(Received for publication, March 17, 1997, and in revised form, May 20, 1997)
From the Departments of Dermatology,
§ Cell Biology and Histology, and ¶ Biochemistry,
Institute of Cellular Signaling, University of Nijmegen, P. O. Box
9101, 6500 HB Nijmegen, The Netherlands
The human epithelial proteinase inhibitor SKALP/elafin and the porcine sodium-potassium ATPase inhibitor SPAI-2 are two highly homologous proteins that share an NH2-terminal transglutaminase substrate domain and a COOH-terminal whey acidic protein (WAP) domain. Here we describe the bovine and simian orthologs of SKALP/elafin as well as two new bovine family members that are designated Trappin-4 and Trappin-5 on the basis of a new nomenclature that we propose (Trappin = TRansglutaminase substrate and WAP motif-containing ProteIN). Sequence analysis of Trappin-4 and Trappin-5 revealed a domain structure that is very similar to SPAI-2 (Trappin-1) and SKALP/elafin (Trappin-2). The transglutaminase substrate motifs are conserved although the number of repeats varies among species and among family members. The sequence of Trappin-4 and Trappin-5 diverges from Trappin-1 and Trappin-2 at the putative reactive site in the WAP domain. The bovine ortholog of Trappin-2 is expressed in tongue and snout epidermis; Trappin-4 is expressed in trachea, ileum, and tongue; and Trappin-5 is expressed at low levels in trachea, as determined by RNase protection and Northern blot analysis. Based on the analysis of 67 transglutaminase substrate repeats as present in all known Trappin gene family members from four different mammalian species a consensus sequence could be established: Gly-Gln-Asp-Pro-Val-Lys (GQDPVK). Using biotinylated hexapeptide probes we found that the GQDPVK sequence is a very efficient transglutaminase substrate both for guinea pig liver transglutaminase and for epidermal transglutaminase, and it acts as acyl donor as well as acceptor. We propose that the Trappin protein family forms a new group of enzyme inhibitors with various specificities of the WAP domain, which share transglutaminase substrate motifs that can act as an anchoring sequence.
Skin-derived antileukoproteinase (SKALP)1 (1) and sodium-potassium ATPase inhibitor-2 (SPAI-2) (2) are two molecules that share an NH2-terminal domain that functions as a transglutaminase (TGase) substrate, and a COOH-terminal whey acidic protein (WAP) domain that harbors an inhibitory activity toward at least two distinct enzymes. Porcine SPAI-2 was the first of these molecules to be described and is expressed mainly in the intestine (3). Human SKALP, otherwise known as elafin (4), or elastase-specific inhibitor (5), is a potent inhibitor of the leukocytic proteinases elastase and proteinase-3 (6, 7). We found that SKALP/elafin is expressed in several human stratifying squamous epithelia, except for epidermis where it is only expressed in the context of inflammation, such as psoriasis or wound healing (8-10). We mapped the genomic localization of human SKALP/elafin to chromosome 20q12-13 (11). Interestingly, this region contains various other genes involved in TGase-mediated cross-linking processes such as the tissue TGase gene (12), epidermal TGase (13, 14), and the genes coding for semenogelin I and semenogelin II (15), which are also epithelial TGase substrates. SKALP/elafin is distinct from SPAI-2 on the basis of its inhibitory activity, the amino acid sequence of the putative active site, and the epithelial expression pattern. The cDNAs and genes for porcine SPAI-2 (2, 3), porcine SKALP/elafin (16), human SKALP/elafin (7, 17), and a new porcine family member (16) that is more similar to SPAI-2 than to SKALP/elafin, have been cloned and revealed a high degree of conservation in the gene structure and the intronic sequences but a strong sequence divergence in the second exon.
The SKALP/SPAI-2 gene family members are composed of two evolutionary
building blocks that are found in other proteins as well (Fig.
