and in response to injury - journal of biological chemistry · functions in several tissues both in...

Epilysin: A Novel Human Matrix Metalloproteinase

(MMP-27) Expressed in Testis and Keratinocytes

and in Response to Injury

Jouko Lohi1, Carole L. Wilson, Jill D. Roby, and William C. Parks

Departments of Pediatrics (Allergy and Pulmonary Medicine) and Cell Biology and Physiology,

Washington University School of Medicine, St. Louis, MO 63110

Running title: Human Epilysin Gene

1To whom correspondence should be addressed:

Jouko Lohi, M.D., Ph.D. Telephone: +358-9-191-26469

Department of Pathology Fax: +358-9-191-26475

Haartman Institute, University of Helsinki Email: [email protected]

P.O. Box 21 (Haartmaninkatu 3)

FIN-00014 University of Helsinki

FINLAND

Key words: cDNA cloning, wound healing

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 19, 2000 as Manuscript M001599200 by guest on July 6, 2018

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Summary

We have cloned a new human matrix metalloproteinase (MMP-27, epilysin) from human

keratinocyte and testis cDNA libraries. Like most MMPs, epilysin contains a signal sequence, a

prodomain with a PRCGVTD sequence, a zinc-binding catalytic domain with an

HEIGHTLGLTH sequence, and a hemopexin-like domain. In addition, epilysin has a furin

activation sequence (RRKKR) but has no transmembrane sequence. The exon-intron

organization and splicing pattern of epilysin differs from that of other MMP genes. It has only 8

exons, and 5 exons are spliced at sites not used by other MMPs. Another novel feature of

epilysin is that exon 4 is alternatively spliced to a transcript that does not encode the N-terminal

half of the catalytic domain. Northern hybridization of tissue RNA indicated that epilysin is

expressed at high levels in testis, and at lower levels in lungs, heart, colon, intestine, and brain.

RNAse protection assay with various cell lines indicated that epilysin was selectively expressed

in keratinocytes. Recombinant epilysin degraded casein in a zymography assay, and its

proteolytic activity was inhibited by EDTA and by batimastat, a selective MMP inhibitor.

Immunohistochemical staining showed expression of epilysin protein in the basal and suprabasal

epidermis of intact skin. In injured skin, prominent staining for epilysin was seen in basal

keratinocytes both at and some distance from the wound edge, a pattern that is quite distinct from

that of other MMPs expressed during tissue repair. These findings suggest that this new MMP

functions in several tissues both in tissue homeostasis and in repair.

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Introduction

The matrix metalloproteinases (MMPs)4 comprise a family of enzymes that share several

common structural features and that function both in the turnover and degradation of

extracellular matrix proteins and in the processing, activation, or deactivation of a variety of

soluble factors (1). MMPs, or matrixins, are a subgroup of the much larger metalloproteinase

superfamily, which also includes astacin and ADAM proteinases, among others. To date 23

different MMPs have been cloned, and additional members continue to be identified (2).

To be classified as a matrix metalloproteinase, a protein must have conserved features of

two domains, namely the pro- and the catalytic domains. The prodomain of a typical MMPs is

about 80 amino acids, and all MMPs, except MMP-23 (3), contain the consensus sequence

PRCXXPD. As for all metalloproteinases, the catalytic domain contains an active site Zn2+ that

binds three conserved histidines in the sequence HEXXHXXGXXHS/TXXXXXXM, which also

contains a conserved methionine to the carboxy side of the zinc-binding site (metzincins) (4). In

an inactive state, the conserved cysteine residue in the prodomain provides the fourth

coordination site for the catalytic zinc ion. In addition, with the exception of matrilysin (MMP-

7), endometase/matrilysin-2 (MMP-26), and MMP-23, MMPs have a hinge region, which is

often proline-rich, and a so-called hemopexin-like C-terminal domain (3,5,6). Other domains

found in MMPs are specialized to subgroups of enzymes. For example, four membrane-type

MMPs (MMP-14, -15, -16, -24) have transmembrane and cytosolic domains, whereas MT4-

MMP and MT6-MMP (MMP-17 and –25, respectively) have C-terminal hydrophobic extensions

that act as a glycosylphosphatidylinositol (GPI) anchoring signal (7-9). The two gelatinases

(MMP-2 and MMP-9) have gelatin-binding domains. MMP-23 lacks the hemopexin domain and

has a novel cysteine array motif and an immunoglobulin-like C2 type fold domain (3,10). In

addition to a common domain structure, MMPs share a similar gene arrangement suggesting that

they were generated by duplications of an ancestor gene. At least eight of the known human


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MMP genes (MMPs 1, 3, 7, 8, 10, 12, 13, and 20) are clustered on chromosome 11 at 11q21-23.

Other known MMP genes are scattered along chromosomes 1, 8, 12, 14, 16, 20, and 22

(3,11,12).

MMPs are secreted or bound or anchored to the cell membrane, and all function extracellularly

or within the secretion pathway. As demonstrated in defined in vitro studies, almost all MMPs

can cleave or degrade some protein components of the extracellular matrix, and many are able to

act on a wide variety of proteins (13). Notable exceptions to this rule are stromelysin-3 (MMP-

11) and MMP-23, which have no known extracellular matrix substrates (3,14). In addition and

quite important, MMPs can process or degrade non-matrix proteins. For example, matrilysin is

responsible for activation of the pro-form of α-defensins (15), a class of secreted antimicrobial

peptides, and several MMPs can cleave and inactive the serpin α1-proteinase inhibitor (16,17),

which is an in vivo substrate for gelatinase-B (MMP-9) (18). In addition, several MMPs, such as

MMP-1, -2, -3, -7, and –11, among others, directly modulate the activity of several growth

factors, such as TNFα, IGF-1, EGFs, and FGFs (19-24). Thus, matrix degradation is neither a

sole or common functional feature of MMPs.

Many of the secreted MMPs, including MMPs 1, 3, 9, 10, 11, and 13, are not expressed

in normal, healthy, resting tissues, and, with some exceptions, their production and activity are

maintained at nearly undetectable levels. In contrast, some level of MMP expression is seen in

any repair or remodeling process, in any diseased or inflamed tissue, and in essentially any cell

type grown in culture (25,26). Although the qualitative pattern and quantitative levels of MMPs

vary among tissues, diseases, tumors, inflammatory conditions, and cell lines, a reasonably safe

generalization is that activated cells express MMPs. Some MMPs, including MMPs 7, 19, 24, 25,

and 26, are expressed in healthy tissues (27-31).


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In the present study, we report on the cloning and initial characterization of a novel

human MMP, MMP-27, which we call epilysin. We isolated the cDNA for this protein from

keratinocyte and testis libraries, and we show that it has the essential domains of a prototypic

MMP, as well as several unique features. Because of its ability to degrade a protein substrate

was fully inhibited by EDTA and a hydroxamate MMP inhibitor, epilysin is indeed a

metalloenzyme. Our data suggests that epilysin is expressed in intact tissues, and upregulated in

response to injury. Thus, this new MMP may function in both tissue homeostasis and tissue

repair.


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Experimental Procedures

Cloning of Human Epilysin cDNA. Exon/Intron Mapping. A search of the GenBank

data base with the peptide string FDGXXXXLAHAXXPGXXXXGDXHFDXXEXW, which is

conserved among MMPs, returned a homologous sequence within a 82 kb human genomic DNA

clone (Accession AC006237). Nested primers were designed to amplify a 161 bp cDNA

fragment by RT-PCR using HT-1080 human fibrosarcoma line RNA as a template. The

amplified product corresponds to bases 726-886 (see Fig. 1). By screening a human foreskin

keratinocyte cDNA library (HL1110b, Clontech, Palo Alto, CA) by plaque hybridization with

the 32P-labeled 161 bp cDNA fragment, we obtained 3 positive clones, which were then

sequenced. The longest clone of 1.5 kb contained exons 3 to 8 of epilysin (see below for exon

numbering). This cDNA clone was then used to screen a pooled human testis cDNA library

(HL5033t, Clontech). Among the more than 20 positive clones was a clone that contained the

coding regions of exons 1 and 2. Exon-intron boundaries were determined by comparing the

cDNA sequences with the genomic sequence in data base.

Computer Analyses. Screening of GenBank data base was performed using the

TBLASTN program and the NCBI server (URL: www.ncbi.nlm.nih.gov) (32). Epilysin signal

peptide was identified and its cleavage site was predicted using SignalP server (URL:

genome.cbs.dtu.dk/services/SignalP/) (33). The amino acid sequences of human MMPs 1, 3, 11,

14, and 19 were aligned with epilysin using CLUSTAL W (34). Phylogenetic tree was drawn

based on a CLUSTAL W alignment of the amino-acid sequences of the catalytic domains of all

known human MMPs.

