Calreticulin Biosynthesis and Processing in Human Myeloid Cells : Demonstration of Signal Peptide Cleavage and N-Glycosylation

Calreticulin is a soluble endoplasmic reticulum protein comdodecyl sulfate-polyacrylamide gel electrophoresis analysis, prising the major storage reservoir for inositol trisphossuggesting that the potential site for N-glycosylation at asphate-releasable calcium. Although its highly conserved priparagine-327 was unmodified. However, oxidative derivatimary structure and a wide range of functions have been well zation of carbohydrate components with digoxigenin described, less attention has been paid to its biosynthesis, showed that human calreticulin produced in either HL-60 particularly in human tissues. We report analyses of synthecells or Sf9 insect cells is glycosylated, indicating that glycosis, proteolytic processing and glycosylation of human calsylated and nonglycosylated human calreticulin have indisreticulin. In both HL-60 and PLB-985 myeloid cell lines tinguishable electrophoretic mobilities. Direct measurement calreticulin was immunoprecipitated as a single 60-kD speby phenol-H2SO4 confirmed the presence of carbohydrate cies without evidence of precursor forms. However, in vitro on recombinant human calreticulin. These data show that cell-free synthesis produced a 62-kD primary translation human myeloid calreticulin undergoes cotranslational signal product, which in the presence of microsomal membranes, peptide cleavage and posttranslational N-linked glycosylawas processed by cotranslational signal peptide cleavage to tion. Although glycosylation of calreticulin has been shown a 60-kD species that comigrated with mature calreticulin in rat liver and bovine liver and brain, it has been reported produced in myeloid cells. Neither tunicamycin treatment of to be lacking in other tissues including human lymphocytes. the cells nor endoglycosidase digestion of calreticulin req 1997 by The American Society of Hematology. sulted in any forms other than the 60-kD protein on sodium


C
and the association of calreticulin with these receptors thereby inhibits their binding to the promoter elements of ALRETICULIN is a high-capacity calcium-binding protein comprising the major intracellular storage reservoir for inositol-1,4,5 trisphosphate-releasable calcium in steroid-responsive genes. 14,15Calreticulin is present in cytotoxic granules of T lymphocytes where it may regulate cal-most nonmuscle cells. 1-4Its structure is highly conserved and its range of expression is extremely broad with respect to cium-sensitive cytolysin activity. 168][19][20] In cells infected with rubella virus calreticulin binds to viral RNA, thereby modu-regulin, ERp60, CRP55, CAB-63, CaBP3, calsequestrin-like protein), calreticulin has a single high-affinity and multiple lating viral replication. 21,22Calreticulin on the cell surface may serve as a lectin that triggers melanoma cell spreading. 23low-affinity calcium binding sites. 1,8Although the calciumbinding properties of calreticulin have been known for some Binding of calreticulin to various coagulation factors interferes with blood clotting. 24We have shown that calreticulin time, recent work in many systems has established a wide range of other functions not directly related to calcium stor-functions as a molecular chaperone for newly synthesized myeloperoxidase in myeloid cells 25 and a similar function age, as well as localizations of the protein outside of the endoplasmic reticulum in keeping with some of its novel has been recently reported for calreticulin in other cell systems. 26][11] Binding of calreticulin to a conserved cytoplasmic domain Despite calreticulin's broad range of functions and widespread expression, its biosynthesis and processing have re-of integrin a subunits may be involved in regulation of cell shape and interaction with the extracellular matrix. 12,13Ste-ceived relatively little attention.The cDNA sequence predicts a typical N-terminal signal sequence and, depending roid hormone receptors bind DNA through a peptide domain very similar to the calreticulin-binding sequence of integrins on species, one or two sites of potential N-linked glycosylation. 1,6,7,17,27,28In the current work we cloned the cDNA for human myeloid cell calreticulin, developed a high-level ex- From the Department of Medicine, Department of Veterans Affairs pression system in insect cells, and generated a high-affinity Medical Center and University of Iowa, Iowa City; and the Departpolyclonal antibody against the purified recombinant protein.
