Vol. January
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
31, 1991
EXPRESSION OF HUMAN ALZHEIMER AMYLOID IN INSECT CELLS
983-989
PRECURSOR PROTEIN
Narayan Ramakrishna*., Pothana Saikumarr, Anna Potempska, Henryk M. Wtsniewski, and David L. Miller Department of Molecular Biology NYS Institute for Basic Research in Developmental Disabilities 1050 Forest Hill Road, Staten Island, NY 10314 ‘Wistar Institute, 36 Spruce St., Philadelphia, PA 19104 Received
November
26,
1990
SUMMARY: The amyloid beta-peptide is a major constituent of amyloid deposited in the brains of patients with Alzheimer’s disease and is derived from a larger precursor protein/s (APP-695, 751, 770). A human cDNA encoding full-length APP-751 was inserted into the genome of Aurogropha califomica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of APP-751. Analysis of infected cell proteins indicate that APP-751 is localized in the membrane fraction; however, a significant amount of the protein was cleaved and released into the medium. The NHZ-terminal sequence of recombinant APP-751 from the membrane fraction was identical to that of mammalian APP. Immunoblot analysis suggests that the a 1991 AcademicPress, Inc. secreted form results from cleavage within the P-peptide.
Alzheimer’s disease (AD) is characterized by the deposition of amyloid in neuritic plaques and in the walls of cerebral and meningeal blood vessels. A major constituent of these deposits is the p-peptide. It is derived from a large membrane-spanning glycoprotein, the p-amyloid peptide precursor (APP), which is found in many mammalian tissues. Aberrant degradation of APP likely causes the formation of p-peptide. The APP gene encodes at least three different proteins (APP-695, 751, 770) through alternative splicing of a single pre-mRNA; two of them (APP-751, 770) carry a domain with a Kunitztype protease inhibitor (KPI) sequence (1). The membrane-associated forms have been shown to be processed into extracellular (secreted) forms (2) lacking the intracellular domain. APP-751 (but not APP-695) has been shown to be mitogenic for 3T3 fibroblasts in vitro (3). The secreted form of APP-751 is protease nexin-II, a potent anti-chymotrypsin (4). Bodmer et al (5) have demonstrated that the secreted form of APP-751 binds transforming growth factor beta. Studies of the interactions and turnover of p-APP require sizeable amounts of protein, which cannot be prepared conveniently from mammalian tissues; therefore, we have developed a baculovirus expression system capable of producing milligram quantities of APP-751. In this work, we report the high level expression of APP75 1, its secreted form, and the COOH-terminal peptide in insect cells. *To
whom
request
for
reprints
and
correspondence
should
be
addressed.
Vol.
174,
No.
2, 1991
BIOCHEMICAL
MATERIALS
AND
Construction
of the transfer
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
METHODS vector
ah-APP-751
An APP-751 cDNA clone was isolated from a human retina cDNA library supplied by Dr. S.P. Bhat, Dept. of Ophthamology, Univ. of California, Los Angeles. Plasmid pND-1 was provided by Dr. Didier Negre of the Roche Institute of Molecular Biology and was prepared as described (6). pND-1 DNA was digested with Bam HI and Hind111 and was ligated to oligonucleotide containing restriction sites for BamHI, NruI, EcoRV, ClaI, BamHI, Sal1 and HindIII. The resulting plasmid pK7 was digested with NruI and Clal and was ligated to the NruI-ClaI fragment from the APP-cDNA creating pNDI-APP-751. The partial BamHI fragment of 2Skb contains the entire APP-751 cDNA starting at -5 and ending 325 bases after the termination codon. Subcloning of the partial BamHI fragment in pVL1393 DNA linearized with BamHI resulted in pAc-APP-751 with the APP-751 sequence in both orientations, pAc-APP-751, in which the APP-751 is in the correct orientation with reference to the polyhedrin promoter and direction of transcription, was identified by digestion with BglII. pAc-APP-751 DNA, isolated from transformed E. coli MV1190 cells, was purified by Sephadex column chromatography and used for transfection (7). Cells and viruses Spodopterufnrgiperdu lPLB-Sf21-AE clonal isolate 9 (Sl9) cells obtained from ATCC were cultured at 27°C in TNMFH medium (7) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (GibcoBRL) in monolayer or suspension culture. Plasmid pVL1393 and wild type baculovirus AcNPV were kindly provided by Dr. M.D. Summers of Texas A&M University. A recombinant baculovirus (AC-APP-751) containing a cDNA encoding full-length APP-751 under transcriptional regulation of the polyhedrin promoter was produced by cotransfecting pAc-APP-751(2pg) with wild-type Autograph califomicu nuclear polyhedrosis virus (AcNPV, strain E2) DNA (l/.&g) prepared as total DNA from infected cells. After 5 days the medium was tested by a plaque assay. Recombinant virus was detected by hybridization (8). Hybond-N filters (Amersham) were processed according to the manufacturer’s protocol and probed with the NruI-ClaI fragment from pNDIAPP-751, which had been labeled by random priming (8). After identifying the recombinant, the medium was serially diluted, and applied to growing Sf9 cells. The resulting infected cells were probed to identify recombinants. We identified a recombinant at lo-’ dilution which was checked by plaque assay using wild-type virus as the reference. Infection
