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Similarity with Serine Proteases (Primary Structure/Sequence Homology/Seminal Protease/Chymotrypsin-Like and Trypsin-Like Activity) KENNETH W

Similarity with Serine Proteases (Primary Structure/Sequence Homology/Seminal Protease/Chymotrypsin-Like and Trypsin-Like Activity) KENNETH W

Proc. Nati. Acad. Sci. USA Vol. 83, pp. 3166-3170, May 1986 Biochemistry

Human prostate-specific antigen: Structural and functional similarity with (primary structure/sequence homology/seminal /-like and -like activity) KENNETH W. K. WATT*, PAUL-JANE LEE*, THABISO M'TIMKULUt, WAI-PAN CHAN*, AND RUEYMING LOORt Departments of * Chemistry and tDiagnostics, Cetus Corp., 1400 Fifty-Third Street, Emeryville, CA 94608 Communicated by Russell F. Doolittle, December 31, 1985

ABSTRACT The complete sequence of the MATERIALS AND METHODS prostate-specific antigen (PA) from seminal plasma has PA was purified from human seminal plasma as described (1). been determined from analyses of the generated by PA was finally purified on a large-pore Vydac (Hesperia, CA) cyanogen bromide, hydroxylamine, endoproteinases Arg-C C4 column and eluted either with a linear gradient between and Lys-C. The single polypeptide chain of PA contains 70% (vol/vol) buffer A (buffer A = 0.1% trifluoroacetic acid 240-amino acid residues and has a calculated Mr of 26,496. An in H20) and 37% (vol/vol) buffer B (buffer B = 0.1% N-linked carbohydrate side chain is predicted at asparagine-45, trifluoroacetic acid in acetonitrile) in 280 min or 10% and O-linked carbohydrate side chains are possibly attached to (vol/vol) buffer B and 80% (vol/vol) buffer B in 60 min. serine-69, threonine-70, and serine-71. The primary structure The enzymatic activity ofPA was assessed on commercially of PA shows a high degree of sequence homology with other purified including insulin A, insulin B, gelatin, serine proteases ofthe family. The residues myoglobin, , fibrinogen (Sigma), and recombinant of , , and serine comprising the charge- interleukin 2 (Ala-IL2, Cetus). The substrates (1 mg/ml) were relay system of typical serine proteases were found in similar dissolved in either 0.1 M ammonium bicarbonate or 50 mM positions in PA (histidine-41, aspartic acid-96, and serine-192). Tris HCl, pH 7.8, containing PA at 0.1 mg/ml. In some cases, At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant a mass ratio (:substrate) of1:20 was used. After an 18-hr interleukin 2, and-to a lesser extent-gelatin, myoglobin, at 37°C, the peptides were separated by HPLC using ovalbumin, and fibrinogen. The cleavage sites ofthese proteins a 60-min linear gradient of 0-80% (vol/vol) buffer B. To by PA were chemically analyzed as the a-carboxyl side of some determine the bond specificity of PA, hydrazinolysis hydrophobic residues, , , , and phenylal- was performed on each ofthe PA/substrate digestion mixtures, anine, and of basic residues histidine, , and . The substrate alone, and intact PA as described (9). The free PA with the carboxyl-terminal amino acids were lyophilized and analyzed chymotrypsin-like activity of exhibited chromo- on a Beckman 121MB amino acid analyzer. genic substrate N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phen- The kinetics ofPA hydrolytic activity on synthetic substrates ylalanine p-nitroanilide yielded a specific activity of 9.21 ,uM were studied by monitoring the absorbance change at room per min per mg of PA and Km and krt values of 15.3 mM and temperature using a Hewlett-Packard 8450A spectrophotome- 0.075 s-1, respectively. "Trypsin-like" activity of PA was also ter. The following substrates were used: Na-benzoyl-L-tyrosine detected with Na-benzoyl-DL-arginine p-nitroanilide and gave ethyl ester (Bz-Tyr-OEt), Na-succinyl- nitroani- a specific activity of 1.98 ,uM per min per mg of PA. Protease lide (N-Suc-Phe-NA), Na-benzoyl-arginine-13-naphthylamide inhibitors such as phenylmethylsulfonyl fluoride, diisopropyl (Bz-Arg-NHNpt), Na-benzoyl-DL-arginine-p-nitroanilide (Bz- fluorophosphate, L-1-tosylamido-2-phenylethyl chloromethyl Arg-p-NA), Na-tosyl-L-arginine methyl ester (Tos-Arg-OMe) ketone, , , soybean as well (Sigma), and Na-succinyl-L-alanyl-L-alanyl-L-prolyl-Lphenyl- as Zn2+ and spermidine were effective inhibitors of PA -p-nitroanilide (N-Suc-Ala-Ala-Pro-Phe-p-NA) (Vega enzymatic activity. Biochemical, Tucson, AZ). The Km and the kg of PA on N-Suc-Ala-Ala-Pro-Phe-p-NA were calculated from Line- Human prostate-specific antigen (PA) is a glycoprotein with weaver-Burke plots. Inhibitors at 2 mM in 0.1 M CaCl2/0.1 M a Mr of 33,000-34,000 containing approximately 7% (wt/wt) Tris HCl (pH 8.0) were used to affect the PA activity on these PA is in the cells substrates. The following inhibitors were tested: phenylmethyl- carbohydrate (1, 2). detected only epithelial sulfonyl fluoride (PhMeSO2F), diisopropylfluorophosphate of the prostatic ductal element by a specific antibody to PA (DFP), L-1-tosylamido-2-phenylethyl chloromethyl ketone (1, 3). Seminal plasma also contains PA identical to that ofthe (Tos-PheCH2Cl), aprotinin, soybean trypsin inhibitor, zinc prostate gland (3). PA is synthesized in the epithelial cells of acetate, spermidine (Sigma), and leupeptin (Boehringer Mann- the prostate gland and secreted into the seminal fluid (3). heim). Recently, PA has been demonstrated to be clinically impor- The primary structure ofPA was established by automated tant for the detection and monitoring ofprostate cancer (4, 5). sequence analyses of intact polypeptide and by character- The PA molecule was independently identified as p30 (6) and ization of CNBr, hydroxylamine, endoproteinase Arg-C, and was tested as a marker for postcoital detection in rape endoproteinase Lys-C peptides. CNBr reaction was conduct- investigations (7). Although the clinical utility ofPA has been shown, its biological function and chemical structure are not Abbreviations: N-Suc-Ala-Ala-Pro-Phe-p-NA, Na-succinyl-L- well characterized (8). We report here the complete amino alanyl-L-analyl-L-prolyl-L-phenylalanine-p-nitroanilide; Bz-Arg-p- acid sequence of PA and describe the characteristics of its NA, Na-benzoyl-DL-arginine-p-nitroanilide; PhMeSO2F, phenyl- enzymatic properties. methylsulfonyl fluoride; DFP, diisopropylfluorophosphate; Tos- PheCH2Cl, L-1-tosylamido-2-phenylethyl chloromethyl ketone; Bz- Tyr-OEt, Na-benzoyl-L-tyrosine ethyl ester; N-Suc-Phe-NA, Na- The publication costs of this article were defrayed in part by page charge succinyl-phenylalanine nitroanilide; Bz-Arg-NHNpt, Na-benzoyl- payment. This article must therefore be hereby marked "advertisement" arginine-,f-naphthylamide; Tos-Arg-OMe, Na-tosyl-L-arginine in accordance with 18 U.S.C. §1734 solely to indicate this fact. methyl ester; PA, prostate-specific antigen. 3166 Downloaded by guest on September 30, 2021 Biochemistry: Watt et al. Proc. Nail. Acad. Sci. USA 83 (1986) 3167

