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Bispecific and human disease-related anti-keratin rabbit monoclonal antibodies
Experimental Cell Research 312 (2006) 411 – 422 www.elsevier.com/locate/yexcr
Research Article
Bispecific and human disease-related anti-keratin rabbit monoclonal antibodies Guo-Zhong Tao a, Ikuo Nakamichi a, Nam-On Ku a, Jing Wang b, Maria Frolkis b, Xiaosong Gong b, Weimin Zhu b, Robert Pytela b, M. Bishr Omary a,* a
Palo Alto VA Medical Center and Stanford University School of Medicine, 3801 Miranda Avenue, Mail code 154J, Palo Alto, CA 94304, USA b Epitomics, Inc. 863 Mitten Road, Burlingame, CA 94010, USA Received 30 August 2005, revised version received 1 November 2005, accepted 5 November 2005
Abstract Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites. Published by Elsevier Inc. Keywords: Intermediate filaments; Phosphorylation; Bispecific antibodies; Rabbit monoclonal antibody; Molecular mimicry; K8 glycine-61; Keratin mutations
Introduction Intermediate filament (IF) proteins are a major component of the cytoskeleton of most eukaryotic cells [1– 4]. They are expressed in tissues in a cell-type preferential manner such as desmin in muscle, neurofilaments in neuronal cells and keratins in epithelial cells. In epithelial cells, IFs consist of types I and II keratins (K) that include more than 20 unique gene products (termed K1 – K20) which form obligate non-covalent type I (K9 – K20)
Abbreviations: IEF, isoelectric focusing; IF, intermediate filament; K, keratin; MAb, monoclonal antibodies; S or Ser, serine; OA, okadaic acid; p, phospho; PV, pervanadate. * Corresponding author. Fax: +1 650 852 3259. 0014-4827/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.yexcr.2005.11.010
and type II (K1 – K8) heteropolymers [5 –7]. Although many epithelia may express up to five or more individual keratins, one keratin pair typically predominates. For example, keratins 8 and 18 (K8/18) are the major IF proteins of simple-type epithelia (as found in liver, pancreas and gastrointestinal tract) with variable levels of K7, K19 and K20 depending on the specific cell type [2,5,8]. The function of keratins includes protection of cells from mechanical and non-mechanical forms of stress [1,6] which is reflected by a variety of epithelial tissue-specific diseases that are caused, or predisposed to, by mutations in epidermal and non-epidermal keratins [9– 11]. Mutations in K8 – K18 have been identified in patients with end-stage liver disease, with the two most common mutations identified to date being K8 R340H and G61C [9,11]. An
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important aspect of understanding the function of keratins is to understand their regulation, which involves dynamic posttranslational modifications, in particular phosphorylation, and modulation by an accumulating group of associated proteins [3,12]. For example, the three major phosphorylation sites of human K8 are S23, S73 and S431 [13], and there is also evidence for K8 and K19 tyrosine phosphorylation though specific phospho-Tyr sites have not been identified [14]. In terms of keratin phosphorylation, K8/18 are the best studied, and an important tool in studying K8/18 phosphorylation has been the generation and use of mouse monoclonal and rabbit polyclonal sitespecific anti-phospho/non-phospho K8/18 antibodies [15]. The N- and C-terminal domains of keratins (and all other IF proteins), which are termed the ‘‘head’’ and ‘‘tail’’ domains, respectively, flank the central ‘‘rod’’ domain and are the regions where keratin phosphorylation takes place due to accessibility of these regions to cytoplasmic kinases [6,13]. Until recently it has been very difficult to raise monoclonal antibodies (MAb) in rabbits though rabbit antibodies have potential favorable properties compared to mouse antibodies, including high affinities and recognition of antigens that may be poor mouse immunogens [16 – 19]. The most efficient and practical way to immortalize antibody-secreting B lymphocytes is by fusing them with myeloma cells under selection conditions that allow the
growth of only fused cells [20]. Since myeloma-like tumors are not naturally found in rabbits, it has not been possible to use this approach for producing rabbit MAb. Attempts have been made to use mouse myeloma cell lines as fusion partners [18] or to use in vitro-transformed lymphoid cell lines from rabbits, but these approaches were hampered by the instability of the resulting hybridomas. A different approach was taken by producing a myeloma-like tumor in transgenic rabbits expressing the v-abl and c-myc oncogenes under the control of the immunoglobulin heavy and light chain enhancers, respectively [21]. This generated a plasmacytoma cell line, termed 240E, which was fused with rabbit lymphocytes to produce stable hybridomas. In the present study, we tested the possibility of using a combined reverse immunologic and biochemical approach that sought to generate rabbit MAb to a phosphokeratin-enriched immunogen. Then we proceeded in a systematic ‘‘reverse’’ fashion to identify potential unique epitopes that are recognized by the generated antibodies. This approach was used successfully to generate mouse anti-phospho-keratin antibodies and to characterize Ser73 as a unique and highly conserved human K8 phosphorylation site [13,22]. Our findings herein describe the generation of several rabbit anti-K8 and K18 antibodies and the detailed characterization of two highly unique antibodies.
Fig. 1. Antigen purification and testing of immune rabbit anti-sera. (A) Human K8/18 were isolated by immunoprecipitation (i.p.) from HT29 cells that were pretreated with the dimethylsulfoxide vehicle (C), okadaic acid (OA) or pervanadate (PV). Precipitates or total cell lysates were analyzed by (i) SDS-PAGE, followed by Coomassie blue staining, or by (ii) blotting using anti-K8 pS73 (mouse MAb LJ4) or anti-pY antibodies. Asterisks (lane 5, upper panel) highlight the slower K8 and K18 migration due to hyperphosphorylation. Arrowhead points to degraded K8 species. (B, C) Identical samples to those used in panel A were analyzed by blotting using polyclonal antibodies (1:5000 dilution) from rabbits immunized with K8/18 purified from OA-treated (OA serum) or PVtreated (PV serum) cells. Blots using the preimmune sera afforded minimal if any reactivity (not shown). Double asterisks point to mouse IgG (derived from the Ab used for the immunoprecipitation of K8/18).
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Materials and methods Cell lines and antibodies HT29 (human colon) and BHK-21 (hamster kidney) cells (American Type Culture Collection; Manassas, VA) were cultured at 37-C in appropriate media as recommended by the supplier. Mouse MAb that were used included [15] anti-K8 (M20), anti-K8 rod domain (TS-1) and anti-K18 (DC10) (NeoMarkers; Fremont, CA); anti-ubiquitin (Santa Cruz Biotech.; Santa Cruz, CA); anti-human K18 (L2A1) [23]; anti-human K8 pS73 (LJ4) which also recognizes mouse K8 pS79 [22]; and anti-human/mouse K8 pS431 (5B3) [24]. A polyclonal rabbit anti-K8/18 (Ab 8592) was also used [22]. Antigen purification and two-dimensional gel electrophoresis
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bridoma culture supernatants, then visualized by enhanced chemiluminescence. ELISA-positive clones were transferred from 96-well plates to 24-well plates then used for immunoblot screening. Each positive clone was subcloned, and single-colony subclones were re-screened by ELISA then immunoblotting followed by further propagation of the positive clones. Immunofluorescence and immunohistochemical tissue staining Normal human or FVB/n mouse livers were frozen in O.C.T. compound (Miles; Elkhart, IN) and sectioned for subsequent immunofluorescence staining using acetonefixed tissues [15]. Human liver explants from two patients who underwent transplantation for end-stage liver disease
HT29 cells (at 80– 90% confluency) were treated with okadaic acid (OA; 1 Ag/ml, 2 h), pervanadate (PV; 1 mM, 90 min) or dimethylsulfoxide (0.1% as a control for the OA vehicle) [25]. OA was solubilized in dimethylsulfoxide and used as a 1 mg/ml stock solution. Cells were processed for K8/18 immunoprecipitation [15] by solubilizing (60 min, 4-C) in 1% Nonidet 40 in PBS containing 0.1 mM phenylmethylsulfonyl fluoride, 25 Ag/ml aprotinin, 10 AM leupeptin, 10 AM pepstatin, 5 mM EDTA and 0.5 Ag/ml OA. Non-solubilized residue was removed (16,000 g; 30 min; 4-C), and the solubilized material was incubated with anti-K8/18 MAb L2A1 that was covalently preconjugated to protein A Sepharose beads in a slowly rotating tube (2 h, 4-C). After five washes, K8/18 were eluted from the beads using an 8 M urea-containing buffer, followed by overnight dialysis in PBS to remove the urea. Keratin concentration was measured by a BCA protein assay kit (Pierce; Rockford, IL) and adjusted to 0.2 mg/ml in PBS for rabbit immunizations. Two-dimensional gel separations were carried out using isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension [26]. Generation and screening of rabbit MAb Rabbits were immunized subcutaneously using standard procedures. After insuring an adequate polyclonal immune response, rabbit splenocytes were harvested and fused with the rabbit plasmacytoma cell line 240E [21]. Individual hybridomas supernatants were screened by ELISA and immunoblotting. For screening, total lysates of HT29 cells (pretreated with OA or PV) were used as antigen. ELISA was carried out using alkaline phosphatase-conjugated secondary goat anti-rabbit IgG (Pierce, cat #31340) and substrate PNPP (Pierce, cat #34045). For immunoblotting, antigen-containing protein samples were separated by 10% SDS-PAGE [27], transferred to polyvinylidene difluoride membranes, blotted [28] with hy-
Fig. 2. Analyses of rabbit monoclonal antibodies by immunoprecipitation (i.p.) and fluorescence staining. (A) Antibodies from the indicated GT subclones were conjugated to protein A Sepharose beads then used to immunoprecipitate K8/18. The immunoprecipitates were resolved by SDSPAGE followed by Coomassie blue staining. (B) Human and mouse normal liver sections were double stained using GT1 (or GT3) and mouse anti-K18 MAb DC10. All six GT antibodies (#1, 2, 3, 6, 8, 10) recognize human liver by staining (only GT1 and GT3 are shown), but only GT1 also recognizes mouse keratins.
