J Biochem. 109, 30-35 (1991)

Isolation and Characterization of Recombinant Human Prorenin in Chinese Hamster Ovary Cells1 Yasuyuki Ishizuka,* Akihito Shoda,' Shigeki Yoshida,* Yukio Kawamura,** Kazutomo Haraguchi," and Kazuo Murakami* 'Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305; and "National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305 Received for publication, September 15, 1990

Recombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-prorenin by employing a simple two-step procedure which consisted of ammonium sulfate precipitation and immunoaffinlty chromatography using a monoclonal antibody specific for the profragment of human prorenin. About 100-fold purification with 35% recovery was achieved after the two steps. Purified rh-prorenin migrated as a single protein band with apparent molecular weights of 46,000-47,000 and about 50,000 on SDS-PAGE and gel filtration (HPLC), respectively, although it consisted of multiple components (pi values, 5.6-6.4) that could be resolved by isoelectric focusing (IEF). The treatment of rh-prorenin with endo-0-Nacetylglucosaminidase converted the rather broad protein band to a sharp band on SDS-PAGE and reduced the number of multiple pi peaks on IEF. Amino-terminal sequence analysis of both the purified rh-prorenin and rh-renin revealed Leu-Pro-Thr-Asp- and Leu-Thr-Leu-Gly-, respectively, which agreed with those predicted from the base sequences of their cDNA. These data suggested that microheterogeneity of rh-prorenin is due to the carbohydrate moiety, but not to the protein moiety. Purified rh-prorenin was almost inactive, but was cleaved at the carboxyl end of a dibasic pair Lys~2-Arg~' by trypsin and converted to active renin. However, at the early stage during trypsin activation, new intermediate forms between rh-prorenin and rh-renin were formed, suggesting multiple activation steps of rh-prorenin in addition to the one step activation.

Renin is an unique aspartic proteinase which initiates a renin-angiotensinogen reaction leading to a very potent vasoactive compound, angiotensin II (2, 2). Renin is mainly synthesized in kidney as an inactive precursor, prorenin. Prorenin can be activated in vitro by limited proteolysis (3) [trypsin (4), chymotrypsin (5), pepsin (6), plasmin (7), and kallikrein (4, 8, 9)] or by acidification (10, 11). However, only a few preliminary studies have been reported on the biosynthetic processing (12) and activation mechanism (13) of human prorenin. Moreover, prorenin or prorenin-like inactive renin is present not only in kidney but also in many organs including placenta (14), uterus (25), ovary (16, 17), testis (18), adrenals (19), and anterior pituitary (20). The function of prorenin and renin in these organs is not completely known. An obstacle to the solution of these problems is that isolation and characterization of prorenin in these organs including kidney is extremely difficult, mainly due to its very low concentration and its instability. To overcome the difficulty, we (21) and other investigators (22-24) produced recombinant human prorenin (rhprorenin) from culture medium of Chinese hamster ovary 1 This work was supported by grants from the Ministry of Education, Science and Culture of Japan and from Chichibu Cement Co. Abbreviations: AI, angiotensin I; CHO, Chinese hamster ovary; IEF, isoelectric focusing; PMSF, phenylmethylsulfonyl-fluoride; rh-prorenin, recombinant human prorenin; rh-renin, recombinant human renin.

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(CHO) cells transfected with the cDNA for human preprorenin. Moreover, we (21), Carilli et al. (25), andHeinrikson et al. (24) purified rh-prorenin from the culture medium of CHO cells. However, the details of physicochemical properties and activation mechanism of pure rh-prorenin have not been reported. In the present study, we report a simple method for the improved purification of rh-prorenin and we describe the characterization and activation mechanism of pure rh-prorenin. MATERIALS AND METHODS Materials—BSA and trypsin were of the highest grade available from Sigma Chemical Co. Enzyme—Endo-/9-iV-acetylglucosaminidase D from Diplococcus pneumoniae (endo D), endo-/9-iV-acetylglucosaminidase H from Streptomyces griseus (endo H), glucopeptidase A from almond, and neuraminidase from Streptococcus sp. were purchased from Seikagaku Kogyo. Endo/9-iV-acetylgluco8aniinidase F from Flavobacterium meningosepUcum (endo F) was purchased from Boehringer Mannheim Biochemica. Cell Culture—Chinese hamster ovary (CHO) cell line transfected with a gene encoding preprorenin was prepared as described by Poorman et al. (26). The cell lines were cloned by induction of methotrexate to secrete substantial quantities of rh-prorenin. The cells were grown to confluJ. Biochem.

