THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 34, Issue of December 5, pp. 18133-18137,1988 Printed in U.S.A.

Cloning and Sequencing of cDNA Encoding a Rat Salivary Cysteine Proteinase Inhibitor Inducible by @-Adrenergic Agonists*

(Received for publication, June 27, 1988)

Phyllis A. Shaw$#, James L. Cox$, Tibor BarkaSIl, and Yukio NaitoII From the Departments of $Anatomy and TPathology, Mount Sinai School of Medicine, City University of New York, New York, New York 10029 and the IIDepartment of Biological Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467, Japan

The &adrenergic agonist isoproterenol induces a unique secretory protein (LM) in the salivary glands of developing and adult rats. In order to study the regulation of growth and gene expression by catechol- amines, we have isolated and sequenced several cDNA clones encoding the LM protein. Each of the LM cDNA clones described identifies, by Northern blot analyses, a single mRNA species of approximately 900 bases in size. The mRNA encoding this secreted protein was not detected in submandibular glands or brains of un- treated adult rats. Sequence analyses of the LM cDNA clones revealed a striking similarity to the family 2 of cysteine proteinase inhibitors. Furthermore, when pu- rified LM protein was used to assay for inhibition of cysteine proteinases, the data demonstrated that it is indeed a type of cysteine proteinase inhibitor. This inhibitor, termed rat cystatin S, provides the first ex- ample of cysteine proteinase inhibitors that can be induced by &adrenergic agonists.

The adrenergic agonist isoproterenol (IPR),’ acting via pl- adrenergic receptors (l), can profoundly affect the growth and differentiation of the submandibular and parotid glands of mice and rats. IPR stimulates DNA synthesis and replication of acinar cells (2-4), causes conspicuous hypertrophic/hyper- plastic enlargements of salivary glands (5-9), and, postnatally, it induces an apparent acceleration of acinar cell differentia- tion (7, 10-12). IPR is a potent secretogogue for proteins and mucoproteins. The drug not only stimulates overall protein synthesis in the submandibular gland (13, 14), but also in- duces the synthesis of specific proteins that are absent or occur at very low concentrations in unstimulated glands. Thus, adrenergically stimulated rodent salivary glands pro- vide a favorable system to study the regulatory mechanisms of secretion, cell replication, hypertrophic/hyperplastic growth, differentiation, and the regulation of expression of specific genes by catecholamines.

~~~ ~ ~

* This work was supported by National Institutes of Health Grants DE08174 (to P. A. S.) and DE 07637 (to T. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper ~ Q S been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 504206.

$ T o whom correspondence and reprint requests should be ad- dressed.

The abbreviations used are: IPR, DL-isoproterenol-HC1; LM, low molecular weight mobile protein; E-64, L-trans-epoxysuccinyl-leu- cylamido-(4-guanidino)butane; NMC, 7-(4-methyl)coumarylamide; SDS, sodium dodecyl sulfate.

Administration of IPR to rats produces structural (15) and compositional alterations of the submandibular gland as well as of saliva. Menaker et al. (16, 17) have described a conspic- uous protein, characterized by its relatively high electropho- retic mobility in polyacrylamide gels, in the submandibular saliva of IPR-treated rats and have called this protein LM protein. For descriptive purposes, this operational term will be retained. The LM protein (M, = 13,000) is rich in aspartic acid, glutamine, and serine, but, in distinction from the class of proline-rich salivary gland proteins (21, 22), it is low in proline (18-20). By administration of IPR, the LM protein can be induced in the submandibular glands of adult and developing rats (23, 24). The function(s) of the LM protein were hitherto unknown.

In this paper, we describe the cloning and sequencing of several cDNAs encoding the LM protein. The deduced amino acid sequence of LM protein revealed a high degree of se- quence similarity to low molecular weight, naturally occurring cysteine proteinase inhibitors, suggesting that LM protein is an inhibitor belonging to the cystatin superfamily. Direct inhibitor assays confirmed that LM protein, isolated from the saliva of IPR-treated rats, is indeed a potent inhibitor of some cysteine proteinases. To our knowledge, this is the first dem- onstration of the induction of a cysteine proteinase inhibitor in any tissue. It forms the basis of further studies of the regulation of expression of cystatin genes by catecholamines and the role of the sympathetic nervous system, in general, in modulating the activities of cysteine proteinases that have been implicated in various physiologic and pathologic proc- esses (25-33).

