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Recombinant breast carcinoma-associated mucins expressed in a baculovirus system contain a tumor specific epitope
Immunotechnology 4 (1998) 97 – 105
Recombinant breast carcinoma-associated mucins expressed in a baculovirus system contain a tumor specific epitope Peisheng Hu 1,a, Stephen E. Wright a,b,c,* a
Department of Internal Medicine, Texas Tech Uni6ersity Health Sciences Center, 1400 Wallace Bl6d., Amarillo, TX 79106, USA b Department of Cell Biology and Biochemistry, Texas Tech Uni6ersity Health Sciences Center, 1400 Wallace Bl6d., Amarillo, TX 79106, USA c Veterans Affairs Medical Center, 6010 Amarillo Bl6d. West, Amarillo, Texas 79106, USA Received 8 January 1998; received in revised form 24 February 1998; accepted 24 February 1998
Abstract Mucins are highly immunogenic glycoproteins that are abundantly expressed by breast and other adenocarcinomas. In order to progress in the understanding of the structure – immunity relationship of the breast tumor associated mucin and normal tissue mucin, two forms of breast carcinoma associated mucin, muc7-BV and pem-BV, were expressed in a baculovirus expression system. The muc7-BV was constructed by inserting the seven tandem repeats of mucin core cDNA fragment into transfer vector pAc360, forming a fusion protein containing 14 amino acids of the baculovirus polyhedrin N-terminus. The pem-BV was constructed by cloning full-length mucin cDNA into the transfer vector pVL1392. The recombinant mucins were purified using immunoaffinity chromatography. The purified muc7-BV and pem-BV had molecular weights of 28 and 59 kd, respectively. No carbohydrate was detected on these recombinant mucins and is speculated to explain why both forms of recombinant mucin showed strong affinity to tumor-specific monoclonal antibody SM3. These recombinant mucins may have the potential value to develop vaccines against breast and other adenocarcinomas and to induce cytotoxic T-lymphocyte lines for immunotherapy of the same. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Recombinant proteins; Mucins immunology; Baculoviridae genetics; Epitopes immunology; Adenocarcinoma immunology Abbre6iations: bp, base pair(s); m.o.i., multiplicity of infection; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; TBST, 10 mM Tris, pH 7.5, 0.15 M NaCl, 0.05% Tween-20; ECL, enhanced chemiluminesence; CNBr, cyanogen bromide; pfu, plaque forming unit(s); IgG, immunoglobulin G; TBS20,50, Tris buffered saline, with 20 or 50 mM Tris, respectively; BV, baculovirus; w/v, weight/volume; TBSMMC, 50 mM Tris–HCl, 150 mM NaCl, 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, pH 7.5; PNA, peanut agglutinin; WGA, wheat germ agglutinin; ECL, erythrina cristaggalli lectin; SBA, soybean agglutinin; RCA, ricinus communis toxin; ConA, concanavalin A; PBS, phosphate buffered saline; MAb, monoclonal antibody; ELISA, enzyme linked immunosorbent assay; HMP, human milk protein; mtr, mucin tandem repeat. * Corresponding author. Tel.: + 1 806 3547871; fax: + 1 806 3545549. 1 Present address: Department of Pathology, University of Southern California Health Sciences Center, Los Angeles, CA 90033, USA. 1380-2933/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S1380-2933(98)00009-8
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1. Introduction Mucins are large molecular weight (\ 0.25 × 106 daltons) heterogeneous glycoproteins, which contain 50–90% O-linked carbohydrate [1]. Breast carcinoma-associated mucin has been the focus of basic and clinical studies because of many antigenic differences between carcinomas and adjacent normal epithelial tissues, that have been attributed to the aberrant glycosylation of this glycoprotein [1]. The entire mucin core protein has been cloned and sequenced [1 – 3]. The encoded protein consists of three distinct regions: (a) the amino terminus, consisting of a putative signal peptide and a region containing degenerate tandem repeat sequences; (b) the major portion of the protein consisting only of the tandem repeat region, the carboxyl terminus of which contains degenerate tandem repeats; and (c) a unique region containing a 31-amino acid transmembrane sequence and a 69-amino acid cytoplasmic tail [1,2]. Tandem repeats are a general characteristic of mucin core proteins. Each tandem repeat unit is rich in serine, threonine, glycine, alanine and proline and consists of a 60 bp2 sequence encoding a 20-amino acid sequence: GSTAPPAHGVTSAPDTRPAP [1–3]. The same tandem repeat, termed MUC1, is found in breast, ovarian and pancreatic mucins [1], but different repeat units have been found in the core proteins of other mucins [4]. These mucins are members of a family of genes expressed in organs with glands. Mucins generally contain between 40 and 100 tandem repeat sequences, thus resulting in structural polymorphisms [2]. Recently, attention has been focused on these glycoproteins since many antibodies, raised from malignant epithelial cells, recognize epitopes on these molecules. It appears that the mucins expressed by breast and other carcinomas are in an aberrantly glycosylated form [5] and that the carbohydrate side chains of the cancer-associated mucin [6] are shorter than the side chains of the mucin produced by normal cells [7]. The aberrant glycosylation of the cancer-associated mucin results in the exposure of antigenic epitopes on the core protein which are masked in the fully glycosylated form [5]. This is evidenced by the develop-
ment of a tumor-specific monoclonal antibody, SM3, which recognizes tumor-specific mucins and the fully deglycosylated native mucin, but not the fully glycosylated native mucin [5,8]. Other epitopes in the tandem repeat domains are expressed in both the normal and tumor-associated mucins [9]. In addition to the humoral identification of a tumor-specific mucin epitope in mice, humans have also been found to produce antibodies that are specific for mucin expressing tumor cells and recognize a MUC1 peptide [10]. The tumor-specific recognition of tumor-associated mucins by cytotoxic thymic lymphocyte (CTL) lines derived from patients with pancreatic [11], breast [12] and ovarian [13] cancer has also been demonstrated. The CTL lines recognize an epitope present within the last seven amino acids of the 20-amino acid core peptide of the breast tumor mucin molecule: the epitope is the same recognized by the SM3 monoclonal antibody. These data imply that cancer-specific mucin is a common tumor antigen on multiple carcinomas and thus has the potential for use as an immunogen for these malignancies. Since recombinant baculovirus expressing foreign glycoproteins in insect cells produce shortened carbohydrate side chains [14], it was the intent of this study to use the recombinant baculovirus expressed mucin as a model for exposure of tumor associated epitopes.