ment of Medicine, South Texas Veterans Health Care System, Audie These reagents were then used to characterize calreticulin Zyma (Nyon, Switzerland).Address reprint requests to Robert A. Clark, MD, Department of MATERIALS AND METHODS Medicine, University of Texas Health Science Center, 7703 Floyd  Curl Dr, San Antonio, TX 78284-7870.Calreticulin cloning, expression, purification, and antibody preparation.The cDNA for human myeloid calreticulin was cloned from The publication costs of this article were defrayed in part by page charge payment.This article must therefore be hereby marked a previously described HL-60 cell cDNA library in the l-Zap bacteriophage vector (Stratagene, La Jolla, CA). 29The library was ''advertisement'' in accordance with 18 U.S.  The viral clone producing the largest amount of recombinant methionine codon has an appropriate consensus sequence for a eukaryotic initiator site (CCGCCATGC).The predicted protein is 417 protein was optimized for multiplicity of infection (approximately 10:1) and time of cell culture (3 days).Large batches of infected amino acids in length, which based on N-terminal sequencing of mature calreticulin, 1,17,30,31 includes a 17-residue signal sequence.
Sf9 cells (usually 6 flasks of 225 cm 2 with a total of about 10 9 cells) were cultured, procured by pipette disruption of the monolayer, There is a single potential site for N-linked glycosylation at asparagine-327 (of the mature protein) within the motif NET.Comparing collected at 500g for 6 minutes, washed in phosphate-buffered saline (PBS) pH 7.2 and resuspended in 10 to 20 mL of lysis buffer (relax-the sequence to the calreticulin cDNA initially reported as that of the human Ro/SS-A autoantigen, 17 there are several polymorphisms, ation buffer 34 with 12.5 mmol/L EGTA, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 0.5% NP-40).The cells were then kept but the predicted amino acid sequence is identical.As previously noted the conservation of amino acid sequence among human, rabbit, on ice for 10 minutes and vortexed for about 5 seconds once each minute.The lysate was cleared by centrifugation at 200g, 4ЊC for 6 rat, and murine calreticulins is 92% to 94%. 1 Recombinant calreticulin was expressed in the baculovirus system minutes.The supernatant was subjected to chromatography on a DEAE-Sepharose (Pharmacia, Piscataway, NJ) column that was (Invitrogen Corp, San Diego, CA).The full-length cDNA was excised from pBluescript and cloned into the EcoRI site of the equilibrated and run at 4ЊC with 0.1 mol/L potassium phosphate buffer pH 7.1 plus 1 mmol/L EDTA, 0.02% NaN 3 , and a linear pVL1393 baculovirus transfer vector.Forward orientation of the insert was shown by restriction enzyme mapping.Spodoptera frugi-gradient from 50 to 550 mmol/L NaCl.Fractions containing the 60-kD recombinant protein (see Fig 1A) were dialyzed against 20 mmol/ perda Sf9 cells were cotransfected with the transfer vector and linearized baculovirus DNA according to the manufacturer's directions.
L Tris-HCl pH 7.5, lyophilized, and dissolved in running buffer for chromatography on a Sephacryl S-300 (Pharmacia) column run at Recombinant plaques were identified by microscopic visualization and carried through three cycles of plaque purification.Of eight 4ЊC with 20 mmol/L Tris-HCl pH 7.5 plus 0.15 mol/L KCl.Fractions containing the recombinant protein were pooled, dialyzed against clones selected for further study, three induced the synthesis in Sf9 cells of a prominent 60-kD protein (Fig 1A) that stained blue with 10 mmol/L Tris-HCl pH 7.5, lyophilized, and stored at  (Fig 1D).Native human calreticulin was purified from HL-moieties the protein was incubated in Endo F buffer with or without one U of N-glycosidase F for 4.5 hours at 37ЊC before labeling with 60 cells by the method of Krause et al. 32 A polyclonal antibody (presently available from Affinity Biore-digoxigenin.To confirm that the 60-kD digoxigenin-labeled protein was calreticulin the samples were labeled with digoxigenin and then agents, Golden, CO) to the purified recombinant calreticulin was raised in a rabbit by subcutaneous injection every 3 to 4 weeks of immunoprecipitated with anticalreticulin, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibody to about 50 mg of protein in incomplete Freund's adjuvant.The antiserum recognizes a single 60-kD species in immunoblots of lysates of either calreticulin or digoxigenin.