I C~~~e~ Is a eC ed by dissolving the protein (5 mg/mi) in 70% (vol/vol) formic ii. d SI, GI. I.. b.1 C., Si. L.4 M. So, Si.Pahey Si, eta Laow letI AlafSor Are SI, re Ala Vel acid with the addition of solid CNBr (2-fold excess by weight) for 24 hr at room temperature. Hydroxylamine cleavage was Inleet PA carried out to Borustein and Balian (10). Digestion CC VI according -7 --7 7 --7-7 --7-7-7- -7 -7 -7 -7 --7 --7 of PA (5 mg/mi) with endoproteinase Mrg-C and endopro- Arg-C II a teinase Lys-C (Boehringer Mannheim) was performed in 0.1 k - Lye-C IV - Lye-C V O-e e--LoLv-C OX M ammonium bicarbonate containing 0.2 mg of enzyme per ml at 370C for 18 hr. All peptides were purified by HPLC on a Vydac C4 column using the F3CCOOH/CH3CN buffer Cys Sly Sly Val Lew Val MsI Pro Sin lrp Vei Lee Oh, Ala Ala Ms Cy [lie Ae Meo Ly, e Vtale Ie See system with a linear gradient from 100% buffer A to 80% Inlec PA (vol/vol) buffer B in 60 min. Amino acid analyses were CN VI ______performed using a Beckman 121MB amino acid analyzer after 24,48, and 72 hr ofacid hydrolysis (5.7 M HCl, 0.1% phenol). Lye-C IX *I Lye-C XCIb-. Automated sequence analyses were conducted on a Beckman 890C sequencer and Applied Biosystems (Foster City, CA) 470A gas-phase sequencer as reported (11). Comparison of Inlec PA the primary structure of PA with the kallikrein family of CC VI was carried out according to Doolittle (12) and Dayhoff (13). * as w e in La. Tyr Meo Plt Sot Loe Lee Lye PA, leg Phl Leu lee Pto Sly Msp Msp ot Sot CI" My Lee pin Lee See RESULTS Inlacl PA -e Amino Acid Composition. The amino acid composition of PA is tabulated in Table 1. Our results agree well with a previous report and with the theoretical composition derived - ~Lye-C Mill, Lye-C X