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were also used for immunofluorescence staining. The normal human livers were from patients who had transplantation (typically for malignant disease), and available surgical human tissue use was approved by the Panel of Human Subjects Committee. For immunohistochemistry, formalinfixed, paraffin-embedded human tissue sections were subjected to antigen retrieval then stained with primary rabbit antibodies followed by horseradish peroxidase-conjugated goat anti-rabbit antibodies. Cell transfection, immunofluorescence staining and immune dot-blot K8 cDNA expression constructs were described previously [24,29,30] and included wild type, partial deletions and point mutations of the major in vivo phosphorylation
sites (S23A, S73A and S431A) or of mutations identified in patients with liver disease [11]. Lipofectamine 2000 (Invitrogen life technologies; San Diego, CA) was used for transient transfections into BHK cells as recommended by the supplier. Transfections also included co-transfection with WT K18 in order to stabilize the expressed K8 via the typical non-covalent heteropolymerization of type I/II keratins [31]. After 48 h, transfected cells were fixed using 100% methanol ( 20-C, 3 min). In some cases, the cells were treated with OA (1 Ag/ml, 2 h) before fixation. For the immune dot-blot, transiently transfected BHK cells were collected then homogenized in a detergent-free buffer containing 5 mM EDTA in PBS (pH 7.4). Equal fractions of the total homogenates (1 Al of 1 mg/ml of total protein homogenate) without spinning were spotted onto a nitrocellulose membrane followed by drying, rinsing with PBS, blocking with
Fig. 3. Characterization of keratin versus phospho-keratin binding of the GT monoclonals. (A) Total homogenates were prepared from HT29 cells pretreated with the dimethylsulfoxide vehicle (control), OA or PV. Equal amounts of protein were analyzed by blotting with the indicated GT antibodies. (B) K8/18 were isolated by immunoprecipitation from a mixture of HT29 cells treated with vehicle, OA or PV. K8/18 were separated by isoelectric focusing (IEF) in the first (horizontal) dimension and by SDS-PAGE in the second (vertical) dimension. Dotted boxes represent non-phosphorylated K8 or K18 isoforms.
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5% non-fat dry milk in PBS followed by immunoblotting using standard procedures.
Results Antigen purification and generation of rabbit MAb In order to generate anti- K8/18 antibodies to phospho and non-phospho epitopes, we purified K8/18 (as a source of antigen) by immunoprecipitation from HT29 cells that were preincubated with OA or PV. HT29 cells co-express low levels of K19 which co-immunoprecipitates with K8/18 due to heterodimerization of the type I and type II keratins [32]. Enrichment of K8/18/19 after immunoprecipitation was confirmed by Coomassie staining (Fig. 1A, upper panel), and the phosphatase-specific inhibitory effects of OA and PV were verified by blotting with anti-K8 pSer73 (Fig. 1A, middle panel) or anti-pTyr Ab (Fig. 1A, lower panel). Both OA and PV generate a unique hyperphosphorylated form of K8 (termed HK8) that is recognized exclusively by a previously described mouse anti-K8 pS73 MAb [14,22]. OA had a more pronounced effect on K8 S73 phosphorylation as compared with the PV-induced tyrosine phosphorylation of K8 (Fig. 1A), and PV has an indirect effect on K8 S73 phosphorylation [14]. As anticipated, the polyclonal antisera from the immunized rabbits (Figs. 1B, C) reacted strongly with K8/18, and the OA antisera preferentially recognized keratins from OA-treated cells (i.e., phospho-keratins). After the fusions, we screened 7680 clones (one ‘‘OA’’ and one ‘‘PV’’ rabbit spleen were used per fusion) using ELISA and immunoblot assays and obtained ten positive stable independent clones (termed GT1-GT10) after subcloning. The GT1-10 subclones were each derived from unique individual hybridoma clones that were then subcloned after ELISA and immunoblot testing (not shown). MAb GT1-10 were further screened by immunoprecipitation and immunofluorescence staining. Six of the ten GT antibodies (GT1, 2, 3, 6, 8, 10) efficiently immunoprecipitate K8/18 (Fig. 2A; GT1, 6, 8 and GT2, 3, 10 were from the fusions of rabbits immunized with K8/18 isolated from OA- and PV-treated cells, respectively). MAb GT1, 2, 3, 6, 8, 10 all stained K8/18 in human liver [examples are shown for GT1 and GT3 (Fig. 2B), but the remaining antibodies gave similar patterns (not shown)]. Among the six antibodies, only GT1 recognizes both human and mouse keratins (Fig. 2B). Keratin versus phospho-keratin and immunohistochemical characterization of the GT rabbit monoclonals We tested, by immunoblotting, whether GT1, 2, 3, 6, 8, 10 preferentially recognize K8, K18 or K19 and then used 2D gel analysis to determine if these antibodies are specific to phospho-keratins. Total lysates from normal, OA- or PVtreated cells were blotted with the six GT MAb (Fig. 3A).
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GT1 showed a dramatic increase in reactivity towards lysates from OA and PV treated samples (OA > PV), suggesting that it is an anti-phospho-keratin MAb. Subclones of GT2 and GT8 eventually lost their ability to produce antibody (Fig. 3A) so they were not further studied. The reactivity of GT3, 6, 10 increased slightly in OA- and PV-treated samples as compared to control. Based on these results, we focused further characterization on GT1, 3, 6, 10. We then blotted 2D-resolved K8/18 precipitates to determine whether GT1, 3, 6, 10 recognize phospho, nonphospho or total K8 or K18. The 2D-resolved K8/18 contained a 1:1:1 mix of K8/18 immunoprecipitates that were isolated from normal, OA- or PV-treated cells, respectively (Fig. 3B), in order to insure the presence of adequate levels of phospho and non-phospho keratins. As compared with the control mouse MAb M20 (which recognizes the total K8 pool), GT1 does not recognize the most basic spot (dotted box; Fig. 3B) which corresponds to
Fig. 4. Immunohistochemical staining of human tissues with rabbit MAb. Sections of human normal and cancer tissues were stained using MAb GT1 (a – d), GT3 (e, f) or GT6 (g, h). Insets in panels a and g show control staining that includes addition of the secondary but not the primary antibodies. All images were obtained using a 40 objective except for panels e and h which were obtained using a 20 objective. Note in panel f the positive tubule staining (keratin+) and absent glomerular staining (vimentin+/keratin ).