Cell Culture. Human foreskin fibroblasts (HFF), immortalized human keratinocytes

(HaCaT) (35), and human fibrosarcoma HT-1080 cells (CCL-121, American Type Culture

Collection, Rockville, MD) were grown to confluence in Eagle's minimal essential medium


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(MEM) containing 10% heat-inactivated fetal calf serum (GIBCO-BRL, Gaithersburg, MD), 100

IU/ml penicillin, and 50 µg/ml streptomycin. Human colon adenocarcinoma HT-29 cells

(HTB38, ATCC) were maintained in RPMI medium. MMP expression was stimulated by

treatment with 16 nM phorbol ester (PMA, Sigma Chemical Co., St. Louis, MO) for 24 h.

Primary human keratinocytes were isolated from normal full thickness adult skin and cultured on

collagen-coated dishes as described (36). U937 cells (ATCC, CRL 1593), a human monocyte-

like cell line, were cultured and differentiated to stimulated macrophage-like cells by a 24-h

treatment with 4 nM PMA and 5 µg/ml lipopolysaccharide (LPS, Sigma) as described (37). Total

RNA was isolated using RNAzol B (Tel-test, Inc., Friendswood, TX).

Northern Blot Analysis. Nylon filters containing 2 µg of poly (A)+ RNA from various

human tissues (Clontech) were prehybridized with ExpressHyb hybridization solution

(Clontech), and then hybridized in the same solution with 32P-labeled epilysin probe, generated

by random priming using a 1.5 kb cDNA fragment as a template. Loading was normalized by

hybridization with a β-actin cDNA probe. Hybridization and washes were performed according

to manufacturer’s instructions.

RNAse Protection Analysis and Determination of Alternative Splicing. Expression of

epilysin mRNA in cultured cells was analyzed by ribonuclease protection technique using Direct

Protect kit (Ambion Inc., Austin, TX) and two different RNA probes. A PCR fragment of 161 bp

(bases 726-886 in epilysin cDNA) and another fragment of 539 bp (bases 370-908) were cloned

to pGEM-T-Easy (Promega), and plasmid DNA was linearized with Sal I. Antisense RNAs of

257 nt and 635 nt, respectively, were transcribed with T7 RNA polymerase in the presence of [α-

32P]UTP and hybridized with 10 µg of total cellular RNA. After an overnight hybridization,

unpaired RNA was degraded by treatment with RNAse A and RNAse T1 (10 U/ml and 400

U/ml, respectively) at 37°C for 30 min followed by isopropanol precipitation. Protected RNA

fragments were fractionated by 5% SDS-PAGE containing 6 M urea and visualized by


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autoradiography. Alternative splicing was first detected when RT-PCR reactions to amplify

regions containing bases 370-960 and 440-960 (forward primers 5’-

GTGGGTGTCCCAGCTACCTGTC-3' and 5’-TGCGGGGTTACAGATACCAACAG-3',

reverse primer 5’-CTCTTGTAGTAGGGCGCCATGAG-3') using HaCaT cDNA as a template

gave three specific products of different size. Specificity of the amplification products was

determined by Southern blotting using a [γ-32P]ATP labeled internal oligonucleotide probe 5’-

GCGGCGAAGCGCACTTCGACCAAGATGAGC-3', and the amplification products were

purified from gel and sequenced. The presence of alternatively spliced mRNA in HaCaT cells

was verified by RNAse protection analysis using the 635 nt RNA probe described above.

Baculoviral Recombinant Epilysin. For generation of recombinant protein, nucleotides

536 to 1732 of epilysin cDNA, coding for amino acids 123 to 520 of epilysin protein were

amplified using primers 5’-CGGGATCCGACGATGACGATAAGTTTGCAAAGCAAGGT-

AACAAATGGTACAAGC-3' (forward, epilysin sequence underlined) and 5’-

CGGAATTCTCAGAACAGGGCGCTCCCCGAGTTG-3’ (reverse). The amplification product

was digested with Bam HI and Eco RI and cloned into the pAcSecG2T baculovirus transfer

vector (Pharmingen, San Diego, CA). The resulting expression construct codes for a fusion

protein of Schistosoma japonicum glutathione S-transferase (GST) and amino acid residues 123

to 520 of epilysin corresponding to the putative furin-activated enzyme. This vector also

provides the signal peptide from the baculovirus protein gp67 to direct secretion of the fusion

protein. Sf9 cells were transfected with the construct and BaculoGold DNA (Pharmingen) to

produce recombinant baculovirus. Following two rounds of virus amplification, High Five insect

cells (Stratagene) were infected and harvested along with conditioned medium 5 days later. Cell

pellets were lysed in 10 mM Tris-HCl buffer, pH 7.5, containing 130 mM NaCl, 1% Triton X-

100, 10 mM NaF, 10 mM NaPi, and 10 mM NaPPi. GST-epilysin fusion protein was purified

from cell lysates and conditioned medium by glutathione Sepharose affinity chromatography

according to manufacturer's protocol (Pharmingen). Protein eluting from the affinity resin was


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analyzed by SDS-PAGE and Western blotting with a rabbit polyclonal anti-GST antibody

(Upstate Biotechnology, Lake Placid, NY). Unexpectedly, most of the GST-epilysin fusion

protein was found in cell pellets and not in the conditioned medium.

Recombinant Epilysin Produced in E. coli and Casein Zymography. To express a fusion

protein consisting of Schistosoma japonicum glutathione S-transferase (GST) and the pro- and

catalytic domains of epilysin (amino acid residues 23 to 284, see Fig. 1) in E. coli, a 802-bp

fragment of the MMP-27 cDNA was amplified by PCR using primers 5’-

CGGGATCCCAGCCCGCGGAGCGCGGA-3’ (forward, epilysin sequence underlined) and 5’-

GGAATTCTCACCCATACAGGCTCTGCACGGCCAGC-3’ (reverse), and the PCR-product

was digested with Bam HI and Eco RI and cloned into the pGEX-6P-2 vector (Amersham-

Pharmacia, Uppsala, Sweden). The resulting expression vector was then transformed into

BL21(DE3)pLys strain of E. coli. Overnight bacterial culture derived from a single bacterial

colony was diluted 1:10 and incubated at 37°C for 2 h. Expression of the fusion protein was then

induced by adding isopropyl-1-thio-β-D-galactopyranoside (IPTG) (0.5 mM final concentration)

followed by further incubation at 25°C for 4 h. Recombinant protein obtained in inclusion bodies

was solubilized by sonication in the presence of N-lauroyl-sarcosine and affinity purified with

glutathione Sepharose as described (38). Recombinant fusion protein bound to glutathione

Sepharose was then digested with PreScission protease according to manufacturer’s instructions

(Pharmacia) to remove the GST tag. Between the GST domain and epilysin, the fusion protein

has the recognition sequence (LEVLFQGP) for PreScission Protease. Recombinant epilysin

(pro- and catalytic domains) was then eluted with 50 mM Tris-HCl buffer, pH 7.5, containing

500 mM NaCl, 5 mM dithiothreitol and 0.1% Brij-35. Caseinolytic activity was measured by

zymography using a 4-16% SDS-PAGE blue casein zymogram gel (Novex, San Diego, CA)

according to manufacturer’s protocol. After electrophoresis, the zymogram gel was washed and

incubated in 50 mM Tris-HCl buffer, pH 7.5 containing 1-2.5% Triton X-100 and either 1) 10


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mM EDTA or 2) both 5mM CaCl2 and 1 µM ZnCl2 or 3) 10 µM batimastat in the presence of

5mM CaCl2 and 1 µM ZnCl2.

Preparation of Antibodies and Immunoblotting Assay for Epilysin. An 8-chain branching

multiple antigenic peptide (MAP) of 16 amino acids, DQDERWSLSRRRGRNL, corresponding

to the middle of the catalytic domain of epilysin (amino acid residues 219-234, see Fig. 1), was

used as an antigen (Research Genetics, Huntsville, USA). Rabbits were first immunized with 0.5

mg of the peptide in complete Freund’s adjuvant and three booster injections with 0.5 mg of the

peptide in incomplete Freund’s adjuvant were given 2, 6, and 8 weeks later by a commercial

operation (Research Genetics). Antibodies were purified from whole serum, harvested at 10

weeks after primary injection, by affinity chromatography with the peptide coupled to NHS

Sepharose-4B according to manufacturer’s instructions (Pharmacia). For immunoblotting,

confluent cultures of HaCaT cells were washed with serum free medium and were incubated

under serum free conditions for an additional 48 h. The medium was then collected and

concentrated 70-fold using a Centricon microconcentrator (Amicon, Beverly, MA). 10 µl of

concentrated conditioned medium was mixed with an equal amount of Laemmli sample buffer

containing 10% β-mercaptoethanol and resolved by SDS-PAGE through a 4-15% gradient gel.

Recombinant baculoviral GST-epilysin fusion protein was used as a positive control. Proteins

were then electrophoretically transferred to nitrocellulose (Schleicher & Schuell, Dassel,

Germany) using a semi-dry blotting apparatus at 2.5 mA/cm2 for 30 min. Membranes were

blocked with 5% milk in PBS/Triton X-100 (0.5%) and incubated with 0.15 µg/ml of affinity

purified antibodies in 50 mM Tris-HCl buffer containing 500 mM NaCl, 0.1% Tween 20 and

0.1% bovine serum albumin, pH 8.5. After five washes in the same buffer, the bound antibodies

were detected using biotinylated anti-rabbit-IgG antibodies and peroxidase-conjugated

streptavidin (Dakopatts, Copenhagen, Denmark) and enhanced chemiluminescence Western

blotting detection system (Amersham International PIC, Amersham, UK) as described (39).