2. Experimental procedures
2.1. Materials The transfer vectors pAc360 and pVL1392 [15], and the wild type autographa californica nuclear polyhedrosis virus (AcNPV, E2 strain) were obtained from Dr M. Summers, Texas A & M University. Spodoptera frugiperda (Sf9) insect cells were purchased from American Type Culture Collection. The pMuc7 [16] containing seven tandem repeats of mucin core sequence (mtr), pBS-PEMtm [2] containing full length of mucin sequence with 32 mtr and the monoclonal antibody SM3, were obtained from Dr S.J. Gendler, Imperial Cancer Research Fund, London, UK. Protein A-Sepharose, CNBr activated sepharose 4B,
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horseradish peroxidase conjugated goat antimouse or anti-rabbit IgG were purchased from Sigma. Grace’s insect cell culture medium, gentamicin and amphotericin B were purchased from GIBCO. Yeastolate and lactalbumin hydrolysate were obtained from Difco. Ex-400 medium was from J.R. Scientific. Low melting gel agarose was from FMC Bioproducts. Restriction endonucleases, T4 ligase and other molecular biology tools were purchased from New England Biolabs. The linearized wild type virus (AcNPV) DNA was obtained from Invitrogen. Breast milk was donated by a human. Human milk protein (HMP) was prepared as a 40% saturated ammonium sulfate precipitate.
2.2. Cell and 6iral culture [15] Sf9 cells were grown as monolayers or in suspension in spinner flasks at 28°C in Ex-400 medium with the antibiotic amphotericin B (2.5 mg/ml). Viral stocks were made by infecting attached cell cultures at a low multiplicity of infection (m.o.i.) and harvesting 2 – 4 days after infection.
2.3. Construction of recombinant transfer 6ector and recombinant baculo6irus The recombinant vector pAc360-muc7 was constructed from the transfer vector pAc360 and plasmid pMal-muc7 [17]. pAc360 is a vector containing the polyhedrin promoter and 35 bp of 5%-polyhedrin coding sequences upstream of the unique BamHI site. This vector is designed to express especially high levels of fused proteins in the baculovirus system [15]. The BamHI fragment of pMal-muc7, which contains seven tandem repeats of mucin cDNA coding sequence [16], was filled in with Klenow fragment, isolated and blunt-ligated into the blunt-ended BamHI site of the baculovirus transfer vector pAc360. The muc7-cDNA was inserted into the polyhedrin gene with correct reading frame to code a fusion protein containing 14 amino acids of the polyhedrin N-terminus. The recombinant vector pVL1392-pem was constructed from plasmid pBS-PEM-tm and transfer vector pVL1392.
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The vector pVL1392 is derived from vector pAc360 by inserting a multiple cloning region into the unique BamHI site and modifying the polyhedrin translation initiation codon ATG to ATT. This modification allows the translation initiation to begin at the mucin initiation codon and prevents the production of a fusion protein. The plasmid pBS-PEM-tm was digested with NotI and XbalI. The fragment containing the entire mucin coding sequence was then inserted into the same sites of pVL1392 to allow expression of a nonfused full length mucin. The recombinants were then transformed into Escherchia coli DH5a strain. The clones that were ampicillin resistant were selected and their plasmid DNA was obtained by a rapid minilysis procedure [18]. The DNA was analyzed by restriction enzyme and the correct orientation recombinants were selected and their DNA was purified using QIAGEN Plasmid Kits. These recombinant transfer plasmids were then used for inserting mucin DNA into the genome of ACNPV at the polyhedrin gene locus by homologous recombination. This was achieved by cotransfection of wild type virus (AcNPV) linear DNA (Invitrogen) with recombinant transfer vector pAc360-muc7 or pVL1392-pem onto monolayers of Sf9 cells using a calcium phosphate procedure as previously described [19]. Five days after cotransfection, the virus containing supernatant medium was collected, serially diluted and then used to infect fresh monolayers of Sf9 cells. During plaque identification, 0.01% neutral red was used in the agarose overlay, to assist in the visualization of recombinant virus [20]. The recombinant baculoviruses were identified by visually screening for polyhedrin-negative plaques and were further purified by three serial cycles of plaque purification.
2.4. Enhanced chemiluminescence (ECL) immunoblot analysis Sf9 cells were grown in 24-well culture plates (1×105/well with 1 ml medium) and both cells and medium were harvested at 24–96 h post-infection. The cells were solubilized in SDS-sample buffer [21].
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The medium was precipitated by adding 50% volume of acetone and the precipitate was solubilized in SDS-sample buffer. The samples were boiled for 5–10 min and electrophoresed (E) through an 8% SDS-polyacrylamide gel (PAG). Proteins were then transferred to nitrocellulose membranes [18]. Filters were blocked in 4% bovine serum albumin (BSA) in TBST buffer (10 mM Tris, pH 7.5, 0.15 M NaCl, 0.05% Tween20), washed with TBST, incubated with mAb SM3 [5] or rabbit anti-rMucin [17] at room temperature (1:1000; 1:20000 dilution, respectively, 40 min). Filters were then incubated with horseradish peroxidase conjugated goat anti-mouse or rabbit, respectively, IgG (1:30000, 30 min). After wash, the filters were transferred to cyclic diacylhydrazides solution (ECL buffer, Amersham) for 1 min and exposed to X-ray film for 1 – 15 min.
2.5. Immunoaffinity chromatography Anti-rMucin IgG, purified by a protein A column as described [22], was coupled to CNBr activated Sepharose 4B (Sigma) per the manufacturer’s instructions. The antibody was coupled to Sepharose at 5 mg/ml of gel with an average coupling efficiency of 92%.
2.6. Purification of muc7 -BV and pem-BV A total of 150 ml of Sf9 cells at 1.0×106/ml grown in suspension culture, were pelleted and then infected with recombinant baculovirus — five plaque-forming units (pfu)/cell. At 72 h post-infection, the cells were harvested by centrifugation at 1000 ×g. The cell pellet was resuspended in solubilization buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 10 mM EGTA, 1% Nonidet P-40) in the presence of a mixture of protease inhibitors (5 mg/ml aprotinin, 1 mM phenylmethanesulfonyl fluoride) and sonicated and centrifugated at 12000×g for 30 min. The supernatant then was incubated with anti-rMucin IgG gel overnight at 4°C on a rotator. The gel was washed thoroughly with TBS20 (20 mM Tris, 0.15 mM NaCl, pH 7.5) and eluted with 0.1 mM Gly, pH 2.3. The elute was neutralized to pH 7.5 and concentrated in a Centracon-30 (Amicon).
