C-Mannosylation of Human RNase 2 Is an Intracellular Process Performed by a Variety of Cultured Cells
1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês
10.1074/jbc.272.42.26687
ISSN1083-351X
AutoresJoachim Krieg, Wolfgang Gläsner, Anna M. Vicentini, Marie‐Agnès Doucey, Andreas Löffler, Daniel Heß, Jan Hofsteenge,
Tópico(s)Antimicrobial Peptides and Activities
ResumoC 2-α-Mannosyltryptophan was discovered in RNase 2 from human urine, representing a novel way of attaching carbohydrate to a protein. Here, we have addressed two questions related to the biosynthesis of this modification: (i) isC-mannosylation part of the normal intracellular biosynthetic route, and (ii) how general is it, i.e. which organisms perform this kind of glycosylation? To answer the first question, RNase 2, which is identical to the eosinophil-derived neurotoxin, was isolated from intracellular stores of cultured human HL-60 cells. The enzyme was C-mannosylated at Trp-7, showing that the modification occurs intracellularly, before secretion of the protein. The second question was investigated by immunological and chemical analysis of RNase 2 purified from the supernatant of transiently transformed cells from different organisms. This revealed that C-mannosylation occurs in cells from man, green monkey, pig, mouse, and hamster. The observation that pig kidney cells contain the machinery for C-mannosylation of Trp-7 of human RNase 2 but that the homologous RNase from porcine kidney is not a substrate, since it does not contain a tryptophan at position 7, strongly suggests that C-mannosylated proteins other than RNase 2 exist. Recombinant RNase 2 isolated from insect cells, plant protoplasts, and Escherichia coli was notC-mannosylated. These results not only form the basis for further studies on the biochemical aspects ofC-mannosylation but also have implications for the choice of cells for production of recombinant glycoproteins. C 2-α-Mannosyltryptophan was discovered in RNase 2 from human urine, representing a novel way of attaching carbohydrate to a protein. Here, we have addressed two questions related to the biosynthesis of this modification: (i) isC-mannosylation part of the normal intracellular biosynthetic route, and (ii) how general is it, i.e. which organisms perform this kind of glycosylation? To answer the first question, RNase 2, which is identical to the eosinophil-derived neurotoxin, was isolated from intracellular stores of cultured human HL-60 cells. The enzyme was C-mannosylated at Trp-7, showing that the modification occurs intracellularly, before secretion of the protein. The second question was investigated by immunological and chemical analysis of RNase 2 purified from the supernatant of transiently transformed cells from different organisms. This revealed that C-mannosylation occurs in cells from man, green monkey, pig, mouse, and hamster. The observation that pig kidney cells contain the machinery for C-mannosylation of Trp-7 of human RNase 2 but that the homologous RNase from porcine kidney is not a substrate, since it does not contain a tryptophan at position 7, strongly suggests that C-mannosylated proteins other than RNase 2 exist. Recombinant RNase 2 isolated from insect cells, plant protoplasts, and Escherichia coli was notC-mannosylated. These results not only form the basis for further studies on the biochemical aspects ofC-mannosylation but also have implications for the choice of cells for production of recombinant glycoproteins. Post-translational modification of proteins by covalent attachment of carbohydrate is a common and widespread phenomenon. Two kinds of glycosylation have been known for a long time:N-glycosylation, where the sugar residues are linked to Nδ of Asn, and O-glycosylation, where the linkage is to Oγ of Thr or Ser. Both have been extensively characterized with respect to their biosynthetic pathways as well as their distribution in nature. They are found in many organisms, ranging from bacteria to man, and occur in a variety of proteins (1Lis H. Sharon N. Eur. J. Biochem. 