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Expression of Epilysin in CHO Cells. Immunofluorescence Staining. Chinese hamster

ovary (CHO) cells were transfected with a cDNA construct coding for epilysin with a C-terminal

10-aa influenza virus hemaglutinin tag under transcriptional control by CMV promoter in

pcDNA3 vector (InVitrogen, San Diego, CA) using FuGENE 6 transfection reagent according to

manufacturer’s instructions (Boehringer Mannheim GmbH, Mannheim, Germany). 24 h after

transfection, transfected cell clones were selected for neomycin resistance as described (40). For

immunofluorescence staining, a pool of transfected cells was plated on glass coverslips, and 3

days later, the cells were fixed with 3% paraformaldehyde in PBS (phosphate buffered saline,

0.14 M NaCl in 10 mM phosphate buffer, pH 7.4). After fixing the coverslips were washed three

times with PBS and blocked with 5% bovine serum albumin (BSA) in PBS for 30 min. Affinity-

purified epilysin antibody was then added (1:100 dilution) in 0.5% BSA in PBS and incubated 1

h at room temperature with mild shaking. After three washes with the same buffer, FITC

conjugated anti-rabbit IgGs (Jackson Immunoresearch Laboratories, West Grove, PA) were

added and incubated for 1 h. Coverslips were then washed 5 times with PBS and mounted on

glass slides using Vectashield anti-fading agent (Vector Laboratories, Inc., Burlingame, CA).

Immunohistochemistry. Individual, 4-mm-wide, full-thickness biopsies of human skin

used for keratinocyte culture were placed into the wells of 6-well cluster dishes and covered with

DMEM containing antibiotics. 24 h later, tissues were fixed in 10% buffered-formalin and

processed for paraffin embedding. Deparaffinized 5 µm sections were processed for

immunohistochemistry using alkaline phosphatase as described (41). Endogenous peroxidase

activity was blocked by incubation in 0.3% H202 for 30 min at room temperature. Affinity

purified anti-human epilysin antibody was diluted 1:1000. Bound antibody was detected using a

Vectastain ABC Elite kit (Vector Laboratories) following the manufacturer's instructions.

Peroxidase activity was detected using 3,3'-diaminobenzidine tetrahydrochloride as chromogenic

substrate. Sections were counterstained with Harris hematoxylin. For negative controls, sections

were processed with preimmune serum.


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Results

Cloning and Sequencing of a cDNA Encoding Human Epilysin. Comparison with Other

MMPs. To identify undiscovered MMPs, we searched the GenBank database using the

TBLASTN program and a peptide query sequence FDGXXXXLAHAXXPGXXXXGDXHFD-

XXEXW, which defines a partial consensus sequence of a metalloproteinase catalytic domain.

Among the more than 100 hits was a human genomic DNA clone (Accession number

AC006237) that was submitted by Whitehead Institute/MIT Center for Genome Research and

had been sequenced as a part of the Human genome project sequencing chromosome 17. There

was no annotation that the sequence would code for proteins. After translation of the genomic

DNA in three forward reading frames, several peptide sequences typical of MMPs, including a

propeptide sequence PRCGVTD and a catalytic domain sequence HEIGH, were identified, and

these sequences were separated by putative intronic sequences.

To assess if this genomic region was transcribed to an mRNA, two sets of primers were

designed; the forward and reverse primers were directed to different suspected exons. As a

source of RNA, we used the human fibrosarcoma cell line HT-1080, as these cells are known to

express a wide variety of MMPs (40,42). cDNA was synthesized using random hexamer primers

and was amplified by PCR. Two-stage PCR with nested primers produced an amplified DNA

fragment of expected size (161 bp), and the nucleotide sequence of this fragment was identical to

that of presumed exonic portions of the genomic sequence (data not shown).

To obtain the full-length cDNA for this novel MMP, we screened a human keratinocyte

cDNA library using the PCR product as a probe. (RNAse protection analysis of various cell lines

revealed that epilysin is expressed in cultured human keratinocytes; see Fig. 6.) Among the three

positive clones, we isolated and characterized a 1.5 kb cDNA that contained sequence coding for

part of the prodomain, the entire catalytic and hemopexin-like domains, a stop codon TGA, and a


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85 bp of 3’ UTR. To determine the 5’ end of this MMP transcript, we screened a testis cDNA

library with a probe corresponding to the 5’-end of the 1.5 kb keratinocyte cDNA insert.

(Hybridization of tissue-RNA blots revealed that epilysin is expressed in human testis at high

levels; see Fig. 5.) Of the positive clones, we identified one containing a cDNA insert that coded

for the missing portion of the prodomain and for about 170 bases of 5’ UTR. The open reading

frame, starting from the first ATG codon, contains 1560 nucleotides and codes for a 520-aa

protein with a calculated molecular weight of 59 kDa (Fig. 1). We named this new MMP

epilysin. Using the accepted consecutive-number nomenclature, epilysin would be assigned

MMP-27.

The domain structure and organization of epilysin is predictable for an MMP (Fig. 1, 2).

Using an analysis program available at the SignalP server (33), we identified a typical

hydrophobic signal sequence of 22 amino acids at the amino terminus of epilysin (Fig. 1). The

signal sequence is followed by a prototypic MMP prodomain with the conserved cysteine-switch

sequence PRCGVTD (Fig. 1,2). In this sequence, a proline that is present in all other human

MMPs except MMP-19 (43) is replaced by a threonine (Fig. 2). In addition, after the cysteine

switch sequence, there is an 11-aa insertion, which is not present in other known MMPs,

followed by an RRKKR furin recognition sequence (Fig. 1, 2). The catalytic domain is highly

conserved relative to other MMPs, and as for secreted MMPs, an 8-aa insertion present only in

MT-MMPs (e.g., MMP-14) is lacking from epilysin (Fig. 2). The catalytic center with three

histidine residues, HEIGHTLGLTH, is unique in that no other MMP has threonine within this

sequence. A 39-aa hinge region is followed by a typical hemopexin-like domain. There is no

hydrophobic transmembrane sequence typical of membrane-inserted MMPs or a hydrophobic

extension typical of GPI anchored proteins. In addition, epilysin has two putative N-

glycosylation sites: one in the N-terminal part of the catalytic domain and another in the second

pexin-like repeat of the hemopexin domain (Fig. 1). The calculated molecular weight of the


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proenzyme without the signal sequence is 56 kDa; the active, furin-processed enzyme is

estimated to be 45 kDa. These weights do not include any contribution in mass by glycosylation.

Comparison of the epilysin amino acid sequence with other MMPs by CLUSTAL W

program (Fig. 2) and construction of a phylogenetic tree on the basis of the catalytic domain

sequences (Fig. 3) indicates that epilysin is most closely related to some other recently cloned

MMPs, including MMP-19, MMP-23, MT4-MMP (MMP-17), MT6-MMP (MMP-25), and

stromelysin-3 (MMP-11) (3,8,43-45). The number of identical and similar residues with MMP-

19 catalytic domain is 46% and 60%, respectively.

Structural Organization of the Human Epilysin Gene. We mapped the exon/intron

junctions and determined an exon-intron map of the gene by comparing the cDNA and genomic

sequences (Fig. 4A). Exon-intron boundaries and the sizes of exons and introns are summarized

in Table 1. All exons were contained within the genomic BAC clone except exon 1, and hence,

we do not yet know the size of intron 1 (Table 1). The exon-intron structure of epilysin is unique

compared to other MMP genes. Whereas most MMP genes have 10 exons, the epilysin gene has

only 8 exons, similar to that of stromelysin-3 (MMP-11) (Fig. 4B) (46). Furthermore, only three

of the seven splice sites (splice sites between exons 1 and 2, 5 and 6, and 6 and 7) are at positions

conserved among most MMP genes. None of the unique splice sites are similar to those some

other “nontraditional” MMP genes characterized to date, such as MMP-7, MMP-11, or MMP-14.

Overall, the organization of the epilysin gene is similar to that of MMP-19, with one overt

difference being that exon 8 of epilysin corresponds to exons 8 and 9 of MMP-19 (Fig. 4B) (47).

The exon/intron boundaries conform to the GT/AG rule for splice sites (48) (Table 1).

Analysis of Epilysin Expression in Human Tissues and Cell Lines. To analyze the

expression of epilysin in different human tissues, we hybridized a Northern blot containing

mRNA from various human tissues with a 1.5 kb epilysin cDNA probe. At least three different


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transcripts of 2.6, 2.0 and 1.2 kb were detected in many tissues (Fig. 5), and this heterogeneity is

likely due to alternative splicing (see below). The 2.6 kb transcript was most abundant in all

tissues, and among tissues, the relative levels of three transcripts were about the same. Epilysin

mRNA is highly expressed in testis, and at lower levels in lungs, heart, colon, intestine, and brain

(Fig. 5).