2.7. Lectin binding analysis of recombinant mucins Recombinant muc7-BV and pem-BV were separated with SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then treated with 20 ml of 0.5% blocking reagent (w/v) (Boehringer Mannheim Biochemicals) in TBSMMC (50 mM Tris–HCl, 150 mM NaCl, 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, pH 7.5). After washing three times with TBSMMC the membranes were incubated for 30 min at room temperature with biotin conjugated lectins (Jacalin, 10 mg/ml; PNA, 10 mg/ml; WGA, 10 mg/ ml; ECL,10 mg/ml; SBA, 10 mg/ml; Pierce) (Con A, 5 mg/ml; RCA60, 2 mg/ml; Boehringer Mannheim) in a 1:50000 dilution. After membranes were washed three times with TBSMMC, peroxidase-conjugated streptavidin (0.25 mg/ml, Sigma) was added in a 1:50000 dilution in TBSMMC and incubated for 30 min at room temperature. The membranes were then washed again and transferred to a cyclic diacylhydrazides solution (ECL buffer, Boehringer Mannheim) for 1 min and exposed to X-ray film for 1–15 min.
2.8. Carbohydrate detection of recombinant mucin GlycoTrackTM kit (Oxford GlycoSystem) was used for detecting carbohydrate of recombinant mucins. Briefly, 1 mg of recombinant muc7-BV or pem-BV was spotted onto nitrocellulose membrane using a minifold vacuum apparatus (Schleicher and Schuell). The membranes were washed three times with phosphate buffered saline (PBS) 50 mM sodium phosphate, 150 mM NaCl, pH 7.2 and incubated with 10 mM sodium metaperiodate in 0.1 M sodium acetate buffer containing 5 mM EDTA, pH 5.5 for 20 min at room temperature. After washing three times with PBS, the membranes were incubated with biotin–hydrazide in 0.1 M sodium acetate, pH 5.5 for 60 min at room temperature and washed three times with TBS50 (50 mM Tris, 150 mM NaCl, pH 7.2). The membranes were then treated with 0.5% blocking reagent in TBS50 for 30 min and incubated with streptavidin conjugated with alkaline phosphatase for 60 min. After washing with TBS50, the filters
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were transferred to lumi-phos 530 solution (ECL buffer, Boehringer Mannheim) for 1 min and exposed to X-ray film for 1 – 15 min. To inhibit Nor O-linked glycosylation, 3 mg/ml tunicamycin (Boehringer Mannheim) or 1 mM phenyl-Nacetyl-a-D-galactosaminide (Sigma), respectively, in TNM-FH media were used on infected cell monolayers for 72 h, as described [23]. The cells were then harvested and dissolved in SDS sample buffer and subjected to ECL immunoblot by antirMucin or monoclonal antibody SM3, as described above.
3. Results
3.1. Construction of muc7 -BV and pem-BV recombinant baculo6iruses Transfer vectors pAc360-muc7 and pVL1392pem were constructed as described in Section 2. The expression of either pem or muc7 was under control of the strong polyhedrin promoter. After co-transfection of Sf9 insect cells with linear AcMNPV DNA and pVL1392-pem or pAc360muc7, 10–20% of plaques were polyhedrin-negative recombinant baculoviruses. The recombinant viruses were initially identified by visual screening and confirmed by DNA hybridization and immunoblot analysis (see Section 2).
3.2. Expression of recombinant muc7 -BV and pem-BV The recombinant full length mucin and fusion protein of mucin seven repeats expressed in the baculovirus system were named pem-BV and muc7-BV, respectively. The expression of recombinant mucin was assayed in baculovirus-infected Sf9 cells by ECL-immunoblot analysis using rabbit anti-rMucin or mAb SM3, as described in Section 2. Twelve polyhedrin-negative and DNAhybridization positive plaques of pAc360-muc7 recombinant baculovirus were analyzed and all of them expressed a 28-kDa fusion peptide which is not seen in the wild type AcNPV-infected cells. However, in pVL-1392-pem recombinant baculovirus, five of 12 plaques expressed a protein
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recognized by both mAb SM3 and anti-rMucin. Four of five plaques producing mucin expressed a protein of : 59 kDa and another plaque expressed a 90-kDa protein recognized by mAb SM3. This 90-kDa peptide disappeared after the virus was amplified and a 59-kDa peptide, which is identical to those in other plaques, appeared. The appearance of the lower molecular weight mucin is presumed to be due to recombination between the mucin repeats with deletion of the intervening repeats. Since both sizes of the mucin produced were recognized by mAb SM3, the tumor-specific epitope is present in the 59-kDa, so no further analysis was performed. To study the efficiency of expression of recombinant mucin, Sf9 cells were infected with pure recombinant viruses at 10 m.o.i. (10 pfu of virus/cell) and cells and supernatants were harvested at various times following infection. By including a known amount of purified muc7-BV or pem-BV on immunoblots, we were able to estimate the amount of recombinant mucins expressed in the cells or in the medium at different time intervals. Both muc7-BV and pem-BV reached the detectable level at 48 h post-infection and achieved maximal level of expression by 96 h post-infection. Approximately 10 and 2.5 mg of mucin are produced in 106 muc7BV and PEM-BV infected Sf9 cells, respectively. No detectable amount was found in medium of either muc7-BV (Fig. 1) or pem-BV infected Sf9 cells.
Fig. 1. Analysis of expression of muc7-BV by immunoblot with mucin-specific mAb SM3. (A) Supernatant. (B) Cell. Lanes 1, 2, 3 and 4: 24, 48, 72 and 96 h, respectively.
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antibodies specific to mucin tandem repeats (Fig. 2B, lane 2).
3.4. ELISA of recombinant mucins protein with mAb SM3
Fig. 2. Analyses of purified mucins expressed from recombinant baculovirus infected Sf9 cells. (A) muc7-BV. Lane 1. Immunoblot with mucin-specific mAb SM3. Lane 2. Coomassie blue stained 10% SDS-PAG. Lane 3. Molecular weight standards. (B) pem-BV. Lane 1. Coomassie blue stained 10% SDS-PAG. Lane 2. Immunoblot with mucin-specific MAb SM3.
To determine whether the undenatured form of recombinant mucins expose the tumor-specific epitope [17], an ELISA assay using purified recombinant mucins was performed. Fig. 3 shows the reactions of monoclonal antibody SM3 with muc7-BV, pem-BV, muc7-EC [17] (a fusion protein which contains seven mucin tandem repeats expressed in E. coli ) and human milk protein (HMP). mAb SM3 showed strong affinity to all those recombinant mucins but not the HMP (Fig. 3). The strong binding activity to SM3 suggests that the recombinant mucin fusion protein exposed the tumor epitope in the baculovirus expression system.