from the sequence reported here. 10511 It0 III 1CC Amino Acid Sequence. The complete amino acid sequence us of PA is given in Fig. 1. The sequence was primarily constructed through analysis of the reduced and alkylated chain, of fragments derived from partial endoproteolytic Ley-C X . Lye-C X11e

cleavages, CNBr fragmentation, endoproteinase Arg-C, and 10 1Sn 100 1le SW Lys-C cleavages. Amino-terminal sequence analysis with Oht Ohr Cys Tyr Ale Mt Sly ITy Sly Mt lIe Slie Ptro S SimGluM Lee Low Tyt Myp 1. OtO Lye Lye Lee o-phthalaldehyde blocking at proline residues (11) yielded the sequence of R1-R87. The chemical cleavage of residues and enzymatic digestion with endoproteinase Lys-C Lye-C Xllm VUL

gave the sequences of R1-R170, R174-R198, and R225- Ole ~~~~100 100 lie 15S R240. The sequence between residues 199 and 224 was Gin Cys Vol S Lee lIe Val lite Sot Ms. Myp tel Cye Ale SIn Cal CMe Pro 51. Lye Val Oht Lye sP* ito furnished by an additional chemical cleavage with cmIII hydroxylamine at the unique asparagine- sequence (R199-R200). The sequence of the tripeptide (R171-R173) Lye-C ViIl S&V Table 1. Amino acid composition of human prostate antigen Lee Cy% Ale Sty lee Tp NTh Sly Sly Lye Sot Oht Cys Sot Sty Myp See Sly Sly Po Lee Vel Lye Ms- Sly Residues per 240 residues total cC IV ______Amino acid Ref. 2* This From sequence reportt Lye-C VI old Lye-C XI Cys 7.8 9.9t 10 HY Asx 16.9 16.9 16 Thr 12.8 13.4 13 Ser 21.8 18.8 19 Vol Le. 61n Sly IIs Oht Sot ITep Sty Sot SI. Pt. Cye Ale Lee (Pro) Silm A- PtC So LeeUr lyt Oht Lye Vet Gix 25.1 22.8 23 Lye-C XI -a- Pro 16.6 16.0 16 4-4-4-4-4-4--4 -4 -4 -4 -4 -4-4-4)-4 -4-4 -4-4444A Gly 25.7 20.0 20 Ala 14.4 11.9 12 Val 20.4 21.2 22 I--S4-44*- - 234 4-4-4-104-44

Met 3.7 4.2 5 Cal I M Tyt Arg Lye ITy lb* Lye Myp Tht lie Val Ala Ms. Pto Ile 8.1 8.7 8 CCCIV -1 -7-777-7-7 -7 -7- - Leu 24.4 25.6 26 I Are-C

