Role of N-Glycosylation in Human Angiotensinogen
1998; Elsevier BV; Volume: 273; Issue: 33 Linguagem: Inglês
10.1074/jbc.273.33.21232
ISSN1083-351X
AutoresAnne‐Paule Gimenez‐Roqueplo, Jérôme Célérier, G. Lucarelli, Pierre Corvol, Xavier Jeunemaı̂tre,
Tópico(s)Cardiovascular, Neuropeptides, and Oxidative Stress Research
ResumoHuman angiotensinogen, the specific substrate of renin, is a heterogeneous glycoprotein constitutively secreted by the liver. Different glycosylation levels may be responsible for its heterogeneity. It contains four putative asparagine-linked glycosylation sites (Asn-X-Ser/Thr). Systematic site-directed mutagenesis (Asn replaced with Gln) of these four sites was undertaken, and 11 (single, double, triple, and quadruple (N-4)) mutants were produced in COS-7 and/or CHO-K1 cells and characterized. All of the sites were N-glycosylated with preferential glycosylation of the Asn14 and the Asn271. The suppression of the Asn14 glycosylation site led to 5 times lower Km and a 10 times lowerkcat. Angiotensinogen heterogeneity was much lower for the N-4 mutant protein, which produced a single form at 48 kDa. Pulse-chase experiments showed slight intracellular retention (15%) of the deglycosylated protein after 24 h. Interestingly, the N-4 mutant had a higher catalytic efficiency (kcat/Km = 5.0versus 1.6 μm−1 · s−1) than the wild-type protein. The thermal stability of the N-4 protein was unaffected by deglycosylation, suggesting that it was correctly folded. This deglycosylated recombinant human angiotensinogen could be of value for x-ray crystallography studies. Human angiotensinogen, the specific substrate of renin, is a heterogeneous glycoprotein constitutively secreted by the liver. Different glycosylation levels may be responsible for its heterogeneity. It contains four putative asparagine-linked glycosylation sites (Asn-X-Ser/Thr). Systematic site-directed mutagenesis (Asn replaced with Gln) of these four sites was undertaken, and 11 (single, double, triple, and quadruple (N-4)) mutants were produced in COS-7 and/or CHO-K1 cells and characterized. All of the sites were N-glycosylated with preferential glycosylation of the Asn14 and the Asn271. The suppression of the Asn14 glycosylation site led to 5 times lower Km and a 10 times lowerkcat. Angiotensinogen heterogeneity was much lower for the N-4 mutant protein, which produced a single form at 48 kDa. Pulse-chase experiments showed slight intracellular retention (15%) of the deglycosylated protein after 24 h. Interestingly, the N-4 mutant had a higher catalytic efficiency (kcat/Km = 5.0versus 1.6 μm−1 · s−1) than the wild-type protein. The thermal stability of the N-4 protein was unaffected by deglycosylation, suggesting that it was correctly folded. This deglycosylated recombinant human angiotensinogen could be of value for x-ray crystallography studies. Angiotensinogen is the specific substrate of the aspartyl proteinase renin (EC 3.4.23.15). This reaction is the first step of the renin-angiotensin system, which regulates blood pressure, salt homeostasis, and vascular tone. In humans, renin cleaves angiotensinogen at its N terminus, generating the inactive decapeptide, angiotensin I (Ang I), 1The abbreviations used are: Ang Iangiotensin IAGTangiotensinogenPAGEpolyacrylamide gel electrophoresishAGThuman AGTCHOChinese hamster ovaryELISAenzyme-linked immunosorbent assay. which is further hydrolyzed by Ang I-converting enzyme (EC 3.4.15.1) to give active angiotensin II. The generation of Ang I in plasma, and probably also in tissue, is a first-order kinetic reaction and is the rate-limiting step of the renin-angiotensin enzymatic cascade (1Gould A.B. Green D. Cardiovasc. Res. 1971; 5: 86-89Crossref PubMed Scopus (187) Google Scholar). angiotensin I angiotensinogen polyacrylamide gel electrophoresis human AGT Chinese hamster ovary enzyme-linked immunosorbent assay. Interest in the angiotensinogen (AGT) gene was renewed when it was implicated in blood pressure regulation in animals (2Kim H.S. Krege J.H. Kluckman K.D. Hagaman J.R. Hodgin J.B. Best C.F. Jennette C. Coffman T.M. Maeda N. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1995; 91: 3612-3615Google Scholar) and humans (reviewed in Ref. 3Corvol P. Jeunemaitre X. Endocr. Rev. 1997; 18: 662-677PubMed Google Scholar). The M235T polymorphism was rapidly identified as the most interesting of several genetic variants due to its association with plasma angiotensinogen concentration and essential hypertension (4Jeunemaitre X. Soubrier F. Kotelevtsev Y. Lifton R.P. Williams C.S. Charru A. Hunt S.C. Hopkins P.N. Williams R.R. Lalouel J.M. Corvol P. Cell. 1992; 71: 169-178Abstract Full Text PDF PubMed Scopus (1714) Google Scholar). This association probably results from the strong linkage disequilibrium between the M235T polymorphism and the G−6A substitution in the promoter (5Inoue I. Nakajima T. Williams C.S. Quackenbush J. Puryear R. Powers M. Cheng T. Ludwig E.H. Sharma A.M. Hata A. Jeunemaitre X. Lalouel J.M. J. Clin. Invest. 1997; 7: 1786-1797Crossref Scopus (510) Google Scholar). However, rare missense mutations within the coding region may also affect function. One such mutation at the renin cleavage site, L10F, was first identified in a preeclamptic woman and caused a doubling in the catalytic efficiency of the renin angiotensinogen reaction (6Inoue I. Rohrwasser A. Helin C. Jeunemaitre X. Crain P. Bohlender J. Lifton R.P. Corvol P. Ward K. Lalouel J.M. J. Biol. Chem. 1995; 270: 11430-11436Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). We have studied a family with another rare missense mutation, Y248C, which generates an extraN-glycosylation site leading to the production of an additional 61-kDa form when the cDNA is expressed in CHO cells (7Gimenez-Roqueplo A-P. Leconte I. Cohen P. Simon D. Guyene T.T. Célérier J. Pau B. Corvol P. Clauser P. Jeunemaitre X. J. Biol. Chem. 1996; 271: 9838-9844Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The parallelism between the intracellular retention observed in vitro and the decrease in plasma angiotensinogen concentration in affected heterozygous individuals highlighted the involvement of glycosylation in the intracellular processing of angiotensinogen. Plasma human angiotensinogen is a heterogeneous glycoprotein mainly produced by hepatocytes. Pure human angiotensinogen usually gives at least two major bands at about 60 kDa, separated by 3 or 4 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (for a review, see Ref. 8Tewksbury D.A. Laragh J.H. Brenner B.M. Hypertension: Pathophysiology, Diagnosis and Management. Raven Press, New York1990: 1197-1216Google Scholar). A similar level of heterogeneity has been reported for other mammalian species (9Skeggs L.T. Lentz K.E. Hochstrasser H. Kahn J.R. J. Exp. Med. 1963; 118: 73-98Crossref PubMed Scopus (56) Google Scholar, 10Campbell D.J. Bouhnik J. Coezy E. Pinet F. Clauser E. Ménard J. Corvol P. Endocrinology. 1984; 114: 776-785Crossref PubMed Scopus (24) Google Scholar, 11Hilgenfeldt U. Schott R. Mol. Cell. Endocrinol. 1987; 51: 211-218Crossref PubMed Scopus (8) Google Scholar, 12Fernley R.T. John M. Niall H.D. Coghlan J.P. Eur. J. Biochem. 