3.5. Carbohydrate analysis of recombinant mucins 3.3. Purification of muc7 -BV and pem-BV Purification of muc7-BV and pem-BV was obtained in a single step by immunoaffinity chromatography, as described in Section 2. Since slight degradation was found in immunoassays, an attempt was made to process the cells as rapidly as possible to minimize any possible degradation. The overall yields obtained from immunoaffinity purification were :10% for pem-BV and 5% for muc7-BV. The affinity purified recombinant mucins were analyzed on a 10% SDS-PAG and stained by Coomassie blue and immunoblotted. Coomassie blue staining of the SDS-PAG shows that muc7-BV gave bands of molecular weight of 28- and 65-kDa (Fig. 2A, lane 2). Immunoblot staining shows that both polyclonal anti-rmucin and mAb SM3 (Fig. 2A, lane 1) only recognize the 28-kDa species, indicating that the 65-kDa band may be a contaminant during the purification. For the purified pem-BV, a main 59-kDa band was stained by both Coomassie blue (Fig. 2B, lane 1) and mAb SM3 (Fig. 2B, lane 2), while the two small degradation products were not stained by the
Three different methods were used to determine whether the mucins expressed in the bac-
Fig. 3. ELISA of purified recombinant mucins with mAb SM3. Ninety-six well polystyrene plates coated with 2 mg/ml of mucin were incubated with SM3 at the indicated concentrations for 1 h, followed by horseradish peroxidase conjugated with antibody to mouse Ig for 1 h, then substrate, 2,2%-azinodo-{3-ethylbenzthiazoline sulfonate} (ABTS) (Kirkegaad and Perry Laboratories) per the manufacturer’s instructions and read in a 96-well reader. Key: muc7-BV ( ); muc7-EC ("); pem-BV ( ); HMP (2).
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recombinant mucin expressed in baculovirus may not contain either O- or N-linked carbohydrate. This was further supported by directed carbohydrate analyses using a glycoprotein detection kit, which used biotin–hydrazide to label periodate oxidized carbohydrate (Fig. 4B) and also by reaction with a streptavidin–alkaline phosphatase conjugate and enhanced chemiluminescence (ECL) lectin binding assays (Table 1).
4. Discussion
Fig. 4. Carbohydrate analyses of muc7-BV. (A) Inhibitors of O- and N-linked glycosylation. Lane 1. No inhibitor. Lane 2. Phenyl-N-acetyl-a-D-galactosaminide. Lane 3. Tunicamycin. (B) Carbohydrate detection by GlycoTract™ (Oxford Glycosystem).
ulovirus system are glycoproteins and analyze their possible carbohydrate structure. Glycosylation inhibition assays were performed on recombinant baculovirus infected Sf9 cell media, as described under Section 2. Treatment with either O-glycosylation inhibitor (phenyl-N-acetyl-a-Dgalactosaminide, Fig. 4A, lane 2) or N-linked glycosylation inhibitor (tunicamycin, Fig. 4A, lane 3), did not change the mobility of mucin expressed from muc7-BV infected Sf9 cells on SDS-PAGE. Similar patterns were also found in pem-BV (data not shown). This suggests that the
The production of recombinant mucin in high yields from baculovirus expression systems offers a new way to study mucin functions and to develop diagnostic tests and immunogens for carcinomas that express tumor-specific mucin. The purpose of this study was to determine whether the baculovirus expression system would produce mucin containing a tumor-specific epitope and the role of carbohydrate in masking the tumor-specific epitope. Many eukaryotic proteins have been successfully expressed in the baculovirus system [15]. In most cases the baculovirus expressed proteins were produced in substantial quantities and with carbohydrate side chains if the foreign gene coded for a glycoprotein. In this study, we have expressed breast tumor associated mucin in a baculovirus system. The expression levels of recombinant mucins were fairly high compared to other proteins [15].
Table 1 Lectin analysis of recombinant mucin expressed in baculovirus system Lectin
Specificity
muc7-BV
pem-BV
Milk protein
Jacalin PNA WGA ECL SBA RCA60 ConA
a-D-Galactose b-D-Gal-(13)-D-GalNAc b-D-GlcNAc b-D-Gal-(14)-D-GlcNAc a-D-GalNAc D-GalNAc, b-D-Gal a-D-Man, a-D-Glc
− − − − − − −
− − − − − − −
+ + + + + + +
The samples were separated on SDS-PAGE, transferred onto nitrocellulose membrane, detected by biotinylated lectins and reported by peroxidase conjugated streptavidin using enhanced chemiluminescence (ECL), as described in Section 2. PNA, peanut agglutinin; WGA, wheat germ agglutinin; ECL, erythrina cristaggalli lectin; SBA, soybean agglutinin; RCA, ricinus communis toxin; ConA, concanavalin A.
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Moreover, it is clear from above data that the mucin expressed in the baculovirus system demonstrated strong affinity to tumor-specific monoclonal antibody SM3, indicating the presence of a tumor-specific epitope. The reason for the exposure of the tumor-specific epitope, is attributed to no detectable carbohydrate in the recombinant mucin expressed in the baculovirus system. The mAb SM3 did not recognize the HMP (Fig. 3), which is glycosylated, whereas it recognized baculovirus expressed, unpurified mucin when analysed from cells (Fig. 1). In addition, analysis with an inhibitor of O-linked glycosylation did not alter the molecular weight of intracellular mucin (Fig. 4A), consistent with lack of glycosylation of the baculovirus expressed mucin in the insect cells. These two assays with unpurified mucin support the results of lack of detectable carbohydrate with immunoaffinity purified baculovirus expressed mucin by immunoblotting with mAb SM3 (Figs. 2 and 3) and lectin assays for carbohydrate (Table 1). Others [24,25] have found glycoproteins not glycosylated when expressed in insect cells by baculovirus. The mucin expressed by baculovirus will be tested as a tumor-specific mucin in antigenic and immunogenic studies, in comparison with nonglycosylated tumorspecific mucin produced in bacteria [17]. The advantages of a baculovirus system over that of bacteria [17] already identified, are that the yield of mucin is 4– 24 times better, the protien is not degraded and a pure, non-fusion protein is expressed from one baculovirus recombinant.
Acknowledgements This work was supported by the Department of Veterans Affairs Medical Research Funds to SEW. We thank those mentioned in the text for materials, Aoyun Yun for technical assistance, Melody Robinson, Harrington Cancer Center and Ralph Leone, VAMC, for photography, Gregoria T. Avila, Kaye Ezzell, Stephanie Stevenson, Kimberly Hamilton, Diana Wright
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and Jennifer Moore for clerical assistance and Dr K.E. Dombrowski for review of the manuscript.
