fig. 4E), which indicates that the presence of cystatin M/E
normally regulates legumain activity in skin in vivo. For
comparison, immunohistochemical cystatin M/E staining of
dorsal skin is shown in wild-type and cystatin M/E deficient
mice. This confirms the absence of cystatin M/E expression at
the protein level in the ichq mice (Fig. 4F), whereas the wild-type
skin shows cystatin M/E expression in the infundibular
epithelium of the hair follicle and the stratum granulosum of
the interfollicular epidermis (Fig. 4G). The localization of free
legumain activity in the infundibular part of the hair follicle
in cystatin M/E deficient mice coincides with the reported
localization of tissue pathology in these mice such as excessive
cornification (6,12), which leads to plugging of the hair follicle
and ichthyosis as shown before.
Absence of loricrin monomers in cystatin M/E
deficient mice
Abnormal cornification in ichq mice could be the result of
abnormal expression or processing of structural components of
the cornified envelope. In previous studies, no abnormalities
were found in structural proteins such as cytokeratins 1 and 14,
involucrin and filaggrin (18). We now analyzed extracts of ichq
and wild-type mice for the presence of loricrin and involucrin,
two major components of the stratum corneum. We could
confirm the reported normal presence of involucrin in ichq skin
(data not shown), but remarkably, loricrin monomers and
dimers were nearly absent in cystatin M/E deficient mice as
revealed by western blot analysis (Fig. 5B). We took care to
prepare the skin extracts in buffer with and without the specific
synthetic legumain inhibitor Cbz-Ala-Ala-Aasn-EP-COOEt to
preclude the action of excess free legumain in the cystatin M/E
deficient skin during extraction. No low molecular weight
degradation products of loricrin were detected in lanes 1 and 2
of Figure 5B, nor did we observe high molecular weight
loricrin complexes, although these probably cannot be
extracted, due to their poor solubility. Immunohistochemical
analysis, however, showed that loricrin was abundantly present
in the upper layers of the interfollicular epidermis of dorsal skin
of cystatin M/E deficient mice (Fig. 5C), arguing against
loricrin degradation as a mechanism to explain the findings on
western blot. Moreover, in the epidermis of the mutant mice,
three of five cell layers were found positive for loricrin expres-sion,
whereas in only one of three cell layers, positive staining
was detected in the epidermis of wild-type mice (Fig. 5D).
In Figure 5A, a Coomassie blue stained blot is shown to check
for equal loading of the samples in Figure 5B. No gross
differences were seen in most of the high molecular weight
bands, which represent the major soluble structural proteins
of mouse skin. Interestingly, two abundant protein bands of
13 and 15kDa were observed in the extract of cystatin M/E
deficient mice (boxed in Fig. 5A), which were less pronounced
in wild-type skin. In order to exclude the possibility that these
bands represented accumulated loricrin breakdown products
not detected by the anti-mouse loricrin serum (directed against
a single epitope), we subjected these bands to in-gel tryptic
digestion followed by matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry. The lower
13kDa band was identified as S100 calcium binding protein A9
(or calgranulin B). This is a protein known to be induced in
epidermis during inflammation (19), and it can also act as a
modulator of intracellular calcium homeostasis during follicular
differentiation (20). The upper 15kDa band was identified as
epidermal fatty acid binding protein (E-FABP). Interestingly,
E-FABP has been described as an epidermal protein that is
strongly upregulated following skin barrier disruption (21), which
is in accordance with the observed increase in transepidermal
water loss described above. These findings also demonstrate
that these low molecular weight bands are not derived from
breakdown of loricrin monomers and dimers.