Phenol-sulfuric acid quantitation of carbohydrate.The carbohy-Sf9 cells expressing calreticulin (Fig 1C), human neutrophils, and cultured human myeloid cell lines HL-60 and PLB-985.Immunopre-drate content of proteins was directly measured by the phenol-sulfuric acid method 42 using standard curves run with dextrose.Fetuin cipitation of biosynthetically labeled cells yields a prominent 60 kD species and in some experiments a trace 90-kD protein (see Results).
and transferrin served as control glycoproteins and bovine serum albumin as a nonglycosylated protein (all from Sigma).Samples of Protein biosynthesis in cultured cell lines.The human myeloid leukemia cell lines HL-60 35 and PLB-985 36 were cultured and bio-0.5 mg of protein in 0.5 mL of PBS were mixed with 0.5 mL aqueous phenol.Color was developed by rapid addition of 2.5 mL synthetic labeling was performed as we have reported in detail 25,37 using a 30-minute preincubation in methionine-free medium fol-concentrated sulfuric acid, followed by incubation at 50ЊC for 15 minutes, cooling and determination of absorbance at 490 nm.lowed by pulse-labeling with 35 S-methionine (Ç1,000 Ci/mmol; Amersham, Arlington Heights, IL).The duration of the pulse and subse-Replicate experiments.In all cases the experiments shown are representative of the results obtained in at least 3 separate studies.quent chases with excess unlabeled methionine are indicated in individual protocols.Following labeling the cells were disrupted in RESULTS antiprotease buffer and labeled proteins were immunoprecipitated with whole antisera to either calreticulin (above) or myeloperoxi-Biosynthesis of calreticulin by HL-60 cells.Cultured dase 37 using our previously described protocol. 25Immunoprecipitates HL-60 myeloid cells were pulse-labeled with 35  sults were obtained with PLB-985 cells (data not shown, but tunicamycin induces differentiation, 38,39 the period of preincubation with the inhibitor was limited to 4 hours.In other studies the immu-see Fig 4).In an attempt to detect an early precursor of the noprecipitated calreticulin and myeloperoxidase were treated at 37ЊC mature 60-kD species, pulses as short as 2 minutes were used for 16 hours in Endo F buffer (0.1 mol/L sodium phosphate buffer (Fig 2B).However, only the 60-kD product was observed, pH 6.5 containing 50 mmol/L EDTA, 0.5% NP-40, 0.2% SDS) suggesting that cleavage of the 17-amino acid signal peptide plus 1% b-mercaptoethanol with either endoglycosidase H or Noccurs cotranslationally.Further evidence for this and for glycosidase F (Boehringer Mannheim) before gel electrophoresis as the lack of any other posttranslational processing was the we have described. 40nding that postlabeling chases of up to 2 hours with cold Cell-free biosynthesis.Cell-free protein synthesis was carried methionine did not result in the appearance of any other out in the rabbit reticulocyte lysate system (Promega, Madison, WI) forms of calreticulin, at least as detectable in the SDS-PAGE in the presence of 35 S-methionine according to the manufacturer's system (Fig 2C).In some anticalreticulin immunoprecipidirections and our published technique. 37The reaction mixture included no RNA (control sample), poly-A-enriched RNA prepared tates a 90-kD species was detected (eg, Fig 2A and C).We from HL-60 cells using the FastTrack System (Invitrogen), or synhave recently shown that this coprecipitating protein is the thetic transcripts generated from the calreticulin cDNA in pBlueapopro form of myeloperoxidase and that calreticulin funcscript using T3 polymerase and with polyadenylation and capping tions as a chaperone for myeloperoxidase. 