1986; 154: 597-601Crossref PubMed Scopus (10) Google Scholar, 13Moffett R.B. Biochim. Biophys Acta. 1987; 912: 73-98Crossref Scopus (9) Google Scholar, 14Printz M.P. Printz J.M. Dworschack R.T. J. Biol. Chem. 1977; 252: 1654-1662Abstract Full Text PDF PubMed Google Scholar). Treatment of pure human plasma angiotensinogen with trifluoromethane sulfonic acid or glycopeptidase F reduces its apparent molecular weight by 10 kDa, but angiotensinogen still displays two or three bands in SDS-PAGE (15Campbell D.J. Charlton P.A. Grinham C.J. Mooney C.J. Pendlebury J.E. Biochem. J. 1987; 243: 121-126Crossref PubMed Scopus (10) Google Scholar). These data suggest that glycosylation plays a predominant role in angiotensinogen heterogeneity. However, the effect of human angiotensinogen glycosylation on protein secretion, stability, and reaction with renin is not fully understood. Hilgenfeldt et al. (16Hilgenfeldt U. Mol. Cell. Endocrinol. 1988; 56: 91-98Crossref PubMed Scopus (25) Google Scholar) reported differences in the half-lives of the two major glycoforms in rats; the highly glycosylated form was secreted faster by the liver and eliminated more slowly by the kidney than the less glycosylated form. Human angiotensinogen has four potential sites for asparagine-linked glycosylation (Asn-X-Ser/Thr): Asn14-Lys15-Ser16, Asn137-Cys138-Thr139, Asn271-Ser272-Thr273, and Asn295-Phe296-Ser297. The first site (Asn14-Lys15-Ser16) is closest to the renin cleavage site (Leu10-Val11) and may play a key role in the recognition of human angiotensinogen by human renin (17Cumin F. Le-Nguyen D. Castro B. Ménard J. Corvol P. Biochim. Biophys. Acta. 1987; 913: 10-19Crossref PubMed Scopus (68) Google Scholar). However, it is not known if all four Asn-X-Ser/Thr sequences are actually glycosylated. We describe herein the site-directed mutagenesis of singleN-glycosylation sites and combinations of them in 11 mutants of human AGT (hAGT). Analysis of these mutant proteins, synthesized in eukaryotic cells, indicates that oligosaccharide residues are linked to each site, accounting for the heterogeneity of the protein in vitro. We found that Asn14 and Asn271 were preferentially glycosylated and that glycosylation of Asn14has a large effect on the kinetics of the reaction of human angiotensinogen with human recombinant renin. We also showed that a fully deglycosylated angiotensinogen is produced efficiently by Chinese hamster ovary (CHO) cells and is a good renin substrate. A specific angiotensinogen enzyme-linked immunosorbent assay (ELISA) (18Cohen P. Badouaille G. Gimenez-Roqueplo A-P. Mani J.C. Guyene T-T. Jeunemaitre X. Ménard J. Corvol P. Pau B. Simon D. J. Clin. Endocrinol. Metab. 1996; 81: 3505-3512PubMed Google Scholar) was provided by Sanofi Recherche (Montpellier, France). Modification enzymes were purchased from New England Biolabs. Neuraminidase and O-glycosidase, cell culture reagents and fetal calf serum were obtained from Boehringer Mannheim (Meylan, France). Phenylmethylsulfonyl fluoride, aprotinin, pepstatin A, antipain, leupeptin, and Triton X-100 were purchased from Sigma. Molecular masses were estimated by comparison with14C-methylated proteins from Amersham Pharmacia Biotech and prestained SDS-PAGE molecular mass markers from Bio-Rad. The cDNA encoding the entire polypeptidique sequence of hAGT has been described elsewhere (19Gaillard I. Clauser E. Corvol P. DNA ( N. Y. ). 1989; 8: 87-99Crossref PubMed Scopus (120) Google Scholar). The corresponding protein has a Met in position 235, the most frequent variant in Caucasian populations. The cDNA was inserted (7Gimenez-Roqueplo A-P. Leconte I. Cohen P. Simon D. Guyene T.T. Célérier J. Pau B. Corvol P. Clauser P. Jeunemaitre X. J. Biol. Chem. 1996; 271: 9838-9844Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) into the eukaryotic expression vector pECE (20Ellis L. Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (696) Google Scholar). The human AGT cDNA contains four N-linked potential glycosylation sites (Asn-X-Ser/Thr) at amino acid positions 14, 137, 271, and 295. For single-stranded mutagenesis, the 1.7-kilobase pair insert was inserted between the XbaI and HindIII sites of M13mp19 to produce single-stranded DNA. Site-directed mutagenesis to convert each Asn into a Gln was performed using Kunkel's method (21Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-495Crossref PubMed Scopus (4900) Google Scholar) with the Mutagene M13 in vitro mutagenesis kit (Bio-Rad), recombinant single-stranded M13 DNA as template and an antisense mutagenesis oligonucleotide (Asn14 → Gln, 5′-ACA GGT ACT CTC TTG GTG GAT GAC GAG-3′; Asn137 → Gln, 5′-CCG GGA GGT GCA CTG CTT GTC CTT CCA-3′; Asn271 → Gln, 5′-CAC TGA GGT GCT CTG GTC CAC CCA GAA-3′; or Asn295 → Gln, 5′-AGT CAC CGA GAA CTG GTC CTG GAT GTC-3′) to produce the four mutant proteins with single residue changes: N14Q, N137Q, N271Q, and N295Q. Each mutated M13mp19 clone was sequenced (Sequenase II kit, U.S. Biochemical Corp.) and used for a second, third, and fourth round of mutagenesis to obtain double mutants (N14Q/N137Q and N271Q/N295Q) triple mutants (N14Q/N137Q/N271Q, N137Q/N271Q/N295Q, N14Q/N271Q/N295Q, and N137Q/N271Q/N295Q) and a quadruple mutant, in which all four Asn thought to be involved in N-glycosylation were mutated (N14Q/N137Q/N271Q/N295Q). This mutant was called N-4 (see Fig.1). Each hAGT mutant was sequenced and inserted into pECE. The 11 mutant hAGT cDNAs and the wild-type hAGT cDNA were used to transiently transfect COS-7 cells using the DEAE-dextran method (22Thibonnier M. Auzan C. Madhun Z. Wilkins P. Berti-Mattera L. Clauser E. J. Biol. Chem. 1994; 269: 3304-3310Abstract Full Text PDF PubMed Google Scholar). COS-7 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 10 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a 5% CO2, 95% air atmosphere. Cell lines producing wild-type angiotensinogen, the four separate single mutant proteins, and the N-4 mutant protein were established so that large amounts (10–40 mg) of proteins could be produced. CHO-K1 cells were transfected with a neomycin-resistant plasmid (pSV2 neo) by the calcium phosphate precipitation procedure (23Southern P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-340PubMed Google Scholar). Recombinant cells were selected using 500 μg/ml Geneticin® (Life Technologies, Inc.). Pure cell lines overproducing wild-type angiotensinogen or mutant proteins were obtained by the limiting dilution method, as described previously (7Gimenez-Roqueplo A-P. Leconte I. Cohen P. Simon D. Guyene T.T. Célérier J. Pau B. Corvol P. Clauser P. Jeunemaitre X. J. Biol. Chem. 1996; 271: 9838-9844Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). CHO-K1 cells were grown in Ham's F-12 medium containing 10% fetal calf serum, 10 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Mutant and wild-type angiotensinogen proteins were metabolically labeled 48 h after transfection. Cells were incubated in 35 × 10-mm dishes in methionine- and cysteine-free Ham's F-12 medium (Eurobio) for 1 h. A pulse of 50 μCi of [35S]methionine and [35S]cysteine (Amersham Pharmacia Biotech) was given for 10 min in methionine- and cysteine-free Ham's F-12 medium containing 10% dialyzed fetal calf serum. Various chase times were used in serum-free medium at 37 °C. The cells were solubilized at 4 °C in 20 mm Tris-HCl buffer, pH 7.4, 150 mm NaCl, 10 mm EDTA, 1.2% (v/v) Triton X-100, 0.1% (w/v) bovine serum albumin. Protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml antipain, 1 μg/ml leupeptin) were added. After centrifugation, the supernatants were incubated overnight with 1 μl of normal rabbit serum (Nordic Immunological Laboratories) and 5 mg of protein A-Sepharose (Protein-A Sepharose CL-4B; Pharmacia, Uppsala, Sweden). The