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Amino Acid Sequence of Mouse Submaxillary Gland Renin (Homology with Pepsin/Active Site) KUNIO S

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Amino Acid Sequence of Mouse Submaxillary Gland Renin (Homology with Pepsin/Active Site) KUNIO S Proc. Natl. Acad. Sci. USA Vol. 79, pp. 4858-4862, August 1982 Biochemistry Amino acid sequence of mouse submaxillary gland renin (homology with pepsin/active site) 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, chymosin, 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 enzyme 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 proteases (2, 3), renin is unique in that it has 0.1% trifluoroacetic acid eluted with a linear gradient of ace- little, ifany, general protease 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 renins (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.
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