1993; 218: 1-27Crossref PubMed Scopus (790) Google Scholar). Recently, a new kind of linkage between a carbohydrate and a protein was discovered in human ribonuclease 2 (RNase 2), namely a C-glycosidically linked mannosyl residue (2Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (232) Google Scholar, 3de Beer T. Vliegenthart J.F.G. Löffler A. Hofsteenge J. Biochemistry. 1995; 34: 11785-11789Crossref PubMed Scopus (96) Google Scholar). In this case the C-1′ atom of the mannosyl residue is directly linked to the C-2 atom of the indole moiety of Trp-7 (Scheme 1). RNase 2 from urine is completely identical in primary structure to eosinophil-derived neurotoxin (EDN), 1The abbreviations used are: EDN, eosinophil-derived neurotoxin; (C 2-Man-)Trp,C 2-α-mannopyranosyltryptophan; ESIMS, electrospray ionization-mass spectrometry; FCS, fetal calf serum; HPLC, high performance liquid chromatography; LC-ESIMS, liquid chromatography interfaced with ESIMS; PAA, polyacrylamide; r-RNase 2, recombinant RNase 2; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. 1The abbreviations used are: EDN, eosinophil-derived neurotoxin; (C 2-Man-)Trp,C 2-α-mannopyranosyltryptophan; ESIMS, electrospray ionization-mass spectrometry; FCS, fetal calf serum; HPLC, high performance liquid chromatography; LC-ESIMS, liquid chromatography interfaced with ESIMS; PAA, polyacrylamide; r-RNase 2, recombinant RNase 2; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. which is located in the cytotoxic granules of the eosinophil and may play a role in the anti-parasitic action of these cells (4Snyder M.R. Gleich G.J. D'Alessio G. Riordan J.F. Ribonucleases, Structures and Functions. Academic Press, Inc., New York1997: 425-444Google Scholar). EDN is a potent neurotoxin, causing muscle stiffness and ataxia when injected intracerebrally into experimental animals (Gordon phenomenon, see Refs. 5Gordon M.H. Br. Med. J. 1933; 1: 641-644Crossref PubMed Scopus (44) Google Scholar and 6Durack D.T. Sumi S.M. Klebanoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1443-1447Crossref PubMed Scopus (163) Google Scholar). This is associated with the loss of Purkinje cells and vacuolation of white matter in the cerebellum, brain stem, and spinal cord (6Durack D.T. Sumi S.M. Klebanoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1443-1447Crossref PubMed Scopus (163) Google Scholar). The putative glycosyltransferase that carries out the modification of RNase 2 must have a considerable degree of specificity as it transfers a mannosyl residue to Trp-7 while fully ignoring the tryptophan at position 10 (2Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (232) Google Scholar). Originally, (C 2Man-)Trp was found in peptides obtained from RNase 2 from human urine. Subsequently, using NMR it was shown in the entire, intact protein (7Löffler A. Doucey M.-A. Jansson A.M. Müller D.R. de Beer T. Hess D. Meldal M. Richter W.J. Vliegenthart J.F.G. Hofsteenge J. Biochemistry. 1996; 35: 12005-12014Crossref PubMed Scopus (66) Google Scholar). Since the modification was also found in RNase 2 isolated from human erythrocytes, it was concluded that C-mannosylated RNase 2 from urine does not represent a metabolized form of the excreted protein but that it constitutes a genuine post-translational modification (7Löffler A. Doucey M.