Connective Tissue Research, 1992, Vol. 28, pp. 205-212 Reprints available directly from the publisher Photocopying permitted by license only 0 1992 Gordon and Breach Science Publishers S.A. Pnnted in the United States of America
MONOCLONAL ANTIBODY BRL 12 REACTS WITH BONE KERATAN SULPHATE PROTEOGLYCAN
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CLIVE J. JOYNER, AMARJIT S. VIRDI, JON N. BERESFORD, JONATHAN H. BENNETT, MAUREEN E. OWEN, and JAMES T. TRIFFITT MRC Bone Research Laboratory, Nufield Department of Orthopaedic Surgery, University of Oxford, Nufield Orthopaedic Centre, Oxford, England (Received September 3, 1991; in revised form February 19, 1992; accepted February 24, 1992)
Our previous studies suggest that a monoclonal antibody, BRL 12, reacts with a specific product of differentiated cells of the osteoblastic lineage. In the present study, the bone constituent recognized by this antibody has been characterized by gel filtration, ion exchange chromatography, protein blotting and immunolocalization. Our findings show that the antibody reacts with an epitope associated with the core protein of rabbit keratan sulfate proteoglycan (KSPG), a molecule which shares considerable homology with the sialoprotein present in the bone tissue of other mammalian species.
KEYWORDS: bone, extracellular matrix, differentiation, monoclonal antibody
INTRODUCTION The study of cellular differentiation depends on an ability to distinguish between cells of different lineages and varying degrees of specialization. Monoclonal antibodies to a range of bone cells and matrix components have been produced in order to obtain markers which are relatively specific for cells of the osteoblastic lineage.l-6 Previously we have described a monoclonal antibody (BRL12) which, based on immunohistochemical evidence, recognizes an antigen present exclusively in osteogenic tissues.’ Binding of this antibody was demonstrated in osteoid, in the mineralized bone matrix and in ossifying growth plate cartilage in the rabbit. In the present study the bone constituent recognized by the antibody is identified.
METHODS AND MATERIALS Monoclonal Antibody Production and Screening The methods used for monoclonal antibody production and screening are described in detail in reference 7. In brief, the monoclonal antibody BRL 12 was obtained from mice immunized with rabbit bone extract (RBE), prepared from the long bones of 2-3 kg New Zealand White rabbits, by using guanidine hydrochloride (4 M) and EDTA (0.5 M) solution, at Address for correspondence: C. J. Joyner, MRC Bone Research Laboratory, Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Oxford OX3 7LD, England. Fax: 865 742348. Tel. Oxford 221657. 205
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neutral pH by the method of Termine et al. 1980.8 Following initial screening by enzymelinked immunosorbent assay (ELISA), the BFU 12 secreting clone was selected for further study because of the specific reaction of BRL 12 with osteogenic tissues determined by immunohistochemistry.7 In the present study similar immunohistochemical staining methods have been used to determine the distribution of antigen in cornea and bone using tissues from young New Zealand White rabbits (Body weight, 540g). Monoclonal antibody class and subclass were determined by using a mouse monoclonal typing kit (Serotec, Kidlington, Oxfordshire, UK.)
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Gel Permeation Chromatography RBE (10 mg) was chromatographed on a column (90 X 1.5 cm) of Sepharose CL-6B (Pharmacia, Milton Keynes, UK.) under dissociating conditions (4M guanidinium chloride, 0.05 M Tris, pH 7.4) using a flow rate of 10 ml/h. Albumin and cytochrome c were used as molecular weight standards to calibrate the column. Fractions (2 ml) were collected and the absorbance measured at 276 nm. Immunological reactivity of the fractions with BRL 12 was determined by using ELISA. The elution position of osteocalcin was determined by using a rat anti-rabbit osteocalcin antiserum prepared in our laboratory and by HPLC on a C18 p Bondapak column.10 Ion Exchange Chromatography RBE (25 mg) was applied to a column (5 cm x 2.5 cm) of DEAE-cellulose (DE 52, Whatman, Maidstone, Kent, England.) equilibrated with 0.05 M Tris-HC1 buffer pH 7.2. After elution of the unbound components the absorbed material was eluted with a linear gradient of 0 to 0.9 M NaCl in the column buffer at a flow rate of 10 ml/h. Fractions (1 ml) were collected, the absorbances measured at 276 nm and their reactivity with BRL 12 assessed by using ELISA. Enzyme-Linked Immunosorbent Assay 96 well microassay plates (Nunc, Roskilde, Denmark) were coated by incubation at 37°C for 1 h with aliquots (100 pl) of the ion exchange or gel filtration column fractions. Plates were