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Recombinant breast carcinoma-associated mucins expressed in a baculovirus system contain a tumor specific epitope Peisheng Hu 1,a, Stephen E. Wright a,b,c,* a
Department of Internal Medicine, Texas Tech Uni6ersity Health Sciences Center, 1400 Wallace Bl6d., Amarillo, TX 79106, USA b Department of Cell Biology and Biochemistry, Texas Tech Uni6ersity Health Sciences Center, 1400 Wallace Bl6d., Amarillo, TX 79106, USA c Veterans Affairs Medical Center, 6010 Amarillo Bl6d. West, Amarillo, Texas 79106, USA Received 8 January 1998; received in revised form 24 February 1998; accepted 24 February 1998
Abstract Mucins are highly immunogenic glycoproteins that are abundantly expressed by breast and other adenocarcinomas. In order to progress in the understanding of the structure – immunity relationship of the breast tumor associated mucin and normal tissue mucin, two forms of breast carcinoma associated mucin, muc7-BV and pem-BV, were expressed in a baculovirus expression system. The muc7-BV was constructed by inserting the seven tandem repeats of mucin core cDNA fragment into transfer vector pAc360, forming a fusion protein containing 14 amino acids of the baculovirus polyhedrin N-terminus. The pem-BV was constructed by cloning full-length mucin cDNA into the transfer vector pVL1392. The recombinant mucins were purified using immunoaffinity chromatography. The purified muc7-BV and pem-BV had molecular weights of 28 and 59 kd, respectively. No carbohydrate was detected on these recombinant mucins and is speculated to explain why both forms of recombinant mucin showed strong affinity to tumor-specific monoclonal antibody SM3. These recombinant mucins may have the potential value to develop vaccines against breast and other adenocarcinomas and to induce cytotoxic T-lymphocyte lines for immunotherapy of the same. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Recombinant proteins; Mucins immunology; Baculoviridae genetics; Epitopes immunology; Adenocarcinoma immunology Abbre6iations: bp, base pair(s); m.o.i., multiplicity of infection; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; TBST, 10 mM Tris, pH 7.5, 0.15 M NaCl, 0.05% Tween-20; ECL, enhanced chemiluminesence; CNBr, cyanogen bromide; pfu, plaque forming unit(s); IgG, immunoglobulin G; TBS20,50, Tris buffered saline, with 20 or 50 mM Tris, respectively; BV, baculovirus; w/v, weight/volume; TBSMMC, 50 mM Tris–HCl, 150 mM NaCl, 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, pH 7.5; PNA, peanut agglutinin; WGA, wheat germ agglutinin; ECL, erythrina cristaggalli lectin; SBA, soybean agglutinin; RCA, ricinus communis toxin; ConA, concanavalin A; PBS, phosphate buffered saline; MAb, monoclonal antibody; ELISA, enzyme linked immunosorbent assay; HMP, human milk protein; mtr, mucin tandem repeat. * Corresponding author. Tel.: + 1 806 3547871; fax: + 1 806 3545549. 1 Present address: Department of Pathology, University of Southern California Health Sciences Center, Los Angeles, CA 90033, USA. 1380-2933/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S1380-2933(98)00009-8
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1. Introduction Mucins are large molecular weight (\ 0.25 × 106 daltons) heterogeneous glycoproteins, which contain 50–90% O-linked carbohydrate [1]. Breast carcinoma-associated mucin has been the focus of basic and clinical studies because of many antigenic differences between carcinomas and adjacent normal epithelial tissues, that have been attributed to the aberrant glycosylation of this glycoprotein [1]. The entire mucin core protein has been cloned and sequenced [1 – 3]. The encoded protein consists of three distinct regions: (a) the amino terminus, consisting of a putative signal peptide and a region containing degenerate tandem repeat sequences; (b) the major portion of the protein consisting only of the tandem repeat region, the carboxyl terminus of which contains degenerate tandem repeats; and (c) a unique region containing a 31-amino acid transmembrane sequence and a 69-amino acid cytoplasmic tail [1,2]. Tandem repeats are a general characteristic of mucin core proteins. Each tandem repeat unit is rich in serine, threonine, glycine, alanine and proline and consists of a 60 bp2 sequence encoding a 20-amino acid sequence: GSTAPPAHGVTSAPDTRPAP [1–3]. The same tandem repeat, termed MUC1, is found in breast, ovarian and pancreatic mucins [1], but different repeat units have been found in the core proteins of other mucins [4]. These mucins are members of a family of genes expressed in organs with glands. Mucins generally contain between 40 and 100 tandem repeat sequences, thus resulting in structural polymorphisms [2]. Recently, attention has been focused on these glycoproteins since many antibodies, raised from malignant epithelial cells, recognize epitopes on these molecules. It appears that the mucins expressed by breast and other carcinomas are in an aberrantly glycosylated form [5] and that the carbohydrate side chains of the cancer-associated mucin [6] are shorter than the side chains of the mucin produced by normal cells [7]. The aberrant glycosylation of the cancer-associated mucin results in the exposure of antigenic epitopes on the core protein which are masked in the fully glycosylated form [5]. This is evidenced by the develop-
ment of a tumor-specific monoclonal antibody, SM3, which recognizes tumor-specific mucins and the fully deglycosylated native mucin, but not the fully glycosylated native mucin [5,8]. Other epitopes in the tandem repeat domains are expressed in both the normal and tumor-associated mucins [9]. In addition to the humoral identification of a tumor-specific mucin epitope in mice, humans have also been found to produce antibodies that are specific for mucin expressing tumor cells and recognize a MUC1 peptide [10]. The tumor-specific recognition of tumor-associated mucins by cytotoxic thymic lymphocyte (CTL) lines derived from patients with pancreatic [11], breast [12] and ovarian [13] cancer has also been demonstrated. The CTL lines recognize an epitope present within the last seven amino acids of the 20-amino acid core peptide of the breast tumor mucin molecule: the epitope is the same recognized by the SM3 monoclonal antibody. These data imply that cancer-specific mucin is a common tumor antigen on multiple carcinomas and thus has the potential for use as an immunogen for these malignancies. Since recombinant baculovirus expressing foreign glycoproteins in insect cells produce shortened carbohydrate side chains [14], it was the intent of this study to use the recombinant baculovirus expressed mucin as a model for exposure of tumor associated epitopes.