Tyr 4.1 4.7 5 '-Lye-C -4e Lye-C It 4 - Lye-C III Phe 6.7 6.4 5 Lys 10.7 12.0 12 ICY His 11.2 11.0 11 Trp, 2.4 5.5t 7 Arg 9.5 9.5 10 FIG. 1. Schematic presentation of the evidence for sequence Total 242.3 238.5 240 assembly of human PA. Peptides were generated by cleavage with CNBr, endoproteinases Mrg-C or Lys-C, or hydroxylamine (HY). *Recalculated from the original data on the basis of 240 residues. Arrows above the designated peptides indicate sequenced residues. tThe data represent averages of duplicate analyses of four different -,intact PA; ---, CNBr peptides; -, Arg-C peptides; ==, Lys-C PA preparations after 24, 48, and 72 hr of acid hydrolysis. peptides; --+, HY peptide; e-, 2 major internal proteolytic cleavages; tCysteine values were obtained as cysteic acid after performic acid and +-, carboxyl-terminal analysis by hydrazinolysis. The vertical oxidation. values were obtained after a 24-hr hydrolysis arrows indicate the three proteolytic cleavage sites contributing to with 5.7 M HCl/10% (vol/vol) mercaptoacetic acid. 10-20% of the total naturally occurring forms. Downloaded by guest on September 30, 2021 3168 Biochemistry: Watt et al. Proc. Natl. Acad. Sci. USA 83 (1986) was determined from the sequence analysis of the internal However, two minor sequences of Lys-Leu-Gln-Cys and fragment, during the sequencing of the intact molecule, as a Ser-Thr-Cys-Ser were also found. These minor sequences are result of the major but partial endoproteolytic cleavage consistent with cleavages in PA at lysine-148 between residue 148 and 149. The peptide Arg-CI overlapped and lysine-185, respectively. Some of the molecules con- the two carboxyl-terminal Lys-C peptides (Fig. 1). This tained a third cleavage following arginine-85 (Fig. 1). These sequence, together with the carboxyl-terminal proline resi- three cleavage sites were present in 10-20% of the total due obtained by hydrazinolysis of the intact molecule, naturally occurring forms; the remaining 80orepresented the showed that the primary structure of PA ends at residue 240. single chain of PA without any internal clips. There were Endoproteolytic Cleavages. Automated Edman degradation differences in the relative amounts of PA containing cleav- ofreduced and alkylated HPLC-purified PA always gave the ages at lysine-148, lysine-185, and arginine-85. In general, the amino-terminal tetrapeptide sequence of Ile-Val-Gly-Gly. pattern of cleavage was lysine-148 > lysine-185 > arginine- 85. Therefore, PA is a mixture of molecules containing one, two, three, or four chains attached covalently by 0.1 bridges that arise from one, two, or three endoproteolytic cleavages. Six out of a possible total of eight forms of PA, 0.10 resulting from incomplete cleavages at the three cleavage sites, have been identified and sequenced (Fig. 2). 0.08 Proteolytic Activity. Fig. 3 shows that PA was able to hydrolyze insulin A and B chain, gelatin, recombinant 0.06 interleukin 2, and, to a lesser extent, myoglobin, ovalbumin, and fibrinogen. The degree ofdigestion was estimated in each 0.04 HPLC chromatogram (Fig. 3) by the appearance of new peaks and the disappearance of the parent substrate after PA digestion. Only insulin B appeared to be completely digested. Hydrazinolysis performed with each PA/protein digestion Elution time, min mixture indicated that the new carboxyl-terminal residues after digestion were hydrophobic residues such as tyrosine, PEAK A' 00 ta)o leucine, valine, phenylalanine, and basic residues of lysine, sub- (2)t ~~~to co arginine, and histidine. Hence, PA possesses the same strate specificity as chymotrypsin and trypsin. (2) The hydrolytic activity of PA was also studied with 0.0 a e synthetic substrates in 0.1 M Tris HCl, 0.1 M CaCl2 (pH 8.0) at 25TC. PA exhibited a very low but detectable activity

PEAK A Insulin B Insulin A Gelatin Ala-IL-2

(2) i X1)

GD4 PEAKPEAK B: NO CLEAVAGE PA-treated PA-treated PA-treated 0 cm C>gY r->00D It PA-treated