Increased processing of TGase 3 in skin of ichq mice
could explain abnormal cornification
The strongly diminished presence of loricrin monomers and
dimers in skin extracts of ichq mice, and the observation that

Figure 2. Protease inhibition by recombinant cystatin M/E. (A) Inhibition of free legumain activity in human kidney extract. Nanomolar concentrations of recom-binant
cystatin M/E and the specific synthetic legumain inhibitor inhibited hydrolysis of the fluorogenic substrate. Representative curves of three experiments are
shown. (B) Hydrolysis of the fluorogenic substrate by the plant protease papain was almost completely inhibited by nanomolar concentrations of recombinant
cystatin M/E. Inhibition of a panel of lysosomal cathepsins was incomplete, even when 2 mM recombinant cystatin M/E was used.
this phenomenon is not due to degradation, led us to consider
misregulated crosslinking as an alternative explanation. It is
known that loricrin and small proline-rich proteins (SPRs) are
cross-linked by cytosolic TGase 3 enzyme primarily to form
homodimers and heterodimers (22). Proteolytic cleavage of
TGase 3 is required to achieve maximal specific activity of the
enzyme (23). We now addressed the question of whether
aberrant processing of the TGase 3 zymogen in the
interfollicular epidermis of cystatin M/E deficient mice could
be responsible for the observed defects in epidermal cornifica-tion.
TGase 3 activation during keratinocyte differentiation
involves cleavage of the 77 kDa zymogen by a hitherto
unknown protease resulting in the release of 30 and 47 kDa
fragments (Fig. 6A), which then associate non-covalently to
form the active enzyme (24). To confirm the aberrant
processing of the TGase 3 zymogen due to unrestricted
legumain activity in the interfollicular epidermis of cystatin
M/E deficient mice, we used a cleavage-site-specific antibody
that was generated against a synthetic peptide (FGATS) that
corresponds to the cleavage site of mouse TGase 3 (Fig. 6A).
Western blot analysis revealed the abundant presence of
proteolyzed (and hence activated) TGase 3 in skin extracts of
cystatin M/E deficient mice at day 6, whereas no appreciable
levels of this 30 kDA fragment could be detected in skin extract
of wild-type littermates (Fig. 6B). This indicates that prema-ture,
high levels of activated TGase 3 are present at the time
when the skin lesions develop.
The next step was to investigate if legumain could process
human recombinant TGase 3 in vitro into its active form by
proteolysis of the 77 kDa zymogen into the 30 and 47 kDa
fragments. As we were unsuccessful in generating active
recombinant legumain, and most tissues do not contain

Figure 3. Immunohistochemical localization of legumain in mouse tissues. (A) Expression of legumain in the epidermis of dorsal skin of an adult wild type mouse;
(B) in the inner root sheet epithelium of a hair follicle; and (C) high expression around the hair follicle at the junction of dermis and subcutaneous fat. (D)
Legumain expression in the ciliated epithelium of the trachea and the chondrocytes in the cartilage below and in (E) bronchial epithelial cells (the lung tissue
is slightly positive). (F) Immunohistochemical staining of dorsal skin in an ichq mutant mouse (9 days of age) shows normal epidermal staining. Scale bars: B
and F, 12.5 mm; C–E, 25 mm; A, 50 mm.
measurable free legumain activity, we used human kidney
extract that is reported by others as a tissue where free legumain
is relatively abundant. As shown in Figure 6C (lanes 1 and 4),
recombinant human TGase 3 was slowly processed to its
active form by human kidney extract as detected by the
monoclonal antibodies C2D and C9D that are directed against
the human zymogen and the 30 and 47 kDa fragments,
respectively. For comparison, we show cleavage of TGase 3
by dispase, a neutral bacterial protease that is commonly
used to activate TGase 3 in vitro. Dispase generated a fragment
(Fig. 6C, lane 3) with the same electrophoretic properties
as those generated using kidney extract (Fig. 6C, lane 4).
Proteolytic processing of TGase 3 by kidney extract could
be completely inhibited by the specific legumain inhibitor
Cbz-Ala-Ala-Aasn-EP-COOEt (Fig. 6C, lanes 2 and 5). As it
is known that legumain affects processing of lysosomal
cathepsins (11), we considered the possibility that legumain
mediates processing of TGase 3 in an indirect manner. Indeed,
when we used E-64, a synthetic broad-spectrum inhibitor
of lysosomal cysteine proteases that is not effective against
legumain, a complete inhibition of TGase 3 processing
was also found (Fig. 6C, lane 6). This finding was addressed
in further detail by investigating the in vitro processing
of TGase 3 by a panel of purified lysosomal cysteine proteases.