and extraction
of proteins
Sl9 cells (1~10~) were infected with AC-APP-751 virus at a multiplicity of infection of 10. Mockinfected and wild-type virus infected cells served as controls. After a 1-hr adsorption period, the inoculum was replaced with fresh Grace’s medium supplemented with 10% fetal bovine serum. Cells were disrupted with a Polytron in 50mM Tris-HCl, 1mM EDTA, 1OmM D’lT, 1mM PMSF, pH=7.4 (10’ cells per ml) and the homogenate was spun down at 100,OOOgfor 30 min.. The resulting pellet was extracted with 1% Triton X-100 in 20mM Tris-HCl pH = 8.0,lmM EDTA, 1OmM DTT, 1mM PMSF for 1 h at room termperature and centrifuged as above. The extract was applied to a heparin-agarose column. Unbound material was washed off with 0.15M NaCl in 0.025% Brij-35, 20mM Tris-HCl pH=8.0 and the bound proteins (mostly APP-751) were eluted with 1M NaCl in Tris/Brij. The secreted form of APP was isolated from the cell culture medium. The medium was spun down at 100,OOOgfor lh to remove cell debris. The supernatant was applied to a heparin-agarose column, which was washed with 0.15 M NaCl in 50 mM Tris-HCl pH=7.4. APP-751 was eluted with 1M NaCl in Tris. Electronhoresis
and immunoblotting
Antibodies were raised to synthetic peptides derived from the APP-695 amino acid sequence. Two antibodies, anti-aa45-62, and anti-aa672-695, were raised in rabbits. Anti-aa597-620, 4G8, is a mouse monoclonal antibody to the P-peptide sequence (9). Electrophoretic separation of the proteins in the presence of SDS (10) was performed on minigels. Proteins were stained with Coomassie Blue or transferred to nitrocellulose (11). After the electrotransfer the blots were immersed in PBSA containing 0.1% BSA and 0.05% Tween-20. Binding of primary antibodies, diluted in PBSA/BSA/Tween, was performed overnight at room temperature. Bound antibodies were detected with goat anti-rabbit or goat anti-mouse IgG coupled to alkaline phosphatase and visualized by the reaction with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3indolyl phosphate. Isolation of immunoreactive proteins from the polyacrylamide gels was performed according to Feick and Shiozawa (12). NH2-terminal sequencing was performed on an Applied Biosystems model 470A sequencer with a model 120 on-line PTH amino acid analyzer. Northern
blot analvsis
Total RNA was isolated by guanidinium thiocyanate extraction (13) and separated by electrophoresis on a 1% agarose gel containing 2% formaldehyde (14) in 2OmM MOPS, 5mM sodium acetate and 1mM EDTA and transferred to nitrocellulose filters (Schleicher & Schuell) using 20 x SSC overnight at room temperature. The blots were hybridized with an oligolabelled 0.8kb DNA fragment of the APP structural gene. Hybridization was done in 50% formamide, 6 x SSC for 18hr at 42’C and washing was done in 0.1 x SSC, 0.1% SDS at 55’C. 984
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APP mRNA was synthesized by T7 transcription of pRB6, which contains most of the structural gene for APP751 (15). Amounts of AF’P-751 mRNA were measured by densitometric scanning of autoradiograms; synthetic AF’P-mRNA served as the standard.
RESULTS
AND
DISCUSSION
Exuression of human APP-751 in insect cells To transfer the APP-751 gene into the AcNPV genome, Sf9 cells were cotransfected with the plasmid pAc-APP-751 and genomic DNA of wild-type AcNPV. Recombinant viruses, produced by allelic replacement of the polyhedrin gene by homologous recombination, were identified as described in Materials and Methods. Two recombinant viruses designated Cl0 and D9 were analysed. At 48hr post-infection whole-cell homogenates were prepared from cells infected with the wild-type AcNPV, from cells infected with the two recombinant viruses, and from uninfected cells. Analyses of these extracts on SDS-PAGE followed by immunoblotting with three anti-APP antibodies are shown in Fig. 1. All three antibodies revealed the presence of an abundant protein of - 116 kDa in extracts from cells infected with recombinant viruses C9 and DlO (Fig. 1 lanes 3 and 4). This protein was not detected in extracts from cells infected with AcNPV or from uninfected cells (lanes 1 and 2). Antibodies to the Kunitz protease inhibitor domain also revealed the 116 kDa protein only in recombinant virus-infected cell extracts (data not shown). Purified APP from rat brain (predominantly APP-695) has a similar electrophoretic mobility and immunoreactivity (Fig. 1, lane 5) (16). APP-751 (indicated by arrows, Panel D lane 3) was made in amounts comparable to the polyhedrin protein (indicated by arrow, lane 2), which, in wild-type virus infected cells, accumulates to 25% of total cellular protein (17). Clones Cl0 and D9 showed similar expression levels. The following experiments employed clone ClO. Cellular localization of recombinant APP-751 To determine the cellular location of the recombinant APP-751 the cells were fractionated into cytosolic, Triton-insoluble and Triton-soluble fractions and analysed (16)
A
B
c
D --
116kD-
1
2
3
4
5
12
(. ,.