co -co PEAK C C0 (1) * a Myoglobin Ovalbumin Fibrinogen (2)-- -1- (3)i

(4)i i- -|i

FIG. 2. Separation of naturally occurring forms of human PA as PA-treated PA-treated PA-treated a result of endoproteolytic cleavages. A very shallow organic gradient of 0.025% per min [30-37% (vol/vol) buffer B in 280 min] was used to elute the HPLC-purified PA sample on a Vydac C4 column. Four pools designated A', A, B, and C were obtained, lyophilized, and subjected separately to Edman degradation. The different PA forms were derived from the stepwise yield of phenyl- thiohydantoin amino acid derivatives in each pool. The bold arrow denotes a higher yield of cleavage, and the small arrow denotes a FIG. 3. Elution profiles ofvarious proteins and their PA digestion lower yield in each pool. Thus, peptide 1 in peaks A', B, and C; products on reverse phase HPLC. The top panels represent the peptide 2 in peak A'; peptide 3 in peak A' is peptide 1 in peak A and control experiments when the proteins were eluted on a Vydac C4 peptide 2 in peak C; peptide 4 in peak A'; peptide 2 in peak A is column with the same gradient used to separate the digestion peptide 4 in peak C; and peptide 3 in peak C represent six out of a products of these proteins after PA treatment. The extent of PA possible total of eight forms of PA resulting from three endoproteo- digestions was highlighted by the appearance of newly generated lytic sites in the PA molecule held together by internal disulfide peptide peaks shown in the bottom panels. A linear gradient of linkages. 0-80%o (vol/vol) buffer B in 60 min was used. Downloaded by guest on September 30, 2021 Biochemistry: Watt et al. Proc. Nati. Acad. Sci. USA 83 (1986) 3169 toward Bz-Tyr-OEt, N-Bz-Arg-NHNpt, N-Suc-Phe-NA, and that found for other N-linked glycoproteins. Serine-69, threo- Tos-Arg-OMe. The enzyme hydrolyzed the synthetic nine-70, and serine-71 are possible sites for 0-linked carbohy- chymotrypsin and trypsin p-nitroanilide ester substrates, drate side chains, because these three residues (69-71) gave namely, N-Suc-Ala-Ala-Pro-Phe-p-NA and N-Bz-Arg-p-NA, significantly lower yields from Edman degradation when com- at a rate of 9.21 ttM per min per mg and 1.98 14M per min per pared to that of serine-80, which appears later in the sequence. mg, respectively. A Km value of 15.3 mM and a kcat of 0.075 Comparisons of PA amino acid sequence with the published were obtained with N-Suc-Ala-Ala-Pro-Phe-p-NA as the sequences of serine proteases show a very strong homology substrate. The chymotrypsin type of activity of PA toward between PA and the various in the kallikrein family this substrate was about 200 times less than that of (Fig. 4). The homology is observed throughout the entire chymotrypsin (as measured by kcat). The kcat/Km values for molecule, suggesting that their three-dimensional structures are chymotrypsin and PA were 2.18 x l01 M'1S' and 4.88 very similar. The sequence identity ofPA with kallikrein is 57% MiS-1, respectively. Protease inhibitors such as PhMe- and with y-,nerve growth factor it is 56%. Less homology was SO2F, DFP, Tos-PheCH2Cl, aprotinin, leupeptin, soybean seen with other serine proteases, including tonin (54%), epider- trypsin inhibitor, as well as Zn2+ and spermidine when mal growth factor-binding protein (53%Y), a-nerve growth factor preincubated with PA for 16 hr at 250C, were found to inhibit (51%), trypsin (42%Y), chymotrypsin (35%), and Streptomyces the enzymatic activity of PA. At 2 mM concentrations, griseus protease B (26%). The extensive homologies suggest PhMeSO2F and DFP gave an average of90% inhibition of PA that these proteases are descendants of a common ancestral activity with N-Suc-Ala-Ala-Pro-Phe-p-NA, whereas the gene. same concentration of Zn2+ completely abolished this chy- Ten haif-cystine residues are located in PA and occupy motrypsin type of activity of PA. homologous positions to those of other serine proteases. Based on the known disuflfide arrangements for trypsin, DISCUSSION chymotrypsin, and y-nerve growth factor, it is reasonable to We have determined the complete amino acid sequence of assume a similar geometry for the five disulfide bonds of PA human PA. PA is a single polypeptide chain of 240 amino acids as a subset of the six disuflfides present in bovine trypsin. with a calculated Mr of 26,4% for the peptide portion. A Thus, cysteine-7 is linked to cysteine-152 (corresponding to potential polysaccharide side chain has been located on residues 13 and 143 in trypsin); cysteine-26 to cysteine-42 asparagine-45 with a sequence of Asn-Lys-Ser consistent with (residues 31 to 47 in trypsin), which forms the histidine loop

IN Is a 3 3 W V C CE ESN S AQ P V L A T C 6 V L P AQ AS V A I S IS F C V L P L CA Kgli kirv in I Il a CC E D0 A66 AN Bovine Trypsi IIV T CC CA N T AVP S L BNS ClAN F CD66SL I N S AYWVS CAA boAine CA I 6lE C AY V Toni, I CCC P SV P S L C S L I N E N A A P NC C V I T Q E0 C I P S TGl L C C EC N S P V L V V L T D CE V L A EEAACAS P AlT A T CC A L C CAY L ABAP BY CAN AQ AW VA CA BYT TBQAV AT A66C6 AL S0 B SMSB N S P BEA C Y6 L 0L tA AD sN Ss sN SO PA i5 so CII NB.....A..... L L ANAR S L PANP CD0T AC A P P L D Kailikrein A C C NONY C AWALA A N L F C NCE N T A CF F V P P BF L S Bovine Trypsin A C YA C 551 A N LAG A N I N A A CA6N A AF I SA DCK I Av " p S N Bovine CA A C G VAT T SOYAAV A C C P A A A S S S C C A A KCL C I A C A N S EK V S L Tovin A JAY SA N T AVL L C A NAL F C ACE PP A A A ALAV A S PBSN P CUF-NP A CY A D T YC ALE C N C L PA C C P S A A NR LAV S CS P HSP S f -NAP A C ADD N YCK AWALA6 CNN L F C ACE PS A HAN F A SCK A I P AN P A6 F S a-NAP A CAN D C Y VAW LA6 CNN PLC DC0 P SA A R LADES A I PPH P F N N S