To determine which cell types express epilysin, we screened RNA from several cell lines

known to actively express a variety of MMPs. To obtain maximal specificity and sensitivity, we

developed an RNA protection assay for epilysin mRNA. The human cells we used included HT-

29 colon carcinoma cells (49), U937 monocytic-like cells (50), HT-1080 fibrosarcoma cells (42),

neonatal foreskin fibroblasts (51,52), immortalized HaCaT keratinocytes (53), and normal

primary keratinocytes (53). Because the transcription of many MMP genes is strongly

upregulated by PMA, some cells were treated with this agent for 24 h before RNA was isolated.

U937 cells were treated with a combination of PMA and lipopolysaccharide, which mediates

differentiation to a macrophage-like phenotype accompanied by a potent induction of several

MMPs (37,50).

Although epilysin is expressed in several tissues, of the cell types tested, epilysin mRNA

was detected only in keratinocytes (Fig. 6). HaCaT keratinocytes showed the highest expression

of epilysin mRNA and primary keratinocytes, which were grown on collagen to induce MMP

expression (54), had a somewhat lower level of expression (Fig 6). All other cell lines were

negative for epilysin expression. Because we could amplify a portion of epilysin cDNA from

HT-1080 RNA by RT-PCR, these fibrosarcoma cells express epilysin at low levels.

To determine if epilysin mRNA is translated into a protein, we generated rabbit

polyclonal antibodies against a 16 amino acid MAP peptide in the middle of the catalytic

domain. Anti-epilysin antibodies were affinity purified using the same peptide. The specificity of


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the antibody was tested by immunofluorescence staining of transfected CHO cells and by

immunoblotting of conditioned medium (Fig. 7). CHO cells transfected with an epilysin

expression construct showed a predictable range of recombinant protein production. Whereas

some clones showed prominent fluorescence for epilysin, other selected clones had no staining

(Fig. 7A). In addition, we used immunoblotting to assess if epilysin protein is released by HaCaT

keratinocytes. As a positive control, we used a fusion protein of Schistosoma japonicum

glutathione S-transferase and amino acid residues 123 to 520 of epilysin, corresponding to the

putative furin-activated enzyme. A strongly immunoreactive band of about 58 kDa was detected

in HaCaT conditioned medium and the predicted 75 kDa band was seen in the fusion protein

preparation (Fig. 7B). In addition to the 58-kDa band, we detected in HaCaT conditioned

medium a slightly smaller band of about 55 kDa of much lower intensity (Fig. 7B). As is

discussed below, this smaller band could be a product of alternative splicing or alternative

glycosylation. Together, these findings indicate the epilysin mRNA codes a secreted protein that

is produced and released by keratinocytes.

Detection of Alternative Splicing. During the cloning of epilysin, we generated a probe by

RT-PCR using primers complementary to sequences in the exons 3 and 5 (see Methods). In

addition to the expected PCR product, we obtained a shorter cDNA of nearly equal intensity and

a weak band of intermediate size (Fig. 8A). Similar amplified products were generated using a

different forward primer in exon 3, and all PCR products were positive in Southern blotting (Fig.

8A). We gel purified and sequenced the amplified DNA and found that the longer amplification

product contained sequences for exons 3, 4, and 5, while the shorter amplification product

represented a mRNA species that contained exons 3 and 5 but lacked exon 4. To assess the

relative abundance of the different splice forms, another PCR product was cloned and used to

generate an RNA probe for RNAse protection analysis. This probe covers 179 nt of exon 3, the

entire exon 4 (225 nt), and 135 nt of exon 5. In addition to epilysin specific sequences, the RNA

probe contains 96 nt of vector derived sequence giving the total length of 635 nt. HaCaT RNA


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was then hybridized with this probe and non-hybridized probe was degraded by RNAse

treatment. The protected fragments included two strong bands of approximately 550 nt and 180

nt, and a weaker band of 130 nt. The ~550 nt fragment could be protected by a mRNA transcript

that contains exons 3, 4, and 5 (expected size 539 nt), and the ~180 nt fragment would represent

probe protected by exon 3 (expected size 179 nt). The weaker ~130 nt band could be derived

from probe protected by an mRNA species containing exon 5 but not exon 4 (expected size 135

nt). The 135 nt band was much weaker than the 179 nt band suggesting that another species of

mRNA transcripts containing exon 3 but lacking both exons 4 and 5 is expressed in testis and by

HaCaT cells. RNAse protection using RNA from testis tissue and RNA from HaCaT cells gave

identical results (Fig. 8B).

Production of recombinant epilysin (pro and catalytic domains) in bacterial cells and

analysis of its enzymatic activity. To determine if epilysin cDNA codes for an enzyme with

proteolytic activity, we expressed recombinant epilysin in E. coli as a fusion protein consisting of

GST and the pro and catalytic domains of epilysin. Because the prodomain may be necessary for

the correct re-folding of the recombinant protein, this region was included in the construct. SDS-

PAGE analysis and Coomassie blue staining of proteins bound to glutathione Sepharose revealed

a single major protein of the expected size (56 kDa) and a minor 33-kDa band (Fig. 9, lane1).

After treatment with PreScission protease, the 56-kDa protein was cleaved into two proteins with

apparent molecular weights of 34 and 32 kDa (lane 2), which were identified by immunoblotting

as epilysin and GST, respectively (data not shown). After PreScission cleavage, recombinant

epilysin was eluted from the column matrix with high salt concentration (500 mM NaCl) (lane

3). Zymogram analysis with 4-16% SDS-PAGE impregnated with blue casein indicated that both

the 56-kDa fusion protein and the 34-kDa free epilysin had caseinolytic activity. This proteolytic

activity was completely inhibited by exclusion of calcium and zinc and addition of 10 mM

EDTA in the incubation buffer, indicating dependence on divalent cations, calcium and zinc

(Fig. 9). Incubation of the casein zymogram gel in the presence of 10 µM batimastat, a specific


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MMP inhibitor, also completely inhibited the caseinolytic activity (data not shown). This

compound is a substrate-based inhibitor containing a hydroxamic acid moiety that chelates the

active site zinc cation and renders all MMPs catalytically inactive. PreScission protease (46

kDa) did not have any detectable caseinolytic activity (Fig. 9, lane 2).

Expression in Human Skin. Because epilysin mRNA and protein were detected in

cultured human keratinocytes, we assessed if this MMP is expressed in human skin. For this

study, we incubated small, uniformly-sized pieces of normal adult human skin for 24 h in culture

medium and then fixed and processed the samples for immunohistochemistry using affinity-

purified antibody. During the incubation, epidermal cells migrated down the edge of the cut

surface of the biopsy in an attempt to heal the “wounded” tissue. In other studies, we have

demonstrated that the expression of MMPs in this ex vivo model mirrors that seen in vivo. In the

center of the tissue specimens, at some distance (about 2 mm) from the wound edge, staining for

epilysin protein was seen in the intact epidermis (Fig. 10A). The staining intensity was strongest

in basal keratinocytes and progressively weaker in suprabasal cells. A different pattern of

staining was seen at the wound edge (Fig. 10C). Here, intense staining for epilysin was seen in

migrating keratinocytes at the wound edge and in stationary basal keratinocytes several cells

behind the wound front. In contrast to intact epidermis, epilysin was not detected in suprabasal

keratinocytes near the wound front (Fig. 10C). No dermal cells were stained for epilysin protein

(Fig. 10A, C), and no reactivity was detected in samples processed with preimmune serum (Fig.

10B, D).


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Discussion

Here we report the identification, gene and domain organization, and tissue expression of

a new member of the matrix metalloproteinase gene family. Based on the sequential numerical

nomenclature, this new protein would be designated MMP-27. Because of its prominent

expression in the epidermis and its catalytic activity as an endopeptidase, we call this new MMP

“epilysin”. Epilysin has all the key domains of a typical MMP: a signal peptide, a conserved

cysteine-containing prodomain, a conserved histidine-containing, catalytic domain, a hinge, and

hemopexin-domain. It degrades casein, and its proteolytic activity requires divalent cations and

is inhibited by a synthetic MMP inhibitor. In contrast, epilysin does not include domains

characteristic of other metalloproteinases subfamilies, such as the disintegrin and

thrombospondin-like domains found in ADAMs and tsADAMs, respectively, or a

transmembrane domain as is found in most membrane-type MMPs (55).

The unique exon-intron structure suggests that epilysin diverged early from other MMPs.

However the splicing pattern is very close to that of MMP-19, to which epilysin is also most

closely related at the amino acid sequence level. A notable difference between MMP-19 and

epilysin is that MMP-19 has no furin-recognition site between its pro- and catalytic domains.

Unlike many MMP genes, which are clustered on the long arm of chromosome 11, the locus for

epilysin is present on chromosome 17. In addition, we have recently isolated the cDNA for

mouse epilysin (data not shown). Comparison of the amino acid sequences of the catalytic

domains indicates that the coding regions of the mouse and human epilysin genes are highly

conserved (97% identical residues), suggesting an important function of this region for enzyme

activity.