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the non-phospho K8 isoform, but does recognize all the remaining K8 acidic isoforms that shifted from left to right due to phosphorylation. This supports the findings in Fig. 3A and indicates that GT1 is a phospho-K8 epitope. Interestingly and in contrast to the anti-K8 pS73 Ab used in Fig. 1A, which recognizes only the unique HK8 hyperphosphorylated isoforms [22], GT3 recognizes only the isoforms of the faster migrating K8 species but not HK8. This indicates that GT3 recognizes the non-phospho-S73-related K8 species exclusively. In contrast with GT1 and GT3, MAb GT6 and GT10 recognize the total K18 pool and manifest a pattern similar to the control mouse MAb DC10 (which recognizes the total K18 pool) (Fig. 3B). GT1 and GT3 (anti-K8) and GT6 (antiK18) MAb also work well by immunohistochemistry when tested on a variety of fixed human tissues (Fig. 4). GT1 stains
only epithelial cells of normal colon, colon and gastric cancers (Figs. 4A, B, D) but does not stain brain which expresses neuronal intermediate filaments but not keratins (Fig. 4C). Similarly, GT3 and GT6 specifically stain epithelial cells in different tissues including stomach, kidney and colon (Figs. 4E – H). Molecular characterization of the K8 GT1 and GT3 epitopes The three major in vivo K8 phosphorylation sites are Ser23, Ser73 and Ser431 [13]. We used three K8 cDNA deletion mutants as a first step towards characterizing the GT1 phospho-epitope. The DN and DC mutants remove potential K8 phosphorylation at Ser23 and Ser431, respec-
Fig. 5. GT1 recognizes the tail domain of K8. (A) Three HA-tagged (small circles) human K8 constructs were used: normal wild-type (WT) K8, an Nterminally deleted K8 (DN, which deletes the first 35 amino acids and a C-terminally deleted (DC, which truncates the last 60 amino acids thereby leaving 422 amino acids) K8 [29]. The positions of the three major human K8 phosphorylation sites (S23, S73 and S431) are indicated with arrows. (B) BHK cells were transfected with vector alone (V) or with the constructs shown in panel A. After 48 h, cells were harvested, and total cell lysates were prepared from the transfected cells then subjected to SDS-PAGE and blotting with the indicated antibodies. (C) Transfected BHK cells, as in panel B, were used for double immunofluorescence staining using GT1 and anti-K8 (which recognizes the K8 rod domain; see text) or anti-K8 pS431 mouse MAb. Arrows highlight cells that stain with GT1 but not anti-K8 pS431.
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tively, while maintaining the Ser73 site intact (Fig. 5A). Transfection of wild type (WT), DN and DC K8 into BHK cells followed by immunoblotting with the test Ab showed that deletion of the K8 tail ablates GT1 and anti-K8 pS431 Ab binding, but neither DN nor DC affects GT3 binding (Fig. 5B). A control anti-K8 Ab TS-1 that recognizes amino acids 340 – 365 of the K8 rod domain [33] and the mouse MAb to K8 pS73 both recognized all three K8 constructs (Fig. 5B), as expected. These results were confirmed by immunofluorescence staining which showed (Fig. 5C) that (i) anti-K8 recognizes all three K8 constructs while GT1 does not bind to DC K8, (ii) GT1 and anti-K8 pS431 do not stain DC K8, and most but not all cells are recognized by
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both antibodies (arrows highlight cells recognized by GT1 but not anti-K8 pS431), and (iii) GT3 (and anti-K8 pS73, not shown) binds all three K8 constructs. Collectively, these results indicate that the GT1 epitope resides in the tail domain of K8, and the GT3 epitope is independent of K8 residues 1 –35 and 423– 482. We then tested whether GT1 recognizes K8 pS431 since GT1 clearly binds to a phospho-epitope by immunoblotting (Fig. 2) and has a similar reactivity pattern to the already available anti-K8 pS431 mouse MAb (Figs. 5B, C). For this, we compared the reactivity of GT1 with several other established anti-keratin and anti-phosphokeratin antibodies by immunoblotting (Fig. 6A) and by
Fig. 6. Epitope characterization of MAb GT1 using K8 site-specific phosphorylation mutants. (A) BHK cells were transfected with the indicated K8 WT or mutant constructs. After 48 h, cells were treated with or without OA for 2 h. Total cell lysates were prepared by adding SDS-containing sample buffer, followed by SDS-PAGE then blotting with the indicated antibodies. (B) BHK cells were transfected as in panel A, then used for double immunofluorescence staining with rabbit MAb GT1 and either mouse MAb LJ4 (anti-K8 pS73) (panels a – f) or mouse MAb 5B3 (anti-K8 pS431) (panels g – l). Note that GT1 recognizes K8 pS431 under denaturing conditions and a second confirmation-dependent epitope under non-denatured conditions.
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immunofluorescence staining (Fig. 6B) of BHK cells transfected with WT or mutant Ser23/S73/S431 K8. By immunoblotting, K8 S431A but not other mutations eliminates GT1 and anti-K8 S431 antibody binding to K8, which indicates that GT1 recognizes K8 pS431. Although GT3 recognizes exclusively K8 that is not phosphorylated on S73 (Fig. 3B), mutation of S73 does not affect GT3 binding (Fig. 6A); while, as a control, K8 S73A eliminates binding of the already characterized mouse anti-K8 pS73 MAb [22] (Fig. 6A). This suggests that non-phospho S73 is not the GT3 epitope, but that possibly a near-by motif may be the epitope. In contrast to the results obtained by immunoblotting, where proteins become denatured, the immune staining results of transfected and methanol-fixed cells were surprising in that GT1 recognized WT and S431A K8 equally well (Fig. 6B, panels g and j), whereas the anti-K8 pS431 mouse MAb did not bind to K8 S431A (Fig. 6B, panel k). As a staining control, mouse anti-K8 pSer73 recognized K8 WT
but not S73A (Fig. 6B, panels b and e). These results led us to hypothesize that there is a second epitope in the K8 tail domain that is also recognized by GT1 under nondenaturing conditions. Since the second putative GT1 epitope was not detected by immunoblotting (likely due to loss of the epitope from SDS/heat-related unfolding), immune dot-blot analysis using ‘‘native keratins’’ was carried out in order to confirm the presence of a second epitope. The dot-blot also enables semi-quantitative analysis of the transfected cell lysate, which was used as a source of antigen. As shown in Fig. 7A, both GT1 and mouse anti-K8 pS431 recognize WT K8, and their reactivity increases significantly after treatment of the transfected cells with OA (due to increased keratin phosphorylation). However, as compared to the loss of reactivity of the mouse anti-K8 pS431 to K8 S431A, GT1 maintained a modest reactivity to K8 S431A that was independent of OA treatment. These results indicate that the second GT1 epitope is a nonphosphorylated epitope. Since GT3 reacts only with K8
Fig. 7. Immune dot-blot analysis of GT1 and identification of the GT3 epitope. (A) BHK cells were transfected then cultured in the presence or absence of OA as in Fig. 6A, then homogenized in a detergent-free PBS-EDTA buffer (pH 7.4). Equal amounts of homogenates without denaturation were triple spotted on nitrocellulose membranes, followed by immunoblotting with mouse MAb to K8 that recognize the rod domain (TS-1), pS73 (LJ4), pS431 (5B3) or with rabbit MAb GT1. Note that OA treatment does not enhance binding of GT1 to K8 S431A, which supports the presence of a non-phospho secondary epitope. (B) BHK cells were co-transfected with K18 and the indicated K8 constructs. After 3 days, transfected cells were solubilized in SDS-containing sample buffer then analyzed by immunoblotting with GT3 or with the anti-K8 mouse MAb M20 (as a control). Note that the G61C mutation induces some K8 dimer formation, and that GT3 does not bind to K8 G61C dimers (arrow) or monomers.
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species that are not phosphorylated on S73 (Fig. 3B) and S73A does not affect its reactivity (Fig. 6A), we tested K8 head domain mutations that have been identified in patients with end-stage liver disease [11] that are proximal to K8 S73. As shown in Fig. 7B, only G61C, but not G52V or Y53H, inhibit GT3 binding to K8, which indicates that the K8 G61-containing subdomain is the GT3 epitope. Analysis of the GT3 antibody using human liver tissues We tested the relationship between GT3 antibody and K8 S73 phosphorylation in human liver disease tissues. Hepatocyte ‘‘Mallory body’’ cytoplasmic inclusions that are found in association with several liver diseases [34],
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which stain with anti-keratin and anti-ubiquitin antibodies (Fig. 8, panels a –c), do not stain with GT3 (Fig. 8, panels d –i). However, GT3 does stain the cytoplasmic filaments (e.g., Fig. 8, panels d and g). Interestingly, a significant portion of K8 pS73-positive staining is negative for GT3 (Figs. 8j– l), which supports the biochemical characterization of the GT3 epitope (i.e., GT3 recognition of K8 species that are not phosphorylated on S73; Fig. 3). The areas of GT3 and K8 S73 co-localization (yellow staining; Fig. 8l) likely represent keratin oligomers that contain both phospho- and non-phospho-K8 S73 species. Therefore, the GT3 antibody will be a useful adjunct for studying K8 G61C keratin turnover and its relationship to K8 S73 phosphorylation in human disease states.