1). The COOH-terminal WAP domain is
homologous to the second domain of secretory leukocyte proteinase
inhibitor (SLPI), which inhibits elastase and cathepsin G (18). The
NH2-terminal domain, containing the TGase substrate motifs,
is homologous to the guinea pig seminal vesicle protein-1 (19) and the
human semenogelins (15). We showed that the NH2-terminal
TGase substrate domain, for which the name "cementoin" was coined
by others (20), is actually used in vivo and in
vitro for cross-linking to stratum corneum proteins. Recently, it
was shown by direct sequencing of cross-linked peptides from human
foreskin epidermis that SKALP/elafin is cross-linked in vivo
to loricrin and cytokeratin-1 (21), which are structural proteins of
the terminally differentiating keratinocyte. The cross-linked
SKALP/elafin is proteolytically processed further by unidentified
proteinases to yield low molecular weight COOH-terminal fragments
containing the antiproteinase activity, starting at amino acid
positions 149, 151 and 156 (numbering according to Fig. 2), as shown by
NH2-terminal sequencing of purified SKALP/elafin from
epidermal scale extracts (7, 10). We have found that the COOH-terminal
part of SKALP/elafin is cleared via the plasma and can be recovered
from the urine (22, 23). This mechanism provides the epidermis with an
anchored proteinase inhibitor, which could protect the structural
proteins of the stratum corneum against unwanted proteolysis; in
addition it generates, after cleavage, a gradient of low molecular
weight inhibitors from the epidermis to the dermis, thereby possibly
interfering with polymorphonuclear leukocyte chemotaxis and
polymorphonuclear leukocyte-induced degradation of extracellular matrix
proteins such as elastin and basal membrane components.
To study the evolutionary divergence of SKALP/elafin in various species and to identify potential new members of this gene family we performed reverse transcription-polymerase chain reaction (PCR) on mRNA of epithelial tissues from various mammals, using degenerate primers that encompass the TGase substrate domain and the WAP domain. In this way we identified the putative simian and bovine orthologs of SKALP/elafin, and we have identified two new family members from bovine tissue. On the basis of the family members thus far identified we propose a new unifying nomenclature. From all known SKALP/elafin gene family members in various species we derived a consensus hexapeptide sequence for the TGase substrate motif. Using synthetic peptides we show that this hexapeptide motif is an extremely efficient substrate for various TGases.
Bovine and simian (rhesus monkey) tissues were
obtained from the central animal laboratory, University of Nijmegen,
The Netherlands. Spontaneously shed scales from psoriatic, eczema, and
lamellar ichthyosis (LI) patients were collected and stored at
20 °C. The LI patient was homozygous for a splice site mutation in
intron 5 of the TGase 1 gene as determined by single strand
conformation polymorphism analysis and sequence analysis. This mutation
causes a deficiency for TGase type 1 as recently described (24).
Total RNA from bovine
tongue and rhesus monkey skin were extracted with RNAzol B as suggested
by the supplier (Cinna/Biotex). First strand cDNA was generated
from total RNA with Moloney murine leukemia virus range H
reverse transcriptase (Expand Reverse Transcriptase, Boehringer Mannheim) under conditions as suggested by the supplier, using excess
oligo(dT) primers. The reverse transcriptase reaction products were
used for PCR amplification to obtain the partial cDNA of bovine and
simian orthologs of SKALP/elafin.
Based on
conserved amino acid stretches within SKALP/elafin and porcine SPAI-2
we designed two oligonucleotide forward primers (positions are shown in
Fig. 2): primer SKA1,
5-AGGGCCAGCAGCTTCTTGATC-3
; and primer SKA2,
5
-CAAGA(G/T)CCAGTCAA(A/G)GGT-3
. As reverse oligonucleotide primers we
used primer SKA3, 5
-CAGCACTTCTTGA(C/T)(C/T)CCTGG-3
and the oligo(dT)
primer (Fig. 2). PCRs were carried out using a DNA thermal cycler
(PTC-200, Biozym) in 25-µl mixtures. The following buffer conditions
were used: 10 mM Tris-HCl, pH 8.8, 1.5 mM
magnesium chloride, 50 mM potassium chloride, 0.1% Triton X-100, all four dNTPs (each at 200 µM), 2 units of
PrimeZyme DNA polymerase (Biometra), and 20 pmol of each primer. After
an initial incubation of 6 min at 94 °C amplification was conducted
for 35 cycles as follows: 1 min at 94 °C, 1 min at annealing
temperature, and 2 min at 72 °C. An additional 10 min at 72 °C
was used for the last cycle. Annealing temperatures were 57 °C when
using primers SKA1 and SKA3, 55 °C for primers SKA2 and SKA3,
51 °C for primer SKA2 and the oligo(dT) primer. The PCR products
were purified from agarose gel and cloned into the pGEM-T vector
(Promega) according to the protocol provided by the manufacturer.
Blunted PCR fragments were cloned into a SmaI-digested
pBluescript KS(+) vector (Stratagene). Both strands were sequenced
using the dideoxy chain termination procedure (25).
DNA sequence gel readings were recorded, edited, and assembled using the GCG package (26) provided by the Dutch CAOS/CAMM center. Deduced protein sequences and alignments were analyzed with the same software.
