Primary Structure of Human Urinary Prokallikrein1 Saori Takahashi

J. Biochem. 104, 22-29 (1988) Primary Structure of Human Urinary Prokallikrein1 Saori Takahashi,* Akiko Irie,** and Yoshihiro Miyake*, 1*Department of Biochemistry , National Cardiovascular Center Research Institute and **Clinical Laboratory, National Cardiovascular Center Hospital, Suita, Osaka 565 Received for publication, January 26, 1988 The complete amino acid sequence of human urinary prokallikrein has been determined by amino acid analysis and sequence determination of peptide fragments obtained from chemical and enzymological cleavages of kallikrein and by comparison of the N-terminal sequence of prokallikrein with that of kallikrein, the active form. Prokallikrein was a single chain polypeptide which comprised 238 amino acid residues of kallikrein and 7 amino acid residues of the propeptide. The sequence, Asn-X-Thr(Ser), which is a common glycosylation site was found at positions 78-80, 84-86, and 141-143. Two trypsin-suscep tible sites were identified. One is the Arg(-1)-Ile(1) bond and the other is the Arg (87)-Gln(88) bond. The sequence of human urinary kallikrein was identical with that of human pancreatic and kidney kallikreins (Fukushima, D. et al. (1985) Biochemistry 24, 8037-8043; Baker, A.R. & Shine, J. (1985) DNA 4,445-459), which were predicted from the nucleotide sequences of cDNAs. The primary structure of human urinary kallikrein is homologous to those of the other animal kallikreins and kallikrein-related proteins. Key amino acid residues, His(41), Asp(96), and Ser(190), required for catalytic activity and Asp (184) required for kallikrein-type specificity are completely conserved. The results show that human urinary prokallikrein and kallikrein are of tissue type and they are excreted in urine without any modification. Tissue-type kallikreins represent a group of closely related from human urine and characterized some of their prop serine proteases which liberate lysyl-bradikinin, a vaso erties (18). In addition, it has been shown from the active decapeptide, from kininogens. These enzymes are N-terminal amino acid sequences of the two proteins that found in many mammalian tissues (1-4). Recently, the the inactive form is a proenzyme, prokallikrein, having an primary structures of tissue kallikreins have been predict- additional seven amino acids at the amino terminus of ed from molecular cloning and sequence analysis of cDNAs kallikrein (19). However, the complete amino acid se encoding mouse submaxillary gland kallikrein (5), rat and quence of human urinary prokallikrein has not been human pancreatic kallikreins (6, 7), and human kidney determined. kallikrein (8). In the mouse, these enzymes are encoded by In the present study, we have determined the complete a closely linked multigene family on chromosome 7 (9). amino acid sequence of human urinary prokallikrein, and The urinary kallikrein is particularly interesting from the sequence was compared with those of kallikreins of both physiological and clinical aspects, since it may partici other mammalian species and kallikrein-like proteins. On pate in the regulation of water and electrolyte balance (10), the basis of the present results and our earlier data (18-20) and the amount of total kallikrein and the ratio of active- we identified trypsin-susceptible sites in human urinary to-total kallikrein in urine changed with essential hyperten prokallikrein, and proposed an activation mechanism of tion and high-renin Bartter's syndrome (11-13). Human prokallikrein by trypsin as a model for the physiological urinary kallikrein is a single-chain polypeptide and an activation. acidic glycoprotein, and the partial amino acid sequence was reported (14, 15). Moreover, the existence of an MATERIALS AND METHODS inactive form of kallikrein in human urine has been demonstrated (16, 17). However, the physiological sig Materials-Human urinary kallikrein and prokallikrein nificance and the structural relationship between the two were purified by the method of Irie et al. (18). The purified forms of kallikrein remained to be settled. Recently, we preparations were homogeneous as judged by poly have purified both active and inactive forms of kallikrein acrylamide gel electrophoresis at pH 8.0 and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Lysyl endopep 1 Preliminary results have been presented at the 5th International tidase from Achromobacter lyticus was purchased from Congress on Kinin held in Tokyo, Nov. 29-Dec. 3, 1987. This work Wako Pure Chemical Co., Tokyo, TPCK-treated bovine was supported in part by Grants-in-Aid for Special Project Research pancreatic trypsin from Cooper Biomedical, bovine pancre (No. 62122005) and for Scientific Research (No. 61570151) from the atic chymotrypsin from Sigma, and a Pep RPC HR 5/5 Ministry of Education, Science and Culture of Japan. column (5•~50mm, 100A) for high-performance liquid 2 To whom correspondence should be addressed . chromatography (HPLC) from Pharmacia Fine Chemicals Abbreviations: HPLC, high-performance liquid chromatography; . PTH, phenylthiohydantoin; NGF, nerve