Fig. 8. Immunofluorescence staining of keratins in human liver explants. Liver sections were double stained with antibodies to the indicated antigens then analyzed by confocal microscopy. The double stainings are shown as single staining and merged images (panel c is the merge of a + b; panel f is the merge of d + e; panel i is the merge of g + h; and panel l is the merge of j + k). All images were captured using a 40 objective. The staining shown in panels a – i were from one explant, while the staining shown in panels j – l were from a second cirrhotic liver. Closed arrowheads in panels h and i highlight keratin aggregates; arrows in panels j and l highlight GT3-positive staining that is K8 pS73-negative; open arrowheads in panels k and l highlight K8 pS73-positive staining that is GT3-negative.
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Discussion We describe the application of a combined biochemical and reverse immunologic approach for the generation and detailed characterization of several unique anti-keratin rabbit MAb. This approach includes a detailed biochemical analysis to identify the epitopes of the generated antibodies that display unique properties. The initial goal of immunizing with keratins purified from cells treated with the phosphatase inhibitors OA or PV (Fig. 1) was to enrich for keratin phospho-epitopes and thereby generate phospho-epitope-specific antibodies that may recognize hitherto uncharacteized phosphorylation sites. This goal was partially met in that one of the stable antibodies we generated (GT1) is directed to the phospho-epitope K8 pS431. In addition to producing other general utility antikeratin antibodies such as GT6 and GT10, this approach resulted in the generation of a highly unusual bispecific MAb (GT1) and another unique MAb (GT3) that recognizes K8 S73-non-phosphorylated species by binding to a G61-containing epitope. Hence, we posit that such an approach is likely to be successful not only in generating useful but also novel reagents. The unique features of MAb GT1 and GT3 are summarized in Fig. 9. GT1 is a bispecific rabbit MAb that simultaneously recognizes two epitopes in the tail domain of K8. One epitope is conformation-independent K8 pSer431, which is abolished upon mutation of K8 S431 (Fig. 6), while the precise location of the conformation-dependent second epitope (aside from being in the tail domain) is unknown. The GT1 second epitope is unlikely to be phosphorylated since its binding does not change in response to OA (Fig. 7) and because the tail domain of K8 does not contain any other known phosphorylation sites aside from S431 [24]. However, the second GT1 epitope is predicted to be a negatively charged acidic motif, due to reactivity of GT1 with K8 pS431, and one candidate K8 sequence (IETRDQKLVSESSDV) is located towards to the end of the tail domain [35]. The conformation dependency of the second GT1 epitope is based on its presence under relatively ‘‘native’’ conditions [e.g., in methanol fixed cells (Fig. 6) and in detergent-free extracts (Fig. 7)] and its disruption after denaturation by SDS/heat. K8 pS431 is immunogenic not only in rabbits (as demonstrated by the generation of GT1) but was also immunogenic in mice that were immunized with K8/18 isolated from OA-treated cells [24]. K8 S431 is a major in vivo phosphorylation site that undergoes hyperphosphorylation after cell stimulation with epidermal growth factor or after mitotic arrest [24] or when cells are exposed to extracellular stresses including heat and hyperosmosis (unpublished observations). The in vivo kinases of K8 S431 include MAPK and cdc2 [13,24], while K8 pS431 is a physiologic substrate for protein phosphatase 2A in HT29 cells (unpublished observa-
Fig. 9. Schematic of the epitopes recognized by the rabbit anti-keratin MAb. (A) GT1 is a bispecific anti-K8 rabbit MAb, which recognizes (i) conformation-independent K8 pS431 (highlighted with a red-filled circle) under denaturing conditions such as during immunoblotting of SDSsolubilized K8, and (ii) a second conformation-dependent non-phospho epitope (highlighted in blue) also located within the tail domain of K8, and found in native K8. This second epitope disappears when K8 is denatured (e.g., after SDS/heat treatment). (B) GT3 is a unique rabbit MAb that exclusively recognizes K8 species that are not phosphorylated on S73. GT3 binds to a K8 G61-containing epitope, and interestingly, this binding is completely ablated when S73 becomes phosphorylated. In contrast, the previously described mouse MAb LJ4 [22,36] recognizes K8 pS73 exclusively.
tions). With regard to K8 S73, its phosphorylation behaves as an off– on –off switch during mitosis, apoptosis and a variety of stresses [22,36,37]. Interestingly, and in contrast to other K8/18 phosphorylation sites, only K8 S73 phosphorylation causes a unique slower migration of K8 on SDS-PAGE [22,30]. This suggests that K8 S73 phosphorylation induces a conformational change even after SDS/heat-denatured K8 [22]. MAb GT3 will provide a unique compliment to the LJ4 mouse anti-K8 pS73 MAb, since it exclusively recognizes K8 isoforms that have not been phosphorylated on Ser73 (Figs. 3, 8). Bispecific MAb are typically artificially designed mouse or human antibodies that recognize two independent antigens. They are generally produced by chemical crosslinking, genetic engineering or somatic hybridization, and as such can be considered second-generation monoclonal antibodies [38]. Their primary use is for immunodiagnosis and possible immunotherapy of cancer and
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other diseases [39,40]. For example, bispecific antibodies that crosslink tumors and immune cells can direct immune effector cells toward tumor cells, thereby resulting in the destruction of tumor targets [39,41,42]. The generation of rabbit MAb to predefined epitopes has been very limited (e.g., anti-cdc25c phosphatase antibodies [43]), though such targeted antibodies are likely heading towards ‘‘mainstream status’’ as they are becoming available from several commercial sources. However, we are not aware of any description of a naturally derived bispecific rabbit MAb. It is likely that ‘‘natural’’ bispecific antibodies are more common than one may predict since it is common for antibodies to recognize more than one protein (e.g., by immune blotting), but investigators generally focus on the recognized protein of interest. In fact, the bispecific nature of GT1 simply reflects molecular mimicry of epitopes [44] that has been described for several mouse MAb, including an anti-K19 antibody that recognizes K19 and an interferon-inducible protein [45]. The GT3 MAb is unique from several respects. It exclusively recognizes K8 species that are not phosphorylated on S73, yet it does not bind to a K8 S73-containing epitope but binds to a G61-containing conformational epitope. Our data indicate that S73 phosphorylation leads to a conformational change in K8 that blocks GT3 access to K8 G61 but not to proximal residues including G52, Y53 and S73 (Figs. 6A, 7B). S73 is an important physiologic phosphorylation site, and its phosphorylation induces a K8 conformational change that retards its migration when analyzed by SDS-PAGE [22,34], with another reflection of this conformational change being inaccessibility of GT3 to K8 G61. Since GT3 recognizes only human (52GYGGASGMGGITAVTVNQSLL) but not mouse (52GGFGGAGVGGITAVTVNQSLL) K8 (Fig. 2B), we hypothesize that the GT3 epitope likely includes 57 SGMGGIT (given that S57 and M59 are different in the human and mouse K8 protein sequences; assuming a linear epitope). The utility of the GT3 MAb is exemplified by the staining of human liver cirrhotic liver explants (Fig. 8). MAb GT3 may prove useful for testing the presence of the K8 G61C mutation in human tissues albeit sensitivity would need to be optimized given the heterozygous nature of the G61C variant in patients with liver disease [11].
Acknowledgments This work is supported by NIH DK47918 and Department of Veterans Affairs Merit Awards (M.B.O.) and NIH Digestive Disease Center grant DK56339 Cell Imaging Core. G.-Z.T. is supported in part by a Crohn’s and Colitis Foundation of America Research Award. We are grateful to Dr. Sheri M. Krams for assisting with the liver explant harvesting, Evelyn Resurreccion for tissue sectioning and immunofluorescence staining and to Liying Xie for assisting with subcloning.