2. Experimental procedures
2.1. Materials The transfer vectors pAc360 and pVL1392 [15], and the wild type autographa californica nuclear polyhedrosis virus (AcNPV, E2 strain) were obtained from Dr M. Summers, Texas A & M University. Spodoptera frugiperda (Sf9) insect cells were purchased from American Type Culture Collection. The pMuc7 [16] containing seven tandem repeats of mucin core sequence (mtr), pBS-PEMtm [2] containing full length of mucin sequence with 32 mtr and the monoclonal antibody SM3, were obtained from Dr S.J. Gendler, Imperial Cancer Research Fund, London, UK. Protein A-Sepharose, CNBr activated sepharose 4B,
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horseradish peroxidase conjugated goat antimouse or anti-rabbit IgG were purchased from Sigma. Grace’s insect cell culture medium, gentamicin and amphotericin B were purchased from GIBCO. Yeastolate and lactalbumin hydrolysate were obtained from Difco. Ex-400 medium was from J.R. Scientific. Low melting gel agarose was from FMC Bioproducts. Restriction endonucleases, T4 ligase and other molecular biology tools were purchased from New England Biolabs. The linearized wild type virus (AcNPV) DNA was obtained from Invitrogen. Breast milk was donated by a human. Human milk protein (HMP) was prepared as a 40% saturated ammonium sulfate precipitate.
2.2. Cell and 6iral culture [15] Sf9 cells were grown as monolayers or in suspension in spinner flasks at 28°C in Ex-400 medium with the antibiotic amphotericin B (2.5 mg/ml). Viral stocks were made by infecting attached cell cultures at a low multiplicity of infection (m.o.i.) and harvesting 2 – 4 days after infection.
2.3. Construction of recombinant transfer 6ector and recombinant baculo6irus The recombinant vector pAc360-muc7 was constructed from the transfer vector pAc360 and plasmid pMal-muc7 [17]. pAc360 is a vector containing the polyhedrin promoter and 35 bp of 5%-polyhedrin coding sequences upstream of the unique BamHI site. This vector is designed to express especially high levels of fused proteins in the baculovirus system [15]. The BamHI fragment of pMal-muc7, which contains seven tandem repeats of mucin cDNA coding sequence [16], was filled in with Klenow fragment, isolated and blunt-ligated into the blunt-ended BamHI site of the baculovirus transfer vector pAc360. The muc7-cDNA was inserted into the polyhedrin gene with correct reading frame to code a fusion protein containing 14 amino acids of the polyhedrin N-terminus. The recombinant vector pVL1392-pem was constructed from plasmid pBS-PEM-tm and transfer vector pVL1392.
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The vector pVL1392 is derived from vector pAc360 by inserting a multiple cloning region into the unique BamHI site and modifying the polyhedrin translation initiation codon ATG to ATT. This modification allows the translation initiation to begin at the mucin initiation codon and prevents the production of a fusion protein. The plasmid pBS-PEM-tm was digested with NotI and XbalI. The fragment containing the entire mucin coding sequence was then inserted into the same sites of pVL1392 to allow expression of a nonfused full length mucin. The recombinants were then transformed into Escherchia coli DH5a strain. The clones that were ampicillin resistant were selected and their plasmid DNA was obtained by a rapid minilysis procedure [18]. The DNA was analyzed by restriction enzyme and the correct orientation recombinants were selected and their DNA was purified using QIAGEN Plasmid Kits. These recombinant transfer plasmids were then used for inserting mucin DNA into the genome of ACNPV at the polyhedrin gene locus by homologous recombination. This was achieved by cotransfection of wild type virus (AcNPV) linear DNA (Invitrogen) with recombinant transfer vector pAc360-muc7 or pVL1392-pem onto monolayers of Sf9 cells using a calcium phosphate procedure as previously described [19]. Five days after cotransfection, the virus containing supernatant medium was collected, serially diluted and then used to infect fresh monolayers of Sf9 cells. During plaque identification, 0.01% neutral red was used in the agarose overlay, to assist in the visualization of recombinant virus [20]. The recombinant baculoviruses were identified by visually screening for polyhedrin-negative plaques and were further purified by three serial cycles of plaque purification.
2.4. Enhanced chemiluminescence (ECL) immunoblot analysis Sf9 cells were grown in 24-well culture plates (1×105/well with 1 ml medium) and both cells and medium were harvested at 24–96 h post-infection. The cells were solubilized in SDS-sample buffer [21].
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The medium was precipitated by adding 50% volume of acetone and the precipitate was solubilized in SDS-sample buffer. The samples were boiled for 5–10 min and electrophoresed (E) through an 8% SDS-polyacrylamide gel (PAG). Proteins were then transferred to nitrocellulose membranes [18]. Filters were blocked in 4% bovine serum albumin (BSA) in TBST buffer (10 mM Tris, pH 7.5, 0.15 M NaCl, 0.05% Tween20), washed with TBST, incubated with mAb SM3 [5] or rabbit anti-rMucin [17] at room temperature (1:1000; 1:20000 dilution, respectively, 40 min). Filters were then incubated with horseradish peroxidase conjugated goat anti-mouse or rabbit, respectively, IgG (1:30000, 30 min). After wash, the filters were transferred to cyclic diacylhydrazides solution (ECL buffer, Amersham) for 1 min and exposed to X-ray film for 1 – 15 min.
2.5. Immunoaffinity chromatography Anti-rMucin IgG, purified by a protein A column as described [22], was coupled to CNBr activated Sepharose 4B (Sigma) per the manufacturer’s instructions. The antibody was coupled to Sepharose at 5 mg/ml of gel with an average coupling efficiency of 92%.
2.6. Purification of muc7 -BV and pem-BV A total of 150 ml of Sf9 cells at 1.0×106/ml grown in suspension culture, were pelleted and then infected with recombinant baculovirus — five plaque-forming units (pfu)/cell. At 72 h post-infection, the cells were harvested by centrifugation at 1000 ×g. The cell pellet was resuspended in solubilization buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 10 mM EGTA, 1% Nonidet P-40) in the presence of a mixture of protease inhibitors (5 mg/ml aprotinin, 1 mM phenylmethanesulfonyl fluoride) and sonicated and centrifugated at 12000×g for 30 min. The supernatant then was incubated with anti-rMucin IgG gel overnight at 4°C on a rotator. The gel was washed thoroughly with TBS20 (20 mM Tris, 0.15 mM NaCl, pH 7.5) and eluted with 0.1 mM Gly, pH 2.3. The elute was neutralized to pH 7.5 and concentrated in a Centracon-30 (Amicon).