Figure 6D shows that cathepsin S completely processed
TGase 3 in 30 min (lane 7) similar to the positive control
dispase (lane 2). Partial processing of TGase 3 was found using
cathepsin B and cathepsin L (Fig. 6D, lanes 3 and 6), whereas
cathepsins C and H were not able to do this (Fig. 6D, lanes
4 and 5). The addition of E-64 to the reaction mixtures
completely blocked the processing of the TGase 3 zymogen
(data not shown).
DISCUSSION
In this report, we provide evidence that the asparaginyl
endopeptidase legumain is a physiologically relevant target
protease of cystatin M/E in skin. No detectable free legumain
activity was found in the skin of wild-type mice whereas in
ichq mice, legumain activity was found at exactly the same
location where cystatin M/E is normally expressed. Absence of
cystatin M/E and appearance of legumain activity colocalize
with the observed pathology in ichq mice (i.e. excessive
cornification in hair follicles and epidermis).
Cystatin M/E is an unusual cystatin in its biochemical
properties, chromosomal localization and restricted tissue
distribution. Whereas most cystatins are extracellular inhibitors
of cysteine proteases, cystatin M/E appears to function more
locally and also intracellularly, as will be discussed below.
Little is known on the specific biological functions of cystatin
family members. Deficiency for cystatin B in humans causes
myoclonic epilepsy (25); deficiency for cystatin C in mice
causes no obvious spontaneous phenotype (26). In ichq mice,
deficiency for cystatin M/E causes a dramatic, apparently
tissue-specific phenotype, with pronounced morphological and
ultrastructural abnormalities in epidermis and hair follicles. We
considered a number of known lysosomal cysteine proteases as
likely candidate target enzymes for cystatin M/E. Only the
recently discovered asparaginyl endopeptidase legumain was
found to be inhibited at physiologically relevant concentrations.
Mammalian legumain is found in late endosomes and is
postulated to have a regulatory role in the biosynthesis of
lysosomal enzymes, as recently demonstrated using legumain
deficient mice (11). The body weights of the legumain deficient
mice were significantly decreased, however, they were normally

Figure 4. Cytochemical detection of free legumain activity. (A) In situ enzymatic legumain activity is shown as a green fluorescent precipitate (speckles) in the
proximal tubulus epithelial cells (arrowheads) of pig kidney. Nuclei were counterstained with propidium iodide. (B) Legumain activity is blocked by Cbz-Ala-Ala-
Aasn-EP-COOEt. (C) Immunohistochemical localization of legumain in pig kidney: epithelial cells of the proximal tubuli (arrowheads) showed positive staining;
staining in the glomeruli (GL) was weak to absent. (D) Free in situ protease activity of legumain is visible as yellow speckles in the stratum granulosum in dorsal
skin of ichq mutant mice, and in the hair follicles at the level of the infundibulum (arrows). Red staining is from the nuclei. A magnification of this signal is shown
in the inset. (E) No legumain activity was found in the skin of wild type mice. (F) Immunohistochemical staining of dorsal skin in the ichq mutant mouse shows
absence of cystatin M/E expression, whereas (G) the wild type mouse reveals cystatin M/E expression in the infundibular epithelium of the hair follicle and the
stratum granulosum of the interfollicular epidermis. Scale bars: A–E 100 mm; F,G 50 mm.
born and fertile. Disruption of the legumain gene led to the
enlargement of lysosomes in the proximal tubule cells of the
kidney, which suggests that materials to be degraded are being
accumulated within the lysosomal compartments. It appeared
that the processing of the lysosomal proteases, cathepsins B, H
and L, from the single-chain forms into the two-chain forms
was defective in the kidney cells of these legumain deficient
mice. This observation confirms the earlier assumption that
legumain could act as a protease responsible for processing of
lysosomal cathepsins into their active forms (10). Interestingly,
an increase of lysosomal cysteine protease activity has been
observed in the terminal differentiation process of keratinocytes
(27,28). Recent studies have reported increasing evidence that
lysosomal proteases play important roles in physiological
processes not restricted to lysosomes only (29). For example,
protease activity and regulation outside lysosomes potentially
contributes to propagation of apoptosis, a process that is
distinct from terminal differentiation of the epidermis but
nevertheless shares some molecular and cellular features.