washed with PBS Tween 20 (0.05%, v/v) and incubated (37°C; 30 min.) with culture medium containing BRL 12. The plates were washed as before and incubated (37°C; 30 min.) with rabbit anti-mouse-peroxidase conjugated antibody (Sigma Chemical Co., Poole, Dorset , England) before final washing and incubation with substrate (0-phenylenediamine 0.04% w/v in citrate buffer 0.1 M pH 5.0 containing 0.012% (H202). The reaction was stopped after approximately 2 min. by acidification with H,SO, (4N). The absorbance at 490nm was measured by using an automatic plate reader (Dynatech Labs. Ltd., Billingshurst, Sussex, England.). N.B. The absorbance readings by using this method should not be considered to give a quantitative measure of antigen but do indicate those fractions containing antigen. Protein Blotting Lyophylized samples of RBE (100 pg) and pure KSPG (10 pg) were treated with either 10 mU chondroitinase ABC (Seikagaku Kogyo Co., Tokyo, Japan.) or 40 mU keratanase
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(Seikagaku Kogyo Co., Tokyo, Japan.) in appropriate buffers." The keratanase (keratan sulfate endo-P-galactosidase) was prepared from Pseudomonas sp. Pure rabbit KSPG was kindly provided by L. Fisher (Bone Research Branch, NIDR, NIH, Bethesda). Incubations were for 1 h. at 37°C in the presence of protease inhibitors (10 mM EDTA; 10 mM N-ethyl maleimide; 5 mM phenylmethylsulphonylfluoride;0.36 M pepstatin A). Proteolytic activity in the enzyme preparations was assessed by comparing the electrophoretic pattern of pure albumin (10 pg) before and after incubation under the same conditions. The reactions were stopped by the addition of gel sample buffer (Tris-HC1, 0.08M pH 8.8 containing sodium dodecyl sulfate 4%w/v, dithiothreitol 0.1 M, sucrose 10% w/v and bromophenol blue) and heating to 95°C for 3 min. Protein samples (chondroitinase- and keratanase-treated RBE and KSPG) were applied to gradient (4-20% w/v) SDS polyacrylamide slab gels and electrophoresed for 4 h at a constant current of 60 mA/gel with cooling, using the buffer system of Laemmli.12 Prestained molecular weight markers (BioRad Labs., Watford, Herts., England.) were included in each run. Electrotransfer of proteins from the gels to nitrocellulose membranes (Sartorius, Gottingen, Germany) was performed by using a Transblot apparatus (BioRad Labs., Watford, Herts., England.) according to the method of Towbin et al.13 Following electrotransfer, nitrocellulose membranes were incubated overnight at 37°C in PBS containing Tween 20 (0.05% viv), rinsed in PBS and transferred to medium containing BRL 12 with the following additions: rabbit serum (10% v/v), Tween 20 (0.05% v/v), D-glucose (1 M) and glycerol (10% v/v) for 1 h. at 37OC.14 Binding of BRL 12 mouse immunoglobulin was detected by sequentially incubating the membranes with affinity purified goat anti-mouse immunoglobulin (Dakopatts, High Wycombe, Bucks. England) 1:200 in PBS for 30 min. at 37°C. and mouse monoclonal peroxidase anti-peroxidase antibody (Sigma Chemical Co., Poole, Dorset, U.K.) 1:400 in PBS for 30 min. at 37°C. Peroxidase activity was detected by staining with 3,4,3' ,4'-tetraphenyl hydrochloride (Sigma Chemical Co., Poole, Dorset) 0.5 mg/ml in 0.1 M imidazole in PBS pH 7.2 containing 0.3% v/v hydrogen peroxide for 5 min. Membranes were washed 3 times between each step with PBS containing Tween 20 (0.05% v/v). Immunolocalization of BRL 12 Binding in Bone and Cornea Pieces of femur and cornea from a New Zealand White rabbit (540g wt.) were fixed in 95% methanol and embedded in glycol methacrylate. Sections (5 pm) were cut and stained using the BRL 12 antibody as previously described.7
RESULTS Biochemical Characterization of the Antigen Monoclonal antibody, BRL 12, was prepared using mice immunized with an extract of rabbit bone (RBE) known to contain a multitude of bone proteins, glycoproteins and proteoglycans.8,15,16,17Initial characterization of the component recognized by the antibody was achieved by Sepharose CL6B chromatography and DEAE-cellulose ion-exchange chromatography. Fractions from the Sepharose column were analyzed by ELISA using BRL 12 antibody and immunological reactivity with BRL 12 antibody was found to be mainly associated with components of high relative molecular weight (Figure 1). Fractions from the
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FIGURE 1 Sepharose CL-6B chromatography of rabbit bone extract. Elution profile on Sepharose CL-6B of the RBE used as immunogen, absorbance measured at 276 nm (0).The presence in the eluted fractions of material reactive with antibody BRL 12 was determined by ELISA ( + 1. The location of osteocalcin (OC)was determined by ELISA and confirmed by HPLC. Molecular weight standards were albumin (68000) and cytochrome c (12500).