The predicted molecular weight of secreted pro-epilysin is 56 kDa and if it is cleaved in

the secretion pathway at its furin-recognition site, the released protein would be about 45 kDa.


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These weights, however, do not account for additional mass contributed by glycosylation.

Indeed, by immunoblotting analysis of HaCaT keratinocyte-conditioned medium, we detected a

protein of about 58 kDa and a less prominent band of about 55 kDa. These two bands may be

due to differential glycosylation, such as is characteristic of collagenase-1 (56), or they may

reflect two distinct isoforms. Using RT-PCR and RNAse protection assays, we determined that

epilysin is transcribed into at least two different mRNAs, one of which lacks exon 4. Because

exons 3, 4 and 5, among others, end with split codons for glycine, splicing exon 4 would not

affect the amino acid sequence coded by exon 5 (Table 1). Omission of exon 4 would reduce the

predicted molecular weight of pro-epilysin by about 8 kDa, within the range of the size

difference we detected by immunoblotting. Because our antibody was raised against sequences

coded by exon 5, the epitope would be present in the putative smaller isoform. Although we do

not yet know if the smaller transcript is translated, splicing exon 4 would place the cysteine-

switch of the prodomain much closer to the zinc-coordination site (Fig. 1). This reorganization

may have significance for the structure of the catalytic pocket and for the activation state of the

pro-enzyme.

In our Northern hybridization analyses we could detect at least three different MMP-27

transcripts of about 2.6, 2.0 and 1.2 kb. While the longest transcript of 2.6 kb most probably

corresponds to the sequence presented in Fig. 1 with some more 5’UTR and poly-A tail added,

the other transcripts remain uncharacterized. They could be either products of alternative splicing

or different utilization of polyadenylation sites. At least the 1.2 kb transcript is too short to code

for a full length enzyme. During the screening of the libraries we isolated clones containing

intronic sequences (data not shown). Whether they are cloning artifacts or real functional

transcripts, remains to be shown.

Another unusual feature of epilysin is that it is expressed in normal, intact tissues, such as

testis, intestine, lung, and skin, and this pattern of expression suggests that this MMP may serve


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a role in tissue homeostasis. Similarly, matrilysin (MMP-7) is expressed by the epithelium of

intact mucosal tissues (31,49,57), and we recently reported that matrilysin functions in innate

immunity, a homeostatic function, by activating prodefensin peptides (15). Although matrilysin

is expressed by mucosal epithelium, including that of small intestine, injured colon, airways, and

exocrine glands, it is not expressed in the epidermis (31,49). It is tempting to speculate that

epilysin participates in host defense in intact epidermis by processing antimicrobial proteins.

Indeed, because it is expressed by basal and suprabasal keratinocytes, released epilysin may not

encounter a matrix substrate in intact skin. Thus, though epilysin is a member of the matrix

metalloproteinases gene family, we cannot yet conclude that matrix components are physiologic

substrates for this enzyme.

In addition to tissue homeostasis, epilysin may serve a distinct and additional role in

repair of cutaneous wounds. In response to injury, several MMPs are produced by the epidermis

in functionally distinct subpopulations of keratinocytes (58). For example, collagenase-1 (MMP-

1), stromelysin-2 (MMP-10), and gelatinase-B (MMP-9) are produced by basal keratinocytes at

the migrating front, whereas stromelysin-1 (MMP-3) is expressed by the hyperproliferative cells

just behind these migrating cells (59-63). Distinct from the localization of these MMPs,

prominent staining for epilysin was seen in basal keratinocytes at the migratory front and in

many cells behind the wound edge. Again, this pattern is similar to that for matrilysin in

wounded epithelium. Although it is not found in cutaneous wounds (58), matrilysin is expressed

by migrating and stationary epithelial cells in wounds and ulcerations of mucosal tissues, such as

lung and intestine (64,65). Demonstrating an essential role for matrilysin in mucosal repair,

airway epithelial wounds do not repair in MMP-7-null mice (65). Thus, matrilysin serves at least

two distinct role in mucosal tissues: one in innate defense, the other in epithelial repair and

migration. Epilysin may have equally critical roles in skin. Our future studies will be directed at

determining the function of this new MMP.


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Acknowledgements

We thank Prof. Norbert Fusenig of German Cancer Research Center (Heidelberg, Germany) for

HaCaT cells. The Academy of Finland, the Finnish Cultural Foundation and grants from the

NIH, supported this work. Jouko Lohi was a William S. Keck fellow at Washington University

School of Medicine.


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FOOTNOTES

The nucleotide sequence for the human epilysin cDNA has been deposited in the GenBank

database under GenBankAccession Number AF219624.

4) Abbreviations used: GST, Schistosoma japonicum glutathione S-transferase; MMP,

metalloproteinase; PMA, phorbol 12-myristate 13-acetate; UTR, untranslated region


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FIGURE LEGENDS

FIG. 1. Nucleotide sequence of the human epilysin (MMP-27) cDNA and its deduced amino acid

sequence. The deduced amino acid sequence is shown under the DNA sequence. The first ATG

and the termination codon TGA are in bold. Numbers on the right and left refer to the positions

of nucleic acids and amino acid residues, respectively. Prosequence PRCGVTD, and zinc-

binding site HEIGHTLGLTH are inverted, and the furin recognition sequence RRKKR is boxed.

Predicted signal peptide cleavage site is indicated with an arrow, and the furin cleavage site is

marked with an arrowhead. Two potential N-glycosylation sites are underlined. Vertical bars

indicate the exon limits and exons are numbered as indicated in Table I. Our cDNA clones cover

the sequence between bases 1-1817. First consensus polyadenylation signal AATAAA is found

about 500 bp downstream in the genomic sequence (Accession AC006237), and the sequence

between bases 1818-2332 is derived from this deposited sequence and represents the most

probable 3’end of epilysin mRNA.The nucleotide sequence data are in the GenBank nucleotide

sequence database with the accession number AF219624

FIG. 2. Comparison of the amino acid sequence of epilysin with other human MMPs. Peptide

sequences for human MMPs including collagenase-1 (MMP-1), stromelysin-1 (MMP-3),

stromelysin-3 (MMP-11), membrane-type-1 matrix metalloproteinase (MT1-MMP, MMP-14),

and MMP-19 were retrieved from GenBank and aligned with epilysin (MMP-27) peptide

sequence using CLUSTAL W program. Identical amino acid residues in all six MMPs are

indicated below the sequences. Epilysin domains are indicated above the sequence.


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FIG. 3. Dendogram of the catalytic domains of human MMPs. The amino acid sequences of the

catalytic domains of human MMPs were retrieved from GenBank and aligned with CLUSTAL

W to generate a phylogenetic tree. Epilysin is most closely related to MMPs 19, 23, 11, and 17.

Other clusters of MMPs were formed by MT-MMPs excluding MT4-MMP (MMPs 14, 15, 16,

and 24), gelatinases (MMPs 2 and 9), stromelysin-1 and –2 (MMPs 3 and 10) and collagenases-1

and –2 (MMPs 1 and 8).

FIG. 4. Organization of the human epilysin gene. Comparison with other human MMP genes. A)

Organization of the epilysin gene was drawn based on the comparison of the cDNA sequence

with the sequence of the genomic BAC clone hRPC.161_P_9. Exons are numbered from the 5'-

end of the gene and depicted by black boxes. The noncoding regions of the first and last exons

are depicted by open boxes. The size of the first intron is unknown; it was not present in the

genomic BAC clone. The positions of the transcription start site (ATG), stop codon (TGA), pro-

sequence (PRCGVTD), furin cleavage site (RRKKR) and the catalytic zinc-binding site

(HEIGHTLGLTH) are indicated under the gene graph. Base positions in the BAC clone are

indicated above the gene graph. B) Comparison of exon and domain structures of members of

MMP family. The exons in human epilysin (MMP-27), gelatinase A (MMP-2), collagenase-1

(MMP-1), stromelysin-1 (MMP-3), matrilysin (MMP-7), stromelysin-3 (MMP-11), membrane-

type-1 matrix metalloproteinase (MT1-MMP, MMP-14), and MMP-19 are shown as boxes, with

their sizes in nucleotides underneath. Open boxes indicate untranslated sequences. Filled boxes

indicate different domains of the matrix metalloproteinases: signal peptide, prodomain, catalytic

domain, hinge region, hemopexin-like domain, transmembrane domain, and intracellular domain.

FN, fibronectin like domain of gelatinase A. The locations of the exon-intron splicing sites in the

epilysin gene differ markedly from other MMPs. Only the splice sites between exons 1 and 2, 5

and 6, and 6 and 7 are at conserved positions among most MMP genes, while all the splice sites

of epilysin gene are utilized also in MMP-19 gene.


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33

Fig. 5. Northern blot analysis of epilysin expression in a variety of human tissues. 2 µg of

poly(A)+ RNA from the indicated tissues were analyzed by hybridization with the cDNA for

human epilysin. Migration of RNA size markers is shown on the left. Filters were subsequently

hybridized to a human β-actin probe to control the loading of RNA. At least three different

transcripts of 2.6, 2.0 and 1.2 kb were detected.