In epidermis there is a balanced regulation of protease
activity that, when disturbed, could lead to faulty cornification
processes in the epidermis and upper part of the hair follicle.
Examples of a disturbed protease–antiprotease balance invol-ving
lysosomal cathepsins, leading to abnormal cornification,
include Papillon–Lefevre syndrome (cathepsin C deficiency)
and the furless mouse (cathepsin L deficiency) (30,31).
However, the exact role for these cathepsins in epidermal
differentiation and desquamation is unknown. Our findings on
the legumain dependent, cathepsin-mediated TGase 3 proces-sing
fit very well with the proposed role of legumain in other
cell types. Although it has to be verified experimentally, we
assume that legumain regulates lysosomal cathepsin processing
and TGase 3 processing in normal skin, a process that is
apparently under tight control of cystatin M/E. Deficiency of
cystatin M/E, as found in ichq mice, unleashes legumain
activity thereby causing the observed phenotype.
Much of the barrier function of human epidermis against the
environment is provided by the cornified cell envelope (CE)

Figure 5. Western blot analysis of mouse skin extracts. Proteins were extracted in buffer with (þ) or without () the addition of Cbz-Ala-Ala-Aasn-EP-COOEt.
(A) Coomassie blue staining, and (B) rabbit anti-mouse loricrin antiserum. Each lane contains 20 mg mouse skin extract. Note the diminished amounts of loricrin
monomers and dimers in the skin extracts of ichq mice (d¼dimer, m¼monomer). Two abundant protein bands are found in ichq mouse skin extracts (boxed in A),
which are identified as calgranulin B and E-FABP using MALDI-TOF mass spectrometry. (C) Immunohistochemical staining of loricrin in dorsal skin of an ichq
mutant mouse, and (D) a wild-type littermate. Scale bars: 50 mm.
(32–34), which is assembled by TGase mediated cross-linking
of several structural proteins during the terminal stages of
normal keratinocyte differentiation. Surprisingly, recent studies
on mice in which major CE components were knocked out (e.g.
loricrin, envoplakin and involucrin), have shown no discernable
phenotype (35–37). This suggests that there are compensatory
backup systems and additional unidentified components
involved that maintain the skin barrier function. However,
mutations or absence of desmosomal or cytoskeletal proteins in
differentiated keratinocytes often leads to severe pathology and
disturbance of barrier function (38–42). Deficiency for
regulatory enzymes as TGase1 and steroid sulfatase also leads
to disease in humans (43–45). The severe phenotype of cystatin
M/E deficient mice provides an example of a disturbed
protease–antiprotease balance that causes faulty differentiation
processes in the epidermis and hair follicle. Another example is
the recent identification of the serine protease inhibitor Kazal-type
5 (SPINK5) as the defective gene in Netherton syndrome
(NS, OMIM 256500) (46). It was shown that the proteolytic
processing and distribution of the protein product of SPINK5,
LEKT1, is disturbed in NS patients (46,47). NS is a congenital
ichthyosis associated with erythroderma, a specific hair shaft
defect and atopic features. It was hypothesized that defective
inhibitory regulation by LEKT1 result in increased protease
activity in the stratum corneum, accelerated degradation of
desmoglein-1 and overdesquamation of corneocytes (48).
Colocalization of LEKT1 transcripts with stratum corneum
serine proteases (SCTE and SCCE) in hair follicles suggested
that the regulation of the activity of these proteases by LEKT1
might also affect hair growth and morphogenesis. Targeted
epidermal overexpression of stratum corneum chymotryptic
enzyme (SCCE) results in pathologic skin changes (49), which
suggest that increased activity of proteases present in the skin
may indeed play a significant part in skin pathophysiology. Our
study underscores the importance of the regulation of
proteolysis in the process of keratinocyte terminal differentia-tion.