A -

SB AN 35* IDA IDA N R F A E E PA L LENA ADDS A I DNAL BLSPA C 5DB0 2 L P TAQ FLAP 0 S L L L A LTD A Ca IIhro In LA0 QSEP L P TAQ L L S A A S Nolvie Trypsin T LAN NFlA AC ADAi IINIAL IE C CATLBNS A A SI S L P A S I N L LA S A Bovine CA T LAK PT DCSQ AS A C L P SCA TvoYi DAB P ILI NILL A L S EP A DA1TA6 I D0 L P ACK CAP.BP L INN TT P P CAD P L CLL A A TOY LB1 LAQ LIL]TFS DIALNLNO0 LAN LAR STSEKP A 0lSPSQT I L P ACK v-NAP L A BFL CA BAD NO L L LA: L SE 00 IT P I T L P T C v-NAP CA C A LB NT PAQP DOTLUS ND LA L LA L SEKP A SITS K P I T L P A C

525 105 035 DAN4 ISON 060 PA C P A LAG TACT A DABS TI ICE P EE A LLY DANCEHK LA S N Ka~li~rein C PCE LAGST C C ADS6 SEI I BC PA6P OB9 PEP P DCEI Q C L L A N Bovine Trypsin C A SOC TAQ C L ISA B ANIT A SS A T SAVP DAVLEK C LEA P I L S N Bovine CA S DDPAA A T T C A A TAG SGCLI BAYT BOA T PD0 ALA A S L P L L O N Tonin C PEAS S T CLOS A CI6GSIT B PS ASNAD$N LA C AV AL L S N CAP-NP C PEKP A SA C LA A BA6W LI T P T C ODD50 LA L L PBN ,-NAP C PCK LAGS T C LA A C6W6SS I A PT QFTP D D L A 0L EK L L PBN v-NAP C PCK LA6ST C L AS A BEST TAP I AYPSO LA C AVN L KEL L PBN

165 PA D A C V T L C A K S AC S C6 P L] C N C6 Calikirein T F C A BA P B VT S ANL C CATG Y K D T C D L I C N C6 Novnlie Trypsin S K S A P CK S C IA6 S YIP C S C6 A Bovine CA T C ATE A SN I C A A S S S C D A6 P C EK NG Tonin C KCC T O0 G L CC A E H 0 T C A 6 Dj P L I C CUF-NP C N C V T S0 AL C OA CAE T C A6 0 S P L I C AC6 v-NAP C A C A OA lN CE T OA AL C OA CAE TAC AD S P L I C D C6 v-NAP C S C K A CK AD A AL C A AM 6 S AT C A" D 6 P L I C

t t Pt

PA A 230DO 236 L I TS S P C A L (P) P 5 L AT ESRK I T A N P N AV CaiAkrein 6 I S CANT P C CS A P SI AT L I F L ANW IN BAI T CANP C L I S Novine Trypsin CG S AC OA K N EK P AV C T S N I T I CA S N Bovinve CA ACT .L CI S BW SS T C S P T A AV A L AVN CATL AA Tovi, LAS I T S SOA TP C ACK P EKT PA DI A L I S SEV A: P CGU-NP LA0 T S N P CE P C P P A DI L I SN I AD T N CA i-NA A L I T S NM P C P D P EK L N EK F S I T OA P v-NAP I L I T ,S P CE P C P P S TA7 EL I PS S I ABT A N P

