Proc. Natl. Acad. Sci. USA Vol. 79, pp. 4858-4862, August 1982 Biochemistry

Amino acid sequence of mouse submaxillary gland (homology with /) KUNIO S. MISONO, JIN-JYI CHANG, AND TADASHI INAGAMI Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Communicated by Grant W. Liddle, April 30, 1982 ABSTRACT The complete amino acid sequences ofthe heavy experimental details ofthe sequence determination will be pub- chain and light chain ofmouse submaxillary gland renin have been lished elsewhere. determined. The heavy chain consists of 288 amino acid residues having a Mr of 31,036 calculated from the sequence. The light chain contains 48 amino acid residues with a Mr of 5,458. The se- MATERIALS AND METHODS quence of the heavy chain was determined by automated Edman Materials. Renin was purified from the submaxillary gland degradations of the cyanogen bromide peptides and tryptic pep- of adult male Swiss-Webster mouse and the heavy chain and tides generated after citraconylation, as well as other peptides light chain of renin were isolated as described (7, 8). generated therefrom. The sequence ofthe light chain was derived Separation of Peptide Fragments of the Heavy Chain. Two from sequence analyses of the peptides generated by cyanogen sets ofpeptide fragments were generated from the bromide-cleavage or by digestion with Staphylococcus aureus pro- heavy chain tease. The sequences in the active site regions in renin containing by CNBr cleavage and by tryptic digestion of citraconylated two catalytically essential aspartyl residues 32 and 215 were found heavy chain (Fig. 1). The five methionyl bonds were cleaved identical with those in pepsin, , and penicillopepsin. by CNBr, yielding six primary fragments, CB1 through CB6. Comparison ofthe amino acid sequence of renin with that ofpor- In addition, an acid-labile Asp-Pro bond at residue 171-172 was cine pepsin indicated a 42% sequence identity of the heavy chain partially cleaved during CNBr treatment in 70% formic acid, with the amino-terminal and middle regions and a 46% identity generating subfragments ofCB4 (CB4-1 and CB4-2). The CNBr ofthe light chain with the carboxyl-terminal region ofthe porcine fragments were separated into six peaks by gel filtration on a pepsin sequence. Residues identical in renin and pepsin are dis- column of Sephadex G-50 in 50% acetic acid. The material un- tributed throughout the length ofthe molecules, suggesting a sim- der the breakthrough (first) peak was citraconylated and re- ilarity in their overall structures. chromatographed on a column ofSephadex G-75 in 50 mM am- monium bicarbonate buffer (pH 9.3). A peptide spanning CB5 The formation of the peptide angiotensin I from the macromo- and CB6 regions (CB5-CB6) generated due to incomplete lecular prohormone angiotensinogen is catalyzed in a highly cleavage at the Met-Gln linkage (232-233) was obtained in a specific manner by the renin by both the extracellular peak eluting immediately after the breakthrough peak. and intracellular (1) mechanisms. Because it is the first and the The second peak from the Sephadex G-50 column contained rate-limiting step in the series of reactions leading to the for- CB1 and CB4. These were separated by ion-exchange chro- mation of angiotensin II in the plasma and release of aldoste- matography on a column of QAE-Sephadex A-25 eluted with rone, it is considered to play a central role in blood pressure a NaCl gradient in 50 mM Tris-HC1 buffer (pH 9.0) containing regulation. Its intracellular function may be considered as a 8 M urea. Peptides CB2, CB6, and CB4-1 that eluted together model of peptide hormone formation in endocrine cells. Al- in the third peak from the Sephadex G-50 column were sepa- though the active site ofrenin shares properties closely resem- rated by HPLC on a SynChropak RP-P column (SynChrom) in bling those ofacid (2, 3), renin is unique in that it has 0.1% trifluoroacetic acid eluted with a linear gradient of ace- little, ifany, general activity and that it does not func- tonitrile (0-70% in 60 min). Smaller peptides CB5, CB4-2, and tion in the acidic pH range. The structural basis for these unique CB3 were eluted from the Sephadex G-50 column as the fourth, properties ofrenin is unknown. The extremely limited quantity fifth, and sixth peaks, respectively. of renin in the kidney and lengthy procedures required for its Subfragments of peptide CB1 were generated by digestion purification prevented the determination of its structure. The at 37C with Staphylococcus aureus protease (Miles) or with submaxillary gland of mouse has been known to contain a rel- Lysobacter enzymogenes endoproteinase Lys-C (Boehringer atively large amount ofrenin. Although the physiological func- Mannheim). Digestion with S. aureus protease was done in 0.1 tion ofsubmaxillary gland renin is not entirely understood, this M ammonium bicarbonate buffer (pH 7.8) for2hrat an enzyme/ enzyme is known to have catalytic and antigenic properties substrate ratio of 1:30. In addition to the two glutamyl bonds closely resembling renal (4, 5, 6). We have developed in the peptide CB1, the aspartyl bond at residue 32 was also a rapid large-scale purification method for renin from this gland cleaved. Digestion with Lys-C was carried out in 0.1 M that allows preparations ofthe enzyme in 100-mg quantities (7). Tris-HCl buffer (pH 7.6) containing 3 M urea for 4 hr at an en- Large amounts of renin obtained by this method have allowed zyme/substrate ratio of1:100. Peptides were isolated by HPLC detailed characterization of this enzyme. as described above. In the present communication, we present the complete Cleavage at the five arginyl bonds of the heavy chain was amino acid sequence of mouse submaxillary gland renin and achieved by tryptic digestion ofthe citraconylated heavy chain, compare it with that of an acid protease porcine pepsin. The yielding six fragments CT1 through CT6. Gel filtration of the tryptic digest on a column of Sephadex G-50 in 0.05 M am- The publication costs ofthis article were defrayed in part by page charge monium bicarbonate buffer (pH 9.3) gave six major peaks. Re- payment. This article must therefore be hereby marked "advertise- chromatography of the material under the first breakthrough ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. peak on a column ofSephadex G-75 yielded pure CT4. The sec- 4858 Biochemistry: Misono et aL Proc. NatL Acad. Sci. USA 79 (1982) 4859 HEAY CHAIN LIGHT CHAIN