In addition, our study reveals that the function of cystatin
M/E is to control keratinocyte legumain activity and thereby
probably regulating correct TGase 3-dependent cross-linking of
loricrin molecules, which is an important step in the formation
of the cornified layer (22). In addition to excessive TGase
activity, lack of TGase activity can also lead to disturbed
cornification as witnessed by an autosomal recessive ichthyosis,
termed lamellar ichthyosis (LI1, OMIM 242300) in which
mutations in TGase 1 are responsible for the disease (43,44).
Although this might seem paradoxical at first glance, both loss
and inappropriate gain of TGase activity could explain
ichthyotic changes, although the mechanisms could be
different. Excessive TGase 3 activity could lead to retention
of scales by hypercross-linked corneocytes, whereas deficiency
for TGase 1 could lead to irregular scaling due to lack of
attachment of involucrin to the o-hydroxyceramides (50).
There are, however, other forms that are clinically similar to
lamellar ichthyosis but are not linked to the locus of the TGase
1 gene. The non-redundancy of TGase 3 is indicated by
knockout mice that show an early embryonic-lethal phenotype
(51), but no mutations in this gene have been found in humans
thus far. New loci for autosomal recessive ichthyosis have
recently been identified (reviewed in 51), but they do not link to
the gene for TGase 3. This leaves open the possibility that
mutations in genes that are involved in the processing of
TGases into the active form might be causative for other forms

of ichthyosis. We suggest that cystatin M/E is a candidate gene
for heritable human skin disorders that show faulty cornifica-tion
and desquamation.
In conclusion, the data presented in this study have identified
cystatin M/E and legumain as a functional dyad in skin, and we
provide evidence that legumain and lysosomal cysteine
proteases are involved in the proteolytic activation of TGase
3 during terminal epidermal differentiation.
Figure 6. Processing of TGase 3. (A) The 77 kDa zymogen form of TGase 3 is
proteolyzed into the activated form consisting of 30 and 47 kDa fragments. The
boxed amino acid sequence corresponds to the N-terminus of the 30 kDa frag-ment
that was used to generate the mouse-specific cleavage-site-directed anti-body
(the arrow indicates the cleavage site). (B) Western blot analysis of
mouse skin extracts. Processed TGase 3 is found in skin extract of 6-day-old
cystatin M/E deficient mice (lane 2), whereas hardly any processed TGase 3
is found in skin of wild type littermates (lane 1). Each lane contains 10 mg
mouse skin extract. (C) Processing of recombinant human TGase 3 by human
kidney extract (HKE), and detection by western blot analysis of the zymogen
and proteolyzed fragments of 30 and 47 kDa, which could be recognized by
the monoclonal anti-human TGase 3 antibodies C2D and C9D, respectively.
60 ng recombinant TGase 3 was partially processed by HKE (lanes 1 and 4),
however, no proteolyzed fragments were found in the presence of Cbz-Ala-
Ala-Aasn-EP-COOEt (lanes 2 and 5) or E-64 (lane 6). Processing by dispase
is shown using the C2D antibody (lane 3). (D) Processing of recombinant
TGase 3 by cathepsins, and detection of the zymogen and proteolyzed frag-ments
with the C2D monoclonal antibody. Recombinant TGase 3 (lane 1) is
completely processed in 30 min by dispase (lane 2) and by cathepsin S (lane
7). Partially processed TGase 3 was found using cathepsin B (lane 3) and cathe-psin
L (lane 6). TGase 3 could not be processed by cathepsin C (lane 4) and
cathepsin H (lane 5).