FIG. 6. Expression of epilysin in cultured cells. RNAse protection analysis. Confluent cultures of

colon adenocarcinoma cells (HT-29), histiocytic lymphoma cells (U937), human fibrosarcoma

cells (HT-1080), human foreskin fibroblasts (HFF), immortalized human keratinocytes (HaCaT),

and primary keratinocytes were treated with PMA (40 nM) and LPS (5 µg/ml) for 24 h where

indicated. Total RNA was then extracted and analyzed by RNAse protection for the presence of

epilysin mRNA as described under Methods. Protected RNA fragments were fractionated by 5%

TBE-PAGE containing 6 M urea and visualized by autoradiography. Undigested probe (257 nt)

and protected fragment (161 nt) are indicated on the left. Migration of RNA size markers is

shown on the left. Specific signal for epilysin could be detected only in HaCaT and keratinocyte

samples. Full length probe is also protected to a minor extent because of residual template DNA.

Equal loading of the RNAs was confirmed by separate RNAse protection analysis for cyclophilin

mRNA (cyclo).


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FIG. 7. Expression of epilysin in cultured cells. A) Phase-contrast microscopy of transfected pool

of CHO cells. CHO cells were transfected with an expression construct for recombinant epilysin

and neomycin resistant cells were selected with G418. This transfected pool was then plated on

glass coverslips and fixed with paraformaldehyde 3 days later. B) Immunofluorescence staining.

This field shows one CHO clone expressing epilysin at high levels (marked with arrows). Other

cells seen in phase contrast (A) have low or no expression. C) Western blot. Concentrated

medium from HaCaT cells and purified recombinant fusion protein of Glutathione S-transferase

and catalytic and hemopexin domains of epilysin (GST-M26) were separated by electrophoresis

in 4-15% SDS-PAGE under reducing conditions. The proteins were electrophoretically

transferred to nitrocellulose and immunostained for the presence of epilysin protein.

FIG. 8. Alternative splicing of epilysin. A) RT-PCR and Southern blotting. Epilysin cDNA was

amplified by RT-PCR using two different forward primers in the exon 3 and a reverse primer in

exon 5. PCR reactions were then separated by electrophoresis in a 1.5% agarose gel and stained

with ethidium bromide (lanes 1 and 2). For identification, DNA was transferred onto a nylon

membrane by Southern blotting and probed with an internal oligonucleotide probe in exon 5 of

epilysin (lanes 3 and 4). Lanes 1 and 3: forward primer 370F; lanes 2 and 4: forward primer

440F. M: 100 bp marker, 500 bp band has higher intensity of staining. B) RNAse protection

analysis. Total RNA from human testis or HaCaT cells was hybridized with an anti-sense RNA

probe spanning exons 3, 4, and 5 of epilysin. Unbound probe was then degraded with RNAses

and protected RNA fragments were fractionated by 5% TBE-PAGE containing 6 M urea and

visualized by autoradiography. The protected fragments included a 539 nt fragment

corresponding to mRNA containing all three exons, a 179 bp fragment corresponding to exon 3

and a faint band or 135 nt corresponding to exon 5, as indicated on the right. Migration of RNA

size markers is shown on the left. To avoid overexposure of the film, the amount of the probe

loaded in the firs lane corresponds only ~5% of the probe used in hybridizations.


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FIG. 9. Epilysin is a metalloproteinase capable of degrading casein. Recombinant epilysin was

expressed in E. coli as a fusion protein that consists of glutathione S-transferase (GST), pro-

domain of epilysin and catalytic domain of epilysin. Fusion protein was purified by affinity

chromatography on glutathione Sepharose, and this fusion protein was cleaved with PreScission

protease to release epilysin from GST. Washing of the glutathione Sepharose matrix with 500

mM NaCl selectively released recombinant epilysin while GST and PreScission protease

remained bound to the matrix. Samples of affinity purified fusion protein (lanes 1), fusion

protein cleaved with PreScission protease (lanes 2), and free epilysin eluted from glutathione

Sepharose matrix (lanes 3) were then separated by electrophoresis in 4-15% SDS-PAGE under

reducing conditions and protein stained with Coomassie Blue (left panel) or analyzed by casein

zymography in a 4-16 SDS-PAGE impregnated with blue casein in the presence of calcium and

zinc (middle panel) or in the absence of divalent cations and in the presence of EDTA (10 mM)

(right panel). Migration of molecular weight markers is shown on the left. Migration of the

fusion protein is indicated with an asterisk, epilysin (pro and catalytic domains) with an arrow,

and free GST with an arrowhead, all on the right.

FIG. 10. Expression of epilysin in the epidermis of a healing wound. Four-mm punch biopsies of

normal human skin (n = 4 donors) were cultured on a cell culture dish for 24 h, fixed in buffered

formalin, embedded, and stained for epilysin protein using affinity-purified antibodies. A)

Middle of the biopsy. Staining for epilysin was seen in keratinocytes in all layers of intact

epidermis, with more prominent signal in basal keratinocytes. Staining for epilysin was not seen

in any cell type in the underlying dermis. C) Wound (biopsy) edge. An intense signal for epilysin

protein was seen in basal keratinocytes at the wound edge and in basal keratinocytes some

distance from the migratory front. B, D) Serial sections of those shown in panels in A and C

were processed with pre-immune serum.


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Table IThe Exon-Intron Junctions in the Human MMP-27 Gene

Exon/intron boundaries were determined by comparing the MMP-27 cDNA sequence to a human genomic BAC clonehRPC.161_P_9, accession number AC006237. The nucleotide sequence of each exon (upper-case letters) and intron(lower-case letters) at the exon/intron boundaries is shown. The deduced amino acid sequence at the intron-exonboundaries is indicated under the nucleotide sequence. Exon 1 is not present in the BAC clone, and the size of intron 1 istherefore not known.

Exon Intron - EXON - Intron JunctionExon

Size (bp)Intron

Size (bp)Genomic DNA

Bases*

1 ATG GTC GCG…GAG GCG GAG xxx

Met Val Ala … Glu Ala Glu

>169 - 5’ UTR

111 - Coding

>280 - Total

?

Not Present

in BAC

2 tctcccgcag GCA TTC CTA…GAT GCC ATC AG gtagggtgga

Ala Phe Leu … Asp Ala Ile Ar(g)80 168 8691 - 8770

3 ctcctgacag A GCG TTT CAG…GCA AAG CAA G gtgagcactg

(Ar)g Ala Phe Gln … Ala Lys Gln G(ly)188 5483 8939 - 9126

4 tgggtggcag GT AAC AAA TGG…GAT GGC CCA G gtgctggcac

G(ly) Asn Lys Trp … Asp Gly Pro G(ly)225 2853 14610 - 14834‡

5 ccacctgcag GG GGC GCC CTG…AGC CTG TAT G gtgaggcccc

(G)ly Gly Ala Leu … Ser Leu Tyr G(ly)246 1687 17688 - 17933

6 actcacaaag GG AAG CCC CTA…ATC ACT GTA G gtaagaaggt

(G)ly Lys Pro Leu … Ile Thr Val A(sp)150 310 19621 - 19770

7 ctctgggcag AC AGG CAA CAG…TTC TTC AAA G gtactggctc

(A)sp Arg Gln Gln … Phe Phe Lys G(ly)168 857 20081 - 20248

8 ttccccacag GG GGT CGA TGC…GCC CTG TTC tga

(G)ly Gly Arg Cys … Ala Leu Phe stop

392 - Coding

604 - 3’ UTR

995 - Total

21106 - 22100

* Genomic DNA base positions are derived from BAC clone hRPC.161_P_9, accession number AC006237.

‡ In the genomic sequence of BAC clone hRPC.161_P_9, a C appears at base 14,668, which causes a frameshift. Sequencing of testis and keratinocyte cDNAs confirmed that a G belongs at 14,668 and that the Cresides at 14,669, restoring the open-reading frame. Because of the addition of this single base, thenumbers shown for the end of exon 4 (14,833) through exon 8 (22,099) are increased by one compared tothe base postions shown the in the AC006237 accession file.


