WEN CHANG,l.2* JONATHAN G. LIM,2 INGEGERD HELLSTROM,1'2 AND LARRY E. GENTRY2t ..... Blomquist, M. C., L. T. Hunt, and W. C. Barker. 1984.
Vol. 62, No. 3
JOURNAL OF VIROLOGY, Mar. 1988, p. 1080-1083
0022-538X/88/031080-04$02.00/0 Copyright © 1988, American Society for Microbiology
Characterization of Vaccinia Virus Growth Factor Biosynthetic Pathway with an Antipeptide Antiserum WEN CHANG,l.2* JONATHAN G. LIM,2 INGEGERD HELLSTROM,1'2 AND LARRY E. GENTRY2t
Department of Microbiology and Immunology, University of Washington, Seattle, Washington 98195,1 and Oncogen, 3005 First Avenue, Seattle, Washington 981212* Received 10 June 1987/Accepted 2 November 1987
A synthetic peptide derived from vaccinia virus growth factor (VGF) was used as an immunogen to prepare antiserum able to immunoprecipitate native VGF from both vaccinia virus-infected cell lysate and cell-free medium. Pulse-chase, tunicamycin treatment, and carbohydrate trimming experiments revealed that VGF is synthesized as a 19-kilodalton (kDa) precursor which is rapidly modified to a high-mannose-type 22-kDa protein. This cell-associated form is further processed into a 25-kDa polypeptide which, after proteolytic cleavage, releases the mature VGF into the medium as a 22-kDa glycoprotein. disulfite loop of VGF in which 6 of 11 amino acids are common in all EGF-like growth factors. Peptides were synthesized by solid-phase techniques on a Beckman automated instrument and purified by preparative high-performance liquid chromatography (5). The composition of the peptides was confirmed by amino acid analysis. Purified peptides were coupled to bovine gamma globulin and used to immunize New Zealand White rabbits. Immunization schedules were as described previously (6). Antisera to the VGF peptides were tested by immunoprecipitation of [35S]cysteine-labeled, VV-infected Cercopithecus monkey kidney (BSC-40) cell lysate and culture medium (Fig. 2). In practice, antiserum generated toward VGF70 80 (anti-VGF70-80) did not reveal any specifically identified 35S-labeled proteins (data not shown). AntiVGF20--33, on the other hand, readily identified three polypeptides of 19, 22, and 25 kDa from VV-infected cell lysate by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (Fig. 2A, lane d). These polypeptides were not seen in mock-infected cell lysate (Fig. 2A, lanes a and b) or by immunoprecipitation with rabbit preimmune serum (Fig. 2A, lane c). Anti-VGF2 33 also immunoprecipitated a released form of VGF in the medium of VV-infected cells harvested at 2 and 12 h postinfection (p.i.) (Fig. 2B, lanes d and f ). Preincubation of anti-VGF2033 with as little as 0.1 plg of free peptide VGF2033 specifically blocked the immunoprecipitation of these proteins (Fig. 2A, lanes e and f, and 2B, lanes g and h). Peptide VGF7,-80 did not affect the immunoreactivity of anti-VGF2033 (Fig. 2A lane g, and 2B, lane i). These results indicate that anti-VGF2 33 specifically recognizes multiple forms of VGF immunologically related to one another. Pulse-chase experiments were used to understand the processing of VGF. BSC-40 cells were infected with VV at a multiplicity of infection of 10, pulse-labeled in cysteine-free medium for 15 min with [35S]cysteine, and chased with complete medium. Immunoprecipitation of VV-infected cell lysates labeled and chased for various times is shown in Fig. 3A. After a 15-min pulse-label, two initial VGF polypeptides were detected which migrated on SDS-PAGE at 19 and 22 kDa. The 19-kDa form was less abundant and disappeared rapidly during the chase period. The 22-kDa polypeptide appeared to be more stable and displayed a half-life of 29 min. A third form of VGF, migrating at 25 kDa, appeared transiently after a 20-min chase period. Additionally, a