MATERIALS AND METHODS
Animal studies
Mice were housed in specific pathogen-free facilities at the
Central Animal Laboratory, University of Nijmegen, The
Netherlands. Genotyping of the mice was performed as
described previously (6). TEWL of cystatin M/E deficient
mice (ichq/ichq) and phenotypically normal littermates (þ/þ
and ichq/þ) was measured using a Tewameter TM 210, in
accordance with current guidelines (52).
Recombinant proteins
Recombinant human cystatin M/E was produced both in a
bacterial expression system as a GST fusion protein and as a
fully processed protein in an eukaryotic system using the
baculovirus in insect cells as described previously (15).
Recombinant human TGase 3 was produced as a full length
zymogen in a baculovirus system and as a bacterially expressed
His6-tagged fusion protein as previously described in detail (53).
Antibodies
Purified GST-cystatin M/E fusion protein was used to
immunize a New Zealand White rabbit. Antisera raised against
the GST-cystatin M/E fusion protein were purified by affinity
chromatography as described previously (15). Anti-mouse
legumain antibodies were prepared by immunizing rabbits
with a bacterially expressed His6-tagged fusion protein of
legumain as previously described (11). Purified human
recombinant TGase 3 from Escherichia coli was used to
immunize mice for the establishment of monoclonal antibodies
(C2D and C9D) as described in detail by Hitomi (53). Affinity
purified polyclonal rabbit anti-mouse-FGATS antibodies were
generated as described previously (54). Affinity purified
polyclonal rabbit anti-mouse loricrin antibodies (AF-62) were
purchased from BAbCO (BAbCO, Richmond, CA).

Extraction of proteins and immunoblotting
Human kidney and mouse skin biopsies were frozen in
liquid nitrogen and subsequently grinded using a Micro
Dismembrator U (B. Braun Biotech International, Melsungen,
Germany). Proteins were extracted in buffer containing 50mM
citrate (pH 5.8), 1mM EDTA, 0.1M NaCl and 2mM DTT,
followed by mild sonification of the lysate for 1 min at 4C.
Mouse skin proteins were extracted in the absence and presence
of Cbz-Ala-Ala-AAsn-EP-COOEt. Samples were diluted with
SDS sample buffer (containing a reducing agent) and boiled for
3 min. These protein samples were separated by SDS–PAGE on
a 12% Bis-Tris-Gel and blotted onto polyvinylidenedifluoride
(PVDF) membrane using the NuPAGE system (Invitrogen,
Carlsbad, CA), according to the protocol provided by the
manufacturer. Membranes were stained with Coomassie blue,
or incubated with polyclonal antibodies directed against
mouse loricrin (1 : 5000), mouse anti-FGATS (1 : 100), and the
C2D (1 : 1000) and C9D (1 : 4000) monoclonal antibodies
against human TGase 3. Proteins were detected with the
Phototope-HRP Western Blot Detection Kit (Cell Signaling
Technology, Beverly, MA), according to the manufacturer’s
protocol.
MALDI-TOF mass spectrometry analysis
In-gel digestion coupled with mass spectrometry analysis was
used for the identification of proteins extracted from mouse
skin. To perform digestions on Coomassie blue stained protein
bands that were abundantly found in the skin extract of cystatin
M/E deficient mice, the In-Gel Tryptic Digestion Kit (Pierce,
Rockford, IL) was used in accordance to the protocol provided
by the manufacturer. For MALDI-TOF mass spectrometry,
0.3 ml of the tryptic digest was spotted on the target plate
followed by 0.3 ml of matrix solution (a saturated solution of
a-cyano-4-hydroxy-cinnamic acid in a 50 : 50 mixture of
acetonitrile and 0.1% TFA, v/v). Positive mass ion spectra
were collected in the reflectron mode, with pulsed ion
extraction on a Biflex III instrument (Bruker-Franzen,
Bremen, FRG). Mass calibration was performed externally
using a mixture of peptide standards (Sigma, St Louis, MO). A
total of 150 single laser shots were accumulated for each
sample spot. For identification of proteins, peptide mass lists
were used to search protein databases with the Mascot software
system (Matrix Science, London, UK).