RNase Protection AssayTotal RNA from different bovine
tissues was extracted with RNAzol B as suggested by the supplier. The
following bovine tissues were studied: tongue, kidney, liver, trachea,
lung, ileum, epidermal skin from snout, brain, and esophagus. The
360-base pair blunt fragment encoding the possible bovine ortholog of
human SKALP/elafin (Trappin-2) was cloned into the SmaI site
of the pBluescript KS(+) vector, which contains the T3 promoter for
transcription of antisense RNA. In vitro transcription was
performed using a transcription kit (Boehringer Mannheim) in the
presence of [-32P]UTP (Amersham). Antisense RNA probe
was transcribed from the XbaI-linearized SKALP/elafin
(bovine Trappin-2) template. DNA template was degraded with RNase-free
DNase I, and the labeled RNA probe was purified using Chroma
spin+TE-100 columns (CLONTECH), acidic phenol/chloroform extraction,
and ethanol precipitation. Using the RNase protection kit (Boehringer
Mannheim), the radioactively labeled probe (1 × 105
cpm) was hybridized to 10 µg of total RNA from different bovine tissues or to 10 µg of total RNA from human psoriatic skin biopsies as a negative control. Samples were denatured for 10 min at 80 °C followed by an overnight incubation at 55 °C. Partial RNA·RNA hybrids were treated with DNase-free RNase A and RNase T1
according to the manufacturer's instructions. The protected RNA
fragments were subjected to electrophoresis on a 6% denaturing
polyacrylamide gel containing 7 M urea and were sized using
the sequencing reaction of the PCR product encoding bovine Trappin-2.
Autoradiography was performed on Biomax-MR films (Kodak) at
80 °C
with intensifying screens. Processing of the autoradiographs was
performed using the ImagemasterTM data image system
(Pharmacia Biotech Inc.).
For Northern blot analysis, 10 µg
of total RNA from different bovine tissues was electrophoretically
separated in a 1% agarose gel (dissolved in 10 mM sodium
phosphate buffer, pH 7.0), as described before (27). The gel was
blotted by capillary transfer onto positively charged nylon membrane
(Boehringer Mannheim) using 10 × SSC (1.5 M sodium
chloride, 0.15 M sodium citrate). After transfer, RNA was
fixed to the membrane by ultraviolet irradiation (312 nm, 0.2 J/cm2). The membrane was incubated with ethidium bromide (1 µg/ml) for 15 min prior to photography. Hybridization was performed
in 250 mM phosphate buffer at 60 °C according to Church
and Gilbert (28), using 32P-labeled random primed probes
encoding bovine Trappin-4 and -5 obtained by reverse transcription-PCR.
The blot was washed twice for 15 min at 60 °C using 125 mM phosphate buffer. Autoradiography was performed on
Biomax-MR films at 80 °C with intensifying screens. Processing of
the autoradiographs was performed using the ImagemasterTM
data image system.
Six hexapeptides with an NH2-terminal biotin followed by a C6-spacer were synthesized: GQDPVK, GQDPVR, GNDPVK, GNDPVR, KVPDQG and GKDPVQ (Eurosequence Inc., Groningen, The Netherlands). 250 µg of a hexapeptide (TVQQEL) and a heptapeptide (PGGQQIV), two known acyl donor probes in the TGase assay (29), were biotinylated with 500 µg of NHS-LC-biotin (Pierce) for 2 h at 37 °C in 300 µl of 0.1 M Na2CO3, pH 8.0. The reaction was stopped by the addition of 20 µl of 1 M Tris, pH 8.0 (30).
Cross-linking of the Biotinylated Peptides to Stratum Corneum Proteins by Endogenous TGaseEpidermal scales from a psoriatic
patient (200 mg) were homogenized in 4 ml of buffer containing 50 mM Tris-HCl, pH 7.8, 100 mM sodium chloride,
and 1 mM phenylmethylsulfonyl fluoride (Sigma), and
centrifuged for 30 min at 25,000 × g. The same
protocol was used for scales from patients with eczema and LI. The
supernatants were stored at 20 °C until further use. For
cross-linking experiments 10 µl of scale extract was used with 5 µl
(100 µg/ml) of biotinylated peptide. The following buffer conditions
were used: 50 mM Tris-HCl, pH 7.8, 100 mM
sodium chloride, and calcium chloride at a concentration of 2 mM, in a final reaction volume of 50 µl. In some
experiments 2 µl (0.0313 unit/ml) of guinea pig liver TGase (Sigma)
was added. After 60 min at 37 °C, the reaction was stopped by the
addition of 5 µl of 100 mM EDTA, pH 7.8. Reaction
mixtures containing 10 mM EDTA, pH 7.8, and
heat-inactivated scale extracts were used as controls. The reaction
mixtures were blotted directly onto polyvinylidene difluoride membranes
(Millipore) by a slot-blot manifold or were subjected to SDS-PAGE (12%
Ready gel, Bio-Rad) and blotted onto polyvinylidene difluoride
membrane. Biotinylated proteins were detected with the Western Light
kit (Tropix) according to the manufacturer's instructions. This assay
uses avidin-conjugated alkaline phosphatase and chemiluminescence of a
sensitive alkaline phosphatase substrate. Positive bands were recorded
on X-Omat S1 films (Kodak) and were processed with a maximum scanning
OD range of 1.5 and quantitated using the ImagemasterTM
data image system.