: :Y .. . . . .
-4.m Cd
i3 .$r*
5 .*
>“A
1
2
5
34
4
* dl-
34
5
1
2
3
Fig. 1 Eqmssion of APP-751 in insecr cells. Aliquots of whole cell homogenate 48hr post-infection were separated on 10% SDS-PAGE and immunoblotted with antisera to different epitopes on APP. Panel A, monoclonal antibody 4G8 anti-aa597-624J Panel B, anti-aa672-695; Panel C, anti-aa45-62. Lane 1, uninfected cells; lane 2, infected with wild-type baculovirus; lane 3, infected with recombinant virus ClO; lane 4, infected with recombinant virus D9, lane 5, purified APP from rat brain (16). Panel D: Coomassie Blue staining. Arrow points to the recombinant APP-751 (I ane 3) and polyhedrii protein (lane 1).
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with antisera to the COOH-terminal epitope, aa672-695. Although there was some APP751 in the cytosolic fraction, most of the protein was Triton extractable, which suggested that APP-751 is mostly localized in the membrane fraction of these cells. We have recently performed immuno-gold labelling of the infected cells (manuscript in preparation) using the COOH-terminal antibody, which showed that ASPP-751 is present in the plasma membranes of these cells. These results confirm that recombinant APP-751 is localized in the cell membranes as suggested by the primary structure of the protein (18). Immunoblotting of the proteins in the medium showed that the secreted form (S) is about 12 kDa smaller than the membrane bound form (M). The protein from the medium is recognized well (Fig. 2) with antisera to aa420-440 and the p-peptide domain (aa597612 and aa597-620), but antisera to the COOH-terminal domain (aa672-695) fail to detect the protein (Panel E). This indicates that APP-751 from the medium is missing the COOH-terminal domain. From these results we conclude that the site of cleavage of APP751 is near the epitope of monoclonal antibody 4G8, which is aa613-618 (9). Time course of exmession of APP-751. secreted form and the C-terminal DeDtide The kinetics of expression of both mRNA and protein were followed simultaneously and both intracellular and secreted APP-751 were assayed. The proteins were separated by SDS/PAGE and analyzed by immunoblotting. APP-751 was detectable starting 36 hr postinfection (hpi) and its amount increased until 72 hpi (Fig. 3A). The secreted form of APP751 had a similar time-course of accumulation (Fig. 3B). A 12 kDa peptide was detected in the membrane fraction at 36 hpi and its level increased until 72 hpi. It did not react with antibodies to aa45-62 or to aa597-612. It showed a weak reaction with monoclonal antibody 4G8, and it reacted strongly with COOH-terminal antibodies to aa649-671 and aa672-695 (data not shown). These observations suggest that the NHz-terminal of this peptide is near the epitope of monoclonal antibody 4G8 aa613-619(9). It is interesting that APP overproduced in mammalian cells is also cleaved in this region (19). A
6 cells
24 116kD--’
36 r
medium 46
72
24
36
46
72
hr. oost-infectia
c +
‘h?
-116kD
Infected cell-culture medium obtained 48hr post-infection was Fig.2Analysis of APP-751 in culh~re medium. purified on heparin agarose as in M&vials and Methods. Aliquots from the medium (S) and membrane extract (M) were analysed with antisera to aa45-62 (Panel A), aa420-440 (Panel B), aa.597-612 monoclonal antibody 6ElO (Panel C), aa.597-620 monodonal antibody 4G8 (Panel D) and aa672-695 (Panel E). Arrow indicates the absence of secreted APP-751 in Panel E. Panel A and Panels B tbru E were run under different electrophoretic conditions. Fig. 3 Kinetics of APP-751 accumulation in the medium and inside Sf9 cells. (A) Aliquots of cellular homogenates separated on 15% SDS/PAGE blotted and probed with a&672-695. (B) Aliquots of medium separated on a 10% gel, blotted and probed with anti-45-62. Arrows indicate the positions of 116kDa and 12kDa APP proteins (A) and - 1OOkDa secreted APP-751 (B).
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80 70 60 P
t 50 40
1
30 20 10
i
ot 0
12
24
36
48
60
72
84
96
Hrs. post-infection Fig. 4 Accumulation of cellular APP-751, secreted APP-751 and APP-7Sl mRNA in Sf9 cells infected with recombinant AC-APP-751 virus. Amounts of secreted and intracellular APP-751 at various times post-infection were calculated as described previously (16) from the experiment in Fig. 3. Levels of mRNA were computed from the Northern blots as described in Methods.