25 the presence of 0.5 mmol/L G(5)ppp(3)G (Promega).In some Cell-free synthesis of calreticulin.The in vitro rabbit experiments dog pancreatic microsomes (Promega) were added to reticulocyte lysate system was used to characterize the pristudy processing of the nascent proteins.mary translation product of the calreticulin transcript.Since Digoxigenin labeling of glycoproteins.The glycosylation state we have previously used this system to study myeloperoxiof calreticulin was analyzed by covalent labeling of glycoproteins dase synthesis, 37 calreticulin and myeloperoxidase were anawith digoxigenin 41 using manufacturer's method A of the Glycan lyzed in parallel.In the presence of HL-60 RNA the products Detection Kit (Boehringer Mannheim).Fetuin (Sigma, St Louis, MO) was used as a control glycoprotein.Briefly, Ç1 mg of protein of cell-free synthesis immunoprecipitated by antiserum to was dissolved in 60 mL of 0.1 mol/L sodium acetate buffer pH 5.5.Collectively, the data using the cell-free translation system indicate that the primary translation product of ing that deglycosylation had occurred under the conditions used, as previously described. 40calreticulin mRNA is a 62-kD species, which in the presence of microsomal membranes is converted to the mature 60-kD The foregoing results suggested that human myeloid cell calreticulin lacks N-linked carbohydrate side chains.How-protein by signal peptide cleavage.
Analysis of glycosylation of calreticulin.The cDNA for ever, the alternative explanation that glycosylated and nonglycosylated calreticulin exhibit nearly identical electropho-human calreticulin predicts a single potential site for Nlinked glycosylation at asparagine-327.Whether calreticulin retic mobilities could not be excluded.Thus, we looked for direct evidence of glycosylation through oxidation and cova-is indeed N-glycosylated appears to vary among species and tissues, 1,30 so we sought to determine if human myeloid cell lent derivatization of carbohydrate moieties with digoxigenin.Fetuin (control glycoprotein) and calreticulin purified calreticulin is a glycoprotein.The growth of HL-60 or PLB-985 cells in tunicamycin at concentrations up to 10 mg/mL from either HL-60 cells or Sf9 cells expressing the recombinant protein were oxidized, derivatized with digoxigenin, had no detectable effect on the electrophoretic mobility of the protein that was immunoprecipitated by anti-calreticulin; and then analyzed by SDS-PAGE and immunoblotting with Finally, we determined the amount of carbohydrate present in highly purified (see Fig 1D) Sf9 cell recombinant human calreticulin using the phenol-sulfuric acid method (Table 1).Control glycoproteins fetuin and transferrin had readily measured levels of carbohydrate, whereas a nonglycosylated protein bovine serum albumin contained no detectable carbohydrate by this method.The mean value obtained for calreticulin was 23 mg of carbohydrate/mg of protein, similar to that reported for bovine liver calreticulin of 16 mg/mg protein. 42We were unable to obtain sufficient quantities of highly purified HL-60 cell calreticulin to permit accurate carbohydrate determinations.In the cell-free reticulocyte lysate system either HL-60 DISCUSSION mRNA or synthetic calreticulin transcripts primed synthesis These studies address the mechanisms of synthesis and of a 62-kD calreticulin primary translation product.In the processing of human myeloid calreticulin.In two different presence of microsomal membranes the 62-kD protein was human leukemia cell lines, HL-60 and PLB-985, a 60-kD processed to a 60-kD species, presumably by cleavage of species representing mature calreticulin was the only form the signal peptide.The latter product comigrated in SDSdetected.Neither short periods of pulse labeling nor long PAGE with the protein that was immunoprecipitated from chases revealed any other electrophoretically detectable HL-60 cells.These data indicate that during in vivo synthesis products.These observations indicated that either (1) no nascent calreticulin undergoes cotranslational proteolysis of posttranslational processing occurred or (2) precursor and the signal peptide.Previous work indicated that in vitro synmature forms of calreticulin have very similar mobility under thesis of calreticulin using rabbit mRNA as template proconditions of SDS-PAGE.Further experiments showed that duced a Ç57-kD product, compared with the 55-kD mature for proteolytic processing the first alternative applied in that protein from muscle, 6 whereas synthetic transcripts prepared processing was cotranslational and that for glycosylation the from a human cDNA produced a 60-kD product that was processed to a smaller form in the presence of membranes. 