-A. Jansson A.M. Müller D.R. de Beer T. Hess D. Meldal M. Richter W.J. Vliegenthart J.F.G. Hofsteenge J. Biochemistry. 1996; 35: 12005-12014Crossref PubMed Scopus (66) Google Scholar). It could be argued, however, that human erythrocytes are not representative for other types of cells, because they lack a nucleus and other major cell organelles. As a result of the absence of protein synthesis, erythrocyte proteins are not replaced and age. This raises the possibility that C-mannosylation of RNase 2 in erythrocytes results from an aging process, in contrast to proteinN- and O-glycosylation, which is part of the biosynthetic route of newly made proteins. We have addressed this question by isolating RNase 2 from cultured human promyelocytic cells (HL-60) and examining its C-mannosylation status. In contrast to N- and O-glycosylation, knowledge about the organisms in which C-mannosylation occurs is lacking, because so far only RNase 2 from human sources has been analyzed. This issue is of importance for further studies on the biosynthesis of (C 2-Man-)Trp and in the search for other proteins containing this modification. In addition, it may also have practical implications for the choice of appropriate cells for the production of recombinant proteins with or without this modification. Therefore, we have expressed human RNase 2 in cells from a variety of organisms. The secreted proteins were purified to near homogeneity and analyzed with an antibody specific for (C 2-Man-)Trp of RNase 2, as well as by mass spectrometry and Edman degradation of purified peptides. Human RNase 1 and 2 were purified as described (2Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (232) Google Scholar, 7Löffler A. Doucey M.-A. Jansson A.M. Müller D.R. de Beer T. Hess D. Meldal M. Richter W.J. Vliegenthart J.F.G. Hofsteenge J. Biochemistry. 1996; 35: 12005-12014Crossref PubMed Scopus (66) Google Scholar). Bovine pancreatic RNase B and keyhole limpet hemocyanin were obtained from Sigma. The protease from Staphylococcus aureus was purchased from Promega, Madison, WI, and thermolysin and N-glycosidase F were from Boehringer Mannheim, Germany, and CH-activated and protein A-Sepharose were from Pharmacia, Uppsala, Sweden. Cell culture media and FCS were from Life Technologies, Inc. Antibodies against human RNase 2 have been described previously (7Löffler A. Doucey M.-A. Jansson A.M. Müller D.R. de Beer T. Hess D. Meldal M. Richter W.J. Vliegenthart J.F.G. Hofsteenge J. Biochemistry. 1996; 35: 12005-12014Crossref PubMed Scopus (66) Google Scholar). Antibodies specific for RNase 2 containing (C 2-Man-)Trp at position 7 were obtained by immunizing New Zealand White rabbits with the thermolytic peptide-(5–10) (2Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (232) Google Scholar) coupled with glutaraldehyde to keyhole limpet hemocyanin (350 μg peptide/mg hemocyanin). An initial injection of 430 μg of conjugate in Freund's complete adjuvant was given, followed by 2 booster injections with 430 μg of conjugate in Freund's incomplete adjuvant after 1 and 2.5 months. Specific antibodies were purified as described (7Löffler A. Doucey M.-A. Jansson A.M. Müller D.R. de Beer T. Hess D. Meldal M. Richter W.J. Vliegenthart J.F.G. Hofsteenge J. Biochemistry. 1996; 35: 12005-12014Crossref PubMed Scopus (66) Google Scholar). SDS-polyacrylamide gel electrophoresis and Western blotting were performed as described (7Löffler A. Doucey M.-A. Jansson A.M. Müller D.R. de Beer T. Hess D. Meldal M. Richter W.J. Vliegenthart J.F.G. Hofsteenge J. Biochemistry. 