Total RNA was isolated from infected Sf9 cells harvested at various times postinfection and was analysed by Northern blotting. One major 2.8 kb transcript was detected, which is compatible with the predicted transcript starting at the polyhedrin cap-site, and terminating at the polyhedrin transcription stop site. In wild-type virus-infected cells no cross-hybridizing material was detected (data not shown). The amount of APP-75 1 mRNA increases sharply from 36hpi until 48hpi. Assuming that mRNA is five percent of total RNA isolated from infected cells, we computed that as a fraction of total mRNA, APP-751 mRNA increases from 0.054 percent at 24hpi to 0.616 percent at 36hpi and to 20.73 percent at 48hpi. In a similar study pre-pro-attacin mRNA expression (20) was first detected 15hpi, and it increased until 48hpi. It is clear that infected cells have sufficient mRNA to support a high level of APP-751 synthesis between 48 and 72hpi (Fig. 4). The immunoblots and autoradiograms were quantified by laser densitometry (Fig. 4). That both APP-751 and its mRNA are barely detectable at 24 hpi is consistent with the late expression of the polyhedrin promoter (21). Membrane-associated APP-751 increases two fold from 48 hpi to 72 hpi and decreases at 96 hpi whereas the secreted form increases almost 4-fold and remains at the same level until 96 hpi. These results demonstrate that, under conditions of overproduction, APP-751 is cleaved to release a significant amount of secreted form into the medium with concomitant increase in the amount of COOHterminal peptide in the cells. At 72 hpi, the total amount of APP-751 in this experiment was 4 mg/l08 cells; we have occasionally observed levels of 10 mg/108 cells. Approximately 60% of total APP-751 is membrane-associated and most of the remaining protein is the secreted form. A similar time course of accumulation has been reported for the extracellular domain of the human insulin receptor (22), which reached its maximum 987
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level in the medium at 72 hpi. Other human proteins including the glucocorticoid receptor (23), extracellular domain of nerve growth factor (24) and CD4 (25) have been expressed using this system; however, their levels of expression are at least ten-fold lower than the level we achieved with APP-751. Purification and N-terminal seauence analysis of recombinant APP-751 APP-751 was highly enriched from a membrane extract by a single step of heparin agarose chromatography. The protein was further purified by PAGE. The APP-751 band was eluted from the gel, and its NHa-terminal sequence was determined. Its sequence was found to be Leu-Glu-Val-Phe-Thr-Asp-Gly-Asn-Ala-Gly which is identical to the sequence found for native APP from rat brain (16) and to the sequence of protease nexin II (4). This identifies the protein as APP-751 and shows that insect cells correctly cleave the signal sequence of amyloid precursor protein. In this report we have described a baculovirus derived system that is capable of APP-751 expression at 4 mg/108 cells or 20 mg/liter. We have shown that the protein is localized in membranes and established the authenticity of the recombinant protein by NHz-terminal sequence analysis. We have identified a secreted form of APP-751 lacking the COOH-terminal domain and the trans-membrane region. APP-751 expressed in the baculovirus system will be useful for studies of post-translational modifications such as phosphorylation and glycosylation, and for ligand binding and processing studies as well as for making specific antibodies to native APP-751.
ACKNOWLEDGMENTS: We thank Mr. James Styles for the amino terminal sequencing of recombinant APP and Ms. Gloria Wolfe for help with the RNA blot. Dr. Robert Denman provided synthetic APP-mRNA. Drs. Pankaj Mehta and Bruce Patrick made available the polyclonal antibodies. Monoclonal antibodies were obtained from Dr. Kwang Soo Kim. Dr. E. P. Reddy allowed us the use of laboratory facilities during the isolation of the recombinant virus. Special thanks to Ms. Patricia Casiano for the manuscript preparation. This work was supported by grants No. NS25231 and TO-l-AGO4220 from the NIH and by the New York State Office of Mental Retardation and Developmental Disabilities.
REFERENCES 1. Muller-Hill, B., and Beyreuther, K. (1989) Ann. Rev. Biochem. 58,287-307. 2. Podliiny, M.B., Mammen, A.L., Schlossmacher, M.G., Palmert, M.R., You&in, S.G., and Selkoe, D.J. (1990) Biochem. Biophys. Res. Commun. 167,1094-1101. 3. Schubert, D., Cole, G., Saitoh, T. and Oltersdorf, T. (1989) Biochem. Biophys. Res. Commun. 162,83-88. 4. Van Nostrand, N.E., Wagner, S.L., Suzuki, M., Choi, B.H., Farrow, J.S., Geddes, J.W., Cotman, C.W., and Cunningham, D.D. (1989) Nature 341,546-549. 5. Bodmer, S., Podlisny, M.B., Selkoe, D.J., Heid, I., and Fontana, A. (1990) Biochem. Biophys. Res. Commun. 171,890-897. 6. Cunningham, P.R., Weitzmann, C.J., Nurse, K., Masurel, R., Van Knippenburg, P.H., and Ofengand, J. (1990) Biophys. Biochem. Acta 1050,18-26. 7. Summers, M.D. and Smith, G.E. (1987) A Manual of Methods for Baculovirus Expression Vectors and Insect Cell Culture. Procedures (Texas Agricultural Experiment Station, College Station, TX), Bull. 1555. 8. Steiner, H., Pohl, G., Gunne, H., Hellers, M., Elhammer, A. and Hanssou, L. (1988) Gene 73,449-457. 9. Kim, K.S., Miller, D.L., Sapienza, V.J., Chen, C.J., Bai, C., Grundke-Iqbal, I., Currie, J.R., and Wisniewski, H.M. (1988) Neurosci. Res. Commun. 2,121-130. 10. Laemmli, U.K. (1970) Nature, 227,680~685. 11. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76,4350-4354.