18second alternative was true.Among the standard approaches to analysis of glycosylahuman cells synthesize calreticulin that is not glycosylated tion of proteins are inhibition of glycosylation by tunicannot be excluded.That the baculovirus-insect cell exprescamycin and deglycosylation by endoglycosidases.Our exsion system maintains the fidelity of glycosylation observed perimental design used myeloperoxidase, a known myeloid in HL-60 cells was further shown by direct chemical meacell glycoprotein, to assure that tunicamycin was effectively surement of the carbohydrate content of purified recombinant blocking glycosylation and that endoglycosidase H and calreticulin.Thus, Sf9 cells provide an excellent source of N-glycosidase F, enzymes expected to cleave either highstructurally authentic recombinant human calreticulin.mannose or all N-linked carbohydrates, respectively, were Previous studies of calreticulin glycosylation have applied functioning as predicted.Because there was no shift in the a wide range of methods to various species and tissues.Thus, apparent size of calreticulin after treatment, neither the tuniit has been reported that calreticulin is not glycosylated in camycin nor endoglycosidase studies provided any evidence rat 43 or rabbit 44 muscle, rabbit or chicken liver, 42 or lymphoid for glycosylation of calreticulin, leading to the tentative concells from mouse [45][46][47] or human. 17In contrast, evidence of clusion that asparagine-327, despite its presence in a consencalreticulin glycosylation was found in rat 41,48 and bovine 42,46 sus sequence for glycosylation, was not modified in human liver, bovine brain, 30 and hamster ovary. 49Although the bomyeloid cells.However, the electrophoretic mobility of calvine calreticulin sequence indicates two potential sites for Nreticulin is known to be anomalous, 1 the 46.5-kD mature glycosylation, only asparagine-162 is glycosylated in brain protein showing SDS-PAGE migration compatible with a tissue, whereas the 327 site homologous to the single potenmolecular size of 60 kD.This diminished mobility of tial site in human calreticulin is not modified. 30The only calreticulin is likely a consequence of its unusual zonal strucprevious study of human calreticulin 17 reported that there ture with globular N-terminal and highly acidic C-terminal was no glycosylation, based on digestion with endoglycosidomains separated by a series of proline-rich repeats. 1 dase H and SDS-PAGE, an approach which in our hands Our experiments using covalent derivatization of glycoalso failed to show glycosylation.proteins with digoxigenin showed unequivocally that human Interestingly, in two studies exploring the structure of calcalreticulin is, in fact, glycosylated when synthesized either reticulin N-linked carbohydrates, different results were obin the myeloid HL-60 cell line or as a recombinant protein served in different tissues.In bovine brain a high mannose in Sf9 insect cells.Of course, the possibility that nonmyeloid structure of the form GlcNAc 2 Man 4-9 was found 30 as generally expected for endoplasmic reticulum resident proteins.
In contrast, rat liver calreticulin contained a complex hybrid (calreticulin) of skeletal muscle sarcoplasmic reticulum.J Biol Chem mg/mg).Moreover, the lack of a detectable electrophoretic 264:21522, 1989   shift with deglycosylation is easier to explain for the high 7. McCauliffe DP, Zappi E, Lieu T-S, Michalak M, Sontheimer mannose structure (Ç1.2 kD) than for the larger complex RD, Capra JD: A human Ro/SS-A autoantigen is the homologue of carbohydrate (Ç2.2 kD).Thus, we suggest that human mycalreticulin and is highly homologous with onchocercal RAL-1 antieloid cell calreticulin contains a single N-linked high mangen and an aplysia ''memory molecule ''.J Clin Invest 86:332, 1990   nose carbohydrate moiety.