RESIDUE NUMBER. 100 200 20, 40. 1

CB CB2B-1EwQ&QQQu& C8 C85-CB6 SPI(CBfl SP3(CBI) C84-2 LSPI ILSE? SP(CB-I) iomm LNM\i SP I RXYSM SP2 (CBI) KC-O l l1 KC2(CBI)

CTI CT3 CT5 Cmmmmm==lQ&XCT2 mmom CT4 1 CT6 CT sMM=\\\ I ,mgsr\\~eR~ I KmM

INTACT INTACT , CMN

FIG. 1. Schematic representation of peptide fiagments generated for the sequence analysis of mouse submaxillary gland renin. The hatched section of each bar indicates the portion of the sequence determined. Peptides generated by cleavage with CNBr are designated with the prefix CB; by tryptic digestion of the citraconylated protein, CT; by digestion with Staphylococcus aureus protease, SP; and with Lysobacter enzymogenes en- doproteinase Lys-C, KC. Peptides derivedfrom the light chain are preceded with the letter L. Peptides generatedby secondary cleavages are followed by the name of the parental peptide in parentheses.

ond and third peak from the Sephadex G-50 column contained tion ofthese primary fragments yielded a continuous sequence CT3 and CT1, respectively. The peptides CT2 and CT6 were ofthe heavy chain except for the regions connecting CT1 to CT2 eluted in two partially merged peaks. These peptides were sep- and CB5 to CB6 (Fig. 1). The sequence necessary to overlap arated by HPLC as described above. The sixth peak from the CT1 and CT2 was provided by the three overlapping subfrag- Sephadex G-50 column contained CT5. ments ofCB1: The peptides SP2(CB1) and SP3(CB1) produced Separation of the Peptide Fragments of the Light Chain. by digestion ofCB1 with S. aureus protease and KC2(CB1) gen- The single methionyl bond of the light chain was cleaved by erated by digestion with L. enzymogenes endoproteinase Lys- CNBr to yield two fragments L CB1 and L CB2. The two frag- C. The overlap between CB5 and CB6 was obtained from a con- ments were separated by gel filtration on a column ofSephadex tinuous sequence provided by Edman degradation ofthe CNBr G-25 in 10 mM HCI. Cleavage at the single glutamyl bond by peptide spanning CB5 and CB6 sequences (CBS-CB6), thus S. aureus protease (9) in 0.1 M ammonium acetate buffer (pH completing the amino acid sequence of the heavy chain. The 4.0) yielded two fragments LSP1 and LSP2, which were sep- carboxyl-terminal sequence was confirmed by digestion with arated in the same manner. carboxypeptidase Y to be -Tyr-Pro-Asn. Sequence Analysis. All sequence analyses were done by au- Amino Acid Sequence of the Light Chain. This amino acid tomated Edman degradation in a Beckman 890B Sequencer sequence was determined by automated Edman degradation with 0.55 M Quadrol buffer and a