1996; 35: 12005-12014Crossref PubMed Scopus (66) Google Scholar). HL-60 cells (ATCC 240-CCl) were grown in RPMI 1640 medium, containing 15% FCS. All isolation procedures were performed at 4 °C, and buffers for extraction and chromatography contained a mixture of protease inhibitors, leupeptin (0.2 μg/ml), benzamidine HCl (2 μg/ml), pepstatin A (0.2 μg/ml), p-methanesulfonyl fluoride (0.2 mm), unless indicated otherwise. Harvested cells (7.5 × 109) were washed twice with phosphate-buffered saline, and RNase 2 was extracted with 380 ml of 0.1% trifluoroacetic acid for 2 h with stirring. After centrifugation at 2000 × rpm in a table-top centrifuge for 4 min at room temperature, the supernatant was dialyzed against 20 mm Bis-Tris-HCl, pH 6.0, and loaded onto a column of SP-Sepharose (1.5 × 11 cm) equilibrated in the same buffer. The column was washed, and proteins were eluted with 0.5m NaCl in the same buffer. The fractions containing RNase 2 were diluted 10-fold with 20 mm Bis-Tris-HCl, pH 6.0, and loaded onto a column of heparin-Sepharose (1 × 7.5 cm) equilibrated in the same buffer. The column was washed, and RNase 2 was eluted with 0.6 m NaCl in the same buffer. After 5-fold dilution with 20 mm Tris-HCl, pH 7.5, the RNase 2 containing fractions were purified by immunoaffinity chromatography. An immunoaffinity column was prepared by covalently cross-linking RNase 2-specific antibodies to protein A-Sepharose using dimethylpimelimidate (8Schneider C. Newman R.A. Sutherland D.R. Asser U. Greaves M.F. J. Biol. Chem. 1982; 257: 10766-10769Abstract Full Text PDF PubMed Google Scholar). RNase 2 was bound by recycling over the column for 18 h. The column was washed with 20 mm Tris-HCl, pH 7.5, containing 0.5 m NaCl, followed by 20 mm Tris-HCl, pH 7.5. RNase 2 was eluted with 100 mm glycine, pH 2.8, and neutralized immediately with 1 m Tris base. The fractions containing RNase 2 from the previous step were made 0.1% in trifluoroacetic acid and purified by reversed phase HPLC on a C4 column (1 or 2.1 mm diameter Vydac, Hispania, CA) (solvent A, 0.1% trifluoroacetic acid). A linear gradient of 15–60% solvent B (70% CH3CN, 0.085% trifluoroacetic acid) over 90 min was used at a flow rate of 0.05 or 0.2 ml/min. No protease inhibitors were added in this step. A synthetic gene coding for human RNase 2 in pET11dedn (a gift from Dr. R. J. Youle, National Institutes of Health, Bethesda) was used in all experiments (9Newton D.L. Nicholls P.J. Rybak S.M. Youle R.J. J. Biol. Chem. 1994; 269: 26739-26745Abstract Full Text PDF PubMed Google Scholar). It was modified using polymerase chain reaction-based mutagenesis to introduce the RNase 2 signal sequence for secretion (MVPKLFTSQICLLLLLGLLAVEGSLHV-) and XbaI, SalI,BglII, and BamHI restriction sites on the 3′-side of the stop codon. The mutated edn gene was subcloned into pBluescript (pBluescriptedn). From there theEcoRI/BglII fragment, containing the entire pre-RNase 2 coding sequence, was cloned into a pSMC-type of expression vector, which yielded pSMCi99edn. This vector contains an SV40 origin of replication and the cytomegalovirus immediate early promoter/enhancer (10Asselbergs F.A. Grand P. Anal. Biochem. 1993; 209: 327-331Crossref Scopus (15) Google Scholar). The EcoRI/BamHI fragment obtained from pSMCi99edn was cloned into the expression plasmids pVL1392 and pAcMP2 (Pharmingen, San Diego, CA). The constructs, pVL1392edn and pAcMP2edn, encode pre-RNase 2 with its own signal sequence and contain the very late polyhedrin and late basic protein baculovirus promoter, respectively. The same fragment was cloned into pBluescript and from there into the expression vector pBD1119 (a gift from Dr. B. Dickson, University of Zürich), using KpnI and XbaI restriction sites. The plasmid, pBDssedn, contained the pre-RNase 2 coding region and the constitutive D. melanogasterα-tubulin promoter. An MscI and BamHI restriction site was