Baksh S, Michalak M: Expression of calreticulin in Escherichia
Although the biological role of calreticulin glycosylation coli and identification of its Ca 2/ binding domains.J Biol Chem is uncertain, the apparent variations among different cells 266:21458, 1991   and species us to speculate that tissue-specific functional 9. Dedhar S: Novel functions for calreticulin: Interaction with integrins and modulation of gene expression.Trends Biochem Sci properties of calreticulin could be regulated by the presence 19:269, 1994   or absence of N-linked carbohydrate.The most likely mecha-10.Sontheimer RD, Nguyen TQ, Cheng ST, Lieu TS, Capra JD: nism for this would be through effects on the interaction of The unveiling of calreticulin -A clinically relevant tour of modern calreticulin with other proteins in the endoplasmic reticulum.cell biology.J Invest Med 43:362, 1995 Thus, the chaperone function of calreticulin 25,26 or its poten-11.Meldolesi J, Krause KH, Michalak M: Calreticulin: How tial calcium-signaling role 11 may be controlled in part by the many functions in how many cellular compartments.Cell Calcium presence and specific configuration of carbohydrate moieties.20:83, 1996   The studies presented here provide a basic characterization  USA 91:12770, 1994  J 281:651, 1992  22. Atreya CD, Singh NK, Nakhasi HL: The rubella virus RNA 3. Burns K, Atkinson EA, Bleackley RC, Michalak M: Calreticbinding activity of human calreticulin is localized to the N-terminal ulin: From Ca 2/ binding to control of gene expression.Trends Cell domain.J Virol 69:3848, 1995  Biol 4:152, 1994  23. White TK, Zhu Q, Tanzer ML: Cell surface calreticulin is 4. Nash PD, Opas M, Michalak M: Calreticulin: Not just another a putative mannoside lectin which triggers mouse melanoma cell calcium-binding protein.Mol Cell Biochem 135:71, 1994  spreading.J Biol Chem 270:15926, 1995 5. Khanna NC, Waisman DM: Development of a radioimmunoas-24.Kuwabara K, Pinsky DJ, Schmidt AM, Benedict C, Brett J, say for quantitation of calregulin in bovine tissues.Biochemistry Ogawa S, Broekman MJ, Marcus AJ, Sciacca RR, Michalak M,  25:1078, 1986  Wang F, Pan YC, Grunfeld S, Patton S, Malinski T, Stern DM, 6. Fliegel L, Burns K, MacLennan DH, Reithmeier RAF, Michalak M: Molecular cloning of the high affinity calcium-binding protein Ryan J: Calreticulin, an antithrombotic agent which binds to vitamin

L
. Murphy Division and University of Texas Health Science Center, biosynthesis in cultured human myeloid cells and in cell-San Antonio, TX. free systems, focusing particularly on the primary product Submitted January 2, 1997; accepted February 17, 1997.Supported by Grants from the Medical Research Service of the of translation and its posttranslational modification by prote-Department of Veterans Affairs (Washington, DC) and the Fondation olysis and glycosylation.

Fig 1 .
Fig 1.Recombinant calreticulin expression, characterization, purification, and antibody generation.(A) Lysate of Sf9 cells infected with recombinant baculovirus was chromatographed on DEAE-Sepharose and aliquots of the fractions were analyzed by 7.5% SDS-PAGE and Coomassie Blue staining.Recombinant calreticulin is seen as a prominent 60-kD band in the Sf9 cell lysate (lane marked L) and in column fractions 11 and 12.The lane marked S shows standards with molecular weights as indicated.(B) The same Sf9 cell lysate as in (A) was analyzed by 7.5% SDS-PAGE and staining with Stains-All.The prominent 60-kD band stained blue, whereas other bands stained faintly red and are not visible in the photograph.(C) Purified recombinant calreticulin was analyzed by 7.5% SDS-PAGE, transfer to nitrocellulose, and detection by immunoblotting using a 1:1,000 dilution of rabbit polyclonal antibody to the purified protein.(D) Purified recombinant calreticulin was analyzed by 7.5% SDS-PAGE and silver staining.The left lane is the purified protein and the right lane shows standards with molecular weights as indicated.