31

Isolation and Characterization of Recombinant Human Prorenin ency in a Cell Factory (Nunc) and then switched to serumfree medium (S-clone; Sanko, Japan) containing 100 units penicillin and 100 units of streptomycin per ml. Renin Assay—Renin activity was determined by radio immunoassay (27) of angiotensin I. A sample (20 fil) was incubated for 60 min at 3TC (total volume 200 ^1) with a partially purified hog angiotensinogen preparation (27) in 0.1 M Tris-HCl buffer, pH 7.4, containing 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF). Activation of Prorenin—1) Trypsin activation: A sample (20 //I) was incubated for 20 min at 22'C with 20 //I of a freshly mixed solution of L-l-chloro-3-[4-tosylamido]-4phenyl-2-butane (TPCK)-treated trypsin (5-50//g/ml, Sigma) in 0.1 M Tris-HCl buffer, pH 7.4 containing 1% BSA. The activation was stopped by the addition of 100 //g of soybean trypsin inhibitor (Cooper) in 10 //I of the same buffer. 2) Acid activation: A sample was dialyzed against 50 mM glycine buffer, pH 3.3 at 4"C for 24 h, then quickly neutralized, and the renin activity was determined. Protein Concentration—Protein concentration was determined by the microassay of Bradford (28), using bovine y-globulin (Bio-Rad) as a standard. Isoelectric Focusing (IEF)—IEF was performed using the method of Vesterberg (29) on a 5% polyacrylamide gel plate containing 2.2% ampholine, pH 3.5 to 9.5 or 9.0 to 11.0 in an Multiphor apparatus (Pharmacia LKB Biotechnology). The electrode voltage applied across the shorter edge was increased from 100 to 500 V over 1 h. The pH and renin activity of gel slices cut at 0.15 cm intervals were determined after overnight extraction in 0.3 ml of distilled water at 4'C. Immunoaffinity Chromatography—Monoclonal antibodies, 2-X-C1 or 4-X-E1 (30), specific for the middle region or C-terminus of the human prorenin profragment were coupled to CNBr-activated Sepharose 4B (2 mg antibody/ ml moist gel). The coupled monoclonal antibody (3 ml of gel) was then incubated overnight at 4"C with gentle shaking with 10 mg/9 ml of the recombinant human prorenin. Gel Filtration—Gel filtration was performed on a high pressure liquid chromatography (HPLC) system using a Fig. 1 Immnnoaffinity chromatography of rh-prorenin. From 2.1 liters of the culture medium of CHO cells as described in "MATERIALS AND METHODS," rh-prorenin was precipitated at 75% saturation of ammonium sulfate. The precipitate was dissolved in phosphate-buffered saline (pH 7.4) (PBS) containing 1 mM PMSF and 1 mM EDTA, dialyted against the same buffer and applied to an immunoaffinity column ( l x 5 cm) using monoclonal antibody (2-X-Cl) (30) equilibrated in the same buffer. The column was washed with PBS, pH 7.4, containing 0.1% Tween 20 (a). Elution was performed with PBS, pH 7.4 containing 50% ethylenglycol (b): 50 mM diethylamine buffer, pH 11 (c): PBS, pH 7.4 containing 10% dioxane (d). Four milliliters fractions were collected, and the pH was adjusted to 7.4 by the addition of 1 M Tris-HCl buffer, pH 6.0. All isolation procedures were performed at 4'C. Renin activity was determined before (•) and after (O) trypsin activation.