DEAE-cellulose column were also analyzed by ELISA. Immunoreactive material was found to be highly acidic eluting at NaCl concentrationsbetween 0.4 M and 0.5 M (Figure 2) in a region known to contain proteins rich in sialic acid.'* To characterizefurther the bone constituent recognized by BRL12, the RBE was subjected to Western blot analysis (Figure 3). The electrophoreticproperties of the particular component binding the BRL 12 antibody are shown in Figure 3 lane 2. For comparison, Figure 3 lane 9 shows the separation of RBE stained for total protein with amido black. The antibody reacts with a region of polydisperse high molecular weight material characteristic of proteoglycans. The major proteoglycans present in bone of most species are the chondroitin 4-sulfate proteoglycans decorin (PGI) and biglycan (PGII).19 Treatment of RBE with chondroitinase ABC enzyme, however, had no effect on the pattern of immunostaining (Figure 3, compare lanes 2 and 4), demonstratingthat BRLl2 does not react with native chondroitinor dermatan sulfate chains. In addition, since the electrophoreticmobility of the immunostained material was not changed, it seems unlikely that the antibody is reactive with any part of these proteoglycans. A major proteoglycan present in rabbit bone is keratan sulfate proteoglycan (KSPG) which consists of a core protein of M, 80,000 with 0-linked side chains of keratan sulfate.llJ0 Keratanase treatment of RBE led to a partial cleavage of the high molecular
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weight immunoreactive material (Figure 2 , lane 3), yielding a stained band at 75000 (Figure 3, lane 3) with similar apparent molecular weight to that of the core protein of KSPG. Confirmation that BRL 12 reacts with rabbit bone KSPG was obtained by Western blot analysis using pure KSPG. Pure KSPG bound the antibody (Figure 3, lane 5) and produced a pattern of staining similar to that obtained with the RBE (Figure 3, lane 2). Digestion of pure KSPG with keratanase (Figure 3, lane 6) or chondroitinase ABC (Figure 3, lane 7) produced patterns of staining similar to those obtained by the same enzyme treatment of RBE (Figure 3, lanes 3 and 4).No evidence of proteolytic activity could be detected in either enzyme preparation (data not shown). Immunohistochemical Localization of Antibody Binding Antibody binding was assessed in sections of rabbit bone and rabbit cornea, a tissue containing high amounts of keratan sulfate proteoglycan. Localization of immunostaining in osteoid and the mineralized bone matrix of rabbit femur was confirmed. No specific reaction between BRL 12 and rabbit cornea was detected (results not shown).
DISCUSSION
The results presented demonstrate that BRL 12 antibody reacts with rabbit bone KSPG. Treatment with keratanase removes the majority of the carbohydrate associated with the core
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FIGURE 3 Nitrocellulose blot of total rabbit bone extract and pure rabbit bone KSPG immunostained using BRL12. Samples of total RBE and pure KSPG (undigested, C; digested with keratanase, K; digested with chondroitinase ABC, CH) were subjected to SDS-polyacrylamide gel electrophoresis (4 to 20% gradient) .and blotted onto nitrocellulose. The membrane was then incubated with culture supernatant containing the monoclonal antibody and the resulting immunocomplex visualized using a peroxidase anti-peroxidase method. Lanes 1 and 8: Prestained molecular weight markers (molecular mass X 103). Lane 2: Total RBE, undigested. Immunostaining in a broad region of high molecular weight. Lane 3: Total RBE, keratanase digested. Immunostaining concentrated in a 75 OOO. Lane 4: Total RBE, chondroitinase ABC treated. Pattern of immunostaining similar to band of Mr control (Lane 2). Lane 5 : Pure KSPG, undigested. KSPG is immunostained and appears as a broad band between the top of the gel and the 130 k marker. Lane 6: Pure KSPG, keratanase digested. Immunostaining as in Lane 3 is concentrated in a band of Mr 75 OOO. Lane 7: Pure KSPG, chondroitinase ABE treated. Pattern of immunostaining is similar to untreated control (Lane 5). Lane 9: Total RBE, undigested. Stained for total protein with amido black.
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protein of KSPG.11 Keratanase treatment of KSPG did not, however, prevent antibody binding (Figure 3, lane 6) and it seems likely, therefore, that BRL 12 is reactive with an epitope closely associated with the core of rabbit bone KSPG. The absence of binding of BRL 12 to cartilage and to cornea, tissues which are known to be rich in keratan sulfate, also suggests that the antibody recognizes the core protein rather than the glycosaminoglycan prosthetic groups unless these are peculiarly masked in these tissues. Bone proteoglycans containing keratan sulfate are restricted to a few species, including the quail21 and the rabbit.11.19 It has recently been shown, however, that the core protein of rabbit compact bone KSPG is closely related to that of human bone sialoprotein. 11 Bovine bone sialoprotein was originally described by Herring22 and later identified as a degraded product of a Mr 80,000 protein which was called bone sialoprotein I123 because it elutes as a
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second sialic acid-containing peak on DEAE columns.24 However, the original name, bone sialoprotein (BSP) is now used instead of bone sialoprotein 11. BSP and bone KSPG possess almost identical N-terminal amino acid sequences and a polyclonal antibody to rabbit bone KSPG binds bovine BSP core protein on electrotransfers from SDS-polyacrylamide gels. 11 However, the monoclonal antibody BRL12 shows no cross reactivity with BSP, in the human, cow, or rat (our unpublished results). BSP is highly concentrated in bone matrix and it is believed to be synthesized by osteoblasts.25 Likewise, bone KSPG is considered to be a product of differentiated cells of the osteoblastic lineage. Suspensions of rabbit marrow cells cultured within diffusion chambers in vivo differentiate to form fibrous-osteogenic tissues.26-29 Immunostaining of the tissue in these chambers has confirmed that KSPG is synthesized in this system, just before calcification but after the expression of alkaline phosphatase.’ The physiological role of KSPG is unknown, but it seems likely that BSP and KSPG have related functions. It has been suggested that the BSP may play a role in cell attachment30and spreading at sites of osteogenesis. Amino acid analysis of rat BSP31 has shown the protein to contain the Arg-Gly-Asp sequence which confers cell attachment properties on several other proteins including fibronectin,32 vitronectin,33 fibrinogen34 and osteopontin.35 In addition, BSP has been shown to bind a cell surface receptor on rat osteosarcoma cells. This receptor protein is composed of polypeptides similar in size to those of the vitronectin receptor and both this putative BSP receptor and the vitronectin receptor have been shown to bind to an RGD- containing heptapeptide.36 The expression by osteoclasts of vitronectin receptor37 suggests that exposure of BSP on the bone surface may be an important factor in osteoclast adhesion and, therefore, bone resorption. It has been suggested recently that BSP may be synthesized in some extraskeletal sites, small amounts of BSP mRNA having been detected in cartilage and decidua.38 Nevertheless, BSP is highly enriched in bone and this, together with the evidence obtained using BRL 12, suggest that BSP and KSPG will be useful as a late differentiation stage markers of osteoblastic cells.