MATERIALS AND METHODS~

RESULTS

Selection of LM-specific cDNA Clones-The initial cloning resulted in 307 tetracycline-resistant, ampicillin-sensitive rat submandibular gland cDNAs. Colonies were individually transferred to duplicate nitrocellulose filters, grown overnight, and processed to bind DNA to the filters (39). Screening with 32P-labeledpoly(A)+ RNA from submandibular glands of IPR- treated rats that is enriched for LM mRNA, and from controls that lack LM mRNA enabled the selection of 58 clones that contained cDNA sequences complementary to IPR-induced mRNAs. These colonies were screened by hybridization to the radioactively labeled 23-mer oligonucleotide corresponding to the amino-terminal LM protein sequence (20), and by im-

Portions of this paper (including “Materials and Methods,” Ta- bles I and 11, and Figs. 2,4, and 6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

18133

18134 Cysteine Proteinase Inhibitors Induced by P-Adrenergic Agonists

munoselection of clones synthesizing LM protein by a poly- clonal antibody specific for LM protein. The six clones that met the criteria of being positive in the positive/negative selection scheme and/or hybridizing to the 23-mer corre- sponding to the amino-terminal of LM protein and/or of selection with the antibody to LM protein were used for the preparation of radioactive probes for LM mRNA studies, for cDNA sequencing, for synthesizing oligonucleotides corre- sponding to the carboxyl-terminal of the protein, and for screening the pGEM-3 cDNA library for full length cDNA clones, which were also sequenced.

Determination of the Size of LM mRNA-In Northern blots (Fig. l), each of the 32P-labeled nick-translated cDNAs from the six clones that were positive in, the screening assays hybridized to a single size class of poly(A)' RNA of about 900 bases. Furthermore, each LM cDNA clone detected compa- rable high steady-state levels of LM mRNA in the IPR-treated group (lunes 3 and 4). In contrast, RNA from untreated adult rat submandibular glands (lanes 1 and 2) and brain ( l a n e 5 ) did not hybridize to 32P-labeled nick-translated cDNAs from any of these clones. These findings indicate an induction of LM gene expression by IPR.

Nucleotide Sequence of LM cDNA-The strategy of se- quence analyses is shown in Fig. 2. The sequences were determined from the two complementary strands, using clock- wise and counterclockwise primers flanking the PstI site (cDNA insert site) in pBR322 and pGEM-3 to sequence both strands of the DNA by the dideoxy method. The sequence reported is a composite derived from three overlapping clones in pBR322 (4,6; 10,9; and 9,3), and three overlapping clones in pGEM-3 (la; 5; and 1) (Fig. 2). In the overlapping regions, there was 100% agreement among all 6 LM cDNA clones.