combined benzene/ethyl ofthe intact light chain and its carboxyl-terminal fragments gen- acetate wash program in the presence of Polybrene (3 mg). erated by CNBr cleavage (LCB2) and by digestion with S. au- Phenylthiohydantoin amino acids were determined by two sys- reus protease (LSP2). The carboxyl-terminal sequence was con- tems of HPLC by using a Zorbax CN column (0.46 X 15 cm; firmed by treating the intact light chain successively with DuPont) eluted isocratically with 10 mM sodium acetate buff- carboxypeptidases B and A to be -Leu-Ala-Arg. er, pH 4.2/tetrahydrofuran/acetonitrile/methanol, 66:23.8: 6.8:3.4 (vol/vol), and an Ultrasphere ODS column (0.46 X 25 DISCUSSION cm; Altex) eluted essentially according to Zimmerman et aL (10). Analyses ofthe carboxyl-terminal sequences were done by the The complete amino acid sequences ofthe heavy chain and light method ofAmbler (11) with carboxypeptidases A and B (Wor- chain of mouse submaxillary gland renin are shown in Fig. 2. thington) or by the method of Hayashi et aL (12) with carboxy- The heavy chain consists of288 amino acid residues giving a Mr peptidase Y (Worthington). of31,036 calculated from the amino acid sequence determined here. The light chain contains 48 amino acid residues with a Mr RESULTS of5,458. The molecular weights ofthe two polypeptide chains calculated from their amino acid sequences are in good agree- Amino Acid Sequence of the Heavy Chain. Six CNBr frag- ment with those obtained by independent methods-30,000 for ments CB1 through CB6, accounting for the entire amino acid the heavy chain by sedimentation equilibrium analyses and sequence of the heavy chain, were isolated. Similarly, a com- 5,500 for the light chain by gel filtration in 6 M guanidine-HCl plete set of tryptic peptides CT1 through CT6 were obtained (8). from citraconylated heavy chain. Automated Edman degrada- Inactivation of renin by aliphatic diazo-compounds or the 4860 Biochemistry: Misono et al. Proc. Natl. Acad. Sci. USA 79 (1982)

-6 1 10 20 30 Ser-Ser-Leu-Thr-Asp-Leu-Ile-Ser-Pro-Val -Val-Leu-Thr-Asn-yr-Leu-Asn-Ser-Gln-Tyr-Tyr-Gy-Glu- Ile-Gly-le-Gly-Thr-Pro-Pro-Gln-Thr-Phe-Lys-Val -Ile-

40 46 46A 466 50 60 Phe-Asp-Thr--Ser-Al a-AsnLeu-Trp-Val-Pro-Ser-Thr-Lys- -Ser-Arg-Leu-Tyr-Leu-Ala-Cys-y-I1e-Hi s-Ser-Leu-Tyr-Gl u-Ser-Ser-Asp-

70 80 90 Ser-Ser-Ser-Tyr-Met-Gl u-Asn-Gly-Asp-Asp-Phe-Thr- II e-Hi s-yr-Gly-Ser-Gly-Arg-Val -Lys-Gly-Phe-Leu-Ser-GI n-Asp-Ser-Val -Thr- A 99 101 102 102 103 110 111 113 120 Val -Gly-Gly- I 1e-Thr-Val -Thr-Gl n-Thr-Phe-Gly-Gl u-Val -Thr-Glu-Leu-Pro-Leu-Il e-Pro-Phe-Met-Leu-Al a-Gl n-Phe-Asp-Gly-Val -