ACKNOWLEDGMENTS The authors are grateful to Dr. Tim Hardingham (Kennedy Institute of Rheumatology, London) for his help and advice.
REFERENCES 1 . Stenner, D. D., Romberg, R. W., Tracy, R.
I?, Katzman, J. A ,, Riggs, B. L., and Mann, K. G. (1984). Monoclonal antibodies to native noncollagenous Bone-specific proteins. Proc. Natl. Acad. Sci. USA 81, 2868. 2. Nijweide, €?,and Mulder, R. (1986). Identification of Osteocytes in osteoblast-like cell cultures using a monoclonal antibody specifically directed against osteocytes. Hisrochem. 84, 342. 3. Nakama, T., Nakamura, O., Diakuhara, Y., and Semba, T. (1988). A monoclonal antibody against dentin phosphoryn recognizes a bone protein(s) appearing at the beginning of ossification. Calc. Tiss.Int. 43, 263. 4. Bruder, S. I?, and Caplan, A. I. (1989). First bone formation and the dissection of an osteogenic lineage in the embryonic chick tibia is revealed by monoclonal antibodies against osteoblasts. Bone 10, 359. 5. Nakao, T., Kimoto, S., Tanikawa, K., Kim, K. T., Higaki, M., Kawase, T., and Siato, S. (1989). Identification of osteoblast-specific monoclonal antibodies. Calc. Tiss. Int. 44,220. 6 . Perry, J., Gilligan, M . , Green, E., Docherty, H., and Heath, D. (1990). Monoclonal antibodies to ROS 17/2.8 cells recognize antigens, some of which are restricted to osteoblasts and chondrocytes. J. Bone Mincral Res. 5 , 187. 7. Joyner, C. J., Virdi, A . S., Triffitt, J. T., and Owen, M. E. (1989). Immunohistochemical studies using BRL 12, a monoclonal antibody reacting specifically with osteogenic tissues. Conn. Tiss. Res. 23, 289. 8. Termine, J. D., Belcourt, A. B., Christner, I? J., Conn, K . M., and Nylen, M. U. (1980). Properties of
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dissociatively extracted fetal tooth matrix proteins 11. Separation and purification of fetal bovine dentin phosphoprotein. J. Biol. Chem. 255, 9769. 9. Kohler, G., and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. 10. Triffitt, J. T., and Wilson, J. M.3 (1984). The small molecular weight, carboxyglutamic acid-containing protein of rabbit bone tissue. Arch. Oral Biol. 29, 1015. 11. Kinne, R. W., and Fisher, L. W. (1987). Keratan sulfate proteoglycan in rabbit compact bone is bone sialoprotein 11. J. Biol. Chem. 262, 10206. 12. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. 13. Towbin, H., Staehelin, T., and Gordon, J.(1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350. 14. Birk, H. W., and Koepsell, H. (1987). Reaction of monoclonal antibodies with plasma membrane protein softer binding on nitrocellulose: Renaturation of antigenic sites and reduction of nonspecific antibody binding I. Anal. Biochem. 164, 12. 15. Heinegard, D., Hultenby, K., Oldberg, A., Reinholt, E , and Wendel, M. (1989). Macromolecules in bone matrix. Conn. Tiss. Res. 21, 3. 16. Robey, I? G.(1989). The biochemistry of bone. Endocrinol. Metubol. 18, 859. 17. Triffitt, J. T. (1990). Trace metal complexing organic molecules in bone and teeth. In Trace metals undftuoride in bones and teeth, N. D. Priest and E L. van der Vyver (eds.). Boca Raton, FL, CRC Press, 315-340. 18. Diamond, A. G., Triffitt, J. T., and Herring, G. M. (1982). The acidic macromolecules in rabbit cortical bone tissue. Arch. Oral Biol. 27, 337. 19. Fisher, L. W., and Temrine, J. D. (1985). Noncallagenous proteins influencing the local mechanisms of calcification. Clin. Orthop. Rel. Res. 200, 362. 20. Fisher, L. W., Whitson, S. W., Avioli, L. V., andTermine, J. D.(1983). Matrix sialoprotein of developing bone. J. Biol. Chem. 258, 12723. 21. Fisher, L. W., and Schraer, H. (1982). Keratan sulfate proteoglycan isolated from the estrogen-induced medullary bone in Japanese quail. Comp. Biochem. Physiol. 72, 227. 22. Herring, G. M. (1968). Studies of the protein-bound chondroitin sulphate of bovine cortical bone. Biochem. J. 107, 41. 23. Triffitt, J. T. (1987). The special proteins of bone tissue. Clin.Sci. 72, 399. 24. Franzen, A,, and Heinegard, D. (1985). Isolation and characterization of two sialoproteins present only in bone calcified matrix. Biochem. J. 232, 715. 