protein A-Sepharose was collected by centrifugation to eliminate nonspecific complexes. The resulting supernatants were immunoprecipitated by incubating overnight at 4 °C with 5 mg protein A-Sepharose and a polyclonal human angiotensinogen antibody (dilution 1:1000). The human angiotensinogen antibody, HCL, has been described elsewhere (7Gimenez-Roqueplo A-P. Leconte I. Cohen P. Simon D. Guyene T.T. Célérier J. Pau B. Corvol P. Clauser P. Jeunemaitre X. J. Biol. Chem. 1996; 271: 9838-9844Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 24Genain C. Bouhnik J. Tewksbury D. Corvol P. Ménard J. J. Clin. Endocrinol. Metab. 1984; 59: 478-484Crossref PubMed Scopus (57) Google Scholar). The protein A-Sepharose was washed four times with 20 mm Tris-HCl buffer, pH 7.5, 150 mm NaCl, 0.1% (v/v) Triton X-100, 1 mm EDTA buffer and then with 20 mm Tris-HCl buffer, pH 6.8. The antigen-antibody complex was dissociated by heating (4 min at 95 °C) in Laemmli buffer (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar). The [35S]methionine/cysteine-labeled mutant proteins were resolved by 9% SDS-PAGE, and the gel was subjected to autoradiography. The absence of [35S]methionine/cysteine-labeled intracellular proteins in nontransfected COS-7 was checked by the same procedure. The integrated optical density of each band on the autoradiographs was determined and analyzed using NIH Image, version 1.57 software with a set of 35S radiography standards. The standards were used to produce a calibration curve to convert the optical density values of each pixel of the digitized image into dpm of35S-labeled protein/mm2. The percentage of intracellular retention was calculated by dividing the densities at 6, 24, and 48 h by the densities immediately after the 10-min pulse (t0) for the wild-type and N-4 mutant protein. A COS cell supernatant containing the N-4 mutant protein was denatured by heating at 95 °C for 4 min in the presence of 0.1% (v/v) SDS and 1% (v/v) β-mercaptoethanol. It was then incubated in 50 mm sodium phosphate buffer, pH 6.0, 1% (v/v) Triton X-100 at 37 °C with (i) no addition (mock treatment), (ii) 5 milliunits of neuraminidase (for 18 h), (iii) 5 milliunits of neuraminidase (for 1 h) followed by 2.5 milliunits ofO-glycosidase (for 18 h). Enzymatic deglycosylation was stopped by freezing. The samples were resolved by 9% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore Corp.) by semidry transfer in 25 mm Tris/glycine buffer, pH 7.5, containing 0.5% (v/v) SDS and 20% (v/v) methanol. Human angiotensinogen was detected with HCL antibody (dilution 1:6000). Antigen-antibody complexes were detected by their alkaline phosphatase activity after biotin-streptavidin enhancement. Enzymatic parameters of the renin angiotensinogen reaction were determined using different concentrations of various glycosylated angiotensinogen mutant proteins and a fixed amount (20 pm) of recombinant human renin, which was provided by Hoffmann La Roche (Basel, Switzerland). Renin was produced as prorenin in CHO cells, purified, and activated to mature renin by Arg-C protease treatment as described by Mathews et al. (26Mathews S. Döbeli H. Pruschy M. Bosser R. D'Arcy A. Oefner C. Zulauf M. Gentz R. Breu V. Matile H. Schlaeger J. Fischli W. Protein Expression Purif. 