Purified bovine crystallin proteins B,
-low,
, and
A were prepared as described previously (30, 31).
Recombinant human SKALP/elafin, containing amino acids 151-213
(numbering according to Fig. 2) of the complete amino acid sequence of
SKALP as described by Molhuizen et al. (7), was a kind gift
of Dr. N. Russel, Zeneca, U. K. Bovine serum albumin was from
Boehringer Mannheim. For cross-linking experiments 20 µl of protein
(1 mg/ml) was used with 0.5 nmol of biotinylated GQDPVK peptide and 2 µl of guinea liver pig TGase (0.0313 units/ml) under reaction
conditions as described in the previous paragraph.
TGase assay reaction mixtures
were diluted with SDS-sample buffer (containing dithiothreitol) and
boiled for 2 min. These protein samples were separated by SDS-PAGE on a
12% Tris-glycine gel (Ready gels, Bio-Rad) using Tris-glycine as
electrophoresis buffer (32). The broad range prestained marker from
Bio-Rad was used as molecular marker (myosin (208 kDa),
-galactosidase (116 kDa), bovine serum albumin (79 kDa), ovalbumin
(49 kDa), carbonic anhydrase (34 kDa), soybean trypsin inhibitor (28 kDa), lysozyme (28 kDa), and aprotinin (6.5 kDa)). Gels were
electroblotted onto polyvinylidene difluoride membranes, and protein
staining was performed with Amido Black according to standard
procedures.
Human primary keratinocytes,
cultured according to the Rheinwald-Green system (33), were seeded at
105 cells in keratinocyte growth medium in 60-mm culture
dishes as described by van Ruissen et al. (34). Keratinocyte
growth medium was composed of KBM (Clonetics; 0.15 mM
calcium) supplemented with ethanolamine (Sigma; 0.1 mM),
phosphoethanolamine (Sigma; 0.1 mM), bovine pituitary
extract (Clonetics; 0.4% v/v), epidermal growth factor (Sigma; 10 ng/ml), insulin (Sigma; 5 µg/ml), hydrocortisone (Collaborative
Research Inc.; 0.5 µg/ml), penicillin (Life Technologies, Inc.; 100 units/ml), and streptomycin (Life Technologies, Inc.; 100 µg/ml).
Keratinocyte differentiation was induced by switching confluent
keratinocyte cultures for 48 h to keratinocyte growth medium
containing high (1.8 mM) calcium. Cell extracts were
prepared in 200 µl of water by sonification on ice, followed by
centrifugation for 10 min at 25,000 × g. The
supernatants were stored at 20 °C until further use.
To isolate partial cDNAs of bovine and simian SKALP/elafin orthologs, three oligonucleotide primers (Fig. 2) were designed based on conserved sequences within human SKALP/elafin and porcine SPAI-2. Using total RNA derived from bovine tongue and rhesus monkey skin, first strand cDNA was generated in a reverse transcriptase reaction and amplified by PCR with the designed primers. PCR products were cloned and sequenced. Four clones were identified as members of the SKALP/elafin gene family based on the presence of sequences encoding an NH2-terminal TGase substrate domain and a COOH-terminal WAP-domain. Computer-assisted comparison with published sequences of this gene family was performed to reveal the possible identity of the clones. Fig. 2 shows the alignment of the deduced amino acid sequences of the four clones with the currently known family members. Two sequences can be regarded as the simian and bovine orthologs of SKALP/elafin. Overall, simian SKALP/elafin is 93% identical to human SKALP/elafin, and bovine SKALP/elafin is 71% identical to porcine SKALP/elafin. Within a part of the WAP domain (amino acid residues at positions 163-194 in Fig. 2) these percentages are 97% and 81%, respectively (Table I). The sequences of the putative active sites (residues italicized in Fig. 2) of both simian and bovine SKALP/elafin closely correspond to the known protease binding site of SKALP/elafin (35) and the protease binding site of SLPI (36) which also belongs to the WAP protein superfamily. The homology in the putative active site of both simian and bovine SKALP/elafin suggests that these molecules could be elastase inhibitors, although we have no data from functional studies to substantiate this contention. It was, however, shown recently that the porcine SKALP/elafin ortholog is indeed an inhibitor of at least porcine pancreatic elastase (16).