Poxvirus is a large family of cytoplasmic DNA viruses that contains a double-stranded DNA genome. Vaccinia virus (VV), the prototypic member of the orthopoxviruses, contains a genome of 187 kilobase pairs and has the potential of encoding nearly 200 gene products. Some poxviruses, such as Yaba tumor virus and Shope fibroma virus, induce host cell proliferation to form tumors. Although VV is not tumorigenic, a VV early gene was sequenced and found to encode a 19-kilodalton (kDa) VV growth factor (VGF) polypeptide in vitro, which shares homology with epidermal 'growth factor (EGF) and transforming growth factor (TGF)-alpha (Fig. 1A) (1, 2, 9). Structural analysis of the predicted VGF polypeptide, as deduced from the DNA sequence, suggests that VGF is synthesized as a precursor of 140 amino acids with a potential hydrophobic signal peptide and a transmembrane region close to the C terminus (Fig. iB) (8). It was proposed that, like EGF and TGF-alpha, VGF is synthesized as a membrane-bound precursor and, after proteolytic cleavage, is released into the medium (1, 2). Preliminary characterization of VGF purified from the medium of VV-infected cells has been reported (10). The study revealed that the purified released form of VGF lacked the putative signal peptide and began with aspartic acid at amino acid residue 20. In addition, its amino acid composition predicted that'the C-terminal transmembrane region was absent in released VGF, suggesting proteolytic processing. Although this study demonstrated the existence of N-linked carbohydrate on the released VGF polypeptide, a biosynthetic pathway establishing a clear precursor-product relationship was not identified. To address the biosynthesis and processing mechanism of VGF in more detail, we generated specific antisera to VGF by using synthetic peptides as immunogens. Two synthetic peptides corresponding to sequences of VGF were used. Both peptides reside within the predicted mature secreted form of VGF and were hydrophilic by'hydrophilicity analysis (sequences are underlined in Fig. 1A and enlarged in Fig. 1B). The peptide VGF20-33 starts at the' first N-terminal amino acid of released VGF and shares no homology with other EGF-like growth factor sequences. The peptide VGF70 80, on the other hand, is located within the third * Corresponding author. t Present address: Department of Biochemistry, Medical College of Ohio, Toledo, OH 43699.
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VOL. 62, 1988
NOTES
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FIG. 1. (A) Amino acid sequence homology among EGF-like families: VGF; rTGF, rat TGF; hTGF, human TGF; mEGF, mouse EGF; rEGF, rat EGF; and hEGF, human EGF. Homologous amino acids are boxed. Regions chosen for making synthetic peptides are underlined. Arrows indicate possible proteolytic cleavage sites. (B) Hydropathy analysis of VGF precursor (8). Regions chosen for making synthetic peptides are enlarged.