1996; 7: 81-91Crossref PubMed Scopus (19) Google Scholar). The active renin concentration used in this study was 150 μg/ml, as determined by a commercially available renin immunoradiometric assay (Diagnostics Pasteur, Marnes La Coquette, France). The same renin preparation was used in each experiment and diluted at 20 pm in a 100 mm citric acid/Na2HPO4, 3 mm EDTA buffer, pH 5.7. For the calculation of kinetic parameters, it was assumed that 100% of the mature renin was catalytically active. CHO cells producing wild-type and N14Q, N137Q, N271Q, N295Q, and N-4 mutant proteins were cultured in several 225-cm2 culture flasks for large scale (10–40 mg) production. Cells at 90–95% confluence were transferred to serum-free medium (UltraCHO, BioWhittaker), and supernatants were harvested every 3 or 4 days, filtered through a 0.2-μm membrane, and stored at −80 °C. For kinetic experiments, the supernatant was concentrated at 4 °C by ultrafiltration through a 10-kDa cut-off membrane (YM 10. Amicon, Inc., Beverly, MA) followed by centrifugation through a 30-kDa cut-off membrane (Ultrafree-15 centrifugal filter; Millipore). Angiotensinogen concentration was determined in three ways: (i) direct radioimmunoassay of human angiotensinogen as described previously (24Genain C. Bouhnik J. Tewksbury D. Corvol P. Ménard J. J. Clin. Endocrinol. Metab. 1984; 59: 478-484Crossref PubMed Scopus (57) Google Scholar); (ii) a two-monoclonal antibody (7B2/4G3) sandwich ELISA test, as described by Cohen et al. (18Cohen P. Badouaille G. Gimenez-Roqueplo A-P. Mani J.C. Guyene T-T. Jeunemaitre X. Ménard J. Corvol P. Pau B. Simon D. J. Clin. Endocrinol. Metab. 1996; 81: 3505-3512PubMed Google Scholar); and (iii) an enzymatic assay determining the amount of Ang I generated from angiotensinogen by cleavage with a large excess (1.25 nm) of human recombinant renin, as described by Gimenez-Roqueplo et al. (7Gimenez-Roqueplo A-P. Leconte I. Cohen P. Simon D. Guyene T.T. Célérier J. Pau B. Corvol P. Clauser P. Jeunemaitre X. J. Biol. Chem. 1996; 271: 9838-9844Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). We checked that each enzymatic assay was performed with a saturating renin concentration by increasing the renin concentration and the incubation time. These changes did not lead to an increase in the amount of Ang I peptide produced. We also checked that the angiotensinogen concentrations measured by direct radioimmunoassay and ELISA were not significantly different (±30%, according to the mutant) from those obtained with the enzymatic assay for each mutant. Various concentrations (0.05–10 μm) of each angiotensinogen substrate were incubated in 100 mm citric acid/Na2HPO4, 3 mm EDTA buffer, pH 5.7, with 20 pm purified recombinant human renin for 1 h at 37 °C. The reaction was stopped by raising the pH from 5.7 to 9.2 by adding 20 μl of 4 m Tris-base to each tube and chilling. The rate of Ang I production was determined in triplicate for each reaction mixture by a specific Ang I radioimmunoassay (27Ménard J. Catt K.J. Endocrinology. 1972; 90: 422-430Crossref PubMed Scopus (436) Google Scholar). Kinetic parameter determinations were performed simultaneously for mutant and wild-type human angiotensinogen protein. Less than 10% of the substrate was consumed in each experiment. Various controls were performed. Each concentration of substrate was incubated in the same conditions but without renin to check that no Ang I was produced. Nontransfected CHO cells were transferred to serum-free medium. The supernatant was harvested, concentrated, and incubated with renin to check that no angiotensinogen or Ang I was produced by untransfected CHO cells. Supernatants containing either the wild-type human angiotensinogen or the N-4 mutant protein were incubated for 1 h at various temperatures between 4 and 80 °C. The incubation was stopped by chilling. The angiotensinogen concentration was determined before and after incubation by ELISA and enzymatic assay as described above. The wild-type hAGT cDNA and 11N-glycosylation mutant cDNAs were used to transfect COS-7 cells. The intracellular production of the human angiotensinogen proteins was studied by