|
At present, the nomenclature of the proteins containing an
NH2-terminal TGase substrate domain and a COOH-terminal
WAP-domain is very confusing. Until now porcine SPAI-2 (2) and the
proteinase inhibitor SKALP/elafin/elastase-specific inhibitor were
described in the literature (1, 7, 17, 37). For the
NH2-terminal TGase substrate domain a separate name,
cementoin, was coined by Nara et al. (20). Here we propose
to give these proteins the acronym Trappin (Trappin = TRansglutaminase substrate and
WAP motif-containing
ProteIN) as a new nomenclature for this protein family. Since SPAI-2 and
SKALP/elafin/elastase-specific inhibitor were the first members to be
described, these are designated Trappin-1 and Trappin-2, respectively.
Trappin-3 is a new porcine member of this gene family which was called
pWAP-3 by Tamechika et al. (16). The two other sequences
from bovine tongue we identified (see Fig. 2) are new members of the
SKALP/elafin gene family and are designated Trappin-4 and Trappin-5 on
the basis of the new nomenclature that we proposed. Sequence analysis
of these new members of the Trappin gene family revealed a domain
structure that is very similar to porcine SPAI-2 (Trappin-1) and human
SKALP/elafin/elastase-specific inhibitor (Trappin-2). The sequence of
Trappin-4 and Trappin-5 diverges from SKALP/elafin and SLPI at the
putative reactive site; however, the cysteine residues of the
four-disulfide core structure obtained from the partial cDNA
sequence are conserved. The biological function of these new bovine
Trappin gene family members is not known. The putative reactive site
region in Trappin-4 and Trappin-5 is distinct from SLPI and
SKALP/elafin at the Met181 and Leu182 residues
(numbering according to Fig. 2) and could possibly lead to dissimilar
functions. Diversity in biological functions among SKALP/elafin, SLPI,
and SPAI-2 could be the result of the differences in residues around
the protease cleavage site within the WAP domain (see Fig. 2). (Amino
acid residues of substrates numbered P1, P2, etc., are toward the
NH2-terminal direction, and P1, P2
, etc., are toward the
COOH-terminal direction from the scissile bond, as in the nomenclature
of Schechter and Berger (38)). The preferable residues at P1
and P2
in
-lytic protease, which demonstrates an elastase-like primary
specificity, are, respectively, methionine and leucine (39). As the
scissile peptide bond in SKALP/elafin and SLPI is
Ala180(P1)-Met181(P1
), these proteins hold the
preferable residues at Met181(P1
) and
Leu182(P2
), suggesting a specificity like
-lytic
protease (35). Although porcine SKALP/elafin varies from human
SKALP/elafin and SLPI at the P1 and P2
residue, this protein shows the
ability to inhibit porcine pancreatic elastase (16), suggesting that the conserved Met181 residue is necessary for elastase
specificity. An intact methionine is required for human SKALP/elafin to
act as a high affinity inhibitor of leukocyte elastase (35). The
putative reactive site in the new bovine Trappin gene family members
Trappin-4 and Trappin-5 lacks this methionine residue, which makes it
unlikely that these proteins are elastase inhibitors. Recently,
Trappin-3 was described (16) which is 59% identical to Trappin-1
within a part of the WAP domain (amino acid residues 163-194 according
to Fig. 2). Comparison of our new family members Trappin-4 and -5 with
porcine Trappin-1 and Trappin-3 revealed less than 48% identity.
Trappin-4 and -5 share a 93% identity. On the basis of sequence
identity in the active site, the Trappin family members can be divided in three subgroups: Trappin-1 and -3, Trappin-4 and -5, and Trappin-2 (Table I).