One of these clones contains the complete nucleotide se- quence encoding the mature LM protein (Fig. 3, clone la). The cDNA inserts are 622 nucleotides in length and contain a single open reading frame of 396 nucleotides, encoding a translation product of 132 amino acids. Based on the partial amino-terminal amino acid sequence of the LM protein, the amino-terminal of the LM protein is most likely the leucine, labeled position 1, and the first 14 amino acids represent part of a signal sequence. On this basis, the LM protein is 118 amino acids long with an unmodified molecular size of 13,112 Da. The 3"noncoding sequence is 226 nucleotides in length and includes the polyadenylation signal AATAAA at position

I 2 3 4 5

2 8 S d

I 8 S 4

FIG. 1. A representative Northern blot of 0.5 and 1.0 pg of poly(A)+ RNA from control (lanes 1 and 2) and IPR-treated (lanes 3 and 4) submandibular glands, and 20 pg of total RNA from the brain (lane 5) of untreated rats hvbridized to one of the 32P-nick-translated LM

10 20 30 40 50 60 A T T T C T A C n ; A ~ A C C A T A ~ A G ~ M C A C G A C A ~ T C ~ T C T A G G T C A C IleSerThrAspTyrLeuTyrIleSerPheGluH~sCluThrLeuSerCysLeuClyHis

70 m C T C C G T C G C A T A G A G M G T G C A ~ A G G A G G M C G A G C ~ C A G M G C A ~ M C

BO 90 100 110 120

PheLeuClyClyIleGluLysSerSerMetGluCluGluClyAlaSerGl~laLe~sn

130 140 150 160 170 180 T A n ; C T C T C M T C A G T A T M T C ~ G M C A G n ; A ~ T A C ~ A G C ~ n ; ~ ~ M TyrAlaValAsnGluTyrAsnGluLysAsnSerAspLeu~rLeuSerArgValValClU

G n ; M G G A T G T C C ~ G C A G G ~ ~ C M ~ ~ A n ; T C A ~ ~ A

1

10 20

190 200 210 220 230 240

ValLysAspValGlnLysClnValValAlaGlyThrLysP ePhcPheAspValI1eCau

30

50 6 8

G G C ~ C M T A n ; ~ M G A C A C A ~ ~ A ~ A C C M C P C T C 250 260 270 280 290 300

GlyLysThrIleCysLeuLysThrGlnGlyAspLeuThrAsnCysProLeuAsnGluGlu

31b 320 330 340 350 3 60 ~ A T C A G C A ~ A G C A n ; M ~ ~ ~ ~ T C A T A ~ C ~ ~ A G M T AlaAspClnGlnGluHisCluPheCysSerPheValValHi~AspIleProT~l~sn

380 390 400 410 420

70 80

370 90 100

T A T A ~ ; T ~ A G C ~ C C A ~ T ~ T A G T A T A ~ ~ ~ A ~ ~ M G T C ~ A ~ T TyrIleValLeuLeuSerSerSerCysHisSerIlcEnd

G T A G G A ~ ; c A G A T C T C T C ~ ~ ~ ~ ~ C A C ~ A T C A ~ A T C 430 440 450 460 470 480

110

550 CMGCAPA-ACMATGCCCATA"

560 570 580 590 600

610 620 CTCAATMMTCTCCMCAGCT

FIG. 3. Nucleotide and deduced amino acid sequence of clones encoding rat saliva cystatin S. Numbering of the nucleo- tide sequence proceeds in the 5' to 3' direction beginning at the first nucleotide. Amino acid numbering begins with the putative residue 1 of the mature protein. The polyadenylation signal is underlined.

10 2 0 3 0

4 0

r. V E V K D V Q X Q V V A G T X P P P D V I L G X T I C L X T Q G D L I H C

h 8 hC h s n L R V L R A R Q Q T V G G V N Y F F D V E V G R T I C T K S Q P N L D T C L L Q V V R A R X Q I V A G V N Y P L D V E L G R T T C T X T Q P N L D N C Q I L R A R E I T P 5 : 1 V H Y ~ P ~ E 6 ~ ~ R [ I ~ T ~ S ~ ~ N O D T i

80

FIG. 5. An alignment, according to Salvesen et al. (62), of the amino acid sequences of some of the low molecular mass cystatins of the family 2 type. The sequences are those of rat saliva cystatin S ( rs ) , human cystatin C (hc), human cystatin S (hs), and human cystatin SN (hsn).

604-609. The cDNA .insert does not include the translation initiation consensus sequence.

The putative amino-terminal amino acid sequence deduced from the nucleotide sequence (Fig. 4) is approximately 80% in agreement with the published partial amino-terminal amino acid sequence of the purified LM protein reported by Naito and Suzuki (20). In addition, the calculated amino acid composition is consistent with that of the purified LM protein (Ref. 20 and Table I).