introduced on the 5′- and 3′-side of the edn gene in pET11dedn, respectively. TheMscI/BamHI fragment was cloned into pASK60-strep (11Schmidt T.G. Skerra A. Protein Eng. 1993; 6: 109-122Crossref PubMed Scopus (253) Google Scholar), which had been cut with StuI and BamHI. The plasmid, pASKedn, contained the RNase 2 coding region, in frame with the ompA signal sequence. An NcoI site was introduced at the start codon of the RNase 2 gene in pBluescriptedn, and theNcoI/BamHI fragment was obtained. The plant expression plasmid p35S′-GUS (12Hohn T. Corsten S. Rieke S. Müller M. Rothnie H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8334-8339Crossref PubMed Scopus (76) Google Scholar) was cut with eitherNcoI/Acc65I orAcc65I/BamHI. The three fragments were joined in a three-way ligation, yielding the plasmid p35S′edn, which contained the pre-RNase 2 coding region and the cauliflower mosaic virus 35 S promoter/leader. The sequence of all constructs was verified by dideoxy sequencing (13Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar). HEK293 (ATCC CRL 1573), COS7, LLC-PK1 (ATCC CRL 1392), and CHO cells were grown in Dulbecco's modified Eagle's medium containing 10% FCS. HEK 293, COS7, and CHO cells were transfected with pSMCi99edn (10 μg/10 cm plate) using the Transfection MBS kit (Stratagene). After transfection, the medium was replaced by Dulbecco's modified Eagle's medium containing 0.5% FCS (HEK 293 cells) or 5% FCS (COS7 and CHO cells). LLC-PK1 and NIH 3T3 cells were transfected with pSMCi99edn, using LipofectAMINE (Life Technologies, Inc.). After transfection, the medium was supplemented with either 10% FCS or 10% newborn calf serum. Conditioned media were collected 2 and 6 days post-transfection and stored at −80 °C. The medium was passed over a Sepharose Q column (2.5 × 10 cm) equilibrated in 20 mm Tris-HCl, pH 7.5. r-RNase 2 appeared in the flow-through and was purified by immunoaffinity chromatography and C4 reversed phase HPLC (gradient 0–80% solvent B in 75 min) as described for RNase 2/HL-60, except that no protease inhibitors were added to the buffers. Log phase Sf9 cells were co-transfected with 5 μg of plasmid pVL1392edn or pAcMP2edn and 0.5 μg of linearized BaculoGoldTM DNA (Pharmingen). Cells were incubated at 27 °C for 5 days, and the recombinant virus was amplified 3 times. For large scale protein production, virus-infected Sf9 cells were grown in serum-free medium (SF 900 II, Life Technologies, Inc.). Conditioned medium was harvested after 8 days and stored at −80 °C. The medium was centrifuged and dialyzed against 20 mm Bis-Tris-HCl, pH 6.0, and the RNase was purified essentially as described for RNase 2/HL-60. However, the heparin-Sepharose column was omitted; a 0.25–1 m NaCl gradient was used to elute the enzyme from the SP-Sepharose column, and no protease inhibitors were added to the buffers. Drosophila melanogaster Schneider 2 cells (14Schneider I. J. Exp. Zool. 1964; 156: 91-103Crossref PubMed Scopus (148) Google Scholar) in Schneider II medium (Life Technologies, Inc.), containing 10% heat-inactivated fetal calf serum, were grown in 10-cm tissue culture plates at 25 °C and transfected with 20 μg of pBDssedn using calcium phosphate precipitation. Twelve hours post-transfection serum-free medium was added (SF 900 II medium, Life Technologies, Inc.). The supernatant was harvested 3 days thereafter, and stored at −80 °C. r-RNase 2/Schneider 2 was purified as described for r-RNase 2/Sf9. Protoplasts were prepared from suspension cultures ofOrychophragmus violaceous, transformed with p35S′edn (5 μg/2.1 × 106cells, in 0.7 ml) by electroporation, and transferred to a Petri dish with 10 ml of medium A (15Goodall G.J. Wiebauer K. Filipowicz W. Methods Enzymol. 