FIG. 4. Comparison of amino acid sequence of PA with other serine proteases including kallikrein (14), trypsin (15), chymotrypsin (15), tonin (16), epidermal growth factor-binding protein (17), y and a subunit of 7 S nerve growth factor (18, 19). Gaps have been placed to maximize the homology. Heavily boxed areas contain residues neighboring the vicinity of the active sites of all serine proteases and are highly homologous. Areas that are lightly boxed indicate regions believed to line the substrate binding pocket (20). Residues underlined, numbering 45 and 69-71 of PA, represent potential attachment sites for carbohydrate. Columns of residues underlined are known to be internal and inaccessible to water molecules in the three-dimensional structures of a-chymotrypsin, trypsin, and (21). *Active site residues in PA, histidine-41, aspartic acid-%6, and serine-192 are the components of the "" of serine proteases (22). tlnvariant residues conserved in all known serine proteases including human (23) and bovine prothrombin (23). tAspartic acid and serine residues at position 186 (numbering according to PA) determine the substrate specificity of trypsin and chymotrypsin, respectively. Downloaded by guest on September 30, 2021 3170 Biochemistry: Watt et al. Proc. Natl. Acad. Sci. USA 83 (1986) responsible for proper orientation of histidine residue in the proteases. Two of the cleavage sites in PA, namely, following active site; cysteine-128 to cysteine-198 (residues 122 to 189 lysine-148 and arginine-85, occupy homologous positions to in trypsin); cysteine-163 to cysteine-177 (residues 154 to 168 those of -y-nerve growth factor (18) and are situated on opposite in trypsin), which forms the methionine loop; and cysteine- sides of the substrate region between different 188 to cysteine-213 (residues 179 to 203 in trypsin), which P-strands of the molecule. forms the serine loop linking together the primary and The question still remains regarding the in vivo substrate of secondary binding sites (Fig. 2). PA. Since the initial report in 1962 by Huggins and Neal (28) on PA has been demonstrated to exhibit proteolytic activity the presence of "fibrinolytic" activity in human and canine similar to that of chymotrypsin and trypsin, and this enzy- prostatic fluid, various proteolytic enzymes of human seminal matic activity has been shown not to be due to contaminating plasma have been characterized, including plasminogen activa- enzymes (8). Thus, PA possesses similar proteolytic proper- tors, a neutral protease (seminin), and a type of enzyme ties as those of C1 esterase (24), tonin (16), and Streptomyces (29). However, the physiological substrates and functions of griseus protease B (25). Furthermore, the inactivation of PA these enzymes have not been determined. Whether the physi- by active-site directed reagents of serine proteases, such as ological role of PA is connected to liquefaction of seminal clot PhMeSO2F and DFP, implies that PA is a serine protease or to the fibrinolytic activity in semen, acts as a kininogenase with a catalytic apparatus similar to that of other serine (30), or is involved in the proteolytic processing of biologically proteases. The active site serine residue, serine-192, and the active polypeptides remains to be studied. structurally adjacent histidine-41 and asparagine-96 residues Note. Since the submission of this article, Lilja (31) reported that the ofthe charge-relay system are conserved as in all other serine structural protein ofhuman seminal coagulum, the predominant seminal proteases. The serine residue at position 186, which is located vesicle secreted protein, might be the physiological substrate for PA. six residues before the active site serine-192, is in a homol- ogous position to serine-189 in chymotrypsin that is believed We are greatly indebted to Dr. Russell F. Doolittle for his to determine the substrate specificity for certain hydrophobic assistance in the homology search. We also thank Dr. L. L. Houston residues. The presence of a serine residue at this position, for critical review of the manuscript and Dr. W. Bloch for valuable instead of aspartic acid residue as in trypsin, helps to explain discussions. the characteristics of PA that are similar to chymotrypsin. 1. Wang, M. C., Valenzuela, L. A., Murphy, G. P. & Chu, T. M. (1979) The additional ability of PA to hydrolyze bonds on the Invest. Urol. 17, 159-163. carboxyl side of basic residues 2. Wang, M. C., Loor, R. M., Li, S. L. & Chu, T. M. (1983) IRCS Med. may depend on the partici- Sci. 11, 327-328. pation of carboxyl groups of residues in the 3. Wang, M. C., Papsidero, L. D., Kuriyama, M., Valenzuela, L. A., binding of the substrate (12). Some possible candidates near Murphy, G. P. & Chu, T. M. (1981) The Prostate 2, 89-96. the active site, in a position that could interact with the 4. Kuriyama, M., Wang, M. C., Lee, C. L., Killian, C. S., Papsidero, positive charges of the substrate, are glutamic acid-137, L. D., Inaji, H., Loor, R. M., Lin, M. F., Nishiura, T., Slack, N. H., -139, Murphy, G. P. & Chu, T. M. (1982) J. Natl. Cancer Inst. 68, 99-105. and -140. In addition, glutamic acid-211, which is situated in 5. Kuriyama, M., Wang, M. C., Lee, C. L., Papsidero, L. D., Killian, the secondary binding site of PA, might also be involved. C. S., Inaji, H., Slack, N. H., Nishiura, T., Murphy, G. P. & Chu, All but 1 of the 29 "invariant" amino acid residues of the T. M. (1981) Cancer Res. 41, 3874-3876. serine proteases (15) are conserved in the PA 6. Sensabaugh, G. F. (1978) J. Forensic Sci. 23, 105-115. sequence. 