A search of the NBRF protein data base (via Bionet) revealed that LM protein has a high degree of sequence similarity to a number of cysteine proteinase inhibitors, called cystatins (Table 11). These include the human cystatins, SN (52), C (53), S (54), S5 (55), cystatin C-related protein frag- ment (56), chicken cystatin (57), and bovine cystatin (58). All of these inhibitors are members of cystatin 2 family (59). Fig. 5 illustrates the sequence similarity of the LM protein se- quence to those of the human cysteine proteinase inhibitors,

cDNA clones. cystatin C, cystatin S, and cystatin SN. Even though it is not

Cysteine Proteinase Inhibitors Induced by 0-Adrenergic Agonists 18135

known which parts of the cystatin molecule bind cysteine proteinases, there are sequences which are most highly con- served among the three cystatin families. The highly con- served amino acid sequence, QVVAG, present in all of the cystatin families and considered to have inhibitory activity (60), is also present in the LM protein in the same region. There is suggestive evidence that the amino terminus has a role in inhibitory activity, with Gly-2 (Gly-9 in other amino acid line ups) being one of the very few residues conserved in all of the cystatins; the Gly residue is present near the amino terminus of the LM protein. In addition, the cysteine residues, which are involved in the formation of the two disulfide bonds formed in the cystatins of the family 2 type, are also present in the LM protein (Fig. 5). Thus, the areas which appear to be functionally important in the cystatin families as a whole are represented in LM protein as well.

Proteinase Inhibitor Activity of LM Protein-The sequence similarity between the LM protein and several of the cystatins suggested that LM protein may be a low molecular weight proteinase inhibitor belonging to the cystatin superfamily. Measurements of the inhibitory activity of purified LM pro- tein using active site-titrated papain, ficin, and cathepsin B revealed that the protein is a potent inhibitor of papain and ficin (Fig. 6). The calculated values for mole of enzyme inhibitory site/mol of LM protein (Mr = 13,000) were 1.4 and 0.71 for papain and ficin, respectively. However, LM protein did not inhibit another cysteine proteinase, bovine spleen cathepsin B, or kallikrein, the major serine proteinase in rat submandibular glands.

DISCUSSION

The pronounced hypertrophic/hyperplastic enlargements of the parotid and submandibular glands of rats and mice produced by the administration of the @-adrenergic agonist IPR, discovered by Selye et al. (8), is unparalleled among the models of induced growth; with repeated administration of the drug, the weights of these glands may be doubled in 4 days. The molecular mechanisms underlying these processes are largely unknown. IPR induces the expression of several proteins in rat submandibular glands; the best characterized is the family of proline-rich proteins (21, 22) and the LM protein (18-20, 23, 24). Although the LM protein has been known for more than a decade, its function remained obscure.

Cloning and sequencing of cDNAs encoding the LM protein led us to the discovery that the LM protein is a cysteine proteinase inhibitor belonging to the cystatin superfamily. The superfamily of cystatins encompasses three, evolution- arily related protein families, 1) the stefin family, 2) the cystatin family, and 3) the kininogen family (59). The LM protein belongs to the cystatin family, and conforming with the nomenclature recommended (59), we propose to name it “rat cystatin S.” Human cystatin S, which is the prevalent inhibitor in saliva and tears (61), its molecular variants, and human cystatin SN (52,54,55) show a high degree of sequence similarity to human cystatin C (53). Similarly, rat cystatin S has a high degree of structural homology not only to human cystatin S and human cystatin SN, but also to human cystatin C and cystatin C-related protein (56). Rat cystatin S inhibited papain and ficin, but not bovine spleen cathepsin B. Human cystatin S also failed to inhibit cathepsin B (porcine) (54).

Human cystatin S and SN have been isolated from the saliva of healthy individuals. The concentration of rat cystatin S is not measurable in the saliva of untreated adult rats and is very low in gland extracts (18). Rat cystatin S, however, is rapidly induced by IPR and is rapidly accumulated in the glands of adult rats treated with repeated doses of this p-

adrenergic agonist (9). While human cystatin S was detected immunocytochemically in human parotid and submaxillary glands (54), rat cystatin S could be demonstrated only in the glands of adult rats treated with IPR (24).