1990; 181: 148-160Crossref PubMed Scopus (149) Google Scholar). After 48 h the medium was collected. r-RNase 2 was isolated as described for the enzymes from mammalian cells. Escherichia coli strain BL21 (DE3) (Stratagene, La Jolla, CA) harboring plasmid pET11dedn was grown in LB medium containing ampicillin (100 μg/ml) at 37 °C. Expression of RNase 2 was induced with 500 μmisopropyl-β-d-thiogalactoside, and bacteria were harvested after 3 h. Cells were resuspended in 50 mmTris-HCl, pH 7.4, containing 1 mm EDTA, 100 mmNaCl, and lysed in a French pressure cell at 10,000 p.s.i. Inclusion bodies were collected by centrifugation and cleaned up by two sequential wash/centrifugation steps with 1% Nonidet P-40 and 1m urea. Ten mg of RNase 2 in inclusion bodies were incubated for 2 h at room temperature in 10 ml of 0.1 m Tris-HCl, pH 8.0, containing 2 mm EDTA, 6 m guanidine hydrochloride, 200 mm dithiothreitol. Insoluble material was removed by centrifugation, and the supernatant was diluted 100-fold into a stirred solution of 0.1 m Tris-HCl, pH 8.0, containing 2 mm EDTA, 0.5 ml-arginine, 5.6 mm oxidized glutathione. Refolding proceeded for at least 48 h at 25 °C. The refolding solution was dialyzed against 20 mm Bis-Tris-HCl, pH 6.0, and chromatographed on heparin-Sepharose as described for RNase 2/HL-60, except that no protease inhibitors were added to the buffers. The column was washed with 250 mm NaCl and eluted with 1m NaCl in the same buffer. RNase 2 containing fractions were dialyzed against 20 mm Bis-Tris-HCl, pH 6.0, and applied to a 1-ml Mono S column (Pharmacia) with a flow of 0.5 ml/min. RNase 2 was eluted using a linear NaCl gradient (150–600 mm). RNase 2 containing fractions were pooled, dialyzed against 50 mm NH4HCO3, and stored at −80 °C. E. coli strain KS474 (16Strauch K.L. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1576-1580Crossref PubMed Scopus (292) Google Scholar) harboring plasmid pASKedn was grown in LB medium containing ampicillin (100 μg/ml) at 37 °C, and expression of RNase 2 was induced with 160 μm isopropyl-β-d-thiogalactoside. After 3 h cells were harvested and lysed as described above. Insoluble material was removed by centrifugation, and r-RNase 2/E. coli2 was purified as described for r-RNase 2/Sf9. All RNases were active in an assay using yeast RNA as the substrate (17Fominaya J.M. Garcı́a-Segura J.M. Gavilanes J.G. Biochim. Biophys. Acta. 1988; 954: 216-223Crossref PubMed Scopus (8) Google Scholar). Lyophilized RNase 2 (0.5–1.0 μg) in 35 μl of 50 mm Hepes-NaOH, pH 7.8, containing 10 mm CaCl2 was digested with thermolysin (10% w/w) at 75 °C for 60 min. Peptides were fractionated by reversed phase HPLC on a 2.1-mm diameter C18 column (solvent A, 0.1% trifluoroacetic acid). A linear gradient of 20–41% solvent B (0.08% trifluoroacetic acid, 70% CH3CN) over 50 min at a flow rate of 0.2 ml/min was used. Peptides were detected at 214 nm. The percentage modification of a particular RNase 2 was calculated from the ratio of the peaks of the modified and unmodified peptides, using a calibration curve obtained by digesting and fractionating mixtures containing different ratios of RNase 2/urine and r-RNase 2/E. coli1. LC-ESIMS was performed using an Applied Biosystems model 140 chromatograph equipped with a 0.3-mm diameter C8 column and interfaced with a Sciex API III mass spectrometer (18Hofsteenge J. Löffler A. Müller D.R. Richter W.J. de Beer T. Vliegenthart J.F.G. Marshak D.R. Techniques in Protein Chemistry VII. Academic Press, Inc., New York1996: 163-171Google Scholar) operating in the multi-ion monitoring mode at m/z = 691.5, 838.5, and 1000.5. Methods for protein reduction, carboxymethylation, proteolytic