immunoprecipitation after metabolic labeling (Fig. 2 A), and extracellular secreted angiotensinogen was studied by Western blotting (Fig.2 B). The wild-type angiotensinogen protein had a complex and reproducible pattern in SDS-PAGE. Intracellular angiotensinogen gave five bands of 58, 55.5, 52, 50, and 48 kDa, the upper band (58 kDa) being the fully N-glycosylated protein. Angiotensinogen secreted into the medium gave a fuzzy band between 50 and 70 kDa. All of the single mutants lacked the upper intracellular band (58 kDa), and the heterogeneity (2–3 kDa) of secreted angiotensinogen was slightly less, suggesting that each potentialN-glycosylation site was actually glycosylated. The secreted N137Q single mutant had a different pattern in SDS-PAGE, because the polyclonal antibody did not detect the weakly glycosylated forms of this mutant. The heterogeneity of both intracellular and secreted angiotensinogens was significantly lower when at least three potentialN-glycosylation sites were mutated. Deglycosylation of one site gave a 55.5-kDa band, deglycosylation of two sites gave a 52-kDa band, and deglycosylation of three N-glycosylation sites a 50-kDa band in cells. In the medium, the size heterogeneity of secreted angiotensinogen progressively decreased as the number ofN-deglycosylated sites increased, although accurate molecular mass determination was difficult due to the fuzziness of the bands. The single 48-kDa band observed with the N-4 mutant protein was a fully N-deglycosylated form. Thus, there was a stepwise decrease in the heterogeneity of angiotensinogen, directly related to the number of deglycosylated sites. Analysis of triple mutants, in which only oneN-glycosylation site was conserved, showed that this site was N-glycosylated in each case. The metabolic labeling of each triple mutant detected two bands, the upper band (50 kDa) demonstrating N-glycosylation of the conserved site and the lower band (48 kDa) corresponding to the N-deglycosylated form of angiotensinogen. The intensity of the two bands was compared for each mutant to estimate the number of different oligosaccharide structures at each site. The Asn14-Lys15-Ser16 (137/271/295 mutant) and Asn271-Ser272-Thr273(14/137/295 mutant) sites were more glycosylated than the sites of the two other triple mutants, because the 50-kDa form was predominant. The Asn137-Cys138-Thr139 site was less glycosylated, because the two intracellular bands for triple mutant 14/271/295 were of similar intensity. The Asn295-Phe296-Ser297 C-terminal site was also less glycosylated because the deglycosylated form was predominant for the 14/137/271 mutant. The fully deglycosylated hAGT form (N-4) was correctly synthesized and secreted by COS-7 cells. Metabolic labeling detected a single intracellular band (48 kDa), and Western blot analysis detected two secreted bands: a major product of 48 kDa, in size identical to the intracellular band, and a minor 50-kDa band. Similar data were obtained with pure line CHO cells (data not shown). Western blot analysis showed that there was an extra N-4 mutant protein (50 kDa) secreted, which was not present in cells. Treatment with endoglycosidase F did not affect this pattern (data not shown), so we treated this form with neuraminidase alone or with neuraminidase followed by O-glycosidase. Neuraminidase treatment resolved recombinant N-deglycosylated angiotensinogen into a single 48-kDa band, which was not further affected by O-glycosidase treatment (Fig.3), demonstrating that the 50-kDa form contained only sialic acid residues. The 48-kDa form of the N-4 mutant protein produced in the medium was an entirely unglycosylated angiotensinogen with no O-glycosylation. The effect of N-glycosylation on cellular