growth factor; EGF, epider All other reagents were of guaranteed grade. mal growth factor. Reduction and Carboxymethylation•\Human urinary 22 J. Biochem. Human Urinary Prokallikrein; Complete Amino Acid Sequence 23 kallikrein and prokallikrein were reduced and carboxy quenator (Applied Biosystems, Model 430A). The obtained methylated by the method of Lottspeich et al. (15). The PTH-amino acid were identified by a PTH-analyzer (Ap- preparations (500ƒÊg) were lyophilized and then dissolved plied Biosystems, Model 120A PTH analyzer) directly in 500,u 1 of 6 M guanidine hydrochloride containing 0.2M connected to the sequenator. All peptide solutions were acetic acid, 0.1M Tris, and 5mM EDTA, pH 4.7. The made in 25% trifluoroacetic acid containing 0.1% SDS solution was flushed with nitrogen gas, and 25 pl of 2- before being applied to the sequenator. mercaptoethanol was added. The solution was incubated for Amino Acid Analysis S-Carboxymethylated proteins 20 h at 40•Ž, and then 500,u 1 of 2M Tris-HCl, pH 9.0, and peptides were hydrolyzed under HCl vapor at 110•Ž for containing 60 mg of iodoacetate and 6M guanidine hy 20 h. The hydrolysates were analyzed by the phenylthio- drochloride was added. The reaction mixture was incubated carbamyl method with the Waters Pico-Tag system (21). for 15 min at room temperature. The solution was acidified to about pH 3.0 with 50% formic acid, then immediately RESULTS applied to a Sephadex G-15 column (1 x 50 cm) previously equilibrated with 50 % acetic acid. The protein fractions Amino Acid Compositions of Human Urinary Kallikrein were collected and lyophilized. and Prokallikrein-Table I shows the amino acid composi- Lysyl Endopeptidase Digestion•\S-Carboxymethylated tions of S-carboxymethylated human urinary kallikrein kallikrein (490ƒÊg) was dissolved in 300 pl of 50mM and prokallikrein. Both proteins showed a very similar Tris-HCl, pH 9.0, and then 5 pl of lysyl endopeptidase amino acid composition. However, amino acids of the solution (10 units/ml of distilled water) was added. The propeptide could not be distinguished under the conditions solution was incubated for 2 hat 37•Ž and then 20 hat room employed. The amino acid composition of kallikrein was temperature. The enzyme reaction was terminated by the also found to be very similar to that reported earlier (15). addition of 200 pl of 1% trifluoroacetic acid. Sequencing Strategy-The sequencing strategy for hu- Chymotryptic Digestion of Peptide L9•\Chymotrypsin man urinary kallikrein is shown in Fig. 1. Two sets of was used for fragmentation of peptide L9, a peptide from fragments of S-carboxymethylated kallikrein were pre- the lysyl endopeptidase-digested sample. The peptide (5 pared. One is lysyl endopeptidase-digested fragments and the nmol) was dissolved in 100 pl of 0.1M Tris-HCl, pH 7.4, other is cyanogen bromide-cleaved fragments. Subfrag- and digested with 0.5ƒÊg of chymotrypsin at 37•Ž for 4 h. ments of some peptides from lysyl endopeptidase digestion The resulting peptides were purified by reversed-phase and cyanogen bromide cleavage were obtained by digestion HPLC. with trypsin or chymotrypsin. Tryptic Digestion of Peptide L10•\Peptide L10, a pep- Amino Acid Sequences of Peptides from Lysyl Endopep- tide from the lysyl endopeptidase-digested sample, was tidase Digestion-Peptides from lysyl endopeptidase diges- further digested with trypsin. The peptide (4nmol) was tion of S-carboxymethylated kallikrein were purified by dissolved in 300ƒÊl of 50mM Tris-HCl, pH 8.0 and then 5 reversed-phase HPLC. The elution profile is shown in Fig. ls. With small fractions (Ll, L2, L3, L4, L5, L6, and L7), pl of trypsin (0.1mg/ml) was added to the solution. The the amino acid sequences were directly determined by reaction was carried out at 25•Ž overnight. Chemical Cleavage with Cyanogen Bromide-For the automated Edman degradation and the amino acid composi- cleavage of methionine residues, 450ƒÊg of S-carboxy tion of each peptide was analyzed. Amino acid analysis and methylated sample was dissolved in 300ƒÊl of 50% formic acid, and then 2mg of cyanogen bromide was added. The reaction was allowed to proceed for 20 h at 37•Ž. The TABLE I. Amino acid compositions of human urinary kallikrein and prokallikrein.' reaction mixture was diluted 5-fold with distilled water and lyophilized. The lyophilized material was dissolved in 500 p 1 of 50% acetic acid for chromatography. Chymotryptic Digestion of Cyanogen Bromide-Cleaved Peptide-Proteolytic digestion of cyanogen bromide- cleaved peptides (CB3, CB5, and CB6) with chymotrypsin was carried out in 0.1M Tris-HCl, pH7.5, at 37•Ž for 4 h. The molar ratio of chymotrypsin to peptide was about 1 to 200 Purification of Peptides by Reversed-Phase HPLC-Pep tides from cyanogen bromide cleavage and protease diges tion of S-carboxymethylated kallikrein were separated by reversed-phase HPLC immediately after digestion. HPLC was carried out on the fast protein liquid chromatography (FPLC) system on a reversed-phase column of Pep RPC HR5/5.

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