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Research Article
Bispecific and human disease-related anti-keratin rabbit monoclonal antibodies Guo-Zhong Tao a, Ikuo Nakamichi a, Nam-On Ku a, Jing Wang b, Maria Frolkis b, Xiaosong Gong b, Weimin Zhu b, Robert Pytela b, M. Bishr Omary a,* a
Palo Alto VA Medical Center and Stanford University School of Medicine, 3801 Miranda Avenue, Mail code 154J, Palo Alto, CA 94304, USA b Epitomics, Inc. 863 Mitten Road, Burlingame, CA 94010, USA Received 30 August 2005, revised version received 1 November 2005, accepted 5 November 2005
Abstract Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites. Published by Elsevier Inc. Keywords: Intermediate filaments; Phosphorylation; Bispecific antibodies; Rabbit monoclonal antibody; Molecular mimicry; K8 glycine-61; Keratin mutations
Introduction Intermediate filament (IF) proteins are a major component of the cytoskeleton of most eukaryotic cells [1– 4]. They are expressed in tissues in a cell-type preferential manner such as desmin in muscle, neurofilaments in neuronal cells and keratins in epithelial cells. In epithelial cells, IFs consist of types I and II keratins (K) that include more than 20 unique gene products (termed K1 – K20) which form obligate non-covalent type I (K9 – K20)
Abbreviations: IEF, isoelectric focusing; IF, intermediate filament; K, keratin; MAb, monoclonal antibodies; S or Ser, serine; OA, okadaic acid; p, phospho; PV, pervanadate. * Corresponding author. Fax: +1 650 852 3259. 0014-4827/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.yexcr.2005.11.010
and type II (K1 – K8) heteropolymers [5 –7]. Although many epithelia may express up to five or more individual keratins, one keratin pair typically predominates. For example, keratins 8 and 18 (K8/18) are the major IF proteins of simple-type epithelia (as found in liver, pancreas and gastrointestinal tract) with variable levels of K7, K19 and K20 depending on the specific cell type [2,5,8]. The function of keratins includes protection of cells from mechanical and non-mechanical forms of stress [1,6] which is reflected by a variety of epithelial tissue-specific diseases that are caused, or predisposed to, by mutations in epidermal and non-epidermal keratins [9– 11]. Mutations in K8 – K18 have been identified in patients with end-stage liver disease, with the two most common mutations identified to date being K8 R340H and G61C [9,11]. An
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important aspect of understanding the function of keratins is to understand their regulation, which involves dynamic posttranslational modifications, in particular phosphorylation, and modulation by an accumulating group of associated proteins [3,12]. For example, the three major phosphorylation sites of human K8 are S23, S73 and S431 [13], and there is also evidence for K8 and K19 tyrosine phosphorylation though specific phospho-Tyr sites have not been identified [14]. In terms of keratin phosphorylation, K8/18 are the best studied, and an important tool in studying K8/18 phosphorylation has been the generation and use of mouse monoclonal and rabbit polyclonal sitespecific anti-phospho/non-phospho K8/18 antibodies [15]. The N- and C-terminal domains of keratins (and all other IF proteins), which are termed the ‘‘head’’ and ‘‘tail’’ domains, respectively, flank the central ‘‘rod’’ domain and are the regions where keratin phosphorylation takes place due to accessibility of these regions to cytoplasmic kinases [6,13]. Until recently it has been very difficult to raise monoclonal antibodies (MAb) in rabbits though rabbit antibodies have potential favorable properties compared to mouse antibodies, including high affinities and recognition of antigens that may be poor mouse immunogens [16 – 19]. The most efficient and practical way to immortalize antibody-secreting B lymphocytes is by fusing them with myeloma cells under selection conditions that allow the
growth of only fused cells [20]. Since myeloma-like tumors are not naturally found in rabbits, it has not been possible to use this approach for producing rabbit MAb. Attempts have been made to use mouse myeloma cell lines as fusion partners [18] or to use in vitro-transformed lymphoid cell lines from rabbits, but these approaches were hampered by the instability of the resulting hybridomas. A different approach was taken by producing a myeloma-like tumor in transgenic rabbits expressing the v-abl and c-myc oncogenes under the control of the immunoglobulin heavy and light chain enhancers, respectively [21]. This generated a plasmacytoma cell line, termed 240E, which was fused with rabbit lymphocytes to produce stable hybridomas. In the present study, we tested the possibility of using a combined reverse immunologic and biochemical approach that sought to generate rabbit MAb to a phosphokeratin-enriched immunogen. Then we proceeded in a systematic ‘‘reverse’’ fashion to identify potential unique epitopes that are recognized by the generated antibodies. This approach was used successfully to generate mouse anti-phospho-keratin antibodies and to characterize Ser73 as a unique and highly conserved human K8 phosphorylation site [13,22]. Our findings herein describe the generation of several rabbit anti-K8 and K18 antibodies and the detailed characterization of two highly unique antibodies.
Fig. 1. Antigen purification and testing of immune rabbit anti-sera. (A) Human K8/18 were isolated by immunoprecipitation (i.p.) from HT29 cells that were pretreated with the dimethylsulfoxide vehicle (C), okadaic acid (OA) or pervanadate (PV). Precipitates or total cell lysates were analyzed by (i) SDS-PAGE, followed by Coomassie blue staining, or by (ii) blotting using anti-K8 pS73 (mouse MAb LJ4) or anti-pY antibodies. Asterisks (lane 5, upper panel) highlight the slower K8 and K18 migration due to hyperphosphorylation. Arrowhead points to degraded K8 species. (B, C) Identical samples to those used in panel A were analyzed by blotting using polyclonal antibodies (1:5000 dilution) from rabbits immunized with K8/18 purified from OA-treated (OA serum) or PVtreated (PV serum) cells. Blots using the preimmune sera afforded minimal if any reactivity (not shown). Double asterisks point to mouse IgG (derived from the Ab used for the immunoprecipitation of K8/18).
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Materials and methods Cell lines and antibodies HT29 (human colon) and BHK-21 (hamster kidney) cells (American Type Culture Collection; Manassas, VA) were cultured at 37-C in appropriate media as recommended by the supplier. Mouse MAb that were used included [15] anti-K8 (M20), anti-K8 rod domain (TS-1) and anti-K18 (DC10) (NeoMarkers; Fremont, CA); anti-ubiquitin (Santa Cruz Biotech.; Santa Cruz, CA); anti-human K18 (L2A1) [23]; anti-human K8 pS73 (LJ4) which also recognizes mouse K8 pS79 [22]; and anti-human/mouse K8 pS431 (5B3) [24]. A polyclonal rabbit anti-K8/18 (Ab 8592) was also used [22]. Antigen purification and two-dimensional gel electrophoresis
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bridoma culture supernatants, then visualized by enhanced chemiluminescence. ELISA-positive clones were transferred from 96-well plates to 24-well plates then used for immunoblot screening. Each positive clone was subcloned, and single-colony subclones were re-screened by ELISA then immunoblotting followed by further propagation of the positive clones. Immunofluorescence and immunohistochemical tissue staining Normal human or FVB/n mouse livers were frozen in O.C.T. compound (Miles; Elkhart, IN) and sectioned for subsequent immunofluorescence staining using acetonefixed tissues [15]. Human liver explants from two patients who underwent transplantation for end-stage liver disease
HT29 cells (at 80– 90% confluency) were treated with okadaic acid (OA; 1 Ag/ml, 2 h), pervanadate (PV; 1 mM, 90 min) or dimethylsulfoxide (0.1% as a control for the OA vehicle) [25]. OA was solubilized in dimethylsulfoxide and used as a 1 mg/ml stock solution. Cells were processed for K8/18 immunoprecipitation [15] by solubilizing (60 min, 4-C) in 1% Nonidet 40 in PBS containing 0.1 mM phenylmethylsulfonyl fluoride, 25 Ag/ml aprotinin, 10 AM leupeptin, 10 AM pepstatin, 5 mM EDTA and 0.5 Ag/ml OA. Non-solubilized residue was removed (16,000 g; 30 min; 4-C), and the solubilized material was incubated with anti-K8/18 MAb L2A1 that was covalently preconjugated to protein A Sepharose beads in a slowly rotating tube (2 h, 4-C). After five washes, K8/18 were eluted from the beads using an 8 M urea-containing buffer, followed by overnight dialysis in PBS to remove the urea. Keratin concentration was measured by a BCA protein assay kit (Pierce; Rockford, IL) and adjusted to 0.2 mg/ml in PBS for rabbit immunizations. Two-dimensional gel separations were carried out using isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension [26]. Generation and screening of rabbit MAb Rabbits were immunized subcutaneously using standard procedures. After insuring an adequate polyclonal immune response, rabbit splenocytes were harvested and fused with the rabbit plasmacytoma cell line 240E [21]. Individual hybridomas supernatants were screened by ELISA and immunoblotting. For screening, total lysates of HT29 cells (pretreated with OA or PV) were used as antigen. ELISA was carried out using alkaline phosphatase-conjugated secondary goat anti-rabbit IgG (Pierce, cat #31340) and substrate PNPP (Pierce, cat #34045). For immunoblotting, antigen-containing protein samples were separated by 10% SDS-PAGE [27], transferred to polyvinylidene difluoride membranes, blotted [28] with hy-
Fig. 2. Analyses of rabbit monoclonal antibodies by immunoprecipitation (i.p.) and fluorescence staining. (A) Antibodies from the indicated GT subclones were conjugated to protein A Sepharose beads then used to immunoprecipitate K8/18. The immunoprecipitates were resolved by SDSPAGE followed by Coomassie blue staining. (B) Human and mouse normal liver sections were double stained using GT1 (or GT3) and mouse anti-K18 MAb DC10. All six GT antibodies (#1, 2, 3, 6, 8, 10) recognize human liver by staining (only GT1 and GT3 are shown), but only GT1 also recognizes mouse keratins.