S-methionine were analyzed by sodium dodecyl sulfate-polyacrylamide gel elecand the products were immunoprecipitated with antiserum trophoresis (SDS-PAGE) and fluorography of the dried gels.In some to recombinant human calreticulin and analyzed by SDSexperiments biosynthesis was performed in the presence of tuni-PAGE.With labeling pulses of 15 minutes to 2 hours only camycin (Boehringer Mannheim, Indianapolis, IN) to inhibit protein a single 60-kD product was observed (Fig 2A).Similar reglycosylation.Since prolonged exposure of myeloid cell lines to

Fig 2 .
Fig 2. Immunoprecipitates of biosynthetically labeled calreticulin from HL-60 cells.After 35 S-methionine pulse labeling, the cells were disrupted and the labeled proteins immunoprecipitated with anticalreticulin.The precipitates were analyzed by 7.5% SDS-PAGE and fluorography.The locations of molecular weight markers are indicated.(A) Pulse labeling was performed for 15 to 120 minutes as indicated.(B) Pulse labeling was performed for 0 to 30 minutes as indicated.Long exposure of the film resulted in the appearance of unidentified lower molecular weight bands in the 30-minute pulse lane.(C) Pulse labeling was performed for 30 minutes and then followed by a chase with excess unlabeled methionine for 0 to 120 minutes as indicated.

Fig
Fig 3. Analysis of calreticulin biosynthesis in vitro.Cell-free protein synthesis was performed in the rabbit reticulocyte lysate system in the presence of 35 S-methionine.Either immunoprecipitates (A and B) or total translation products (C) were analyzed by 7.5% SDS-PAGE and fluorography.The locations of molecular weight markers are indicated.(A) Reactions contained either no RNA (Ï) or poly-A-enriched RNA from HL-60 cells (") and either no membranes (Ï) or dog pancreatic microsomes (").The products of protein synthesis were immunoprecipitated with either nonimmune serum (NIS; from the rabbit subsequently immunized with calreticulin), anticalreticulin (CRT), or antimyeloperoxidase (MPO).(B) Reactions were as in (A) with (") or without (Ï) HL-60 cell RNA and dog pancreatic microsomes.HL-60 cell calreticulin biosynthetically labeled as in Fig 2 was run (third lane from left) for comparison with the cell-free products.For all samples products of translation were immunoprecipitated with anticalreticulin.

Fig 4 .
Fig 4. Effect of tunicamycin on the biosynthesis of calreticulin and

FigFig
Fig 6.Immunoblot analysis of digoxigenin-labeled calreticulin.Fetuin (control glycoprotein) and purified calreticulin (CRT) from either HL-60 or Sf9 cells were incubated in Endo F buffer with (") or without (Ï) N-glycosidase F (N-Glyc F), then oxidized with periodate and derivatized with digoxigenin.The proteins were then analyzed by 7.5% SDS-PAGE, transfer to nitrocellulose and immunoblotting with either antidigoxigenin (upper panel) or anticalreticulin (lower panel).The blots are arranged as indicated with paired lanes (minus or plus N-glycosidase F) for each of the three proteins.