130 140 150 Leu-Gly-Met-Gly-Phe-Pro-Ala-Gl n-Al a-Val -Gly-Gly-Val -Thr-Pro-Val -Phe-Asp-Hi s-I1 e-Leu-Ser-Gl n-Gly-Val -Leu-Lys-Glu-Lys-Val - A B 155 155 155 156 160 170 180 Phe-Ser-Val -Tyr-Tyr-Asn-Arg-Gly-Pro-Hi s-teu-Leu-Gly-Gly-Gl u-Val -Val -Leu-Gly-Gly-Ser-Asp-Pro-Glu-Hi s-Tyr-Gln-Gly-Asp-Phe-His-

190 200 210 Tyr-Val -Ser-Leu-Ser-Lys-Thr-Asp-Ser-Tr2-Gl n-Il e-Thr-Met-Lys-Gly-Val -Ser-Val -Gly-Ser-Ser-Thr-Leu-Leu-Cys-Gl u-Gl u-G -Cys-

220 230 234 236 240 Gl u-Val -Val -Val -Asp-Thr-Gly-Ser-Ser-Phe-I le-Ser-Ala-Pro-Thr-Ser-Ser-Leu-Lys-Leu-Il e-Met-Gl n-Ala-Leu-GI -Ala-Lys-Gl u-

250 260 270 Lys-Arg-Leu-Hi s-Glu-Tyr-Val -Val -Ser-Cys-Ser-Gl n-Val -Pro-Thr-Leu-Pro-Asp- Ile-Ser.-Phe-Asn-Leu-Gly-Gly-Arg-Ala-[r-Thr-Leu- A B 278 278 278 27 0 290 300 Ser-Ser-Thr-Asp-Tyr-Val -Leu-Gl n-Tyr-Pro-Asn -pLys-Leu-Cys-Thr-Va1 -A1a-Leu-Hi s-Ala-Met-Asp-Il e-Pro-Pro-Pro-Thr-Gl -Pro-Val -Tr- HEAVY CHAIN L- LIGHT CHAIN 310 320 Val -Leu-Gly-Ala-Thr-Phe-Ile-A-Lys-Phe-Tyr-Thr-Gl u-Phe-Asp-Arg-Hi s-Asn-Asn-Arg-Il e-Gly-Phe-Ala-Leu-Ala-Arg FIG. 2. Amino acid sequence of mouse submaxillary gland renin. The sequences of the heavy chain and light chain of renin are aligned on the basis ofthe sequence homology with porcine pepsin. Residues are numbered accordingto the numbering system of the pepsin sequence (13). Residues identical to those in pepsin are underlined. To accommodate the difference in the lengths of the sequences, numberings of residues are modified. Residues found in renin that are not found in pepsin at the corresponding positions are indicated by the letters A and B after the residue number of the preceding residue. These include 46A, 46B, 102A, 155A, 155B, 278A, and 278B. When residues in pepsin are not found at the corresponding positions in renin, numbers for such residues are skipped-as in the sequences 99-101, 111-113, and 234-236.