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smaller, 17-kDa polypeptide was also observed in the initial 15-min pulse period on SDS-PAGE (Fig. 3A). The fact that this 17-kDa band was not reproducibly blocked by pretreating anti-VGF2033 with free peptide VGF2033 suggested that it is not related to VGF. Analysis of culture medium from pulse-labeled cells revealed a diffusely migrating polypeptide of 22 kDa which accumulated stably in the culture medium throughout the chase period (Fig. 3B). Because of the extremely short half-life of the 19-kDa polypeptide observed in the pulse-chase experiment, it is difficult to determine the precursor-product relationship between the 19- and 22-kDa polypeptides. BSC-40 cells were pretreated with tunicamycin, an inhibitor for N-linked glycosylation, for 1 h before the pulse-chase experiment. After VV infection, cells were pulsed for 5, 10, or 15 min and chased with complete medium in the presence of tunicamycin (Fig. 3C). As observed at the zero point of the chase, the 19-kDa polypeptide was the only form of VGF synthesized in the tunicamycin-treated, VV-infected cells. After the 30-min chase, a minor band of 23 kDa also appeared in the lysate. This 23-kDa form was converted to 19 kDa after O-glycanase treatment (data not shown), suggesting 0linked glycosylation is involved in VGF modification. Appearance of both the 22- and the 25-kDa species was completely blocked by this inhibitor treatment during the 30-min chase, indicating that they are derived from N-linked glycosylation of the 19-kDa polypeptide. Several lines of evidence suggest that the 19-kDa protein observed by immunoprecipitation of cell lysate after a 15min pulse-label represents the initial VGF translation product. First, the 19-kDa polypeptide was the only form synthesized in short-pulse label experiments (Fig. 3C). Second, when cells were metabolically labeled in the presence of tunicamycin, the accumulation of 19-kDa protein as well as the absence of 22- and 25-kDa forms within the cells suggested a precursor-product relationship (Fig. 4A, lanes a and
b). Third, the 19-kDa form was not labeled with [3H]glucosamine, whereas 22- and 25-kDa species were (results not shown). Finally, the size of the 19-kDa protein identified was consistent with the size of VGF translated in vitro from hybrid-selected mRNA (12). An earlier report failed to show the existence of the 19-kDa polypeptide by immunoprecipitation analysis with mouse anti-EGF antiserum (10). This may be accounted for by the extremely labile nature of this initial VGF precursor. To understand the posttranslational modifications of VGF, endoglycosidases with different specificities were used in carbohydrate-trimming experiments. VGF was immunoprecipitated from [35]Scysteine-labeled cell Iysates by using anti-VGF20-33, and the immunocomplex was digested with N-glycanase (Fig. 4A, lane c) or endoglycosidase H (Fig. 4A, lane d). N-glycanase is rather nonselective in removing N-linked carbohydrate down to the asparagine core, A
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FIG. 2. Immunoprecipitation of VV-infected cell lysate and culture medium with antipeptide antiserum. (A) BSC-40 cells were labeled with [35S]cysteine at 30 min p.i. for 2 h. Detergent lysate was made and subjected to immunoprecipitation. After being washed, the immunoprecipitate complex was lysed in SDS-containing sample buffer and separated by 15% SDS-PAGE. Proteins were immunoprecipitated from samples of mock-infected cell lysate (lanes a and b) or VV-infected cell lysate (lanes c to g) with either preimmune serum (lanes a and c) or postimmune serum generated by VGF20-33 (lanes b and d to g). In preadsorption experiments, antiserum was preincubated with free peptide (lanes: e, 0.01 jig VGF20-33; f, 0.1 jig VGF20-33; g, 1 ,ug VGF70--W) for 30 min at 0°C before immunoprecipitation. (B) Culture medium collected from mock-infected cells (lanes a and b) or from VV-infected cells at 2 (lanes c and d) or at 12 h p.i. (lanes e to i) was incubated with either preimmune serum (lanes a, c, and e) or anti-VGF20-33 serum (lanes d and f to i) for immunoprecipitation. Lanes g to i are preadsorption experiments in which antiserum was preincubated with free peptide before immunoprecipitation. Lanes: g, 0.01 ,ug VGF20-33; h, 0.1 ,ug VGF20-33; i, 1 jig VGF70_80. kd, Kilodaltons.