25. Franzen, A. and Heinegard, D. (1985). Proteoglycans and proteins of rat bone. Purification and biosynthesis of major noncollagenous molecules. In The chemistry and biology of mineralized tissues, W. T. Butler (ed.). Birmingham, AL, EBSCO Media, 132-141. 26. Friedenstein, A. J. (1976). Precursor cells of mechanocytes. Int. Rev. Cytol. 47, 327. 27. Ashton, B. A,, Allen, T. D., Howlett, C. R., Eagleson, C. C., Hattori, A,, and Owen, M. E. (1980). Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin.Orthop. Rel. Res. 151, 294. 28. Bab, I. Howlett, C. R., Ashton, B. A., and Owen, M. E. (1984). Ultrastructure of bone and cartilage formed in vivo in diffusion chambers. Clin.Orthop. Rel. Res. 187, 243. 29. Ashhurst, D. E., Ashton, B. A , , and Owen, M. E. (1990). The collagens and glycosaminoglycans of the extracellular matrices secreted by bone marrow stromal cells cultured in vivo in diffusion chambers. J. Orthop. Res. 8, 741. 30. Sommerrnan, M. J., Fisher, L. W., Foster, R. A,, and Sauk, J. J. (1988). Human bone sialoprotein I and I1 enhance fibroblast attachment in vitro. Calc. Tiss. Int. 43, 50. 31, Oldberg, A,, Franzen, A,, and Heinegard, D. (1988). The primary structure of a cell-binding bone sialoprotein. J. Biol. Chem. 263, 19430. 32. Pierschbacher, M. D., and Ruoslahti, E. (1984). Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309,30. 33. Suzuki, S., Oldberg, A,, Hayman, E. G., Pierschbacher, M. D., andRuoslahti, E. (1985). Completeaminoacid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin. EMBO J. 4, 2519. 34. Gartner, T. K., and Bennett, J. S. (1985). The tetrapeptide analogue of the cell attachment site of fibronectin Inhibits platelet aggregation and fibrinogen binding to activated platelets. J. Biol. Chem. 260, 11891. 35. Oldberg, A., Franzen, A,, and Heinegard, D. (1986). Cloning and sequence analyses of rat bone sialoprotein osteoprotein cDNA reveals an arg-gly-asp cell-binding sequence. Proc. Natl. Acad. Sci. USA 83, 8819. 36. Oldberg, A,, Franzen, A,, Heinegard, D., Pierschbacher, M. D., and Ruoslahti, E. (1988). Identification of a bone sialoprotein receptor in osteosarcoma cells. J. Biol. Chem. 263, 19433. 37. Horton, M. A., and Davies, J. (1989). Perspectives: Adhesion receptors in bone. J. Bone Min. Rex 4, 803. 38. Fisher, L. W., McBride, 0. W., Termine, J. D., and Young, M. E (1990). Human bone sialoprotein, deduced protein sequence and chromosomal. J. Biol. Chem. 265, 2347.
MONOCLONAL ANTIBODY BRL 12 REACTS WITH BONE KERATAN SULPHATE PROTEOGLYCAN
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CLIVE J. JOYNER, AMARJIT S. VIRDI, JON N. BERESFORD, JONATHAN H. BENNETT, MAUREEN E. OWEN, and JAMES T. TRIFFITT MRC Bone Research Laboratory, Nufield Department of Orthopaedic Surgery, University of Oxford, Nufield Orthopaedic Centre, Oxford, England (Received September 3, 1991; in revised form February 19, 1992; accepted February 24, 1992)
Our previous studies suggest that a monoclonal antibody, BRL 12, reacts with a specific product of differentiated cells of the osteoblastic lineage. In the present study, the bone constituent recognized by this antibody has been characterized by gel filtration, ion exchange chromatography, protein blotting and immunolocalization. Our findings show that the antibody reacts with an epitope associated with the core protein of rabbit keratan sulfate proteoglycan (KSPG), a molecule which shares considerable homology with the sialoprotein present in the bone tissue of other mammalian species.
KEYWORDS: bone, extracellular matrix, differentiation, monoclonal antibody
INTRODUCTION The study of cellular differentiation depends on an ability to distinguish between cells of different lineages and varying degrees of specialization. Monoclonal antibodies to a range of bone cells and matrix components have been produced in order to obtain markers which are relatively specific for cells of the osteoblastic lineage.l-6 Previously we have described a monoclonal antibody (BRL12) which, based on immunohistochemical evidence, recognizes an antigen present exclusively in osteogenic tissues.’ Binding of this antibody was demonstrated in osteoid, in the mineralized bone matrix and in ossifying growth plate cartilage in the rabbit. In the present study the bone constituent recognized by the antibody is identified.