epoxide 1,2-epoxy-3-(p-nitrophenoxy)propane and concomitant imities ofthe two active site aspartyl residues Asp-32 and Asp- incorporation ofthe reagent moieties in a mutually nonexclusive 215, respectively. The two sequences containing these active fashion indicated the presence oftwo catalytically essential car- site residues, Phe-Asp-Thr-Gly-Ser (31-35) and Asp-Thr-Gly boxyl groups in the active site ofrenin (2). The active site struc- (215-217), are identical in all four species of acid proteases ture involving two catalytically essential carboxyl groups is a shown, suggesting the important contribution ofthese residues characteristic feature commonly shared by acid proteases. Thus, in constituting the catalytic center of these . In addi- *in spite ofthe marked difference in the substrate specificity and tion,-Tyr-75 and Arg-308 in pepsin or Tyr-75 and Lys-308 in the pH optimum ofrenin catalysis from those ofacid proteases, penicillopepsin implicated in the catalytic mechanism (17, 23) certain structural similarities of renin with acid proteases, par- are also identical or functionally conserved in renin. These find- ticularly in the active site regions, have been expected. ings together strongly support the argument that, in spite ofthe The present study revealed that mouse submaxillary gland marked difference in the pH optimum of the catalysis, the cat- renin contains the sequence Ile-Phe-Asp-Thr-Gly-Ser-Ala-Asn alytic mechanism of renin is very similar to that of acid pro- (residues 30 through 37) which is highly homologous with the teases. sequences containing the catalytically essential aspartyl residue The amino acid sequences ofthe heavy chain and light chain 32 (Fig. 3) in pepsin (14), penicillopepsin (18), and chymosin of renin shown in Fig. 2 are aligned to maximize sequence ho- (16). The P3-carboxyl group ofAsp-32 has been shown to be es- mology with porcine pepsin and residues are numbered ac- terified by the acid protease specific inactivator 1,2-epoxy-3- cordingly using the numbering system of the porcine pepsin (p-nitrophenoxy)propane (14, 19, 20). The sequence containing sequence (13). The sequence ofthe heavy chain showed a 42% another active-site aspartyl residue in pepsin (15), Ile-Val-Asp- identity with the amino-terminal and middle regions ofthe por- Thr-Gly-Thr-Ser (residue 213-219), again finds a highly ho- cine pepsin sequence, whereas that of the light chain showed mologous sequence at the corresponding position in the renin a 46% identity with the carboxy-terminal region. The sequence sequence. Asp-215 has been shown to be the site of specific identity with pepsin for the entire molecule was 43% based on esterification by aliphatic diazo-compounds which takes place the alignment shown in Fig. 2. The renin sequence was also in the presence ofcupric ion with a concomitant inactivation of homologous with chymosin (34% identity) and penicillopepsin the enzyme (21). (22% identity). The amino acid residues in renin identical to The hydroxyl group of Ser-35 has been shown to be hydro- those in pepsin or penicillopepsin are distributed throughout gen-bonded to the carboxylate oxygen of Asp-32 in the crystal the length of the molecules, which suggests that the tertiary structure ofpenicillopepsin resolved at 2.8 (22). Ser-35, which structure ofrenin should be similar to those ofpepsin and pen- is conserved in all three acid proteases shown in Fig. 3, is also icillopepsin. The high content of /structure in renin indicated present in renin. The sequence Thr-Gly (216-217), whose pep- by far-UV CD spectrum (7) is compatible with this notion. tide moiety has been implicated in the catalytic mechanism of By aligning the amino acid sequence ofrenin with that ofpep- penicillopepsin (22), again is found in renin. Both ofthese struc- sin, the point of the scission in the polypeptide chain, which tures-Ser-35 and Thr-Gly (216-217)-are within close prox- yields the heavy chain and light chain of active renin from the Biochemistry: Misono et aL Proc. Natl. Acad. Sci. USA 79 (1982) 4861 EPNP