The physiological role of cystatins in human saliva has not been established. That these inhibitors have a protective role in minimizing the potentially harmful effects of proteinases of bacterial or cellular origin in the oral cavity seems to be a reasonable assumption. Cystatin S mRNA was not detected in the submandibular glands of untreated adult rats, suggest- ing that the protein is not synthesized to any significant degree. Large quantities of cystatin S are secreted into the saliva of IPR-treated adult rats. Whether the inhibitor also has an intracellular role in the glands of such animals awaits further studies. The rapid induction of a proteinase inhibitor and organ growth by a @-adrenergic agonist may not be coincidental, since intracellular proteinases are involved in the degradation of proteins, and inhibition of their activity could contribute to the production of cellular and organ hypertrophy. The demonstration that a specific proteinase inhibitor can be induced by @-receptor mechanisms will per- mit future studies of the in uiuo regulation of this cystatin- like gene by catecholamines. Furthermore, this model may help in defining the role of the sympathetic nervous system in modulation of the activities of cysteine proteinase/cysteine proteinase inhibitor systems that have been implicated in various disease states (25-33).

Acknowledgments-We wish to express thanks to Drs. Edward Gresik, Ruth Gubits, Beth Schachter, and H. van der Noen for constructive criticism of the manuscript.

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Svppl~mcnuvy Material to

CUINING AND SEWENCRiG OF cDNA ENCODING A RAT W A R Y CYSTEINE PROTEINASE INHIBITQR INDUCIBLE BY BETA-ADRENERGIC AGONISTS.

Phyllis A S b . Jamcr 1- Cox, Tibor kka and YuLio Ndto

MATERIALS

TBe chmicsh and their satrces were: guaaidime thiocyanate, Fluka AG: oligo dT (deoxythymidylale) ceilulase (TpC 3). Cnllabarative Rrreareb: dtrlrcUulmc (BA 85) rhecu. Schlcidher and Sehuell: terminal dcorynudeotidyl transferase, HBlOl mmplcnt cek avian myclablartosis virus r w c m transniptaw. and the M13 dideoxy sequencins M Bcthesda Research Labonloricr; restciaion enzymes and d ~ i ~ ” ~ l e ~ acid polymerarc I (cndonucicasc free). Bochringer Mannheim o-32Pdeoxynudcoride triphosphates [tetra

(ultra pure), International Biotechnologies. tnc.; DL- i sopro t t rono l -HCI (uiethyiammonium) salu (670 CNmm0k)l and 1211-prolein k (8.2 rcillrg), New England Nuclear; agarose

(3.~~ydrory~-((i~p~ooproWi-smino)mcthyl]be~i rlmbol). catbepin B (from bwinc spleen. EC3.4.22.1). kallikrein (from porune pancreas). ficin. papmn ( T y p e IV, EC 3.4.22.2). 7-mino4 methyl-coumarin. uans-cpoxyruccinyl-Lleuql-amido (4-gunnidino)buIane (€344). S i p % ZPhe-kg-NMC Enzyme Systems FTnduM: oligonncletide CED p4~6phorpmidite wthesidog til, ABN-Fisshu. LM protein was purified as described (IS). Othcr mated& were purchaccd from mmercid sourcu and were of thc highest purity available.

METHODS

fnr~nv~ of Rae - Sprsgne-Dawlcy raw were kept io I) t e m p m e and humidity controlled environmcnt(12

euanguination under ether anesthcsis. the faad was removed for 4 hr. DL-iropraarsnal-HC1 (disolved in hour lightJ12 hwr dark m e ) and had free e- to mtcr and riandard laboratcry chow. Before killing by

0.1% xdium metabisulfitc. 0.8596 NCI) war &en intr.pritoncaUy. “y rats. 6 days of age. wen injected daily with 0.1 “mol@@ body w&ht of IPR-Ha for 7 dayr and were kilied 24 hr after tho Isst injection of

the drug. Thinecn day old ~ontrol rat3 were injened with the solvent alone. Submandibular glands of raw of the above p u p s wen used for the isolation of p l y (A) + RNA

0 f i p ” f u - An oligonudcolide. 5’-TAITUATlCUCCWtA-A-3’ (inosine replaced the ambiguous bare) camplcmentay to the N-taminal Of the secreted form of LM protein (18) was rptherired and wa) used to generate the radioactive probe for LM cDNA colony selcclion. An oiigonudcotidc ( S . ~ A T ~ T G ~ ~ A ~ G ~ G . 3 ’ ) mmplementay 10 part of the 3’ end of LM mRNA. and one (5”CATGCTAGACITCrCTATG-3’) mmplcmcntay to part of the 5’ end of LM mRNA were rynthwized to use for exlemion of LM poly (A)+ RNA for requeocing and for LM tDNA colony selection frnm the @EM-3 <DNA library.