METHODS AND MATERIALS Monoclonal Antibody Production and Screening The methods used for monoclonal antibody production and screening are described in detail in reference 7. In brief, the monoclonal antibody BRL 12 was obtained from mice immunized with rabbit bone extract (RBE), prepared from the long bones of 2-3 kg New Zealand White rabbits, by using guanidine hydrochloride (4 M) and EDTA (0.5 M) solution, at Address for correspondence: C. J. Joyner, MRC Bone Research Laboratory, Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Oxford OX3 7LD, England. Fax: 865 742348. Tel. Oxford 221657. 205
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neutral pH by the method of Termine et al. 1980.8 Following initial screening by enzymelinked immunosorbent assay (ELISA), the BFU 12 secreting clone was selected for further study because of the specific reaction of BRL 12 with osteogenic tissues determined by immunohistochemistry.7 In the present study similar immunohistochemical staining methods have been used to determine the distribution of antigen in cornea and bone using tissues from young New Zealand White rabbits (Body weight, 540g). Monoclonal antibody class and subclass were determined by using a mouse monoclonal typing kit (Serotec, Kidlington, Oxfordshire, UK.)
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Gel Permeation Chromatography RBE (10 mg) was chromatographed on a column (90 X 1.5 cm) of Sepharose CL-6B (Pharmacia, Milton Keynes, UK.) under dissociating conditions (4M guanidinium chloride, 0.05 M Tris, pH 7.4) using a flow rate of 10 ml/h. Albumin and cytochrome c were used as molecular weight standards to calibrate the column. Fractions (2 ml) were collected and the absorbance measured at 276 nm. Immunological reactivity of the fractions with BRL 12 was determined by using ELISA. The elution position of osteocalcin was determined by using a rat anti-rabbit osteocalcin antiserum prepared in our laboratory and by HPLC on a C18 p Bondapak column.10 Ion Exchange Chromatography RBE (25 mg) was applied to a column (5 cm x 2.5 cm) of DEAE-cellulose (DE 52, Whatman, Maidstone, Kent, England.) equilibrated with 0.05 M Tris-HC1 buffer pH 7.2. After elution of the unbound components the absorbed material was eluted with a linear gradient of 0 to 0.9 M NaCl in the column buffer at a flow rate of 10 ml/h. Fractions (1 ml) were collected, the absorbances measured at 276 nm and their reactivity with BRL 12 assessed by using ELISA. Enzyme-Linked Immunosorbent Assay 96 well microassay plates (Nunc, Roskilde, Denmark) were coated by incubation at 37°C for 1 h with aliquots (100 pl) of the ion exchange or gel filtration column fractions. Plates were washed with PBS Tween 20 (0.05%, v/v) and incubated (37°C; 30 min.) with culture medium containing BRL 12. The plates were washed as before and incubated (37°C; 30 min.) with rabbit anti-mouse-peroxidase conjugated antibody (Sigma Chemical Co., Poole, Dorset , England) before final washing and incubation with substrate (0-phenylenediamine 0.04% w/v in citrate buffer 0.1 M pH 5.0 containing 0.012% (H202). The reaction was stopped after approximately 2 min. by acidification with H,SO, (4N). The absorbance at 490nm was measured by using an automatic plate reader (Dynatech Labs. Ltd., Billingshurst, Sussex, England.). N.B. The absorbance readings by using this method should not be considered to give a quantitative measure of antigen but do indicate those fractions containing antigen. Protein Blotting Lyophylized samples of RBE (100 pg) and pure KSPG (10 pg) were treated with either 10 mU chondroitinase ABC (Seikagaku Kogyo Co., Tokyo, Japan.) or 40 mU keratanase
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(Seikagaku Kogyo Co., Tokyo, Japan.) in appropriate buffers." The keratanase (keratan sulfate endo-P-galactosidase) was prepared from Pseudomonas sp. Pure rabbit KSPG was kindly provided by L. Fisher (Bone Research Branch, NIDR, NIH, Bethesda). Incubations were for 1 h. at 37°C in the presence of protease inhibitors (10 mM EDTA; 10 mM N-ethyl maleimide; 5 mM phenylmethylsulphonylfluoride;0.36 M pepstatin A). Proteolytic activity in the enzyme preparations was assessed by comparing the electrophoretic pattern of pure albumin (10 pg) before and after incubation under the same conditions. The reactions were stopped by the addition of gel sample buffer (Tris-HC1, 0.08M pH 8.8 containing sodium dodecyl sulfate 4%w/v, dithiothreitol 0.1 M, sucrose 10% w/v and bromophenol blue) and heating to 95°C for 3 min. Protein samples (chondroitinase- and keratanase-treated RBE and KSPG) were applied to gradient (4-20% w/v) SDS polyacrylamide slab gels and electrophoresed for 4 h at a constant current of 60 mA/gel with cooling, using the buffer system of Laemmli.12 Prestained molecular weight markers (BioRad Labs., Watford, Herts., England.) were included in each run. Electrotransfer of proteins from the gels to nitrocellulose membranes (Sartorius, Gottingen, Germany) was performed by using a Transblot apparatus (BioRad Labs., Watford, Herts., England.) according to the method of Towbin et al.13 Following electrotransfer, nitrocellulose membranes were incubated overnight at 37°C in PBS containing Tween 