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were also used for immunofluorescence staining. The normal human livers were from patients who had transplantation (typically for malignant disease), and available surgical human tissue use was approved by the Panel of Human Subjects Committee. For immunohistochemistry, formalinfixed, paraffin-embedded human tissue sections were subjected to antigen retrieval then stained with primary rabbit antibodies followed by horseradish peroxidase-conjugated goat anti-rabbit antibodies. Cell transfection, immunofluorescence staining and immune dot-blot K8 cDNA expression constructs were described previously [24,29,30] and included wild type, partial deletions and point mutations of the major in vivo phosphorylation
sites (S23A, S73A and S431A) or of mutations identified in patients with liver disease [11]. Lipofectamine 2000 (Invitrogen life technologies; San Diego, CA) was used for transient transfections into BHK cells as recommended by the supplier. Transfections also included co-transfection with WT K18 in order to stabilize the expressed K8 via the typical non-covalent heteropolymerization of type I/II keratins [31]. After 48 h, transfected cells were fixed using 100% methanol ( 20-C, 3 min). In some cases, the cells were treated with OA (1 Ag/ml, 2 h) before fixation. For the immune dot-blot, transiently transfected BHK cells were collected then homogenized in a detergent-free buffer containing 5 mM EDTA in PBS (pH 7.4). Equal fractions of the total homogenates (1 Al of 1 mg/ml of total protein homogenate) without spinning were spotted onto a nitrocellulose membrane followed by drying, rinsing with PBS, blocking with
Fig. 3. Characterization of keratin versus phospho-keratin binding of the GT monoclonals. (A) Total homogenates were prepared from HT29 cells pretreated with the dimethylsulfoxide vehicle (control), OA or PV. Equal amounts of protein were analyzed by blotting with the indicated GT antibodies. (B) K8/18 were isolated by immunoprecipitation from a mixture of HT29 cells treated with vehicle, OA or PV. K8/18 were separated by isoelectric focusing (IEF) in the first (horizontal) dimension and by SDS-PAGE in the second (vertical) dimension. Dotted boxes represent non-phosphorylated K8 or K18 isoforms.
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5% non-fat dry milk in PBS followed by immunoblotting using standard procedures.
Results Antigen purification and generation of rabbit MAb In order to generate anti- K8/18 antibodies to phospho and non-phospho epitopes, we purified K8/18 (as a source of antigen) by immunoprecipitation from HT29 cells that were preincubated with OA or PV. HT29 cells co-express low levels of K19 which co-immunoprecipitates with K8/18 due to heterodimerization of the type I and type II keratins [32]. Enrichment of K8/18/19 after immunoprecipitation was confirmed by Coomassie staining (Fig. 1A, upper panel), and the phosphatase-specific inhibitory effects of OA and PV were verified by blotting with anti-K8 pSer73 (Fig. 1A, middle panel) or anti-pTyr Ab (Fig. 1A, lower panel). Both OA and PV generate a unique hyperphosphorylated form of K8 (termed HK8) that is recognized exclusively by a previously described mouse anti-K8 pS73 MAb [14,22]. OA had a more pronounced effect on K8 S73 phosphorylation as compared with the PV-induced tyrosine phosphorylation of K8 (Fig. 1A), and PV has an indirect effect on K8 S73 phosphorylation [14]. As anticipated, the polyclonal antisera from the immunized rabbits (Figs. 1B, C) reacted strongly with K8/18, and the OA antisera preferentially recognized keratins from OA-treated cells (i.e., phospho-keratins). After the fusions, we screened 7680 clones (one ‘‘OA’’ and one ‘‘PV’’ rabbit spleen were used per fusion) using ELISA and immunoblot assays and obtained ten positive stable independent clones (termed GT1-GT10) after subcloning. The GT1-10 subclones were each derived from unique individual hybridoma clones that were then subcloned after ELISA and immunoblot testing (not shown). MAb GT1-10 were further screened by immunoprecipitation and immunofluorescence staining. Six of the ten GT antibodies (GT1, 2, 3, 6, 8, 10) efficiently immunoprecipitate K8/18 (Fig. 2A; GT1, 6, 8 and GT2, 3, 10 were from the fusions of rabbits immunized with K8/18 isolated from OA- and PV-treated cells, respectively). MAb GT1, 2, 3, 6, 8, 10 all stained K8/18 in human liver [examples are shown for GT1 and GT3 (Fig. 2B), but the remaining antibodies gave similar patterns (not shown)]. Among the six antibodies, only GT1 recognizes both human and mouse keratins (Fig. 2B). Keratin versus phospho-keratin and immunohistochemical characterization of the GT rabbit monoclonals We tested, by immunoblotting, whether GT1, 2, 3, 6, 8, 10 preferentially recognize K8, K18 or K19 and then used 2D gel analysis to determine if these antibodies are specific to phospho-keratins. Total lysates from normal, OA- or PVtreated cells were blotted with the six GT MAb (Fig. 3A).
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GT1 showed a dramatic increase in reactivity towards lysates from OA and PV treated samples (OA > PV), suggesting that it is an anti-phospho-keratin MAb. Subclones of GT2 and GT8 eventually lost their ability to produce antibody (Fig. 3A) so they were not further studied. The reactivity of GT3, 6, 10 increased slightly in OA- and PV-treated samples as compared to control. Based on these results, we focused further characterization on GT1, 3, 6, 10. We then blotted 2D-resolved K8/18 precipitates to determine whether GT1, 3, 6, 10 recognize phospho, nonphospho or total K8 or K18. The 2D-resolved K8/18 contained a 1:1:1 mix of K8/18 immunoprecipitates that were isolated from normal, OA- or PV-treated cells, respectively (Fig. 3B), in order to insure the presence of adequate levels of phospho and non-phospho keratins. As compared with the control mouse MAb M20 (which recognizes the total K8 pool), GT1 does not recognize the most basic spot (dotted box; Fig. 3B) which corresponds to
Fig. 4. Immunohistochemical staining of human tissues with rabbit MAb. Sections of human normal and cancer tissues were stained using MAb GT1 (a – d), GT3 (e, f) or GT6 (g, h). Insets in panels a and g show control staining that includes addition of the secondary but not the primary antibodies. All images were obtained using a 40 objective except for panels e and h which were obtained using a 20 objective. Note in panel f the positive tubule staining (keratin+) and absent glomerular staining (vimentin+/keratin ).
