cloning and tissue distribution of a novel serine protease esp-1 from human eosinophils

Cloning and Tissue Distribution of a Novel SerineProtease esp-1 from Human Eosinophils

Masahiro Inoue,* Naotomo Kanbe,† Motohiro Kurosawa,‡ and Hiroshi Kido**Division of Enzyme Chemistry, Institute for Enzyme Research, University of Tokushima, 3 Kuramoto-cho,Tokushima 770-8503, Japan; †Department of Dermatology, Gunma University School of Medicine,3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan; and ‡Department of Geriatric Medicine,Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan

Received September 29, 1998

We have cloned a novel serine protease designatedas esp-1 from human eosinophils. The amino acid se-quence deduced from the cDNA showed that ESP-1comprises a signal peptide of 18 amino acids, a propep-tide of 23 amino acids, an active form sequence of 273amino acids starting from an Ile-Val-Gly-Gly-Glu mo-tif, the catalytic triad of serine proteases that has beencharacterized as the essential amino acid residues forthe proteolytic activity, and a hydrophobic amino acidstretch in the carboxyl terminus, suggesting this en-zyme is a novel membrane-type serine protease. Thetissue distributions of esp-1 expression revealed thatthis protease is not only expressed in human eosino-phils, but also widely expressed in mononuclear cellsand various tissues other than skeletal muscle andkidney and is most abundant in testis and prostate,and moderately so in lung, spleen and pancreas. © 1998

Academic Press

A large number of differentiated blood cells expresscharacteristic serine proteases, and each protease inthese cells plays a specific role in patho-physiologicalfunctions in acute and chronic inflammation. Althoughgranzymes from cytotoxic T cells and natural killer(NK) cells (2), cathepsins, elastase, collagenase andgelatinase from neutrophils (3), and tryptase and chy-mase from mast cells (4) have been extensively studied,the proteases from eosinophils and basophils have notbeen characterized, because of the limitation of thenumbers of these cells in the blood. In order to charac-terize a novel serine protease(s) in human eosinophilsand to deduce its function in the progression of anallergic disease, we attempted the cloning of a serineprotease by reverse transcription-polymerase chain re-action (RT-PCR) from purified eosinophils of atopicpatients with eosinophilia. In this paper, we first re-port a novel membrane-bound serine protease deduced

from the amino acid sequence in human eosinophils,determine the distribution of the gene in various or-gans, and discuss the role of the enzyme.

MATERIALS AND METHODS

Eosinophil isolation. Peripheral blood was obtained from pa-tients with eosinophilia with informed consent. The isolation of eo-sinophils and neutrophils was carried out according to the methodspreviously described (5).

Cloning of a novel serine protease sequence from eosinophils. To-tal RNA was isolated from human eoshinophils by the guanidium-thiocyanate-chloroform method (6). One mg of total RNA was sub-jected to cDNA synthesis using SuperscriptII (Life Technologies) inthe presence of an Oligo-dT-NotI primer: 59-AACTGGA-AGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTTV-39. Af-ter the cDNA synthesis, PCR was carried out using a combination ofdegenerate oligonucleotides that encode the conserved amino-acidsequence within the active sites of the serine proteases, His and Ser(7). After PCR, the products were separated on a 2% agarose/TAEgel. The 400-600 bp bands were excised, and ligated to TA-Vectorincluding pGEMT-easy (Promega) and PCR2.1 (Invitrogen). Whitecolonies were randomly picked up and their sequences were verified.To obtain the full length cDNA, the modified 59and 39RACE de-scribed below were carried out. One mg of total RNA was subjected tocDNA synthesis using SuperscriptII in presence of the Oligo-dT-NotIprimer and Cap-switch oligoII (CSII):59-AAGCAGTGG-TATCAACGCAGAGTACGCGGG-39. After the cDNA synthesis reac-tion, excess primers were removed with a PCR-purification kit (Quia-gen). The resultant cDNA was used for the following PCR. Modified59Race: PCR was carried out using esp-1 68: 59-CCAAACTGGACC-ATCCACC-39 and a PCR primer: 59-AAGCA-GTGGTAT-CAACGCAGAGT-39 (Clontech). Heminested PCR was carried outusing esp-1 49: 59-CGGAGGGATCACTAAGGTCAC-39and the PCRprimer (Clontech). The PCR products were applied to a 2 % agarose/TAE gel. The 300 bp band was excised and subcloned into PCR2.1,and the sequences of two independent clones were determined.39RACE: First PCR was performed using a NotI primer: 59-CTGGAAGAATTCGCGGCCGCAGG-39and esp-1 424: 59-GGAGA-CAT-GGTTTGTGCTGGC-39. Heminested PCR was performed usingesp-1 456: 59-CGGGAAGGATGCCTGCTTCG-39and the NotI primer.The PCR products were applied to a 2 % agarose/TAE gel. The 400 bpband was excised, and ligated to PCR2.1 (Invitrogen), and then thesequences of the two independent clones were verified.