2.7. Lectin binding analysis of recombinant mucins Recombinant muc7-BV and pem-BV were separated with SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then treated with 20 ml of 0.5% blocking reagent (w/v) (Boehringer Mannheim Biochemicals) in TBSMMC (50 mM Tris–HCl, 150 mM NaCl, 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, pH 7.5). After washing three times with TBSMMC the membranes were incubated for 30 min at room temperature with biotin conjugated lectins (Jacalin, 10 mg/ml; PNA, 10 mg/ml; WGA, 10 mg/ ml; ECL,10 mg/ml; SBA, 10 mg/ml; Pierce) (Con A, 5 mg/ml; RCA60, 2 mg/ml; Boehringer Mannheim) in a 1:50000 dilution. After membranes were washed three times with TBSMMC, peroxidase-conjugated streptavidin (0.25 mg/ml, Sigma) was added in a 1:50000 dilution in TBSMMC and incubated for 30 min at room temperature. The membranes were then washed again and transferred to a cyclic diacylhydrazides solution (ECL buffer, Boehringer Mannheim) for 1 min and exposed to X-ray film for 1–15 min.
2.8. Carbohydrate detection of recombinant mucin GlycoTrackTM kit (Oxford GlycoSystem) was used for detecting carbohydrate of recombinant mucins. Briefly, 1 mg of recombinant muc7-BV or pem-BV was spotted onto nitrocellulose membrane using a minifold vacuum apparatus (Schleicher and Schuell). The membranes were washed three times with phosphate buffered saline (PBS) 50 mM sodium phosphate, 150 mM NaCl, pH 7.2 and incubated with 10 mM sodium metaperiodate in 0.1 M sodium acetate buffer containing 5 mM EDTA, pH 5.5 for 20 min at room temperature. After washing three times with PBS, the membranes were incubated with biotin–hydrazide in 0.1 M sodium acetate, pH 5.5 for 60 min at room temperature and washed three times with TBS50 (50 mM Tris, 150 mM NaCl, pH 7.2). The membranes were then treated with 0.5% blocking reagent in TBS50 for 30 min and incubated with streptavidin conjugated with alkaline phosphatase for 60 min. After washing with TBS50, the filters
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were transferred to lumi-phos 530 solution (ECL buffer, Boehringer Mannheim) for 1 min and exposed to X-ray film for 1 – 15 min. To inhibit Nor O-linked glycosylation, 3 mg/ml tunicamycin (Boehringer Mannheim) or 1 mM phenyl-Nacetyl-a-D-galactosaminide (Sigma), respectively, in TNM-FH media were used on infected cell monolayers for 72 h, as described [23]. The cells were then harvested and dissolved in SDS sample buffer and subjected to ECL immunoblot by antirMucin or monoclonal antibody SM3, as described above.
3. Results
3.1. Construction of muc7 -BV and pem-BV recombinant baculo6iruses Transfer vectors pAc360-muc7 and pVL1392pem were constructed as described in Section 2. The expression of either pem or muc7 was under control of the strong polyhedrin promoter. After co-transfection of Sf9 insect cells with linear AcMNPV DNA and pVL1392-pem or pAc360muc7, 10–20% of plaques were polyhedrin-negative recombinant baculoviruses. The recombinant viruses were initially identified by visual screening and confirmed by DNA hybridization and immunoblot analysis (see Section 2).
3.2. Expression of recombinant muc7 -BV and pem-BV The recombinant full length mucin and fusion protein of mucin seven repeats expressed in the baculovirus system were named pem-BV and muc7-BV, respectively. The expression of recombinant mucin was assayed in baculovirus-infected Sf9 cells by ECL-immunoblot analysis using rabbit anti-rMucin or mAb SM3, as described in Section 2. Twelve polyhedrin-negative and DNAhybridization positive plaques of pAc360-muc7 recombinant baculovirus were analyzed and all of them expressed a 28-kDa fusion peptide which is not seen in the wild type AcNPV-infected cells. However, in pVL-1392-pem recombinant baculovirus, five of 12 plaques expressed a protein
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recognized by both mAb SM3 and anti-rMucin. Four of five plaques producing mucin expressed a protein of : 59 kDa and another plaque expressed a 90-kDa protein recognized by mAb SM3. This 90-kDa peptide disappeared after the virus was amplified and a 59-kDa peptide, which is identical to those in other plaques, appeared. The appearance of the lower molecular weight mucin is presumed to be due to recombination between the mucin repeats with deletion of the intervening repeats. Since both sizes of the mucin produced were recognized by mAb SM3, the tumor-specific epitope is present in the 59-kDa, so no further analysis was performed. To study the efficiency of expression of recombinant mucin, Sf9 cells were infected with pure recombinant viruses at 10 m.o.i. (10 pfu of virus/cell) and cells and supernatants were harvested at various times following infection. By including a known amount of purified muc7-BV or pem-BV on immunoblots, we were able to estimate the amount of recombinant mucins expressed in the cells or in the medium at different time intervals. Both muc7-BV and pem-BV reached the detectable level at 48 h post-infection and achieved maximal level of expression by 96 h post-infection. Approximately 10 and 2.5 mg of mucin are produced in 106 muc7BV and PEM-BV infected Sf9 cells, respectively. No detectable amount was found in medium of either muc7-BV (Fig. 1) or pem-BV infected Sf9 cells.
Fig. 1. Analysis of expression of muc7-BV by immunoblot with mucin-specific mAb SM3. (A) Supernatant. (B) Cell. Lanes 1, 2, 3 and 4: 24, 48, 72 and 96 h, respectively.
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antibodies specific to mucin tandem repeats (Fig. 2B, lane 2).
3.4. ELISA of recombinant mucins protein with mAb SM3
Fig. 2. Analyses of purified mucins expressed from recombinant baculovirus infected Sf9 cells. (A) muc7-BV. Lane 1. Immunoblot with mucin-specific mAb SM3. Lane 2. Coomassie blue stained 10% SDS-PAG. Lane 3. Molecular weight standards. (B) pem-BV. Lane 1. Coomassie blue stained 10% SDS-PAG. Lane 2. Immunoblot with mucin-specific MAb SM3.
To determine whether the undenatured form of recombinant mucins expose the tumor-specific epitope [17], an ELISA assay using purified recombinant mucins was performed. Fig. 3 shows the reactions of monoclonal antibody SM3 with muc7-BV, pem-BV, muc7-EC [17] (a fusion protein which contains seven mucin tandem repeats expressed in E. coli ) and human milk protein (HMP). mAb SM3 showed strong affinity to all those recombinant mucins but not the HMP (Fig. 3). The strong binding activity to SM3 suggests that the recombinant mucin fusion protein exposed the tumor epitope in the baculovirus expression system.