20 (0.05% viv), rinsed in PBS and transferred to medium containing BRL 12 with the following additions: rabbit serum (10% v/v), Tween 20 (0.05% v/v), D-glucose (1 M) and glycerol (10% v/v) for 1 h. at 37OC.14 Binding of BRL 12 mouse immunoglobulin was detected by sequentially incubating the membranes with affinity purified goat anti-mouse immunoglobulin (Dakopatts, High Wycombe, Bucks. England) 1:200 in PBS for 30 min. at 37°C. and mouse monoclonal peroxidase anti-peroxidase antibody (Sigma Chemical Co., Poole, Dorset, U.K.) 1:400 in PBS for 30 min. at 37°C. Peroxidase activity was detected by staining with 3,4,3' ,4'-tetraphenyl hydrochloride (Sigma Chemical Co., Poole, Dorset) 0.5 mg/ml in 0.1 M imidazole in PBS pH 7.2 containing 0.3% v/v hydrogen peroxide for 5 min. Membranes were washed 3 times between each step with PBS containing Tween 20 (0.05% v/v). Immunolocalization of BRL 12 Binding in Bone and Cornea Pieces of femur and cornea from a New Zealand White rabbit (540g wt.) were fixed in 95% methanol and embedded in glycol methacrylate. Sections (5 pm) were cut and stained using the BRL 12 antibody as previously described.7
RESULTS Biochemical Characterization of the Antigen Monoclonal antibody, BRL 12, was prepared using mice immunized with an extract of rabbit bone (RBE) known to contain a multitude of bone proteins, glycoproteins and proteoglycans.8,15,16,17Initial characterization of the component recognized by the antibody was achieved by Sepharose CL6B chromatography and DEAE-cellulose ion-exchange chromatography. Fractions from the Sepharose column were analyzed by ELISA using BRL 12 antibody and immunological reactivity with BRL 12 antibody was found to be mainly associated with components of high relative molecular weight (Figure 1). Fractions from the
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FIGURE 1 Sepharose CL-6B chromatography of rabbit bone extract. Elution profile on Sepharose CL-6B of the RBE used as immunogen, absorbance measured at 276 nm (0).The presence in the eluted fractions of material reactive with antibody BRL 12 was determined by ELISA ( + 1. The location of osteocalcin (OC)was determined by ELISA and confirmed by HPLC. Molecular weight standards were albumin (68000) and cytochrome c (12500).
DEAE-cellulose column were also analyzed by ELISA. Immunoreactive material was found to be highly acidic eluting at NaCl concentrationsbetween 0.4 M and 0.5 M (Figure 2) in a region known to contain proteins rich in sialic acid.'* To characterizefurther the bone constituent recognized by BRL12, the RBE was subjected to Western blot analysis (Figure 3). The electrophoreticproperties of the particular component binding the BRL 12 antibody are shown in Figure 3 lane 2. For comparison, Figure 3 lane 9 shows the separation of RBE stained for total protein with amido black. The antibody reacts with a region of polydisperse high molecular weight material characteristic of proteoglycans. The major proteoglycans present in bone of most species are the chondroitin 4-sulfate proteoglycans decorin (PGI) and biglycan (PGII).19 Treatment of RBE with chondroitinase ABC enzyme, however, had no effect on the pattern of immunostaining (Figure 3, compare lanes 2 and 4), demonstratingthat BRLl2 does not react with native chondroitinor dermatan sulfate chains. In addition, since the electrophoreticmobility of the immunostained material was not changed, it seems unlikely that the antibody is reactive with any part of these proteoglycans. A major proteoglycan present in rabbit bone is keratan sulfate proteoglycan (KSPG) which consists of a core protein of M, 80,000 with 0-linked side chains of keratan sulfate.llJ0 Keratanase treatment of RBE led to a partial cleavage of the high molecular
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FlGURE 2 DEAE-ion exchange chromatography of rabbit bone extract. Elution profile on DEAE ion exchange of the RBE used as immunogen, absorbance measured at 276 nm (0). The presence in the eluted fractions of material reactive with the antibody BRL12 was determined by ELISA (Bar).
weight immunoreactive material (Figure 2 , lane 3), yielding a stained band at 75000 (Figure 3, lane 3) with similar apparent molecular weight to that of the core protein of KSPG. Confirmation that BRL 12 reacts with rabbit bone KSPG was obtained by Western blot analysis using pure KSPG. Pure KSPG bound the antibody (Figure 3, lane 5) and produced a pattern of staining similar to that obtained with the RBE (Figure 3, lane 2). Digestion of pure KSPG with keratanase (Figure 3, lane 6) or chondroitinase ABC (Figure 3, lane 7) produced patterns of staining similar to those obtained by the same enzyme treatment of RBE (Figure 3, lanes 3 and 4).No evidence of proteolytic activity could be detected in either enzyme preparation (data not shown). Immunohistochemical Localization of Antibody Binding Antibody binding was assessed in sections of rabbit bone and rabbit cornea, a tissue containing high amounts of keratan sulfate proteoglycan. Localization of immunostaining in osteoid and the mineralized bone matrix of rabbit femur was confirmed. No specific reaction between BRL 12 and rabbit cornea was detected (results not shown).