TSK-G3000SWXL (Tosoh) column (0.78 X 30 cm). Protein solution (1%) was applied to the column and eluted with 50 mM phosphate buffer, pH 7.0 containing 0.1 M Na 2 S0 4 . The effluent streak was monitored for protein content by measuring the absorbance at 280 nm. Amino-Terminal Sequence Analysis—Samples were prepared for sequencing by electrophoresis of activated prorenin on 10% polyacrylamide gels under nonreducing conditions. Electroeluted proteins were dialyzed with distilled water and then freeze-dried (31). Amino-terminal sequence analysis was performed on an Applied Biosystems 477A gas-phase sequencer. Phenylthiohydantoins from the individual cycles were identified and quantified by reverse-phase HPLC (32). Glycosidase and GlycopepUdase Treatments—All samples were adjusted to a final volume of 60 /zl and heated at 100'C for 5 min. All incubations were performed at 37*C for 16 h. For endo F digestion, the sample volume was adjusted with 0.1 M potassium phosphate buffer, pH 7.5, containing 20 mM EDTA, 0.1% SDS and 1% 2-mercaptoethanol, and endo F (0.25 U) was added. For endo H and glycopeptidase digestions, the sample volume was adjusted with 0.1 M citrate-phosphate buffer, pH 5.25, containing 0.1% SDS, and endo H (10 mU) and glycopeptidase (0.1 mU) were added, respectively. For endo D and neuraminidase digestions, the sample volume was adjusted with 0.1 M acetate buffer, pH 6.5, and endo D (10 mU) and neuraminidase (0.1 U) were added, respectively. Other Analytical Methods—Immunoblotting and SDSPAGE were performed as described previously (21). RESULTS Purification of Recombinant Human Prorenin—Recombinant human prorenin (rh-prorenin) was purified from the culture medium by ammonium sulfate precipitation and immunoaffinity chromatography using a monoclonal antibody specific for the complete profragment (30). The key step is the immunoaffinity chromatography (21), which resulted in a 60-fold purification with 54% yield. About 5060% of prorenin was adsorbed in an immunoaffinity column and eluted with three different elution buffers (Fig. 1): 0.16

50

B a o

1

00 IN

O

I25
SO4 at aflowrate of 0.5 ml/min. The column was calibrated with glutamate dehydrogenase 290,000 (1), lactate dehydrogenase 142,000 (2), enolase 67,000 (3), adenylate kinase 32,000 (4), and cytochrome c 12,400 (5).

B. Amino-termlnsl sequence of rh-renln (trypsln-s.ctlva.ted rh-prorenfn) 1

10

20

Deduced : LTLGNTTS3VILTNYMDTQY Observed : LTLQ«T»3 •VILTMYMDTQY Fig. 4. Sequence analysis of the amino-terminals of rh-prorenin and rh-renin (trypsin-activated rh-prorenin). The rhprorenin (fraction II) and rh-renin were subjected to microsequence analysis. The results are compared with the amino acid sequence deduced from the human renin cDNA sequence (33). e indicates no positive identification.

TABLE I.

Immunoamnlty purification of rh-prorenin. A two-step procedure was used as described in "MATERIALS AND METHODS. Volume Total protein Activity Specific activity Purification Yield Fraction (ml) (mg) (jig Al/ml/h) (mg Al/mg/h) (fold) (%) 260 9.8 1 Culture medium 2,100 0.079

(NrL.),SO,(75%satd.) precipitate Immunoaffinity chromatography Peak I PeakH

Peakm

80 96 120 108

85

0.16 0.37 0.22

167

0.16

15

8.6

34 16

11.0 7.9

2

m

109 116 100

f 20 8 J. Biochem.

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33

Isolation and Characterization of Recombinant Human Prorenin Fig. 5. Effect of BSA on trypsin and acid activations. A: Incubation of rhprorenin (0 1 mg/ml) by various concen.trations of-trypsin with or without-0 1% BSA (24"C, 20 nun) resulted in conversion of inactive prorenin to active renin, accompanied by an increase in rerun activity. B: Acid activation was earned out with or without 0.1% BSA as described in •MATERIALS AND METHODS."

B

Trypsin Activation

Acrid

lOOp

10Or

-B8A

SO

a u

6.10

25

50

100

Isolation and characterization of recombinant human prorenin in Chinese hamster ovary cells.

Recombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-pror...
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