trafficking and secretion in the culture medium was studied by pulse-chase experiments in transiently transfected COS-7 cells (Fig.4 A). All the mutant proteins were efficiently labeled within 10 min of the pulse (t= 0), giving the pattern shown in Fig. 2 A. The wild-type angiotensinogen and the four single mutant proteins were not detectable within cells after 6 h of chase (t = 6). Thus, the mutation of a single N-glycosylation site was not sufficient to affect cellular trafficking. However, a significant amount of the triple and quadruple mutant proteins was retained within the cells. After 24 h of chase (t = 24), it was still possible to immunoprecipitate radiolabeled triple mutant 14/137/271 protein from cells (the Asn295-Phe296-Ser297 N-glycosylation site was weakly glycosylated), whereas the other three triple mutant proteins were entirely secreted into the medium. A significant amount of the unglycosylated angiotensinogen (N-4 mutant) protein was retained in the cells after 6, 24 (Fig.4 A), and 48 h (data not shown) of chase. The amounts of wild-type and N-4 mutant proteins immunoprecipitated from cells are reported in Fig. 4 B. Pulse-chase experiments with a CHO cell line producing the N-4 mutant protein revealed a similar level of retention (15% retained within cells after 24 h of chase) (data not shown). The overall amount of the N-4 mutant protein produced was similar to the amount of the wild-type protein produced (15versus 18 μg/ml, respectively, corresponding to the 72-h production in serum-free medium for 30 × 106cells). The effect of the glycosylation of human angiotensinogen on its hydrolysis by renin was studied using the wild-type and several deglycosylated mutant proteins. The recombinant angiotensinogens were produced in milligram amounts by CHO cells, and the cell supernatants were incubated with 20 pm of pure recombinant human renin, at pH 5.7 for 1 h at 37 °C. The Michaelis-Menten representation for the N14Q, N137Q, N271Q, N295Q, N-4, and wild-type substrates is shown in Fig. 5. The Michaelis constant (Km), maximum velocity (Vmax), catalytic activity (kcat), and catalytic efficiency (kcat/Km) with renin of these proteins are reported in Table I.Table IKinetic constants for the hydrolysis of wild type and N-glycosylation mutants of human angiotensinogen by purified recombinant human reninSubstrateKmVmkcatkcat/Kmμmnmol Ang I · ml−1 · s−1s−1μm−1 · s−1Wild-type (a)1.7 ± 0.255 ± 3.52.8 ± 0.21.6 ± 0.1N14Q mutant (a)0.3 ± 0.016.2 ± 2.80.3 ± 0.11.1 ± 0.4N137Q mutant1.4542.71.9N271Q mutant1.34021.5N295Q mutant0.8281.41.7N-4 mutant (b)0.7 ± 0.162 ± 203.1 ± 15.0 ± 2Data are the means ± S.D. of two (a) or three (b) independent determinations. Km andVm were determined using Lineweaver-Burk representation, and kcat values were obtained by multiplying Vm with renin concentration. Open table in a new tab Data are the means ± S.D. of two (a) or three (b) independent determinations. Km andVm were determined using Lineweaver-Burk representation, and kcat values were obtained by multiplying Vm with renin concentration. The maximum velocity of the reaction with N14Q was 10 times lower than that observed with wild-type and other mutated substrates. However, its affinity for renin was 5 times higher than that of the wild-type, resulting in a catalytic efficiency similar to the wild-type kcat/Km. Thus, the asparagine residue at position 14 plays a key role in the kinetics of the renin-angiotensinogen reaction. In contrast, the kinetic parameters of the N137Q, N271Q, and N295Q hAGT substrates were quite similar to those of the wild-type protein, suggesting that the asparagine residues in positions 137, 271, and 295 had no significant effect on the enzyme reactio
Referência(s)