Amplification of full length cDNA of esp-1. PCR was carried outusing the following parameters: One min denaturation at 95°C,

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followed by 35 cycles of 15 sec denaturation at 95°C, 30 sec annealingat 62°C, and 2.5 min extension at 72°C, with final extension of 7 minat 72°C. The primers, esp-1 -9: 59-GAGGAG-GCCATGGGCGCGC-39and esp-1 1058: 59-CCTGCAAGGCATCAACTGG-AATGTG-39,were used for the amplification of the cDNA. The 1100 bp band on a1% agarose/TAE gel was excised. The purified DNA fragment wassubjected to subcloning into PCR2.1. Five independent clones werepicked up and all their sequences were examined.

Esp-1 expression in eosinophils but not in neutrophils. The purityof CD16-negative granulocytes (eosinophils) was checked by RT-PCRusing the primers of FcgRIII, oligonucleotides, 465: 59-TCA-TTTGTCTTGAGGGTC-39and 466: 59-GTCTCTTTCTGCTTGGTG-39, described elsewhere (8). Expression of the esp-1 gene was alsodetermined by RT-PCR using the primers, esp-1 -9 and esp-1 68,under the following conditions: One min denaturation at 95°C, fol-lowed by 35 cycles of 15 sec denaturation at 95°C, 30 sec annealingat 62°C, and 1.5 min extension at 72°C, with final extension of 7 min.A b-actin primer set purchased from Stratagene was used for RT-PCR according to the manufacturer’s protocol except for an anneal-ing temperature of 62°C instead of 55°C. Separated mononuclearcells, granulocytes, and CD16-positive granulocytes (neutrophils)were used as controls for all RT-PCR. Each panel contained 100 bpladder marker (Life Technologies) in the left lane, with reverse-transcriptase negative RT-PCR on the right. The amount of cDNAused for PCR was approximately 50ng. The PCR products were runon 2% agarose/TAE gels. The gels were stained with an ethidiumbromide solution, followed by transfer of the products to Hybond N1membranes. The Hybond N1 membranes except those with b-actinproducts were hybridized with 32P-labeled oligonucleotide probes:59-ACAAACATTTGAAGC-TCA-39for FcgRIII and esp-1 49 for esp-1.

Tissue distribution of the esp-1 gene. The human multiple tissuecDNA, MTC Panels I and II, was purchased from Clontech and usedfor measurement of the expression level of esp-1. PCR was performedaccording to the manufacturer’s protocol. The pair of primers used inthese reactions was esp-1 778S: 59-AGCTG-GGGAGTGGGC-TGTGGTC-39and esp-1 949AS: 59-ATGGGCTCAGGTAG-GCTCA-GAC-39. PCR was carried out under the following conditions: Onemin denaturation at 95°C, followed by 32 cycles of 15 sec denatur-ation at 95°C, 30 sec annealing and extension at 68°C, with finalextension of 7 min. The PCR products were run on 2% agarose/TAEgels and visualized by ethidium bromide staining, transferred to aHybond N1 membrane (Amersham Pharmacia Biotech) and thenhybridized with a 32P-labeled oligonucleotide probe: 59-AAGC-TGATGGCCCAGAGTGG-3. Amplification of the glyceroaldehyde-3-phosphate dehydrogenase gene was performed according to the man-ufacturer’s protocol.

Northern blot analysis of the esp-1 gene. Total RNA and mRNAwere isolated from Hela S3 cells and used for Northern blot analysis.mRNA (0.5 mg) was separated on a 0.7% formaldehyde-/MOPS aga-rose gel and then transferred to a Hybond N1 membrane. The blotwas hybridized with 32P-labeled full length esp-1 cDNA. The size ofthe esp-1 transcript was estimated in comparison with the sizes of28S and 18S RNA, as markers.

Expression of the ESP-1 protein in HEK293 cells. The esp-1 genein PCR2.1 was amplified by PCR, and then subcloned intoPME18S-FL by blunt-end ligation. The pair of primers used for theamplification was esp-1 -9 and esp-1 970: 59-GGGCTCAGGTAGG-CTCAGACCG-39. The sequence of the esp-1 gene cloned in PME18S-FL(PME-esp1) was confirmed. Two mg of PME-esp-1 was transfectedinto HEK293 cells using FUGENE6 (Boehringer Mannheim). Twodays after the transfection, cells were lysed with 100ml of SDS-gelloading buffer containing 2-mercaptoethanol, directly added to a6-well culture dish, and then sonicated extensively. Fifteen ml ali-quots of cellular lysates derived from PME-esp-1 transfected andmock-transfected cells were separated by 10-20% gradient SDS-PAGE and then transferred to a PVDF membrane (Millipore). Theblot was incubated with affinity-purified specific rabbit antiserum

against the ESP-1 peptide, followed by a horseradish peroxidase-labeled anti-rabbit IgG antibodies. The resultant blot was visualizedwith ECL (Amersham Pharmacia Biotech) according to the manu-facturer’s protocol.