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12. Feick, R.G., and Shiozawa, J.A. (1990) Anal. Biochem. 187,205211. 13. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-1.59. 14. Lahrach, H., Diamond, D., Wozney, J.M. and Roedtker, H. (1977) Biochemistry 16,4743-4751. 1.5. Denman, R.B., Potempska, A., Wolfe, G., Ramakrishna, N., and Miller, D.L. (1990) submitted for publication. 16. Potempska, A., Styles, J., Mehta, P., Kim, KS., and Miller, D.L. (1990) submitted for publication. 17. Luckow, VA., and Summers, M.D. (1988) Biotechnology 6,47-55. 18. Kang, J., Lemaire, H-G., Unterbeck, A., Salbaum, M.J., Masters, C.L., Grzeschik, K-H., Muhhaup, Cr., Beyreuther, K., and Muller-Hill, B. (1987) Nature 325,733-736. 19. Esch, F.S., Keim, P.S., Beattie, EC., Blather, R.W., Culwell, A.R., Oltersdorf, T., McClure, D., and Ward, P. (1990) Science, 248,1112-1124. 20. Gunne, H., Hellers, M., and Steiner, H. (1990) Eur. J. Biochem. 187,699-703. 21. Oakey, R., Cameron, I., Davis, G., Davis, E., and Possee, R.D. (1989) J. Gen. Viral. 70,769-775. 22. Sissom, J., and Ellis, L. (1989) Biochem. Journal. 261,119-X26. 23. Srinivasan, G., and Thompson, E. (1990) Mol. Endocrinol. 4,209-216. 24. Vissavajjhala, P., and Ross, A.H. (1990) Journal Biol. Chem. 265,4746-4752. 25. Webb, N., Madoulct, C., Tosi, P.F., Broussard, D.R., Sneed, L., Nicolau, C., and Summers, M.D. (1989) Proc. Natl. Acad. Sci. USA, 86, 7731-7735.
989
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
31, 1991
EXPRESSION OF HUMAN ALZHEIMER AMYLOID IN INSECT CELLS
983-989
PRECURSOR PROTEIN
Narayan Ramakrishna*., Pothana Saikumarr, Anna Potempska, Henryk M. Wtsniewski, and David L. Miller Department of Molecular Biology NYS Institute for Basic Research in Developmental Disabilities 1050 Forest Hill Road, Staten Island, NY 10314 ‘Wistar Institute, 36 Spruce St., Philadelphia, PA 19104 Received
November
26,
1990
SUMMARY: The amyloid beta-peptide is a major constituent of amyloid deposited in the brains of patients with Alzheimer’s disease and is derived from a larger precursor protein/s (APP-695, 751, 770). A human cDNA encoding full-length APP-751 was inserted into the genome of Aurogropha califomica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of APP-751. Analysis of infected cell proteins indicate that APP-751 is localized in the membrane fraction; however, a significant amount of the protein was cleaved and released into the medium. The NHZ-terminal sequence of recombinant APP-751 from the membrane fraction was identical to that of mammalian APP. Immunoblot analysis suggests that the a 1991 AcademicPress, Inc. secreted form results from cleavage within the P-peptide.
Alzheimer’s disease (AD) is characterized by the deposition of amyloid in neuritic plaques and in the walls of cerebral and meningeal blood vessels. A major constituent of these deposits is the p-peptide. It is derived from a large membrane-spanning glycoprotein, the p-amyloid peptide precursor (APP), which is found in many mammalian tissues. Aberrant degradation of APP likely causes the formation of p-peptide. The APP gene encodes at least three different proteins (APP-695, 751, 770) through alternative splicing of a single pre-mRNA; two of them (APP-751, 770) carry a domain with a Kunitztype protease inhibitor (KPI) sequence (1). The membrane-associated forms have been shown to be processed into extracellular (secreted) forms (2) lacking the intracellular domain. APP-751 (but not APP-695) has been shown to be mitogenic for 3T3 fibroblasts in vitro (3). The secreted form of APP-751 is protease nexin-II, a potent anti-chymotrypsin (4). Bodmer et al (5) have demonstrated that the secreted form of APP-751 binds transforming growth factor beta. Studies of the interactions and turnover of p-APP require sizeable amounts of protein, which cannot be prepared conveniently from mammalian tissues; therefore, we have developed a baculovirus expression system capable of producing milligram quantities of APP-751. In this work, we report the high level expression of APP75 1, its secreted form, and the COOH-terminal peptide in insect cells. *To
whom
request
for
reprints
and
correspondence
should
be
addressed.
Vol.
174,
No.