DISCUSSION
The results presented demonstrate that BRL 12 antibody reacts with rabbit bone KSPG. Treatment with keratanase removes the majority of the carbohydrate associated with the core
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FIGURE 3 Nitrocellulose blot of total rabbit bone extract and pure rabbit bone KSPG immunostained using BRL12. Samples of total RBE and pure KSPG (undigested, C; digested with keratanase, K; digested with chondroitinase ABC, CH) were subjected to SDS-polyacrylamide gel electrophoresis (4 to 20% gradient) .and blotted onto nitrocellulose. The membrane was then incubated with culture supernatant containing the monoclonal antibody and the resulting immunocomplex visualized using a peroxidase anti-peroxidase method. Lanes 1 and 8: Prestained molecular weight markers (molecular mass X 103). Lane 2: Total RBE, undigested. Immunostaining in a broad region of high molecular weight. Lane 3: Total RBE, keratanase digested. Immunostaining concentrated in a 75 OOO. Lane 4: Total RBE, chondroitinase ABC treated. Pattern of immunostaining similar to band of Mr control (Lane 2). Lane 5 : Pure KSPG, undigested. KSPG is immunostained and appears as a broad band between the top of the gel and the 130 k marker. Lane 6: Pure KSPG, keratanase digested. Immunostaining as in Lane 3 is concentrated in a band of Mr 75 OOO. Lane 7: Pure KSPG, chondroitinase ABE treated. Pattern of immunostaining is similar to untreated control (Lane 5). Lane 9: Total RBE, undigested. Stained for total protein with amido black.
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protein of KSPG.11 Keratanase treatment of KSPG did not, however, prevent antibody binding (Figure 3, lane 6) and it seems likely, therefore, that BRL 12 is reactive with an epitope closely associated with the core of rabbit bone KSPG. The absence of binding of BRL 12 to cartilage and to cornea, tissues which are known to be rich in keratan sulfate, also suggests that the antibody recognizes the core protein rather than the glycosaminoglycan prosthetic groups unless these are peculiarly masked in these tissues. Bone proteoglycans containing keratan sulfate are restricted to a few species, including the quail21 and the rabbit.11.19 It has recently been shown, however, that the core protein of rabbit compact bone KSPG is closely related to that of human bone sialoprotein. 11 Bovine bone sialoprotein was originally described by Herring22 and later identified as a degraded product of a Mr 80,000 protein which was called bone sialoprotein I123 because it elutes as a
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MONOCLONAL ANTIBODY BRL 12
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second sialic acid-containing peak on DEAE columns.24 However, the original name, bone sialoprotein (BSP) is now used instead of bone sialoprotein 11. BSP and bone KSPG possess almost identical N-terminal amino acid sequences and a polyclonal antibody to rabbit bone KSPG binds bovine BSP core protein on electrotransfers from SDS-polyacrylamide gels. 11 However, the monoclonal antibody BRL12 shows no cross reactivity with BSP, in the human, cow, or rat (our unpublished results). BSP is highly concentrated in bone matrix and it is believed to be synthesized by osteoblasts.25 Likewise, bone KSPG is considered to be a product of differentiated cells of the osteoblastic lineage. Suspensions of rabbit marrow cells cultured within diffusion chambers in vivo differentiate to form fibrous-osteogenic tissues.26-29 Immunostaining of the tissue in these chambers has confirmed that KSPG is synthesized in this system, just before calcification but after the expression of alkaline phosphatase.’ The physiological role of KSPG is unknown, but it seems likely that BSP and KSPG have related functions. It has been suggested that the BSP may play a role in cell attachment30and spreading at sites of osteogenesis. Amino acid analysis of rat BSP31 has shown the protein to contain the Arg-Gly-Asp sequence which confers cell attachment properties on several other proteins including fibronectin,32 vitronectin,33 fibrinogen34 and osteopontin.35 In addition, BSP has been shown to bind a cell surface receptor on rat osteosarcoma cells. This receptor protein is composed of polypeptides similar in size to those of the vitronectin receptor and both this putative BSP receptor and the vitronectin receptor have been shown to bind to an RGD- containing heptapeptide.36 The expression by osteoclasts of vitronectin receptor37 suggests that exposure of BSP on the bone surface may be an important factor in osteoclast adhesion and, therefore, bone resorption. It has been suggested recently that BSP may be synthesized in some extraskeletal sites, small amounts of BSP mRNA having been detected in cartilage and decidua.38 Nevertheless, BSP is highly enriched in bone and this, together with the evidence obtained using BRL 12, suggest that BSP and KSPG will be useful as a late differentiation stage markers of osteoblastic cells.
ACKNOWLEDGMENTS The authors are grateful to Dr. Tim Hardingham (Kennedy Institute of Rheumatology, London) for his help and advice.
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