12. Rojiani MV, Finlay BB, Gray V, Dedhar S: In vitro interacof the biosynthesis of calreticulin by human myeloid cells.tion of a polypeptide homologous to human Ro/SS-A antigen (calre-The primary translation product is modified by both cotransticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin a subunits.Biochemistry 30:9859, 1991   lational signal peptide cleavage and posttranslational N-13.Leung-Hagesteijn CY, Milankov K, Michalak M, Wilkins J, linked glycosylation.The experimental approaches as well Dedhar S: Cell attachment to extracellular matrix substrates is inhibas the cellular, molecular and immunologic tools developed ited upon downregulation of expression of calreticulin, an intracelluwill be useful for the further definition of the structure, funclar integrin a-subunit-binding protein.J Cell Sci 107:589, 1994 tion and regulation of calreticulin.Finally, our results point 14.Dedhar S, Rennie PS, Shago M, Leung Hagesteijn C-Y, Yang out the limitations of methods for analysis of glycoproteins H, Filmus J, Hawley RG, Bruchovsky N, Cheng H, Matusik RJ, that rely exclusively on the detection of differences in elec-Gigue `re V: Inhibition of nuclear hormone receptor activity by trophoretic mobility.Especially for proteins that display calreticulin.Nature 367:480, 1994 anomalous migration in SDS-PAGE, the use of alternative 15.Burns K, Duggan B, Atkinson EA, Famulski KS, Nemer M, methods, including oxidative derivatization with digoxigenin Bleackley RC, Michalak M: Modulation of gene expression by calreticulin binding to the glucocorticoid receptor.Nature 367:476, and direct chemical measurement of carbohydrate as we have 1994 employed, as well as lectin chromatography, biosynthetic 16.Dupuis M, Schaerer E, Krause K-H, Tschopp J: The calciumcarbohydrate labeling, gas chromatography and mass specbinding protein calreticulin is a major constituent of lytic granules trometry, may be required.in cytolytic T lymphocytes.J Exp Med 177:1, 1993 17. McCauliffe DP, Lux FA, Lieu T-S, Sanz I, Hanke J, Newkirk ACKNOWLEDGMENT MM, Bachinski LL, Itoh Y, Siciliano MJ, Reichlin M, Sontheimer The cloning of the human myeloid calreticulin cDNA was per-RD, Capra JD: Molecular cloning, expression, and chromosome 19 formed while Dr Clark was on sabbatical at the University of Geneva localization of a human Ro/SS-A autoantigen.J Clin Invest 85:1379, and the Fondation pour Recherches Me ´dicales, Geneva, Switzerland.1990 The many valuable contributions of Drs Daniel P. Lew, Werner 18. Rokeach LA, Haselby JA, Meilof JF, Smeenk RJT, Unnasch Schlegel, and Karl-Heinz Krause of the University of Geneva are TR, Greene BM, Hoch SO: Characterization of the autoantigen calgratefully acknowledged.We thank Dr Marek Michalak of the Unireticulin.J Immunol 147:3031, 1991 versity of Alberta, Canada for his generous gift of the rabbit calretic-19.Karska ´K, Tuckova ´L, Steiner L, Tlaskalova ´-Hogenova ´H, ulin cDNA and for helpful suggestions on the manuscript.Michalak M: Calreticulin--The potential autoantigen in celiac disease.Biochem Biophys Res Commun 209:597, 1995 REFERENCES 20.Cheng ST, Nguyen TQ, Yang YS, Capra JD, Sontheimer RD: Calreticulin binds hYRNA and the 52-kDa polypeptide component 1. Michalak M, Milner RE, Burns K, Opas M: Calreticulin.Bioof the Ro/SS-A ribonucleoprotein autoantigen.J Immunol 156:4484, chem J 285:681, 1992 1996 2. Van Delden C, Favre C, Spa ¨t A, Cerny E, Krause K-H, Lew 21.Singh NK, Atreya CD, Nakhasi HL: Identification of calretic-DP: Purification of an inositol 1,4,5-trisphosphate-binding calreticulin as a rubella virus RNA binding protein.Proc Natl Acad Sci ulin-containing intracellular compartment of HL-60 cells.Biochem

Table 1 . Direct Measurement of Carbohydrate Content by the
* Mean { SEM; n Å 3.