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ACCGGCCCAGAGCGCGCAGCTAGGGCACTGGCGAAACCCCGGGACAGTCCCTCTCCGTGCGGGGGCGGCGCAGAG 75CAGTCCCATCCCCGGGGTTCCGGGCGCGGCTGACTGCCGGCTGGTTCCCTGCGCGCAGTAGCTCCCCGAGCCGGG 150

CTGCACCGGAGGCGGCGAGATGGTCGCGCGCGTCGGCCTCCTGCTGCGCGCCCTGCAGCTGCTACTGTGGGGCCA 225 M V A R V G L L L R A L Q L L L W G H

CCTGGACGCCCAGCCCGCGGAGCGCGGAGGCCAGGAGCTGCGCAAGGAGGCGGAGGCATTCCTAGAGAAGTACGG 300 L D A Q P A E R G G Q E L R K E A E A F L E K Y G

ATACCTCAATGAACAGGTCCCCAAAGCTCCCACCTCCACTCGATTCAGCGATGCCATCAGAGCGTTTCAGTGGGT 375 Y L N E Q V P K A P T S T R F S D A I R A F Q W V

GTCCCAGCTACCTGTCAGCGGCGTGTTGGACCGCGCCACCCTGCGCCAGATGACTCGTCCCCGCTGCGGGGTTAC 450 S Q L P V S G V L D R A T L R Q M T R P R C G V T

AGATACCAACAGTTATGCGGCCTGGGCTGAGAGGATCAGTGACTTGTTTGCTAGACACCGGACCAAAATGAGGCG 525 D T N S Y A A W A E R I S D L F A R H R T K M R R

TAAGAAACGCTTTGCAAAGCAAGGTAACAAATGGTACAAGCAGCACCTCTCCTACCGCCTGGTGAACTGGCCTGA 600 K K R F A K Q G N K W Y K Q H L S Y R L V N W P E

GCATCTGCCGGAGCCGGCAGTTCGGGGCGCCGTGCGCGCCGCCTTCCAGTTGTGGAGCAACGTCTCAGCGCTGGA 675 H L P E P A V R G A V R A A F Q L W S N V S A L E

GTTCTGGGAGGCCCCAGCCACAGGCCCCGCTGACATCCGGCTCACCTTCTTCCAAGGGGACCACAACGATGGGCT 750 F W E A P A T G P A D I R L T F F Q G D H N D G L

GGGCAATGCCTTTGATGGCCCAGGGGGCGCCCTGGCGCACGCCTTCCTGCCCCGCCGCGGCGAAGCGCACTTCGA 825 G N A F D G P G G A L A H A F L P R R G E A H F D

CCAAGATGAGCGCTGGTCCCTGAGCCGCCGCCGCGGGCGCAACCTGTTCGTGGTGCTGGCGCACGAGATCGGTCA 900 Q D E R W S L S R R R G R N L F V V L A H E I G H

CACGCTTGGCCTCACCCACTCGCCCGCGCCGCGCGCGCTCATGGCGCCCTACTACAAGAGGCTGGGCCGCGACGC 975 T L G L T H S P A P R A L M A P Y Y K R L G R D A

GCTGCTCAGCTGGGACGACGTGCTGGCCGTGCAGAGCCTGTATGGGAAGCCCCTAGGGGGCTCAGTGGCCGTCCA 1050 L L S W D D V L A V Q S L Y G K P L G G S V A V Q

GCTCCCAGGAAAGCTGTTCACTGACTTTGAGACCTGGGACTCCTACAGCCCCCAAGGAAGGCGCCCTGAAACGCA 1125 L P G K L F T D F E T W D S Y S P Q G R R P E T Q

GGGCCCTAAATACTGCCACTCTTCCTTCGATGCCATCACTGTAGACAGGCAACAGCAACTGTACATTTTTAAAGG 1200 G P K Y C H S S F D A I T V D R Q Q Q L Y I F K G

GAGCCATTTCTGGGAGGTGGCAGCTGATGGCAACGTCTCAGAGCCCCGTCCACTGCAGGAAAGATGGGTCGGGCT 1275 S H F W E V A A D G N V S E P R P L Q E R W V G L

GCCCCCCAACATTGAGGCTGCGGCAGTGTCATTGAATGATGGAGATTTCTACTTCTTCAAAGGGGGTCGATGCTG 1350 P P N I E A A A V S L N D G D F Y F F K G G R C W

GAGGTTCCGGGGCCCCAAGCCAGTGTGGGGTCTCCCACAGCTGTGCCGGGCAGGGGGCCTGCCCCGCCATCCTGA 1425 R F R G P K P V W G L P Q L C R A G G L P R H P D

CGCCGCCCTCTTCTTCCCTCCTCTGCGCCGCCTCATCCTCTTCAAGGGTGCCCGCTACTACGTGCTGGCCCGAGG 1500 A A L F F P P L R R L I L F K G A R Y Y V L A R G

GGGACTGCAAGTGGAGCCCTACTACCCCCGAAGTCTGCAGGACTGGGGAGGCATCCCTGAGGAGGTCAGCGGCGC 1575 G L Q V E P Y Y P R S L Q D W G G I P E E V S G A

CCTGCCGAGGCCCGATGGCTCCATCATCTTCTTCCGAGATGACCGCTACTGGCGCCTCGACCAGGCCAAACTGCA 1650 L P R P D G S I I F F R D D R Y W R L D Q A K L Q

GGCAACCACCTCGGGCCGCTGGGCCACCGAGCTGCCCTGGATGGGCTGCTGGCATGCCAACTCGGGGAGCGCCCT 1725 A T T S G R W A T E L P W M G C W H A N S G S A L

GTTCTGAAGGCACCTCCTCACCTCAGAAACTGGTGGTGCTCTCAGGGCAAAATCATGTTCCCCACCCCCGGGGCA 1800 F *

GAACCCCTCTTAGAAGCCTCTGAGTCCCTCTGCAGAAGACCGGGCAGCAAAGCCTCCATCTGGAAGTCTGTCTGC 1875CTTTGTTCCTTGAAGAATGCAGCATTGTCTTTGTCTGTCCCCACCACATGGAGGTGGGGGTGGGATCAATCTTAG 1950GAAAAGCAAAAAAGGGTCCCAGATCCCTTGGCCCTTTCCTCCGAGGACTTCTATCCTCCCCAGGCCTTTGTTTCT 2025TCGGCTAAAGGTACAGTTCCTTTCAAGAGGTAACAGCACTGGGATCCAAGCAGGGGGATGAAAAACTCAGCAGAG 2100AAATTCGAGACCATTTTGCAAGACTGTGCCCTTCTCCTCAGGACCCCCTGGCTCAGTTCTTGAAAAACGGTGTCA 2175TATTTAGTCAGAGGCCCCACCCCCAGGAAGCATGGATGGGGATGAAGGCACAGGCGTCTCCAACCTCAGAGGCCC 2250TTTGTGGGGTCAGGACACAGAGTGGGAGGGAGACTGATGCAGGCCTACCAGTCCCTGGCTTTTTGTCTGGGGCTG 2325GAATAAA 2332

1 2

2 3

3 4

4 5

5 6

6 7

7 8

1

20

45

70

95

120

145

170

195

220

245

270

295

320

345

370

395

420

445

470

495

520

Fig. 1


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MMP-27 : MVARVGLLLRALQLLLWGHLDAQPAERGGQELRKEAEAFLEKYGY-LNEQVPK----APTSTRFSDAIRAFQWVSQLPVSGVLDRATLRQM : 86MMP-1 : -MHSFP-PLLLLLFWGVVSHSFPATLETQEQDVDLVQKYLEKY-YNLKNDGRQ-VEKRRNSGPVVEKLKQMQEFFGLKVTGKPDAETLKVM : 87MMP-3 : -MKSLP-ILLLLCVAVCSAYPLDGAARGEDTSMNLVQKYLENY-YDLKKDVKQ-FVRRKDSGPVVKKIREMQKFLGLEVTGKLDSDTLEVM : 87MMP-11 : ---MAP-------AAWLRSAAARALLPPMLLLLLQPPPLLARA---LPPDVHHLHAERRGPQPWHAALPSSP--APAPATQEAP-RPASSL : 75MMP-14 : -MSPAPRPSRCLLLPLLTLGTALASLGSAQSSSFSPEAWLQQYGYLPPGDLRT-HTQRSPQS-LSAAIAAMQKFYGLQVTGKADADTMKAM : 88MMP-19 : --------MNCQQLWLGFLLPMTVSGRVLGLAEVAPVDYLSQYGYLQKPLEGS---NNFKPEDITEALRAFQEASELPVSGQLDDATRARM : 80 L

MMP-27 : TRPRCGVTDTNSYAAWAERISDLFARHRTKMRRKKRFAKQGNKWYKQHLSYRLVNWPEHLPEPAVRGAVRAAFQLWSNVSALEFWEA---- : 174MMP-1 : KQPRCGVPD----------------------VAQFVLTEGNPRWEQTHLTYRIENYTPDLPRADVDHAIEKAFQLWSNVTPLTFTKV---- : 153MMP-3 : RKPRCGVPD----------------------VGHFRTFPGIPKWRKTHLTYRIVNYTPDLPKDAVDSAVEKALKVWEEVTPLTFSRL---- : 153MMP-11 : RPPRCGVPDP-------------SDG-LSARNRQKRFVLSGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEV---- : 149MMP-14 : RRPRCGVPDK-------------FGAEIKANVRRKRYAIQGLKWQHNEITFCIQNYTPKVGEYATYEAIRKAFRVWESATPLRFREVPYAY : 166MMP-19 : RQPRCGLEDP--------------------FNQKTLKYLLLGRWRKKHLTFRILNLPSTLPPHTARAALRQAFQDWSNVAPLTFQEV---- : 148 PRCG D W A W L F