Renin (mouse) ---Ile-Phe-Asp-Thr-Gly-Ser-Ala-Asn --- Pepsin (pig) ---Ile-Phe-Asp-Thr-Gly-Ser-Ser-Asn--- Chymosin (bovine) ---Leu-Phe-Asp-Thr-Gly-Ser-Ser-Asp--- Penici 1 1opepsin ---Asn-Phe-Asp-Thr-Gly-Ser-Ala-Asp--- 32 Diazo-compound Renin (mouse) ---Val-Val-AIp-Thr-Gly-Ser-Ser--- Pepsin (pig) ---VIle-Val-Asp-Thr-Gly-Thr-Ser--- Chymosin (bovine) ---Ile-Leu-Asp-Thr-Gly-Thr-Ser--- Penici 1 1opepsin ---Ile-Ala-Asp-Thr-Gly-Thr-Thr--- 215 FIG. 3. Homology of the amino acid sequences containing the active site aspartyl residues 32 and 215 in renin (this report), pepsin (14, 15), chymosin (16), and penicillopepsin (17). Asp-32 and Asp-215 have been shown to be the sites of specific esterification by 1,2-epoxy-3-(p-nitro- phenoxy)propane (EPNP) and aliphatic diazo-compounds, respectively, in pepsin and chymosin. putative single-chain renin precursor (8, 24-27), is localized at Identification of the specific structures in the renin molecule the linkage between residues 279 and 280. In the crystal struc- that account for the unique functional properties ofthis enzyme ture of penicillopepsin (22), the sequence corresponding to needs further investigation. 279-280 is on a hairpin loop (,3-bend) localized on the exterior In contrast to renal renins, mouse submaxillary gland renin of the carboxyl-terminal lobe. Insertion of the two additional is not glycosylated (7, 28). The absence ofthe typical amino acid residues Tyr-278A and Pro-278B in this region of the renin se- sequences ofglycosylation sites Asn-X-Thr or Asn-X-Ser is com- quence appears to facilitate the interruption of the P-sheet patible with the absence of carbohydrate. Heterogeneity was structure and, in addition, to render the linkage between Asn- observed in the amino-terminal sequence of the heavy chain. 279 and Asp-280 more accessible to the cleavage by an as yet In addition to the sequence shown in Fig. 2, small populations unknown peptidase. It is also possible that one or more residues of the heavy chain have sequences commencing with the pen- are present between Asn-279 and Asp-280which may have been ultimate residue serine or with the fourth residue threonine. removed in the active renin. Six cysteine residues in renin are placed at positions identical The authors are greatly indebted to Drs. J. J. N. Tang and K. Ku- to those in pepsin in the two sequences aligned by homology. romizu for helpful discussion and to Mr. William Burkhart for able tech- However, a difference exists in the relative positions ofCys-45 nical assistance. This study has been supported by research Grants HL and Cys-50 where two additional residues are inserted between 22288 and HL 14192 to T.I. from the National Institutes ofHealth and the two. It has been shown previously that renin contains two by a Grant-in-Aid and an Investigatorship Award to K.S.M. from the free cysteine residues and two disulfide bridges, one ofwhich Tennessee Chapter of the American Heart Association. connects the heavy chain and light chain (8). In pepsin, Cys-45 and Cys-50, Cys-206 and Cys-210, and Cys-250 and Cys-283 are 1. Okamura, T., Clemens, D. L. & Inagami, T. (1979) Proc. Nat. Acad. Sci. USA 78, 6940-6943. linked via disulfide bonds (13). In view of the high overall se- 2. Misono, K. S. & Inagami, T. (1980) Biochemistry 19, 2616-2622. quence homology of renin with pepsin, it is likely that the di- 3. Gross, R., Lazar, J. & Orth, H. (1972) Science 175, 656. sulfide bond connecting the two polypeptide chains of renin 4. Cohen, S., Taylor, J. M., Murakami, K., Michelakis, A. M. & involves Cys-250 and Cys-283. The positions of free cysteine Inagami, T. (1972) Biochemistry 11, 4286-4293. residues and the alignment ofdisulfide linkages need to be fur- 5. Michelakis, A. M., Yoshida, H., Menzie, J., Murakarni, K. & In- ther investigated. agami, T. (1974) Endocrinology 94, 1101-1105. 6. Hirose, S., Workman, R. J. & Inagami, T. (1977) Circ. Res. 45, Although the extremely high substrate specificity and the 275-279. neutral optimalpH ofrenin clearly distinguish this enzyme from 7. Misono, K. S., Holladay, L. A., Murakami, K., Kuromizu, K. acid proteases, the structural basis for such unique catalytic & Inagami, T. (1982) Arch. Biochern. Biophys. in press. properties ofrenin is not immediately evident from its primary 8. Misono, K. S. & Inagami, T. (1982)J. Bio. Chem. in press. structure. Comparison ofthe amino acid sequence ofrenin with 9. Houmard, J. & Drapeau, G. R. (1972) Proc. Nate Acad. Sci. USA those of three acid proteases-pepsin, chymosin, and penicil- 69, 3506-3509. 10. Zimmerman, C. L., Appella, E. & Pisano, J. J. (1977) Anal lopepsin-indicated that there are approximately 80 positions Biochem. 77, 569-573. at which residues are identical or homologous among the acid 11. Ambler, R. P. (1972) Methods Enzymol 25, 155-166. proteases but distinctly different in renin. Such residues in 12. Hayashi, R., Moore, S. & Stein, W. H. (1973)J. Biol Chem. 248, renin are relatively evenly distributed throughout its sequence. 2296-2302. 4862 Biochemistry: Misono et al. Proc. NatL. Acad. Sci. USA 79 (1982)

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