RVA Isolation - Total cellulae RNA was isolated and purified by the method of Chirgwin et SI. (34). Paly(A)+ RNA wa) isoIzIed acmrding to Aviv and Lcder (35) by tw~) cydes of oligo(dT) niiuiose miumn chromatography.

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Acad. Sci. U. S. A. 72,1184-1188

Res. 6,3559-3567

5205

189-198

145-149

170,370-374

S. (1985) FEBS Lett. 186,41-45

Pharm. Bull. (Tokyo) 33,5149-5152

(1986) J. BioL Chem. 261,11282-11289

Cysteine Proteinase Inhibitors Induced by @-Adrenergic Agonists 18137

1.

5 5 ' >l'

t aoo 400 600

2 rS Phe Lau Gly Gly Ileu G1u Lys Sar Ser Met C1u G1U Clu Gly IJ4 Phe Leu Gly Gly lleu 01" Tyr Sor Ser Asn G1U Glu --- Gly

10

20 rS Ala Ser Glu Ala Lau AS" Tyr Ala Val Asn Glu Tyr ASn

28

IJ4 "_ Ala Clu Ala Leu --- Tyr Ala Val A m --- Tyr A m

Figure 4. Comparison of the N-terminal ami00 add wqucncc of rat saliva cytatio S with that of purified LM protein (Naita and Sumki. Ref. 20). The dashes indicate that the amino add residue was not determined at that position.

TABLE I

AMINO ACID MMFUSITION

RESIDUE

HIS LYS

ARG ASX THR SER GLX PRO GLY A U CYS VAL MET ILEU LEU TYR

TRP PHE

'ExPerimantal. DEDUCED FROM EDNA, a L8 PROTEIN

7.6 3.8

14.8 1.0

11.6 2.5

14.8

5.4 1.9

4.2 1.6 9.6 1.0 4.0 7.2

4 . 8 3.1

1.0

5.9 3.. 0.8 11.0

9.1 3.4

~~

15.1 1.7 5.9 4.2 4.2 9.3 0.8 5.1 10.2 3.4 5.1 0.8

TABLE I1

COMPARISON OF RAT CYSTATIN S WITH OTHER W W PUSS CYSTATINS

CYSTATIN HOMOLOGY AMINO ACIDS WEIGHT *MAXIMUM NUMBER OF MOLECULAR REF.

cystatin s (rat) 1008 118 13, 112

cystatin C-related 46% 110 fra9Re"t (human)

156)

chicken cystatin 4 2 % 116 13. 148 (57)

Cystatin C (human) 41% 120 13, 248 (53)

Cystatin SN (human) 419 113 11, 288 (55)

cystatin s (human) 391 113 13, 272 (54)

bovine cystatin 37% 112 12, 789 (58)

'Maximum hon0104Y (IMAXI Was calculated usins the IntelliGenetics align program. "x - total number of correctly matched amino acid pairs - [mismatch penalty + (gap penalty + gap size penalty)]. PEP Reference Manual, Release 5.0, Intelligenetice, I n c . , 1987.

INHIBITION OF PROTEINASE ACTIVITY BY LM PROTEIN

100 " 0

0 e""- - , 0 00 0.25 0 50 0 75 1.00 1 25

MICROGRAMS OF LM PROTEIN

Figure 6. Inhibition pattern of fysteine pratclnarc inhibitor LM The inhibitor puayr were performed ps described in Methods. The inhibitor activity of sal iva LM protein w u determined by active-site titration. Papain (open eirclc). f i b (dowd triangle). cathepsin 6, (closed circle). and Wkrcin (open triangle) were incubated with varying concenuatioa of purified LM protcb then the midual e v e snivity of each was measured. The fluorescence mcBsuremenu were convened to percent inhibitioa

*Reported by Naito and SUIuki ( 2 0 )