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the non-phospho K8 isoform, but does recognize all the remaining K8 acidic isoforms that shifted from left to right due to phosphorylation. This supports the findings in Fig. 3A and indicates that GT1 is a phospho-K8 epitope. Interestingly and in contrast to the anti-K8 pS73 Ab used in Fig. 1A, which recognizes only the unique HK8 hyperphosphorylated isoforms [22], GT3 recognizes only the isoforms of the faster migrating K8 species but not HK8. This indicates that GT3 recognizes the non-phospho-S73-related K8 species exclusively. In contrast with GT1 and GT3, MAb GT6 and GT10 recognize the total K18 pool and manifest a pattern similar to the control mouse MAb DC10 (which recognizes the total K18 pool) (Fig. 3B). GT1 and GT3 (anti-K8) and GT6 (antiK18) MAb also work well by immunohistochemistry when tested on a variety of fixed human tissues (Fig. 4). GT1 stains
only epithelial cells of normal colon, colon and gastric cancers (Figs. 4A, B, D) but does not stain brain which expresses neuronal intermediate filaments but not keratins (Fig. 4C). Similarly, GT3 and GT6 specifically stain epithelial cells in different tissues including stomach, kidney and colon (Figs. 4E – H). Molecular characterization of the K8 GT1 and GT3 epitopes The three major in vivo K8 phosphorylation sites are Ser23, Ser73 and Ser431 [13]. We used three K8 cDNA deletion mutants as a first step towards characterizing the GT1 phospho-epitope. The DN and DC mutants remove potential K8 phosphorylation at Ser23 and Ser431, respec-
Fig. 5. GT1 recognizes the tail domain of K8. (A) Three HA-tagged (small circles) human K8 constructs were used: normal wild-type (WT) K8, an Nterminally deleted K8 (DN, which deletes the first 35 amino acids and a C-terminally deleted (DC, which truncates the last 60 amino acids thereby leaving 422 amino acids) K8 [29]. The positions of the three major human K8 phosphorylation sites (S23, S73 and S431) are indicated with arrows. (B) BHK cells were transfected with vector alone (V) or with the constructs shown in panel A. After 48 h, cells were harvested, and total cell lysates were prepared from the transfected cells then subjected to SDS-PAGE and blotting with the indicated antibodies. (C) Transfected BHK cells, as in panel B, were used for double immunofluorescence staining using GT1 and anti-K8 (which recognizes the K8 rod domain; see text) or anti-K8 pS431 mouse MAb. Arrows highlight cells that stain with GT1 but not anti-K8 pS431.
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tively, while maintaining the Ser73 site intact (Fig. 5A). Transfection of wild type (WT), DN and DC K8 into BHK cells followed by immunoblotting with the test Ab showed that deletion of the K8 tail ablates GT1 and anti-K8 pS431 Ab binding, but neither DN nor DC affects GT3 binding (Fig. 5B). A control anti-K8 Ab TS-1 that recognizes amino acids 340 – 365 of the K8 rod domain [33] and the mouse MAb to K8 pS73 both recognized all three K8 constructs (Fig. 5B), as expected. These results were confirmed by immunofluorescence staining which showed (Fig. 5C) that (i) anti-K8 recognizes all three K8 constructs while GT1 does not bind to DC K8, (ii) GT1 and anti-K8 pS431 do not stain DC K8, and most but not all cells are recognized by
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both antibodies (arrows highlight cells recognized by GT1 but not anti-K8 pS431), and (iii) GT3 (and anti-K8 pS73, not shown) binds all three K8 constructs. Collectively, these results indicate that the GT1 epitope resides in the tail domain of K8, and the GT3 epitope is independent of K8 residues 1 –35 and 423– 482. We then tested whether GT1 recognizes K8 pS431 since GT1 clearly binds to a phospho-epitope by immunoblotting (Fig. 2) and has a similar reactivity pattern to the already available anti-K8 pS431 mouse MAb (Figs. 5B, C). For this, we compared the reactivity of GT1 with several other established anti-keratin and anti-phosphokeratin antibodies by immunoblotting (Fig. 6A) and by
Fig. 6. Epitope characterization of MAb GT1 using K8 site-specific phosphorylation mutants. (A) BHK cells were transfected with the indicated K8 WT or mutant constructs. After 48 h, cells were treated with or without OA for 2 h. Total cell lysates were prepared by adding SDS-containing sample buffer, followed by SDS-PAGE then blotting with the indicated antibodies. (B) BHK cells were transfected as in panel A, then used for double immunofluorescence staining with rabbit MAb GT1 and either mouse MAb LJ4 (anti-K8 pS73) (panels a – f) or mouse MAb 5B3 (anti-K8 pS431) (panels g – l). Note that GT1 recognizes K8 pS431 under denaturing conditions and a second confirmation-dependent epitope under non-denatured conditions.
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immunofluorescence staining (Fig. 6B) of BHK cells transfected with WT or mutant Ser23/S73/S431 K8. By immunoblotting, K8 S431A but not other mutations eliminates GT1 and anti-K8 S431 antibody binding to K8, which indicates that GT1 recognizes K8 pS431. Although GT3 recognizes exclusively K8 that is not phosphorylated on S73 (Fig. 3B), mutation of S73 does not affect GT3 binding (Fig. 6A); while, as a control, K8 S73A eliminates binding of the already characterized mouse anti-K8 pS73 MAb [22] (Fig. 6A). This suggests that non-phospho S73 is not the GT3 epitope, but that possibly a near-by motif may be the epitope. In contrast to the results obtained by immunoblotting, where proteins become denatured, the immune staining results of transfected and methanol-fixed cells were surprising in that GT1 recognized WT and S431A K8 equally well (Fig. 6B, panels g and j), whereas the anti-K8 pS431 mouse MAb did not bind to K8 S431A (Fig. 6B, panel k). As a staining control, mouse anti-K8 pSer73 recognized K8 WT
but not S73A (Fig. 6B, panels b and e). These results led us to hypothesize that there is a second epitope in the K8 tail domain that is also recognized by GT1 under nondenaturing conditions. Since the second putative GT1 epitope was not detected by immunoblotting (likely due to loss of the epitope from SDS/heat-related unfolding), immune dot-blot analysis using ‘‘native keratins’’ was carried out in order to confirm the presence of a second epitope. The dot-blot also enables semi-quantitative analysis of the transfected cell lysate, which was used as a source of antigen. As shown in Fig. 7A, both GT1 and mouse anti-K8 pS431 recognize WT K8, and their reactivity increases significantly after treatment of the transfected cells with OA (due to increased keratin phosphorylation). However, as compared to the loss of reactivity of the mouse anti-K8 pS431 to K8 S431A, GT1 maintained a modest reactivity to K8 S431A that was independent of OA treatment. These results indicate that the second GT1 epitope is a nonphosphorylated epitope. Since GT3 reacts only with K8
Fig. 7. Immune dot-blot analysis of GT1 and identification of the GT3 epitope. (A) BHK cells were transfected then cultured in the presence or absence of OA as in Fig. 6A, then homogenized in a detergent-free PBS-EDTA buffer (pH 7.4). Equal amounts of homogenates without denaturation were triple spotted on nitrocellulose membranes, followed by immunoblotting with mouse MAb to K8 that recognize the rod domain (TS-1), pS73 (LJ4), pS431 (5B3) or with rabbit MAb GT1. Note that OA treatment does not enhance binding of GT1 to K8 S431A, which supports the presence of a non-phospho secondary epitope. (B) BHK cells were co-transfected with K18 and the indicated K8 constructs. After 3 days, transfected cells were solubilized in SDS-containing sample buffer then analyzed by immunoblotting with GT3 or with the anti-K8 mouse MAb M20 (as a control). Note that the G61C mutation induces some K8 dimer formation, and that GT3 does not bind to K8 G61C dimers (arrow) or monomers.
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species that are not phosphorylated on S73 (Fig. 3B) and S73A does not affect its reactivity (Fig. 6A), we tested K8 head domain mutations that have been identified in patients with end-stage liver disease [11] that are proximal to K8 S73. As shown in Fig. 7B, only G61C, but not G52V or Y53H, inhibit GT3 binding to K8, which indicates that the K8 G61-containing subdomain is the GT3 epitope. Analysis of the GT3 antibody using human liver tissues We tested the relationship between GT3 antibody and K8 S73 phosphorylation in human liver disease tissues. Hepatocyte ‘‘Mallory body’’ cytoplasmic inclusions that are found in association with several liver diseases [34],
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which stain with anti-keratin and anti-ubiquitin antibodies (Fig. 8, panels a –c), do not stain with GT3 (Fig. 8, panels d –i). However, GT3 does stain the cytoplasmic filaments (e.g., Fig. 8, panels d and g). Interestingly, a significant portion of K8 pS73-positive staining is negative for GT3 (Figs. 8j– l), which supports the biochemical characterization of the GT3 epitope (i.e., GT3 recognition of K8 species that are not phosphorylated on S73; Fig. 3). The areas of GT3 and K8 S73 co-localization (yellow staining; Fig. 8l) likely represent keratin oligomers that contain both phospho- and non-phospho-K8 S73 species. Therefore, the GT3 antibody will be a useful adjunct for studying K8 G61C keratin turnover and its relationship to K8 S73 phosphorylation in human disease states.