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NOTES
whereas endoglycosidase H specifically cleaves high-mannose-type N-linked oligosaccharides. Each enzyme treatment reduced the size of the 22-kDa species to a form comigrating with the 19-kDa precursor identified in tunicamycin-treated cells (Fig. 4A, lanes b to d). Therefore, the intracellular 22-kDa form contains high-mannose-type Nlinked oligosaccharide. In contrast, the 25-kDa species was resistant to endoglycosidase H and sensitive to N-glycanase, implying the existence of complex-type carbohydrate on the 25-kDa VGF species. Endoglycosidase treatment of VGF immunoprecipitated from the culture medium is shown in Fig. 4B. N-glycanase partially converted the released 22-kDa VGF into a smaller
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FIG. 4. Endoglycosidase treatment of VGF immunoprecipitated from VV-infected cell lysate and culture medium. (A) Immunopre0 10 20' 30' 40' 50' 75' 2h 8h A T!ime -;case cipitation of VV-infected cell lysate harvested at 2 h p.i. with antipeptide serum anti-VGF20-33 in the absence (lanes a, c, and d) or L?.S kCCoff? presence (lane b) of tunicamycin. Analyses of immunoprecipitate 4treated with N-glycanase (lane c) and endoglycosidase H (lane d) are shown. (B) Immunoprecipitation of culture medium from VVinfected cells collected at 12 h p.i. in the absence (lanes a and c to e) 25 ~~~~~~~~~~~~~25 or presence (lane b) of tunicamycin. Lanes c to e show enzymatic 19~~~~~~~~~~~~~~~~1 treatment of VGF immunoprecipitated from culture medium of VV-infected cells at 12 h p.i. Lanes: c, N-glycanase; d, endoglycosidase H; e, neuraminidase. kd, Kilodaltons. Arrow indicates size of VGF observed.
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FIG. 3. Pulse-chase analysis of VGF in VV-infected cell lysate (A), in culture medium (B), and in VV-infected cell lysate in the presence of tunicamycin (C). BSC-40 cells were infected with VV at a multiplicity of infection of 10 and pulse-labeled with [35S]cysteine for 15 min. After labeling, the medium was removed, replaced with complete medium, and chased for various times, as indicated. Immunoprecipitates prepared with anti-VGF2033 were lysed in SDS-containing sample buffer and separated by SDS-PAGE. Peptide blocks were included to demonstrate the specificity of the immunoprecipitation. In panel C, BSC-40 cells were pretreated with tunicamycin at 5.0 p.g/ml for 1 h before VV infection. Tunicamycin was also present throughout the pulse-chase period. Cells were pulse-labeled at 2 h p.i. for 5, 10, or 15 min and chased with complete medium for various times, as indicated. kd, Kilodaltons.
form comigrating with the 14-kDa polypeptide secreted from the tunicamycin-treated cells (Fig. 4B, lanes b and c). The reason for this partial sensitivity to N-glycanase is not clear. Endoglycosidase H (Fig. 4B, lane d) showed no effect on the migration of the 22-kDa secreted form. Treatment of secreted VGF with neuraminidase, an enzyme that trims sialic acid residues, resulted in a polypeptide that migrated slower on SDS-PAGE (Figure 4B, lane e). A similar effect of neuraminidase digestion on the electrophoretic mobility of a tumor-associated antigen was reported (M. S. Lan, M. A. Hollingworth, and R. S. Metzgar, 16th UCLA Symp. Mol. Cell. Biol., abstr. no. R309, 1987). We believe our result indicates that there are neuraminidase-sensitive residues present on the secreted VGF molecule. Our results demonstrate that VGF is translated as an initial 19-kDa precursor which is rapidly modified to an N-glycosylated high-mannose-type 22-kDa form within the cells. This 22-kDa species is the major form observed even after a short 15-min pulse-labeling. It is further modified into a larger, more diffusely migrating 25-kDa molecule. This modification most likely involves the removal of terminal mannose residues followed by further carbohydrate addition to form a complex-type oligosaccharide, as reflected by the resistance of the 25-kDa form to endoglycosidase H. The disappearance of the 25-kDa cellular-associated form correlated with the appearance of the 22-kDa mature VGF found in the culture medium. We