The Trappin
gene family members exhibit distinct tissue distributions. Porcine
Trappin-1 (SPAI-2) was previously found to be abundantly expressed in
the intestine, whereas porcine Trappin-3 is found in the intestine at
relatively low levels (16). The mRNA for porcine Trappin-2
(SKALP/elafin) was mainly found in trachea and large intestine. This is
different from human Trappin-2 which is expressed in several human
stratifying squamous epithelia (9), except for epidermis where it is
only expressed in the context of inflammation, such as psoriasis and
wound healing (8, 10). The observed differences could be the result of
different sensitivity of the detection methods or could represent real
species differences. To define tissue distribution patterns of bovine Trappin-2, expression of the Trappin-2 mRNA was studied by RNase protection analysis. Protected bands with the expected length (360 base
pairs) were detected in the tongue and in the epidermis of snout (Fig.
3A). As a negative control we
used RNA from psoriatic skin, which contains human Trappin-2 mRNA.
Because human Trappin-2 has several mismatches compared with the bovine
ortholog it will be degraded by RNase and is hence not visible on the
gel. The expression of Trappin-4 and -5 mRNA in various bovine
tissues was studied by conventional Northern blot analysis. Trappin-4 is abundantly expressed in trachea, and a faint signal was found in
ileum and tongue (Fig. 3B), whereas Trappin-5 was expressed at relatively low levels in trachea (Fig. 3C). Trappin-4 an
-5 are very similar at the DNA level (88% overall identity), and therefore it cannot be totally excluded that the signal of the Trappin-5 probe on the Northern blot is the result of
cross-hybridization.
The high level of sequence divergence in the short amino acid stretch encoding the inhibitor domain of this gene family can be viewed as an example of accelerated evolution as described by Tamechika et al. (16). This is a process assumed to take place in genes after a duplication event (40) and has been interpreted as an effective mechanism to create new reactive site sequences with different substrate specificities. These amino acid changes are established by positive Darwinian selection as first reported by Hill and co-workers (41, 42), who described this phenomenon in the serine proteinase inhibitor (serpin) family. Since the Trappin family members are all expressed in tissues that are exposed continuously to microbial stimuli (oral cavity, trachea, intestine) it raises the possibility that some of them are directed against bacterial proteinases rather than exclusively against self-proteases (as Trappin-2, which is directed against leukocyte elastase and proteinase-3). This contention is supported by the recent finding that the distant family member SLPI, which is homologous to Trappins in its COOH-terminal domain, possesses antiviral and antibacterial activity (43, 44). The high substitution rates in the reactive center region of the Trappins could conceivably provide the host with a defense system against pathogens and parasites and give them the capacity to deal with an increasing number of attacking proteinases.
The TGase substrate motifs in the NH2-terminal part of the Trappins are conserved, although the number of amino acid repeats varies among species and among the Trappin gene family members. Porcine and bovine Trappin-2 display 11 and 12 repeats, respectively, whereas six repeats were found in human and simian Trappin-2. The deduced amino acid sequences of the novel members derived from bovine tongue, Trappin-4 and Trappin-5, both contained five repeats. Combined with 14 repeats found in the published porcine Trappin-1 protein and eight repeats in the new porcine family member Trappin-3, the frequency of amino acids at each position was calculated by comparing the total of 67 repeats in the putative TGase substrate domains of the currently known Trappin protein family members from four different mammalian species (Table II). In this way a consensus hexapeptide sequence of GQDPVK could be deduced, in agreement with earlier studies (2, 7).
|
To characterize further the biochemical properties of
the above mentioned TGase consensus substrate motif, the biotinylated hexapeptide GQDPVK was synthesized, and enzyme kinetic experiments were
performed using a TGase assay (29) to determine optimal reaction
conditions. The TGase cross-linking reaction is based on a
Ca2+-dependent exchange of primary amines for
ammonia at the -carboxamide group of glutamine residues.
Peptide-bound lysine residues or polyamines serve as the primary amines
to form either
-(
-glutamyl)lysine or (
-glutamyl)polyamine
bonds between proteins (45), which are highly resistant to chemical and
enzymatic degradation (46). As human epidermis is known to contain both
TGase activity (47, 48) and various substrate proteins (e.g.
involucrin, loricrin, small proline-rich proteins) (21), an extract of
psoriatic scales was used to study the incorporation of the
biotinylated hexapeptide. A time course incubation at 37 °C of the
biotinylated hexapeptide with scale extract showed that the reaction
rate as measured by chemiluminescence detection was linear up to 3 h (data not shown). For further experiments a reaction time of 60 min
was used for practical convenience. The effect of the peptide
concentration on the rate of cross-linking to epidermal proteins by
endogenous TGase is shown in Fig. 4. An
apparent Km of 0.46 µM was found.