MMP-27 : ---PATGPADIRLTFFQGDHNDGLGNAFDGPGGALAHAFLP---RRGEAHFDQDERWSLS--RRRGRNLFVVLAHEIGHTLGLTHSPAPRA : 256MMP-1 : ----SEGQADIMISFVRGDHRDNS--PFDGPGGNLAHAFQPGPGIGGDAHFDEDERWTN---NFREYNLHRVAAHELGHSLGLSHSTDIGA : 234MMP-3 : ----YEGEADIMISFAVREHGDFY--PFDGPGNVLAHAYAPGPGINGDAHFDDDEQWTK---DTTGTNLFLVAAHEIGHSLGLFHSANTEA : 234MMP-11 : ----HEGRADIMIDFARYWDGDDL--PFDGPGGILAHAFFPKTHREGDVHFDYDETWTIG--DDQGTDLLQVAAHEFGHVLGLQHTTAAKA : 231MMP-14 : IREGHEKQADIMIFFAEGFHGDST--PFDGEGGFLAHAYFPGPNIGGDTHFDSAEPWTVRNEDLNGNDIFLVAVHELGHALGLEHSSDPSA : 255MMP-19 : ----QAGAADIRLSFHGRQSSYCSN-TFDGPGRVLAHADIP---ELGSVHFDEDEFWTEG--TYRGVNLRIIAAHEVGHALGLGHSRYSQA : 228 ADI F FDG G LAHA P G HFD E W HE GH LGL H A

MMP-27 : LMAPYYKRLGRDA--LLSWDDVLAVQSLYGKPLGGSVAVQLPGKLFTDFETWDSYSPQGRRPETQGPKYCHS--SFDAITVDRQQQLYIFK : 343MMP-1 : LMYPSYTFSGDVQ---LAQDDIDGIQAIYGRSQN----------PVQPIG------PQT-------PKACDSKLTFDAITTIR-GEVMFFK : 298MMP-3 : LMYPLYHSLTDLTRFRLSQDDINGIQSLYGPPPDSPE--TPLV-PTEPVPPE----PGT-------PANCDPALSFDAVSTLR-GEILIFK : 310MMP-11 : LMSAFYTFRYPLS---LSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN-EIAPLEPDAP---PDACEA--SFDAVSTIR-GELFFFK : 312MMP-14 : IMAPFYQWMDTEN-FVLPDDDRRGIQQLYGGESGFP----TKM-PPQPRTTSRPSVPDKPKNPTYGPNICDG--NFDTVAMLR-GEMFVFK : 337MMP-19 : LMAPVYEGYRPHFK--LHPDDVAGIQALYGKKSPVIR-----DEEEEETELP--TVPPVPTEPSPMPDPCSS--ELDAMMLGPRGKTYAFK : 308 M Y L DD Q YG P P C D FK

MMP-27 : GSHFWE-VAADGNVSEPRPLQERWVGLPPNIEAAAVSLNDGDFY-FFKGGRCWRFRGPKPVWGLPQLCRAG-GLPRHP---DAALFFPP-L : 427MMP-1 : DRFYMR-TNPFYPEVELNFISVFWPQLPNGLE-AAYEFADRDEVRFFKGNKYWAVQGQNVLHGYPKDIYSSFGFPRTVKHIDAALSEEN-T : 386MMP-3 : DRHFWR-KSLRKLEPELHLISSFWPSLPSGVD-AAYEVTSKDLVFIFKGNQFWAIRGNEVRAGYPRGIHT-LGFPPTVRKIDAAISDKE-K : 397MMP-11 : AGFVWRLRGGQLQPGYPALASRHWQGLPSPVD-AAFEDAQGHIW-FFQGAQYWVYDGEKPVLG-PAPLTE-LGLVRFP--VHAALVWGPEK : 397MMP-14 : ERWFWRVRNNQVMDGYPMPIGQFWRGLPASIN-TAYERKDGKFV-FFKGDKHWVFDEASLEPGYPKHIKE-LGRGLPTDKIDAALFWMP-N : 424MMP-19 : GDYVWT--VSDSGPGPLFRVSALWEGLPGNLD-AAVYSPRTQWIHFFKGDKVWRYINFKMSPGFPKKLNR----VEPN--LDAALYWPL-N : 389 W LP A F G W G P AA

MMP-27 : RRLILFKGARYYVLARGGLQVEPYYPRSLQ-DWGGIPEEVSGALPRPDG-SIIFFRDDRYWRLDQAKLQATTSGRWATELPWMGCWHANSG : 516MMP-1 : GKTYFFVANKYWRYDEYKRSMDPGYPKMIAHDFPGIGHKVDAVFMKDG--FFYFFHGTRQYKFDPKTKRILTLQKA---NSWFNCRKN--- : 469MMP-3 : NKTYFFVEDKYWRFDEKRNSMEPGFPKQIAEDFPGIDSKIDAVFEEFG--FFYFFTGSSQLEFDPNAKKVTHTLKS---NSWLNC------ : 477MMP-11 : NKIYFFRGRDYWRFHPSTRRVDSPVPRRAT-DWRGVPSEIDAAFQDADG-YAYFLRGRLYWKFDPVKVKALEGFPRLVGPDFFGCAEPANT : 486MMP-14 : GKTYFFRGNKYYRFNEELRAVDSEYPKNIK-VWEGIPESPRGSFMGSDEVFTYFYKGNKYWKFNNQKLKVEPGYPKPALRDWMGCPSGGRP : 514MMP-19 : QKVFLFKGSGYWQWDELARTDFSSYPKPIKGLFTGVPNQPSAAMSWQDG-RVYFFKGKVYWRLN-QQLRVEKGYPRNISHNWMHCRPRTID : 478 F Y P G F C

MMP-27 : SALF---------------------------------------------------------------- : 520MMP-1 : -------------------------------------------------------------------- : -MMP-3 : -------------------------------------------------------------------- : -MMP-11 : FL------------------------------------------------------------------ : 488MMP-14 : DEGTEEETEVIIIEVDEEGGGAVSAAAVVLPVLLLLLVLAVGLAVFFFRRHGTPRRLLYCQRSLLDKV : 582MMP-19 : TTPSGGNTTPSGTGITLDTTLSATETTFEY-------------------------------------- : 508

Signal peptide Propeptide

Propeptide Catalytic

Catalytic

Catalytic Hemopexin

Hemopexin

Hemopexin

Fig. 2


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MMP-8

MMP-17

MMP-25

MMP-23

MMP-27

MMP-19

MMP-11

MMP-16

MMP-24

MMP-14

MMP-15

MMP-12

MMP-9

MMP-26

MMP-2

MMP-13

MMP-3

MMP-10

MMP-1

MMP-20

MMP-7

Fig. 3


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FN

449 227 149 129 174 174 174 155 135 137 160 109 901

149174 245 129 156 118 134 163 104 602

157 245 149 126 165 145 134 160 104 387

155 227 149 129 162 299

MMP-2

MMP-1

MMP-3

MMP-7

Catalytic Hemopexin

TM/IC

ProSig Hinge

Exon No. 1 2 3 4 5 6 7 8

130 230 144 134 242 217 258 905MMP-11

MMP-27>280 188 225 246 150 168 99580

340 148 123 309 162 161 139 153 112 >1000MMP-14

Bases

~

B.

10 15 20BAC clone (kb)

RRKKR HEIGHTLGLTH

TGA

PRCGVTD

ATG

1 2 3 4 5 6 7 8exon

A.

Fig. 4

MMP-19195 131 216 246 129 165 195386 128


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kb

4.4

2.4

1.35

Actin

live

rm

usc

le

he

art

bra

inp

lace

nta

lun

g

kid

ne

yp

an

cre

as

sple

en

thym

us

pro

sta

tete

stis

ova

ry

inte

stin

eco

lon

leu

kocy

te

Fig. 5


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300

200

100

nt

257 nt

161 nt

pro

be

no

RN

A

HT

-29

HT

-29

+ P

MA

U9

37

U93

7 +

PM

A+

LP

S

HT

-10

80

+ P

MA

HFF

HF

F +

PM

A

HaC

aT

kera

tin

ocy

tes

cyclo

+ RNAse

Fig. 6


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A

B

Fig. 7

250

98

6450

363016

kDa HaC

aT

GS

T-M

27C


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1 2 3 4M

Fig. 8

A)

B)

500400

300

200

100

nt539 nt

Pro

be

no

RN

A

Tes

tis

RN

A 1

0 u

g

Tes

tis

RN

A 3

ug

HaC

aT R

NA

10

ug

+ RNAse

179 nt

135 nt


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1 2 3 1

98

6450

3630

16

kDa986450

36

30

16

kDa

2 3 1 2 3

Protein Zymography Zymo + EDTA

*

Fig. 9


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Jouko L. Lohi, Carole L. Wilson, Jill D. Roby and William C. Parkskeratinocytes and in response to injury

Epilysin: A novel human matrix metalloproteinase (MMP-27) expressed in testis and

published online December 19, 2000J. Biol. Chem.

10.1074/jbc.M001599200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

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http://www.jbc.org/lookup/doi/10.1074/jbc.M001599200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M001599200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2000/12/19/jbc.M001599200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2000/12/19/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2000/12/19/jbc.M001599200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

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