3.5. Carbohydrate analysis of recombinant mucins 3.3. Purification of muc7 -BV and pem-BV Purification of muc7-BV and pem-BV was obtained in a single step by immunoaffinity chromatography, as described in Section 2. Since slight degradation was found in immunoassays, an attempt was made to process the cells as rapidly as possible to minimize any possible degradation. The overall yields obtained from immunoaffinity purification were :10% for pem-BV and 5% for muc7-BV. The affinity purified recombinant mucins were analyzed on a 10% SDS-PAG and stained by Coomassie blue and immunoblotted. Coomassie blue staining of the SDS-PAG shows that muc7-BV gave bands of molecular weight of 28- and 65-kDa (Fig. 2A, lane 2). Immunoblot staining shows that both polyclonal anti-rmucin and mAb SM3 (Fig. 2A, lane 1) only recognize the 28-kDa species, indicating that the 65-kDa band may be a contaminant during the purification. For the purified pem-BV, a main 59-kDa band was stained by both Coomassie blue (Fig. 2B, lane 1) and mAb SM3 (Fig. 2B, lane 2), while the two small degradation products were not stained by the
Three different methods were used to determine whether the mucins expressed in the bac-
Fig. 3. ELISA of purified recombinant mucins with mAb SM3. Ninety-six well polystyrene plates coated with 2 mg/ml of mucin were incubated with SM3 at the indicated concentrations for 1 h, followed by horseradish peroxidase conjugated with antibody to mouse Ig for 1 h, then substrate, 2,2%-azinodo-{3-ethylbenzthiazoline sulfonate} (ABTS) (Kirkegaad and Perry Laboratories) per the manufacturer’s instructions and read in a 96-well reader. Key: muc7-BV ( ); muc7-EC ("); pem-BV ( ); HMP (2).
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recombinant mucin expressed in baculovirus may not contain either O- or N-linked carbohydrate. This was further supported by directed carbohydrate analyses using a glycoprotein detection kit, which used biotin–hydrazide to label periodate oxidized carbohydrate (Fig. 4B) and also by reaction with a streptavidin–alkaline phosphatase conjugate and enhanced chemiluminescence (ECL) lectin binding assays (Table 1).
4. Discussion
Fig. 4. Carbohydrate analyses of muc7-BV. (A) Inhibitors of O- and N-linked glycosylation. Lane 1. No inhibitor. Lane 2. Phenyl-N-acetyl-a-D-galactosaminide. Lane 3. Tunicamycin. (B) Carbohydrate detection by GlycoTract™ (Oxford Glycosystem).
ulovirus system are glycoproteins and analyze their possible carbohydrate structure. Glycosylation inhibition assays were performed on recombinant baculovirus infected Sf9 cell media, as described under Section 2. Treatment with either O-glycosylation inhibitor (phenyl-N-acetyl-a-Dgalactosaminide, Fig. 4A, lane 2) or N-linked glycosylation inhibitor (tunicamycin, Fig. 4A, lane 3), did not change the mobility of mucin expressed from muc7-BV infected Sf9 cells on SDS-PAGE. Similar patterns were also found in pem-BV (data not shown). This suggests that the
The production of recombinant mucin in high yields from baculovirus expression systems offers a new way to study mucin functions and to develop diagnostic tests and immunogens for carcinomas that express tumor-specific mucin. The purpose of this study was to determine whether the baculovirus expression system would produce mucin containing a tumor-specific epitope and the role of carbohydrate in masking the tumor-specific epitope. Many eukaryotic proteins have been successfully expressed in the baculovirus system [15]. In most cases the baculovirus expressed proteins were produced in substantial quantities and with carbohydrate side chains if the foreign gene coded for a glycoprotein. In this study, we have expressed breast tumor associated mucin in a baculovirus system. The expression levels of recombinant mucins were fairly high compared to other proteins [15].
Table 1 Lectin analysis of recombinant mucin expressed in baculovirus system Lectin
Specificity
muc7-BV
pem-BV
Milk protein
Jacalin PNA WGA ECL SBA RCA60 ConA
a-D-Galactose b-D-Gal-(13)-D-GalNAc b-D-GlcNAc b-D-Gal-(14)-D-GlcNAc a-D-GalNAc D-GalNAc, b-D-Gal a-D-Man, a-D-Glc
− − − − − − −
− − − − − − −
+ + + + + + +
The samples were separated on SDS-PAGE, transferred onto nitrocellulose membrane, detected by biotinylated lectins and reported by peroxidase conjugated streptavidin using enhanced chemiluminescence (ECL), as described in Section 2. PNA, peanut agglutinin; WGA, wheat germ agglutinin; ECL, erythrina cristaggalli lectin; SBA, soybean agglutinin; RCA, ricinus communis toxin; ConA, concanavalin A.
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Moreover, it is clear from above data that the mucin expressed in the baculovirus system demonstrated strong affinity to tumor-specific monoclonal antibody SM3, indicating the presence of a tumor-specific epitope. The reason for the exposure of the tumor-specific epitope, is attributed to no detectable carbohydrate in the recombinant mucin expressed in the baculovirus system. The mAb SM3 did not recognize the HMP (Fig. 3), which is glycosylated, whereas it recognized baculovirus expressed, unpurified mucin when analysed from cells (Fig. 1). In addition, analysis with an inhibitor of O-linked glycosylation did not alter the molecular weight of intracellular mucin (Fig. 4A), consistent with lack of glycosylation of the baculovirus expressed mucin in the insect cells. These two assays with unpurified mucin support the results of lack of detectable carbohydrate with immunoaffinity purified baculovirus expressed mucin by immunoblotting with mAb SM3 (Figs. 2 and 3) and lectin assays for carbohydrate (Table 1). Others [24,25] have found glycoproteins not glycosylated when expressed in insect cells by baculovirus. The mucin expressed by baculovirus will be tested as a tumor-specific mucin in antigenic and immunogenic studies, in comparison with nonglycosylated tumorspecific mucin produced in bacteria [17]. The advantages of a baculovirus system over that of bacteria [17] already identified, are that the yield of mucin is 4– 24 times better, the protien is not degraded and a pure, non-fusion protein is expressed from one baculovirus recombinant.
Acknowledgements This work was supported by the Department of Veterans Affairs Medical Research Funds to SEW. We thank those mentioned in the text for materials, Aoyun Yun for technical assistance, Melody Robinson, Harrington Cancer Center and Ralph Leone, VAMC, for photography, Gregoria T. Avila, Kaye Ezzell, Stephanie Stevenson, Kimberly Hamilton, Diana Wright
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and Jennifer Moore for clerical assistance and Dr K.E. Dombrowski for review of the manuscript.
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