cleavage, solid phase Edman degradation, and ESIMS have been described (2Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (232) Google Scholar, 18Hofsteenge J. Löffler A. Müller D.R. Richter W.J. de Beer T. Vliegenthart J.F.G. Marshak D.R. Techniques in Protein Chemistry VII. Academic Press, Inc., New York1996: 163-171Google Scholar). To be able to analyze the state ofC-mannosylation of small amounts of RNase 2 (1–2 μg) from cultured cells, two analytical tools were established. First, modification-specific antibodies were raised against the peptide comprising residues 5–10 of RNase 2 (FT(C 2Man-)WAQW). The affinity purified α-(5–10) antibodies recognized RNase 2 from human urine (RNase 2/urine; Fig. 1 A, lane 1) but not recombinant RNase 2 (r-RNase 2; Fig. 1 A, lane 2) isolated from the inclusion bodies of E. coli with unmodified Trp at position 7 (r-RNase 2/E. coli1, see below). To exclude that the α-(5–10) antibodies cross-reacted with mannosyl residues in one of the N-glycans of RNase 2/urine, the fragment containing these glycans (residues 13–134) was produced by proteolytic cleavage at Glu-12 and examined by Western analysis. As cleavage of RNase 2/urine proceeded, the signal produced by the α-(5–10) antibodies was lost (Fig. 1 B, lower panel) but that by the αRNase 2 antibodies remained (Fig. 1 B, upper panel). These experiments showed that the α-(5–10) antibodies recognized an epitope located in peptide-(1–12). Because RNase 2/urine and r-RNase 2/E. coli1 differ in this region only with respect to C 2-mannosylation of Trp-7, it was concluded that RNase 2 is only recognized by the α-(5–10) antibodies when it is C-mannosylated at this residue. RNase 2/urine contains abnormally small N-glycans (19Beintema J.J. Hofsteenge J. Iwama M. Morita T. Ohgi K. Irie M. Sugiyama R.H. Schieven G.L. Dekker C.A. Glitz D.G. Biochemistry. 1988; 27: 4530-4538Crossref PubMed Scopus (85) Google Scholar); therefore, it was of interest to examine other, more heavily N-glycosylated RNases. Fig. 1 A (lanes 3 and 4) shows that neither mannosyl residues in the N-glycans of human RNase 1 (20Ribo M. Beintema J.J. Osset M. Fernandez E. Bravo J. de Llorens R. Cuchillo C.M. Biol. Chem. Hoppe-Seyler. 1994; 375: 357-363PubMed Google Scholar) nor of bovine pancreatic RNase B were recognized. The antibodies seemed to recognize (C 2-Man-)Trp in the context of RNase 2 only, since they did not bind to other proteins in total cell extracts (data not shown). This was most likely due to amino acid residues adjacent to Trp-7 being part of the epitope. We found that Ala-8 and Gln-9 were required for antibody binding. These two residues were not required for C-mannosylation of Trp-7 and therefore do not necessarily occur adjacent toC-mannosylated Trp in other proteins. 2J. Krieg, unpublished results. Second, a micro-method was developed for quantitating the degree ofC-mannosylation, using RNase 2/urine (fullyC-mannosylated at Trp-7) and fully unmodified r-RNase 2/E. coli1. Thermolytic digestion of RNase 2/urine and fractionation of the peptides by C18 reversed phase HPLC yielded C-mannosylated peptide-(5–10) (Fig.2 A, peak b). Cleavage of r-RNase 2/E. coli1 resulted in the formation of two peptides (Fig. 2 B, peaks a and c), which were examined by LC-ESIMS. Peak a ((M + H)+ = 691.5) was assigned to residues 6–10 (TWAQW), whereas peak c ((M + H)+ = 838.5) contained residues 5–10 (FTWAQW). Digestion of mixtures containing RNase 2/urine and r-RNase 2/E. coli1 resulted in all three peptides (not shown). By varying the molar ratios of the two RNases, a calibration curve was obtained that related the mole fraction of (C 2-Man-)Trp in the protein mixture to the relative area of peak b (Fig. 2 C). The