Fig. 8. Immunofluorescence staining of keratins in human liver explants. Liver sections were double stained with antibodies to the indicated antigens then analyzed by confocal microscopy. The double stainings are shown as single staining and merged images (panel c is the merge of a + b; panel f is the merge of d + e; panel i is the merge of g + h; and panel l is the merge of j + k). All images were captured using a 40 objective. The staining shown in panels a – i were from one explant, while the staining shown in panels j – l were from a second cirrhotic liver. Closed arrowheads in panels h and i highlight keratin aggregates; arrows in panels j and l highlight GT3-positive staining that is K8 pS73-negative; open arrowheads in panels k and l highlight K8 pS73-positive staining that is GT3-negative.
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Discussion We describe the application of a combined biochemical and reverse immunologic approach for the generation and detailed characterization of several unique anti-keratin rabbit MAb. This approach includes a detailed biochemical analysis to identify the epitopes of the generated antibodies that display unique properties. The initial goal of immunizing with keratins purified from cells treated with the phosphatase inhibitors OA or PV (Fig. 1) was to enrich for keratin phospho-epitopes and thereby generate phospho-epitope-specific antibodies that may recognize hitherto uncharacteized phosphorylation sites. This goal was partially met in that one of the stable antibodies we generated (GT1) is directed to the phospho-epitope K8 pS431. In addition to producing other general utility antikeratin antibodies such as GT6 and GT10, this approach resulted in the generation of a highly unusual bispecific MAb (GT1) and another unique MAb (GT3) that recognizes K8 S73-non-phosphorylated species by binding to a G61-containing epitope. Hence, we posit that such an approach is likely to be successful not only in generating useful but also novel reagents. The unique features of MAb GT1 and GT3 are summarized in Fig. 9. GT1 is a bispecific rabbit MAb that simultaneously recognizes two epitopes in the tail domain of K8. One epitope is conformation-independent K8 pSer431, which is abolished upon mutation of K8 S431 (Fig. 6), while the precise location of the conformation-dependent second epitope (aside from being in the tail domain) is unknown. The GT1 second epitope is unlikely to be phosphorylated since its binding does not change in response to OA (Fig. 7) and because the tail domain of K8 does not contain any other known phosphorylation sites aside from S431 [24]. However, the second GT1 epitope is predicted to be a negatively charged acidic motif, due to reactivity of GT1 with K8 pS431, and one candidate K8 sequence (IETRDQKLVSESSDV) is located towards to the end of the tail domain [35]. The conformation dependency of the second GT1 epitope is based on its presence under relatively ‘‘native’’ conditions [e.g., in methanol fixed cells (Fig. 6) and in detergent-free extracts (Fig. 7)] and its disruption after denaturation by SDS/heat. K8 pS431 is immunogenic not only in rabbits (as demonstrated by the generation of GT1) but was also immunogenic in mice that were immunized with K8/18 isolated from OA-treated cells [24]. K8 S431 is a major in vivo phosphorylation site that undergoes hyperphosphorylation after cell stimulation with epidermal growth factor or after mitotic arrest [24] or when cells are exposed to extracellular stresses including heat and hyperosmosis (unpublished observations). The in vivo kinases of K8 S431 include MAPK and cdc2 [13,24], while K8 pS431 is a physiologic substrate for protein phosphatase 2A in HT29 cells (unpublished observa-
Fig. 9. Schematic of the epitopes recognized by the rabbit anti-keratin MAb. (A) GT1 is a bispecific anti-K8 rabbit MAb, which recognizes (i) conformation-independent K8 pS431 (highlighted with a red-filled circle) under denaturing conditions such as during immunoblotting of SDSsolubilized K8, and (ii) a second conformation-dependent non-phospho epitope (highlighted in blue) also located within the tail domain of K8, and found in native K8. This second epitope disappears when K8 is denatured (e.g., after SDS/heat treatment). (B) GT3 is a unique rabbit MAb that exclusively recognizes K8 species that are not phosphorylated on S73. GT3 binds to a K8 G61-containing epitope, and interestingly, this binding is completely ablated when S73 becomes phosphorylated. In contrast, the previously described mouse MAb LJ4 [22,36] recognizes K8 pS73 exclusively.
tions). With regard to K8 S73, its phosphorylation behaves as an off– on –off switch during mitosis, apoptosis and a variety of stresses [22,36,37]. Interestingly, and in contrast to other K8/18 phosphorylation sites, only K8 S73 phosphorylation causes a unique slower migration of K8 on SDS-PAGE [22,30]. This suggests that K8 S73 phosphorylation induces a conformational change even after SDS/heat-denatured K8 [22]. MAb GT3 will provide a unique compliment to the LJ4 mouse anti-K8 pS73 MAb, since it exclusively recognizes K8 isoforms that have not been phosphorylated on Ser73 (Figs. 3, 8). Bispecific MAb are typically artificially designed mouse or human antibodies that recognize two independent antigens. They are generally produced by chemical crosslinking, genetic engineering or somatic hybridization, and as such can be considered second-generation monoclonal antibodies [38]. Their primary use is for immunodiagnosis and possible immunotherapy of cancer and
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other diseases [39,40]. For example, bispecific antibodies that crosslink tumors and immune cells can direct immune effector cells toward tumor cells, thereby resulting in the destruction of tumor targets [39,41,42]. The generation of rabbit MAb to predefined epitopes has been very limited (e.g., anti-cdc25c phosphatase antibodies [43]), though such targeted antibodies are likely heading towards ‘‘mainstream status’’ as they are becoming available from several commercial sources. However, we are not aware of any description of a naturally derived bispecific rabbit MAb. It is likely that ‘‘natural’’ bispecific antibodies are more common than one may predict since it is common for antibodies to recognize more than one protein (e.g., by immune blotting), but investigators generally focus on the recognized protein of interest. In fact, the bispecific nature of GT1 simply reflects molecular mimicry of epitopes [44] that has been described for several mouse MAb, including an anti-K19 antibody that recognizes K19 and an interferon-inducible protein [45]. The GT3 MAb is unique from several respects. It exclusively recognizes K8 species that are not phosphorylated on S73, yet it does not bind to a K8 S73-containing epitope but binds to a G61-containing conformational epitope. Our data indicate that S73 phosphorylation leads to a conformational change in K8 that blocks GT3 access to K8 G61 but not to proximal residues including G52, Y53 and S73 (Figs. 6A, 7B). S73 is an important physiologic phosphorylation site, and its phosphorylation induces a K8 conformational change that retards its migration when analyzed by SDS-PAGE [22,34], with another reflection of this conformational change being inaccessibility of GT3 to K8 G61. Since GT3 recognizes only human (52GYGGASGMGGITAVTVNQSLL) but not mouse (52GGFGGAGVGGITAVTVNQSLL) K8 (Fig. 2B), we hypothesize that the GT3 epitope likely includes 57 SGMGGIT (given that S57 and M59 are different in the human and mouse K8 protein sequences; assuming a linear epitope). The utility of the GT3 MAb is exemplified by the staining of human liver cirrhotic liver explants (Fig. 8). MAb GT3 may prove useful for testing the presence of the K8 G61C mutation in human tissues albeit sensitivity would need to be optimized given the heterozygous nature of the G61C variant in patients with liver disease [11].
Acknowledgments This work is supported by NIH DK47918 and Department of Veterans Affairs Merit Awards (M.B.O.) and NIH Digestive Disease Center grant DK56339 Cell Imaging Core. G.-Z.T. is supported in part by a Crohn’s and Colitis Foundation of America Research Award. We are grateful to Dr. Sheri M. Krams for assisting with the liver explant harvesting, Evelyn Resurreccion for tissue sectioning and immunofluorescence staining and to Liying Xie for assisting with subcloning.
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