RESULTS AND DISCUSSION

Identification of a Novel Serine Protease in HumanEosinophils

We searched for a novel serine protease(s) by PCRusing the degenerate oligonucleotides in the cDNA ofpurified eosinophils. For this purpose, we selected theconsensus sequences of various serine proteasesaround the active-site residues, His and Ser, for a set ofprimers, and succeeded in finding a novel serine pro-tease (7, 9). This novel serine protease, designated asesp-1, consists of 1082 bp with a Kozak sequence (10),59-ggccatgg-39, and a poly A additional sequence, asshown in FIG. 1. A blast sequence homology searchshowed that the putative amino acid sequence of ESP-1is most similar to that reported for human prostasin(11). The overall identity is 40.85%, suggesting thatthis ESP-1 is not an isoform of the known protease. Theputative amino acid sequence of ESP-1 starts at nucle-otide 10, as judged with the Kozak’s rule (10). Thesignal peptide sequence of ESP-1 is composed of 18amino acids, as determined with a Nakai server, thatpredicts the structure and intracellular localization ofa protein. The N-terminal sequence of the pro-form ofESP-1 starts at Arg19 and ends with Val314. The acti-vated form of ESP-1 starts at the Ile-Val-Gly-Gly-Glusequence at residues 42-46, indicated by the filled ar-row, as judged from the homology of the N-terminalresidues of various activated serine proteases (12). Theactive-site residues that comprise catalytic triad ofserine proteases, such as His, Asp and Ser, are resi-dues 82, 137 and 238, respectively, in ESP-1, as judgedfrom the homology with other serine proteases (9).Potential N-glycosylation sites are located at Asn167

and Asn273. In addition to the structural similarity ofESP-1 to other serine proteases, this ESP-1 proteinpossesses a unique stretch of carboxyl-terminal hydro-phobic amino acids at residues 299-314. These datasuggest that the ESP-1 protein is a membrane-typeserine protease. It will be a challenge to reveal whetherthis protease stays in the ER, Golgi or plasma mem-brane.

esp-1 Gene Expression in Eosinophils but Not inNeutrophils

Random sequencing of the PCR products that encodeboth the His and Ser residues revealed sequences iden-tical to those of caldecrine (13) and mast cell tryptase(14) in addition to the novel sequence of esp-1. To ruleout the possibility of contamination by neutrophils,that contain various kinds of proteases (3), we highly

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fractionated granuloctes by MACS using CD16 mono-clonal antibodies (Miltenyi Biotec GmbH). The frac-tionated cells were subjected to RT-PCR analysis tocheck the messages for FcgRIII (CD16), esp-1 andb-actin. As a result, amplification of CD16- positivegranulocytes, that mainly consist of neutrophils,showed an amplified 142 bp band of CD16, while CD16-negative granulocytes, that mainly consist of eosino-phils, did not (FIG. 2a). The data indicate that there islittle contamination by neutrophils of the CD16-negative granulocytes. In the mononuclear cells, theexpression of FcgRIII turned out to be positive (Fig.2a). This might be due to the presence of NK cells andmacrophages among the mononuclear cells (8). On theother hand, RT-PCR analysis of esp-1 gene expressionrevealed an amplified 193 bp band of esp-1 for CD16-negative granulocytes and mononuclear cells, but notCD 16- positive granulocytes (FIG. 2b, top). The smalldifferences in the levels of amplified products ofFcgRIII and esp-1 between fractionated CD16-negative cells and total granulocytes might be due totoo many cycles of PCR or the incubation with CD16monoclonal antibodies on ice for 1 hr in the process ofseparation of neutrophils and eosinophils, that de-creases the expression levels of esp-1 and FcgRIII butnot that of b-actin. Each blot was hybridized with a32P-labeled probe, that ensures the identification of thecorresponding gene (FIG. 2a and b, bottom). Theamounts of cDNA used in these PCRs were determinedby PCR using b-actin primers, and they turned out tobe identical (FIG. 2c). The overall results indicatedthat the esp-1 gene is expressed in eosinophils andmononuclear cells, but not neutrophils.

Although the physiological role of ESP-1 has notbeen clarified, it may play a pivotal role in the ex-travasculization of eosinophils through destruction ofthe basement membrane, or the activation of otherproteases such as metalloprotease and/or gelatinase.To prove these hypotheses, we aim to obtain functionalrecombinant proteins and monoclonal antibodies toESP-1.