TGase 3 processing
Baculovirus expressed recombinant TGase 3 zymogen was
processed by treatment with dispase, cathepsins and by a
protein extract from human kidney. To prepare the proteolyzed
form, 0.6 mg zymogen was treated with 5mU dispase (Roche
Diagnostics, Mannheim, Germany) in the presence of 5mM
CaCl2 at 37C for 30 min. The same reaction conditions were
used to test proteolytic activity of human cathepsin B (Sigma),
bovine cathepsin C (Sigma), human cathepsin H (Calbiochem,
San Diego, CA), human cathepsin L (Sigma) and bovine
cathepsin S (Calbiochem). To test whether the TGase 3
zymogen could be processed by free legumain activity, 0.6 mg
zymogen was treated with protein extracts from human kidney
in the presence of 5mM CaCl2 at 37C for 0.5 and 24 h.
For controls the same reaction mixtures were prepared but with
the addition of Cbz-Ala-Ala-Aasn-EPCOOEt, or compound E-64
[trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane]. The
reaction mixtures were subjected to SDS–PAGE and electro-blotted
onto PVDF membrane. The membrane was incubated
with the monoclonal mouse anti-human TGase 3 antibodies
(C2D and C9D) and detection was established as described
above.
In situ assay for legumain activity
This assay is based on data that have shown that 5-
nitrosalicylaldehyde forms an insoluble fluorescent complex
with NH2NapOMe, which has been used to visualize cathepsin
B in fibroblasts (55). Unfixed cryostat sections (6 mm) of mouse
skin and pig kidney were air-dried for 10 min. These tissue
sections were subsequently incubated with 100 ml legumain in
situ assay buffer under a coverslip. Enzyme activity was
measured after an incubation period of 30 min at 37C with the
selective fluorescent Suc-Ala-Ala-Asn-4-methoxy-2-naphthy-lamide
(Suc-Ala-Ala-Asn-NHNapOME) substrate. This sub-strate
was originally synthesized for the measurement of
legumain in colorimetric and fluorimetric microplate assays as
described previously (56). Legumain in situ assay buffer was
prepared by the addition of 5-nitro-salicylaldehyde and Suc-
Ala-Ala-Asn-NHNapOME substrate to a final concentration of
1 and 2mM, respectively, in a buffer that contained 121mM
phosphate (pH 5.8), 39.5mM citric acid, 1mM EDTA, 1mM
DTT and 0.01% CHAPS. The specificity of the reaction was
verified by incubation in the absence of substrate or in the
presence of Cbz-Ala-Ala-Aasn-EP-COOEt.
Fluorimetric enzyme assays
Protease inhibitory activity of recombinant cystatin M/E
against cysteine proteases was determined by measuring the
inhibition of papain and lysosomal cathepsins (B, C, H, L and
S) using fluorogenic synthetic substrates, essentially described
by Abrahamson (53). Protease inhibitory activity of recombi-nant
cystatin M/E and the synthetic aza-peptide epoxide
inhibitor against legumain was determined by measuring the
inhibition of free legumain activity in human kidney extract.
Kidney extract was titrated in the absence and presence of
increasing concentrations of recombinant cystatin M/E or the
specific legumain inhibitor as a positive control. Preincubation
time of kidney extract and inhibitor was 30 min at room
temperature. Enzyme activity was measured after an incuba-tion
period of 30 min at 37C with the selective fluorescent
Z-Ala-Ala-Asn-MCA substrate (Peptides International, Louisville,
KY). The buffer that was used contained 0.1M phosphate ( pH
5.7), 2mM EDTA, 1mM DTT and 2.7mM L-cystein.
ACKNOWLEDGEMENTS
We thank Geert Poelen and Debby Smits for their assistance
with the animal experiments. Wiljan Hendriks and Bruce Jenks
are acknowledged for critical reading of this manuscript. This
work was financially supported by Grant 902.11.092 from the
Netherlands Organization for Scientific Research (NWO).

J.C.P. would like to acknowledge grants from the National
Institute of General Medical Sciences (Grant GM54401 and
GM61964).
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