The protective callus layer resulting from terminal differentiation of
the squamous epithelium is thought to be cross-linked by different
TGase activities present in mammalian epidermis. These TGases are
probably involved in the formation of the cornified cell envelope of
terminally differentiating epidermis and of other stratified squamous
epithelia. Three TGases are expressed in the epidermis, a ubiquitous
tissue type TGase (TGC or TGase 2), a membrane-associated
keratinocyte TGase (TGK or TGase 1) present in cultured
epidermal keratinocytes and in many epithelial and nonepithelial
tissues, and the zymogen epidermal TGase (TGE or TGase 3)
known to be expressed in differentiated epidermal cells and hair
follicles, but not in cultured epidermal keratinocytes (49-53).
Incubation of the biotinylated GQDPVK hexapeptide with psoriatic scale
extract leads to its cross-linking to stratum corneum proteins by
Ca2+-activated endogenous TGase (Fig.
5A, sample 1).
Human epidermis and probably also stratum corneum are known to harbor
TGases 1, 2, and 3 (21, 47, 48). From our data, however, it cannot be
concluded which type is responsible for cross-linking the biotinylated GQDPVK hexapeptide to substrate proteins. Since TGase 1 and 3 are not
available in purified form we only tested guinea pig liver TGase (type
2 TGase), and this was found to cause incorporation of the biotinylated
hexapeptide into stratum corneum proteins of a native scale extract
(sample 2) or scale extract that had been heat-inactivated
to eliminate endogenous TGase activity (sample 3); so both
the TGases present in scale extract and type 2 TGase catalyze the
incorporation of biotinylated peptide into stratum corneum proteins.
Specificity of the reaction was checked by the omission of guinea pig
liver TGase (sample 4) or the addition of excess EDTA (not
shown).
The biotinylated GQDPVK hexapeptide appeared to be an extremely efficient TGase substrate that acts both as an acyl donor and as an acyl acceptor probe (Fig. 5B). Substitution of the acyl acceptor residue lysine (K) for arginine (R) (sample 2) or substitution of the acyl donor residue glutamine (Q) for asparagine (N) (sample 3) showed no influence on cross-linking to stratum corneum proteins. Substitution of both the lysine and the glutamine residue for, respectively, arginine and asparagine, totally abolished cross-linking of the biotinylated hexapeptide (sample 4). The efficiency of cross-linking is sequence dependent as a hexapeptide in the reverse order (KVPDQG, sample 5) or the exchange of lysine and glutamine (GKDPVQ, sample 6) virtually eliminated cross-linking to stratum corneum proteins. Whether these negative effects on substrate reactivity are the result of changes in structural conformation of the peptide or are a consequence of changing the chemical nature of the side chains surrounding the substrate lysine and glutamine is not clear. Grootjans et al. (29) showed that some residues directly preceding the substrate lysines have a negative effect on TGase activity. These residues, like Asp, Gly, Pro, His, and Trp, appeared to be largely avoided in a total of 30 characterized acyl acceptor (lysine) substrates. We would speculate that degenerate hexapeptide motifs could also be used in vivo. We have shown previously that a synthetic peptide comprising the NH2-terminal 14 amino acids of processed SKALP/elafin, which contains the degenerate motif GQDTVK, is also incorporated efficiently by TGase, suggesting that slight variations in the surrounding amino acids are tolerated. In the same study we used full-length SKALP/elafin purified from human keratinocytes and showed that the protein is cross-linked to an acyl acceptor probe by the action of type 2 TGase. A recent study by Steinert and Marekov (21) showed that the degenerate AQEPVK and GQDKVK sequences were used for cross-linking to loricrin and cytokeratin 1 in vivo, as determined by amino acid sequencing of purified peptides from human foreskin.