2, 1991
BIOCHEMICAL
MATERIALS
AND
Construction
of the transfer
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
METHODS vector
ah-APP-751
An APP-751 cDNA clone was isolated from a human retina cDNA library supplied by Dr. S.P. Bhat, Dept. of Ophthamology, Univ. of California, Los Angeles. Plasmid pND-1 was provided by Dr. Didier Negre of the Roche Institute of Molecular Biology and was prepared as described (6). pND-1 DNA was digested with Bam HI and Hind111 and was ligated to oligonucleotide containing restriction sites for BamHI, NruI, EcoRV, ClaI, BamHI, Sal1 and HindIII. The resulting plasmid pK7 was digested with NruI and Clal and was ligated to the NruI-ClaI fragment from the APP-cDNA creating pNDI-APP-751. The partial BamHI fragment of 2Skb contains the entire APP-751 cDNA starting at -5 and ending 325 bases after the termination codon. Subcloning of the partial BamHI fragment in pVL1393 DNA linearized with BamHI resulted in pAc-APP-751 with the APP-751 sequence in both orientations, pAc-APP-751, in which the APP-751 is in the correct orientation with reference to the polyhedrin promoter and direction of transcription, was identified by digestion with BglII. pAc-APP-751 DNA, isolated from transformed E. coli MV1190 cells, was purified by Sephadex column chromatography and used for transfection (7). Cells and viruses Spodopterufnrgiperdu lPLB-Sf21-AE clonal isolate 9 (Sl9) cells obtained from ATCC were cultured at 27°C in TNMFH medium (7) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (GibcoBRL) in monolayer or suspension culture. Plasmid pVL1393 and wild type baculovirus AcNPV were kindly provided by Dr. M.D. Summers of Texas A&M University. A recombinant baculovirus (AC-APP-751) containing a cDNA encoding full-length APP-751 under transcriptional regulation of the polyhedrin promoter was produced by cotransfecting pAc-APP-751(2pg) with wild-type Autograph califomicu nuclear polyhedrosis virus (AcNPV, strain E2) DNA (l/.&g) prepared as total DNA from infected cells. After 5 days the medium was tested by a plaque assay. Recombinant virus was detected by hybridization (8). Hybond-N filters (Amersham) were processed according to the manufacturer’s protocol and probed with the NruI-ClaI fragment from pNDIAPP-751, which had been labeled by random priming (8). After identifying the recombinant, the medium was serially diluted, and applied to growing Sf9 cells. The resulting infected cells were probed to identify recombinants. We identified a recombinant at lo-’ dilution which was checked by plaque assay using wild-type virus as the reference. Infection
and extraction
of proteins
Sl9 cells (1~10~) were infected with AC-APP-751 virus at a multiplicity of infection of 10. Mockinfected and wild-type virus infected cells served as controls. After a 1-hr adsorption period, the inoculum was replaced with fresh Grace’s medium supplemented with 10% fetal bovine serum. Cells were disrupted with a Polytron in 50mM Tris-HCl, 1mM EDTA, 1OmM D’lT, 1mM PMSF, pH=7.4 (10’ cells per ml) and the homogenate was spun down at 100,OOOgfor 30 min.. The resulting pellet was extracted with 1% Triton X-100 in 20mM Tris-HCl pH = 8.0,lmM EDTA, 1OmM DTT, 1mM PMSF for 1 h at room termperature and centrifuged as above. The extract was applied to a heparin-agarose column. Unbound material was washed off with 0.15M NaCl in 0.025% Brij-35, 20mM Tris-HCl pH=8.0 and the bound proteins (mostly APP-751) were eluted with 1M NaCl in Tris/Brij. The secreted form of APP was isolated from the cell culture medium. The medium was spun down at 100,OOOgfor lh to remove cell debris. The supernatant was applied to a heparin-agarose column, which was washed with 0.15 M NaCl in 50 mM Tris-HCl pH=7.4. APP-751 was eluted with 1M NaCl in Tris. Electronhoresis
and immunoblotting
Antibodies were raised to synthetic peptides derived from the APP-695 amino acid sequence. Two antibodies, anti-aa45-62, and anti-aa672-695, were raised in rabbits. Anti-aa597-620, 4G8, is a mouse monoclonal antibody to the P-peptide sequence (9). Electrophoretic separation of the proteins in the presence of SDS (10) was performed on minigels. Proteins were stained with Coomassie Blue or transferred to nitrocellulose (11). After the electrotransfer the blots were immersed in PBSA containing 0.1% BSA and 0.05% Tween-20. Binding of primary antibodies, diluted in PBSA/BSA/Tween, was performed overnight at room temperature. Bound antibodies were detected with goat anti-rabbit or goat anti-mouse IgG coupled to alkaline phosphatase and visualized by the reaction with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3indolyl phosphate. Isolation of immunoreactive proteins from the polyacrylamide gels was performed according to Feick and Shiozawa (12). NH2-terminal sequencing was performed on an Applied Biosystems model 470A sequencer with a model 120 on-line PTH amino acid analyzer. Northern
blot analvsis
Total RNA was isolated by guanidinium thiocyanate extraction (13) and separated by electrophoresis on a 1% agarose gel containing 2% formaldehyde (14) in 2OmM MOPS, 5mM sodium acetate and 1mM EDTA and transferred to nitrocellulose filters (Schleicher & Schuell) using 20 x SSC overnight at room temperature. The blots were hybridized with an oligolabelled 0.8kb DNA fragment of the APP structural gene. Hybridization was done in 50% formamide, 6 x SSC for 18hr at 42’C and washing was done in 0.1 x SSC, 0.1% SDS at 55’C. 984
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APP mRNA was synthesized by T7 transcription of pRB6, which contains most of the structural gene for APP751 (15). Amounts of AF’P-751 mRNA were measured by densitometric scanning of autoradiograms; synthetic AF’P-mRNA served as the standard.