7. Graves, H. C. B., Sensabaugh, G. F. & Blake, R. T. (1985) N. Engl. J. Leucine-155 and proline-161 in chymotrypsin are situated at the Med. 312, 338-343. beginning and end of a conserved 8-pleated stretch. In the 8. Ban, Y., Wang, M. C., Watt, K. W. K., Loor, R. & Chu, T. M. (1984) corresponding positions in PA, leucine also occupies position Biochem. Biophys. Res. Commun. 123, 482-488. 150, but proline is substituted by histidine at position 156. This 9. Schroeder, W. A. (1972) Methods Enzymol. 25, 138-143. 10. Bornstein, P. & Balian, G. (1977) Methods Enzymol. 47, 132-145. may alter the secondary structure in this portion of the PA 11. Wintroub, B. U., Klickstein, L. B., Dzau, V. J. & Watt, K. W. K. molecule. Interestingly, histidine residue is also located at this (1984) Biochemistry 23, 227-232. position in tonin (16). Identical sequences to those of other 12. Doolittle, R. F. (1981) Science 214, 149-159. serine proteases are also retained in the 13. Dayhoff, M. 0. (1978) Atlas of Protein Sequence and Structure (Natl. two regions for the Biomed. Res. Found., Washington, DC), Vol. 5, Suppls. 1-3. substrate-binding site of PA, including the primary binding site 14. Tschesche, H., Mair, G., Godec, G., Fiedler, F., Ehret, W. & at residues 190-195 (Gly-Asp-Ser-Gly-Gly-Pro) and the second- Hirschauer, C. (1979) Adv. Exp. Med. Biol. 120, 245-260. ary binding site corresponding to residues 207-209 (Ser-Trp- 15. Dayhoff, M. 0. (1978) Atlas of Protein Sequence and Structure (Natl. Gly). Biomed. Res. Found., Washington, DC), Vol. 5, Suppl. 3, pp. 79-81. 16. Lazune, C., Leduc, R., Seidah, N. G., Thibault, G., Genest, J. & In eukaryotic multicellular organisms, the serine proteases Chretien, M. (1984) Nature (London) 307, 555-558. are synthesized as single chain precursors, and subsequent 17. Lundgren, S., Ronne, H., Rask, L. & Peterson, P. A. (1984) J. Biol. activation involves proteolytic cleavage of the peptide bond Chem. 259, 7780-7784. 18. Thomas, K. A. & Bradshaw, R. A. (1980) Methods Enzymol. 80, 609-620. preceding the residue at position 16. The a-amino group of 19. Isackson, P. J. & Bradshaw, R. A. (1984) J. Biol. Chem. 259,5380-5383. residue 16 forms a salt bridge following activation with the 20. Mason, A. J., Evans, B. A., Cox, D. R., Shine, J. & Richards, R. I. side chain of aspartic acid residue next to the active center (1983) Nature (London) 303, 300-307. 21. Stroud, R. M., Kay, L. M. & Dickerson, R. E. (1971) Cold Spring serine (26). In PA, the preservation of the active-site resi- Harbor Symp. Quant. Biol. 36, 125-140. dues, histidine-41, aspartic acid-96, and serine-192, as well as 22. Blow, D. M., Birktoft, J. J. & Hartley, B. S. (1969) Nature (London) the amino-terminal hydrophobic sequence Ile-Val-Gly-Gly, 221, 337-340. 23. Mole, J. E., Anderson, J. K., Davison, E. A. & Woods, D. E. (1984) J. lends strong support to the notion that PA is also synthesized Biol. Chem. 259, 3407-3412. as a single polypeptide precursor. Furthermore, the invariant 24. Sim, R. B. (1981) Methods Enzymol. 80, 26-42. sequence of Gly-Trp-Gly found in most serine proteases 25. Delbaere, L. T. J., Hutcheon, W. L. B., James, M. N. G. & Thiessen, (residues 140-142 in chymotrypsin) also occurs in W. E. (1975) Nature (London) 257, 758-763. homolo- 26. Stach, R. W., Pignatti, P. F., Baker, M. E. & Shooter, E. M. (1980) J. gous positions in PA (residues 132-134). This sequence is Neurochem. 34, 850-855. found to lie at the beginning of the autolysis loop in chymo- 27. Fehlhammer, H., Bode, W. & Huber, R. (1977)J. Mol. Biol. 111,415-438. trypsin and is believed to serve a critical function in the 28. Huggins, C. & Neal, W. (1942) J. Exp. Med. 76, 527-541. 29. Zaneveld, L. J. D., Polakoski, K. L. & Schumacher, G. F. (1975) in conversion of a flexible structure to a fixed rigid Proteases andBiological Control, eds. Reich, E., Rifkin, D. B. & Shaw, orientation in the activated protease (27). E. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. Analysis of the amino-terminal sequence of purified PA has 683-706. 30. Geiger, R. & Glausnitzer, B. (1981) Hoppe-Seyler's Z. Physiol. Chem. defined the location of three endoproteolytic cleavages. These 362, 1279-1283. cleavages may be autocatalytic like those of other serine 31. Lilja, H. (1985) J. Clin. Invest. 76, 1899-1903. Downloaded by guest on September 30, 2021

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