In addition to cross-linking of the GQDPVK hexapeptide to its natural
substrate proteins in stratum corneum, we also used purified control
proteins to investigate the substrate specificity of GQDPVK for these
proteins. The biotinylated GQDPVK hexapeptide was found to be
cross-linked efficiently by exogenous type 2 TGase to B-crystallin
and
-low-crystallin, structural proteins of the vertebrate eye lens
(Fig. 5C). These proteins are known acyl acceptor substrates
for type 2 TGase, as the COOH-terminal lysine residue of
B-crystallin was identified as the site of linkage (30), and a
lysine residue in the NH2-terminal extension acts as the
sole acyl acceptor substrate in
A3-crystallin, which is a component
of a
-low-crystallin preparation (31). Two glutamine residues acting
as acyl donor were characterized in the NH2-terminal region
of
A3-crystallin by Berbers et al. (54). No cross-linking capacity was found using
A-crystallin and
-crystallin as a
substrate, which is in accordance with experiments described previously
by Groenen et al. (30, 31). Recombinant SKALP/elafin, which
only contains the 57 COOH-terminal amino acids of the full-length
molecule (and thus lacking the TGase substrate domain), and bovine
serum albumin did not show appreciable cross-linking to GQDPVK. To
determine the specificity of the GQDPVK hexapeptide for cross-linking
to stratum corneum proteins by endogenous TGase in psoriatic scale extracts, we introduced two other acyl donor probes: a biotinylated TVQQEL hexapeptide that is patterned on the NH2-terminal
extension of bovine
A3-crystallin (30), and a biotinylated PGGQQIV
heptapeptide, patterned on the amine acceptor sequence in fibronectin
(55). Fig. 5D shows that the GQDPVK hexapeptide reacts to a
greater extent with stratum corneum proteins than the other acyl donor probes (samples 1-3), whereas they all show the same
reactivity toward
-low-crystallin by the action of exogenous type 2 TGase (samples 4-6). This suggests that the GQDPVK motif is
a preferred substrate for cross-linking to stratum corneum proteins by
epidermal TGases.
TGase activity in stratum corneum extracts could be derived from TGase
types 1, 2, and 3. Previous studies, however, have suggested that the
bulk of the soluble TGase activity comes from TGase type 3 (49). To
investigate the relative contribution of the various TGase types in
stratum corneum extracts we used scale extracts from three psoriatic
patients (Fig. 5E, samples 1-3), a patient with
LI (sample 4), and a patient with eczema (sample
5). Surprisingly, the TGase activity measured in these five scale
extracts was very similar. Assuming that TGase 1 would contribute
significantly, a decreased TGase activity was expected in scale extract
from the patient with LI, since this patient was homozygous for a
splice mutation in intron 5 of the TGase type 1 gene as determined by
single strand conformation polymorphism analysis and sequence
analysis.2 This mutation
causes a deficiency for TGase type 1 as described recently (24) and is
probably the origin for disturbed formation of the cornified envelopes
which may explain the phenotype of LI. Despite a defect TGase 1 gene in
LI, our experiments showed that the GQDPVK hexapeptide is still
cross-linked to stratum corneum proteins by endogenous TGases in scale
extract from a LI patient. It is most likely that TGase type 2 or 3 is
responsible for this phenomenon. In a recent publication TGases 1, 2, and 3 were shown to utilize loricrin in vitro as a complete
substrate, but the types of cross-linking were different (56). TGase 1 mostly formed oligomeric complexes by interchain cross-links, whereas
TGase 3 reactivity is involved in the formation of intrachain
cross-links. The participation of TGase 2 in loricrin cross-linking was
quite weak. It is therefore likely that TGase 3 is the active enzyme in
the scale extract of the LI patient. Evidence that the GQDPVK motif can
be used for cross-linking by TGase 1 was obtained by using cultured
normal epidermal keratinocytes, which do not express TGase 3 (49), as a
source of TGase type 1 and substrate (Fig. 6). Subsequent reaction with the
biotinylated GQDPVK hexapeptide and analysis by SDS-PAGE show
incorporation in proteins predominantly between 30 and 80 kDa (Fig. 6,
lane 2). For comparison the incorporation pattern in stratum
corneum proteins from psoriatic epidermis is shown (Fig. 6, lane
4). Cross-linking of GQDPVK to B-crystallin by TGase 2 is
demonstrated in lanes 5 and 6.
To conclude, we have identified novel Trappin family members, and we have characterized some of the biochemical properties of the GQDPVK motif with respect to TGase cross-linking in vitro. In addition to the TGase substrate domain, Trappin family members possess a COOH-terminal WAP motif that harbors putative proteinase inhibitory activity. The constitutive expression of Trappin gene family members in a number of normal epithelia which are subjected to continuous mechanical and microbial stress or inflammatory stimuli (e.g. oral epithelia, esophagus, trachea, ileum) is in line with a role for these molecules as proteinase inhibitors with different substrate specificities for self and (possibly) non-self proteases. Future research will be directed at investigation of the role of other Trappin family members in epithelial homeostasis and human diseases.
We acknowledge Dr. Shigehisa Hirose (Tokyo Institute of Technology, Japan), whose group has previously cloned and described two other family members, for cooperation in coming to a consensus nomenclature and for the suggestion to use the name Trappin gene family.