Proc. Nati. Acad. Sci. USA Vol. 83, pp. 3166-3170, May 1986 Biochemistry Human prostate-specific antigen: Structural and functional similarity with serine proteases (primary structure/sequence homology/seminal protease/chymotrypsin-like and trypsin-like activity) KENNETH W. K. WATT*, PAUL-JANE LEE*, THABISO M'TIMKULUt, WAI-PAN CHAN*, AND RUEYMING LOORt Departments of *Protein Chemistry and tDiagnostics, Cetus Corp., 1400 Fifty-Third Street, Emeryville, CA 94608 Communicated by Russell F. Doolittle, December 31, 1985 ABSTRACT The complete amino acid sequence of the MATERIALS AND METHODS prostate-specific antigen (PA) from human seminal plasma has PA was purified from human seminal plasma as described (1). been determined from analyses of the peptides 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 proteins including insulin A, insulin B, gelatin, serine proteases ofthe kallikrein family. The active site residues myoglobin, ovalbumin, fibrinogen (Sigma), and recombinant of histidine, aspartic acid, 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 (enzyme:substrate) of1:20 was used. After an 18-hr interleukin 2, and-to a lesser extent-gelatin, myoglobin, digestion 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 peptide bond specificity of PA, hydrazinolysis hydrophobic residues, tyrosine, leucine, valine, and phenylal- was performed on each ofthe PA/substrate digestion mixtures, anine, and of basic residues histidine, lysine, and arginine. 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-phenylalanine 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, aprotinin, leupeptin, soybean trypsin inhibitor as well (Sigma), and Na-succinyl-L-alanyl-L-alanyl-L-prolyl-Lphenyl- as Zn2+ and spermidine were effective inhibitors of PA alanine-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 serine protease 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 methionine 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.