Expression of the esp-1 Gene in Various HumanTissues

MTC-panels I and II were used to study the expres-sion levels of esp-1 in various human tissues in com-parison to the results of Northern blot analysis, for twomajor reasons: Rare messages can be detected, andsemi-quantitation of rare messages is possible. Theresults showed that this gene was expressed not only ineosinophils but also was highly expressed in testis andprostate, moderately so in lung, pancreas and spleen,weakly so in thymus, colon and peripheral blood leu-kocytes (PBL) (FIG. 3, top). Esp-1 expression was de-tected in neither kidney nor skeletal muscle (FIG. 3,top). Hybridization using a 32P-labeled oligonucleo-

FIG. 1. The nucleotide and deduced amino acid sequences ofthe esp-1 gene. A putative signal sequence is underlined. Aster-isks(*) indicate potential sites of the active-site residues of aserine protease. The closed arrow shows an Ile-Val-Gly-Gly-Glusequence, the possible N-terminal sequence of an active serineprotease. A putative polyadenylation signal is dashed-underlined.Sharps (#) indicate potential glycosylation sites.


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tides probe showed the amplified products were of theesp-1 gene (FIG. 3, middle). The amounts of cDNA usedfor PCR were constant, since the expression levels ofthe glyceroaldehyde-3-phosphate dehydrogenese mes-sage were almost identical among the various humantissues tested (FIG. 3, bottom). We have not identifiedthe cells containing ESP-1 in these high expressiontissues yet. Therefore we could not completely rule outthe possibility that the ESP-1 expressed in these tis-sues was derived from mononuclear cells or eosino-phils. In general, normal testis and prostate, however,

are not tissues which contain large quantities of mono-nuclear cells and/or eosinophils.

Since esp-1 was highly expressed in the male repro-ductive system, one can think that this ESP-1 is re-lated to the fertilization process (15).

Determination of the Size of the esp-1 Transcript

The size of the esp-1 message was determined byNorthern blot analysis of mRNA extracted from HelaS3 cells. We have cloned an identical gene to esp-1 from

FIG. 2. Expression of esp-1 mRNA in eosinophils. Fractionated mononuclear cells and granulocytes, that mainly consist of eosinophils:CD16(2) and neutrophils: CD16(1) were subjected to RT-PCR using primers of FcgRIII (CD16) (a), esp-1 (b), and b-actin (c). Top: ethidiumbromide staining of the gel. The 100bp markers are on the left and the RT-negative control in the right lane. Arrows indicate the RT-PCRproducts. Bottom: hybridaization with a 32P-labeled oligonucleotide probe.


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Hela S3 cDNA libraries (unpublished data). As de-duced from the sizes of 28S and 18S RNA, the fulllength of esp-1 mRNA is approximately 1.2-1.4 kb(FIG. 4).

Transfection of the esp-1 Gene to HEK293 Cells

The esp-1 gene under the control of the promoter,SRa, was introduced to HEK293 cells (Human Em-broynic Kidney cells). Mock-transfected HEK293cells were used as a negative control. Western blotanalysis of the cellular lysates showed that only theesp-1 gene transfected cells gave rise to a band cor-responding to an approximate molecular mass of 35kDa (FIG. 5). We further examined the myc-taggedesp-1 gene expressed in Cos7 cells, that gave rise toa protein band with an approximately identical mo-lecular mass on Western blotting with anti-myc Ab(9E10) (data not shown). Although these data indi-cate that esp-1 is correctly transcribed and trans-lated to a protein with a molecular mass of 35kDa,the molecular masses deduced from the amino-acidcompositions of the pro-form and active-form are33.1 and 30.6 kDa, respectively. This may be due tothe glycosylation of this protease that contains twopotential glycosylation sites at residues 167 and 273.However, the functional recombinant enzyme is nec-essary to reveal whether this 35 kDa protein is theactive-form or the pro-form of ESP-1.

FIG. 3. Expression of esp-1 mRNA in various human tissues.Top: PCR products of esp-1. The 100bp markers are on the left.Middle: Southern blot hybridaization of the top panel. Bottom: PCRproducts of glyceroaldehyde-3-phosphate dehydrogenase.

FIG. 4. Northern blot analysis: Determination of the size of esp-1mRNA. The arrows on the left indicate the positions of 28S and 18SRNA. The arrow on the right indicates the esp-1 mRNA.

FIG. 5. Western blot analysis of the ESP-1 protein in esp-1transfected HEK293 cells. The arrows on the left indicate the markerproteins (APPRO).


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ACKNOWLEDGMENTS

We wish to thank to Mr. Mitsuhiro Miyake (Tokushima Univer-sity) for his technical help and Dr. Kazuo Maruyama (Tokyo Medicaland Dental University School of Medicine) for supplying the plasmidvector, pME18S-FL.

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cloning and tissue distribution of a novel serine protease esp-1 from human eosinophils

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