believe that this 25-kDa polypeptide is the final processed membrane-bound form before proteolytic cleavage and release of the 22-kDa VGF occur. This released form of VGF contains complex-type N-linked carbohydrate as well as sialated residues. Our results extend the carbohydrate analysis of VGF reported previously (10) and demonstrate a clear precursor-product relationship among the various VGF-related polypeptides. Recently, other poxviruses, such as Shope fibroma virus and myxoma virus, have been reported to contain a gene that encodes a polypeptide which shares amino acid homology to EGF (3, 11). Although these viruses cause very different cytopathological effects on the hosts, the growth factor gene they carry may produce a gene product with similar tertiary
NOTES
VOL. 62, 1988
structure to EGF, as implicated by the conserved cysteine residues among the EGF-like family. It is likely that these viral growth factors provide some advantages common to all poxviruses as well as subtle differences specific to each virus. The role of VGF in VV biology is not yet totally clear. One report suggested the involvement of VGF in virus adsorption (4). Others have suggested a better model in which VGF plays an important role in virus virulence. VGF may result in a greater number of metabolically active cells surrounding primary infectious centers, leading to enhanced virus spread in infected animals (R. M. Buller, 6th Poxvirus Meet., abstr. no. 80, 1986). Clearly, more work needs to be done to understand the relationship between the biochemical nature of VGF and its biological function in VV. We are grateful to Cara Watkins and Stepheny Ashe for technical help, to Peter Linsely and Anthony Purchio for helpful discussions, to Ron Manger for reviewing the manuscript, and to Phyllis Yoshida for manuscript preparation. We especially thank Shiu-Lok Hu for encouragement and critical comments. LITERATURE CITED 1. Blomquist, M. C., L. T. Hunt, and W. C. Barker. 1984. Vaccinia virus 19-kilodalton protein: relationship to several mammalian proteins, including two growth factors. Proc. Natl. Acad. Sci. USA 81:7363-7367. 2. Brown, J. P., D. R. Twardzik, H. Marquardt, and G. J. Todaro. 1985. Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor. Nature (London) 313:491-492. 3. Chang, W., C. Upton, S.-L. Hu, A. F. Purchio, and G. McFadden. 1987. The genome of Shope fibroma virus, a tumorigenic pox-
4.
5. 6.
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9. 10. 11.
12.
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virus, contains a growth factor gene with sequence similarity to those encoding epidermal growth factor and transforming growth factor alpha. Mol. Cell. Biol. 7:535-540. Eppstein, D. A., Y. V. March, A. B. Schreiber, S. R. Newman, G. J. Todaro, and J. J. Nester, Jr. 1985. Epidermal growth factor receptor occupancy inhibits vaccinia virus infection. Nature (London) 318:663-665. Gentry, L. E., and A. Lawton. 1986. Characterization of sitespecific antibodies to the erbB gene product and EGF receptor: inhibition of tyrosine kinase activity. Virology 152:421-431. Gentry, L. E., L. R. Rohrschneider, J. E. Casnellie, and E. G. Krebs. 1983. Antibodies to a defined region of pp60src neutralize tyrosine-specific kinase activity. J. Biol. Chem. 258: 11,219-11,228. King, C. S., J. A. Cooper, B. Moss, and D. R. Twardzik. 1986. Vaccinia virus growth factor stimulates tyrosine protein kinase activity of A431 cell epidermal growth factor receptors. Mol. Cell. Biol. 6:332-336. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. Reisner, A. H. 1985. Similarity between the vaccinia virus 19K early protein and epidermal growth factor. Nature (London) 313:801-803. Stroobant, P., A. P. Rice, W. J. Cheng, I. M. Kerr, and M. D. Waterfield. 1985. Purification and characterization of vaccinia virus growth factor. Cell 42:383-393. Upton, C., J. L. Macen, and G. McFadden. 1987. Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha. J. Virol. 61:1271-1275. Wittek, R., E. Barbosa, J. A. Cooper, C. F. Garon, H. Chan, and B. Moss. 1980. Inverted terminal repetition in vaccinia virus DNA encodes early mRNAs. Nature (London) 285:21-25.