RESULTS
AND
DISCUSSION
Exuression of human APP-751 in insect cells To transfer the APP-751 gene into the AcNPV genome, Sf9 cells were cotransfected with the plasmid pAc-APP-751 and genomic DNA of wild-type AcNPV. Recombinant viruses, produced by allelic replacement of the polyhedrin gene by homologous recombination, were identified as described in Materials and Methods. Two recombinant viruses designated Cl0 and D9 were analysed. At 48hr post-infection whole-cell homogenates were prepared from cells infected with the wild-type AcNPV, from cells infected with the two recombinant viruses, and from uninfected cells. Analyses of these extracts on SDS-PAGE followed by immunoblotting with three anti-APP antibodies are shown in Fig. 1. All three antibodies revealed the presence of an abundant protein of - 116 kDa in extracts from cells infected with recombinant viruses C9 and DlO (Fig. 1 lanes 3 and 4). This protein was not detected in extracts from cells infected with AcNPV or from uninfected cells (lanes 1 and 2). Antibodies to the Kunitz protease inhibitor domain also revealed the 116 kDa protein only in recombinant virus-infected cell extracts (data not shown). Purified APP from rat brain (predominantly APP-695) has a similar electrophoretic mobility and immunoreactivity (Fig. 1, lane 5) (16). APP-751 (indicated by arrows, Panel D lane 3) was made in amounts comparable to the polyhedrin protein (indicated by arrow, lane 2), which, in wild-type virus infected cells, accumulates to 25% of total cellular protein (17). Clones Cl0 and D9 showed similar expression levels. The following experiments employed clone ClO. Cellular localization of recombinant APP-751 To determine the cellular location of the recombinant APP-751 the cells were fractionated into cytosolic, Triton-insoluble and Triton-soluble fractions and analysed (16)
A
B
c
D --
116kD-
1
2
3
4
5
12
(. ,.
: :Y .. . . . .
-4.m Cd
i3 .$r*
5 .*
>“A
1
2
5
34
4
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34
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2
3
Fig. 1 Eqmssion of APP-751 in insecr cells. Aliquots of whole cell homogenate 48hr post-infection were separated on 10% SDS-PAGE and immunoblotted with antisera to different epitopes on APP. Panel A, monoclonal antibody 4G8 anti-aa597-624J Panel B, anti-aa672-695; Panel C, anti-aa45-62. Lane 1, uninfected cells; lane 2, infected with wild-type baculovirus; lane 3, infected with recombinant virus ClO; lane 4, infected with recombinant virus D9, lane 5, purified APP from rat brain (16). Panel D: Coomassie Blue staining. Arrow points to the recombinant APP-751 (I ane 3) and polyhedrii protein (lane 1).
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with antisera to the COOH-terminal epitope, aa672-695. Although there was some APP751 in the cytosolic fraction, most of the protein was Triton extractable, which suggested that APP-751 is mostly localized in the membrane fraction of these cells. We have recently performed immuno-gold labelling of the infected cells (manuscript in preparation) using the COOH-terminal antibody, which showed that ASPP-751 is present in the plasma membranes of these cells. These results confirm that recombinant APP-751 is localized in the cell membranes as suggested by the primary structure of the protein (18). Immunoblotting of the proteins in the medium showed that the secreted form (S) is about 12 kDa smaller than the membrane bound form (M). The protein from the medium is recognized well (Fig. 2) with antisera to aa420-440 and the p-peptide domain (aa597612 and aa597-620), but antisera to the COOH-terminal domain (aa672-695) fail to detect the protein (Panel E). This indicates that APP-751 from the medium is missing the COOH-terminal domain. From these results we conclude that the site of cleavage of APP751 is near the epitope of monoclonal antibody 4G8, which is aa613-618 (9). Time course of exmession of APP-751. secreted form and the C-terminal DeDtide The kinetics of expression of both mRNA and protein were followed simultaneously and both intracellular and secreted APP-751 were assayed. The proteins were separated by SDS/PAGE and analyzed by immunoblotting. APP-751 was detectable starting 36 hr postinfection (hpi) and its amount increased until 72 hpi (Fig. 3A). The secreted form of APP751 had a similar time-course of accumulation (Fig. 3B). A 12 kDa peptide was detected in the membrane fraction at 36 hpi and its level increased until 72 hpi. It did not react with antibodies to aa45-62 or to aa597-612. It showed a weak reaction with monoclonal antibody 4G8, and it reacted strongly with COOH-terminal antibodies to aa649-671 and aa672-695 (data not shown). These observations suggest that the NHz-terminal of this peptide is near the epitope of monoclonal antibody 4G8 aa613-619(9). It is interesting that APP overproduced in mammalian cells is also cleaved in this region (19). A
6 cells
24 116kD--’
36 r
medium 46
72
24
36
46
72
hr. oost-infectia
c +
‘h?