hyperbolic shape of this curve resulted mainly from the difference in extinction coefficient between (C 2-Man-)Trp and Trp (18Hofsteenge J. Löffler A. Müller D.R. Richter W.J. de Beer T. Vliegenthart J.F.G. Marshak D.R. Techniques in Protein Chemistry VII. Academic Press, Inc., New York1996: 163-171Google Scholar). To examine whether cells that actively divide and synthesize proteins carry outC-mannosylation, RNase 2 was purified from the human promyelocytic cell line HL-60, which yielded 10 μg of RNase 2 from 7.5 × 109 cells (21% recovery). The protein migrated on SDS-PAA gels as a broad smear (Fig.3 A, lane 1), which in a subclone of HL-60 cells has been attributed to heterogeneity ofN-linked glycans (21Tiffany H.L. Li F. Rosenberg H.F. J. Leukocyte Biol. 1995; 58: 49-54Crossref PubMed Scopus (25) Google Scholar). 3In this publication RNase 2 has been indicated with eosinophil-derived neurotoxin. This was confirmed for the cells used here by treatment with N-glycosidase F, which resulted in RNase 2/HL-60 that co-migrated with unglycosylated r-RNase 2/E. coli1 (Fig. 3 A, lanes 2 and 3). Western analysis of N-glycosidase F-treated RNase 2/HL-60 using the α-(5–10) antibodies gave a positive result (Fig. 3 A, lane 5), with RNase 2/urine and r-RNase 2/E. coli1 serving as the positive and negative controls, respectively (Fig.3 A, lanes 4 and 6). This demonstrated the presence of (C 2-Man-)Trp in RNase 2/HL-60. The position of the mannosylated Trp in the protein was established by chemical analyses. Comparison of the thermolytic peptide maps of RNase 2/HL60 and RNase 2/urine by LC-ESIMS in the single ion-monitoring mode at m/z = 1000.5 demonstrated the presence of modified peptide-(5–10) (Fig. 3, B and C, upper traces). The sequence of this peptide was determined by Edman degradation to be FT(C 2-Man-)WAQW. In addition, a small amount of unmodified peptides-(5–10) and -(6–10) was detected by LC-ESIMS atm/z = 838.5 and 691.5 (Fig. 3, B andC, lower and middle traces). Quantitation by the method described above showed that 90% of the RNase 2/HL-60 molecules contained (C 2-Man-)Trp at position 7 (TableI).Table IC-Mannosylation of human RNase 2 from cultured cellsSourceOrganismPeptide (5–10)ProteinPhenylthiohydantoin-(C 2-Man-)TrpLC-ESIMSm/z = 1000.5Binds to α-(5–10) antibodies(C 2-Man-)Trp%UrineMan+++100Cells HL-60Man+++90 HEK293Man+++73 COS7Monkey+++68 LLC-PK1Pig+++55 CHOHamster+++49 3T3Mouse+++81 Sf9Insect−−1-bAt m/z = 838.5 and 691.5 (see Fig. 2), the unmodified peptides-(5–10) and -(6–10) were detected.−0 Schneider 2Insect−−1-bAt m/z = 838.5 and 691.5 (see Fig. 2), the unmodified peptides-(5–10) and -(6–10) were detected.−0 ProtoplastsPlant−−1-bAt m/z = 838.5 and 691.5 (see Fig. 2), the unmodified peptides-(5–10) and -(6–10) were detected.−0E. coli11-aE. coli1 and -2 refer to the enzyme isolated from inclusion bodies and the periplasmic space, respectively.−−1-bAt m/z = 838.5 and 691.5 (see Fig. 2), the unmodified peptides-(5–10) and -(6–10) were detected.−0E. coli2−−1-bAt m/z = 838.5 and 691.5 (see Fig. 2), the unmodified peptides-(5–10) and -(6–10) were detected.−01-a E. coli1 and -2 refer to the enzyme isolated from inclusion bodies and the periplasmic space, respectively.1-b At m/z = 838.5 and 691.5 (see Fig. 2), the unmodified peptides-(5–10) and -(6–10) were detected. Open table in a new tab Treatment of HL-60 cells with butyric acid leads to differentiation toward eosinophils (22Fischkoff S.A. Pollak A. Gleich G.J. Testa J.R. Misawa S. Reber T. J. Exp. Med. 1984; 60: 179-196Crossref Scopus (101) Google Scholar, 23Fischkoff S.A. Brown G.E. Pollak A. Blood. 1986; 68: 185-192Crossref PubMed Google Scho
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