-116kD
Infected cell-culture medium obtained 48hr post-infection was Fig.2Analysis of APP-751 in culh~re medium. purified on heparin agarose as in M&vials and Methods. Aliquots from the medium (S) and membrane extract (M) were analysed with antisera to aa45-62 (Panel A), aa420-440 (Panel B), aa.597-612 monoclonal antibody 6ElO (Panel C), aa.597-620 monodonal antibody 4G8 (Panel D) and aa672-695 (Panel E). Arrow indicates the absence of secreted APP-751 in Panel E. Panel A and Panels B tbru E were run under different electrophoretic conditions. Fig. 3 Kinetics of APP-751 accumulation in the medium and inside Sf9 cells. (A) Aliquots of cellular homogenates separated on 15% SDS/PAGE blotted and probed with a&672-695. (B) Aliquots of medium separated on a 10% gel, blotted and probed with anti-45-62. Arrows indicate the positions of 116kDa and 12kDa APP proteins (A) and - 1OOkDa secreted APP-751 (B).
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80 70 60 P
t 50 40
1
30 20 10
i
ot 0
12
24
36
48
60
72
84
96
Hrs. post-infection Fig. 4 Accumulation of cellular APP-751, secreted APP-751 and APP-7Sl mRNA in Sf9 cells infected with recombinant AC-APP-751 virus. Amounts of secreted and intracellular APP-751 at various times post-infection were calculated as described previously (16) from the experiment in Fig. 3. Levels of mRNA were computed from the Northern blots as described in Methods.
Total RNA was isolated from infected Sf9 cells harvested at various times postinfection and was analysed by Northern blotting. One major 2.8 kb transcript was detected, which is compatible with the predicted transcript starting at the polyhedrin cap-site, and terminating at the polyhedrin transcription stop site. In wild-type virus-infected cells no cross-hybridizing material was detected (data not shown). The amount of APP-75 1 mRNA increases sharply from 36hpi until 48hpi. Assuming that mRNA is five percent of total RNA isolated from infected cells, we computed that as a fraction of total mRNA, APP-751 mRNA increases from 0.054 percent at 24hpi to 0.616 percent at 36hpi and to 20.73 percent at 48hpi. In a similar study pre-pro-attacin mRNA expression (20) was first detected 15hpi, and it increased until 48hpi. It is clear that infected cells have sufficient mRNA to support a high level of APP-751 synthesis between 48 and 72hpi (Fig. 4). The immunoblots and autoradiograms were quantified by laser densitometry (Fig. 4). That both APP-751 and its mRNA are barely detectable at 24 hpi is consistent with the late expression of the polyhedrin promoter (21). Membrane-associated APP-751 increases two fold from 48 hpi to 72 hpi and decreases at 96 hpi whereas the secreted form increases almost 4-fold and remains at the same level until 96 hpi. These results demonstrate that, under conditions of overproduction, APP-751 is cleaved to release a significant amount of secreted form into the medium with concomitant increase in the amount of COOHterminal peptide in the cells. At 72 hpi, the total amount of APP-751 in this experiment was 4 mg/l08 cells; we have occasionally observed levels of 10 mg/108 cells. Approximately 60% of total APP-751 is membrane-associated and most of the remaining protein is the secreted form. A similar time course of accumulation has been reported for the extracellular domain of the human insulin receptor (22), which reached its maximum 987
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level in the medium at 72 hpi. Other human proteins including the glucocorticoid receptor (23), extracellular domain of nerve growth factor (24) and CD4 (25) have been expressed using this system; however, their levels of expression are at least ten-fold lower than the level we achieved with APP-751. Purification and N-terminal seauence analysis of recombinant APP-751 APP-751 was highly enriched from a membrane extract by a single step of heparin agarose chromatography. The protein was further purified by PAGE. The APP-751 band was eluted from the gel, and its NHa-terminal sequence was determined. Its sequence was found to be Leu-Glu-Val-Phe-Thr-Asp-Gly-Asn-Ala-Gly which is identical to the sequence found for native APP from rat brain (16) and to the sequence of protease nexin II (4). This identifies the protein as APP-751 and shows that insect cells correctly cleave the signal sequence of amyloid precursor protein. In this report we have described a baculovirus derived system that is capable of APP-751 expression at 4 mg/108 cells or 20 mg/liter. We have shown that the protein is localized in membranes and established the authenticity of the recombinant protein by NHz-terminal sequence analysis. We have identified a secreted form of APP-751 lacking the COOH-terminal domain and the trans-membrane region. APP-751 expressed in the baculovirus system will be useful for studies of post-translational modifications such as phosphorylation and glycosylation, and for ligand binding and processing studies as well as for making specific antibodies to native APP-751.
ACKNOWLEDGMENTS: We thank Mr. James Styles for the amino terminal sequencing of recombinant APP and Ms. Gloria Wolfe for help with the RNA blot. Dr. Robert Denman provided synthetic APP-mRNA. Drs. Pankaj Mehta and Bruce Patrick made available the polyclonal antibodies. Monoclonal antibodies were obtained from Dr. Kwang Soo Kim. Dr. E. P. Reddy allowed us the use of laboratory facilities during the isolation of the recombinant virus. Special thanks to Ms. Patricia Casiano for the manuscript preparation. This work was supported by grants No. NS25231 and TO-l-AGO4220 from the NIH and by the New York State Office of Mental Retardation and Developmental Disabilities.
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