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• W2157299631 abstract "We determined the specificity of two hamster monoclonal antibodies and a sheep polyclonal antiserum against heparan sulfate proteoglycan isolated from rat glomerular basement membrane. The antibodies were characterized by enzyme-linked immunosorbent assay on various basement membrane components and immunoprecipitation with heparan sulfate proteoglycan with or without heparitinase pre-treatment. These experiments showed that the antibodies specifically recognize approximately 150-, 105-, and 70-kDa core proteins of rat glomerular basement membrane heparan sulfate proteoglycan. Recently, we showed that agrin is a major heparan sulfate proteoglycan in the glomerular basement membrane (Groffen, A. J. A., Ruegg, M. A., Dijkman, H. B. P. M., Van der Velden, T. J., Buskens, C. A., van den Born, J., Assmann, K. J. M., Monnens, L. A. H., Veerkamp, J. H., and van den Heuvel, L. P. W. J. (1998) J. Histochem. Cytochem. 46, 19–27). Therefore, we tested whether our antibodies recognize agrin. To this end, we evaluated staining of Chinese hamster ovary cells transfected with constructs encoding full-length or the C-terminal half of rat agrin by analysis on a fluorescence-activated cell sorter. Both hamster monoclonals and the sheep antiserum clearly stained cells transfected with the construct encoding full-length agrin, whereas wild type cells and cells transfected with the construct encoding the C-terminal part of agrin were not recognized. A panel of previously characterized monoclonals, directed against C-terminal agrin, clearly stained cells transfected with either of the constructs but not wild type cells. This indicates that both hamster monoclonals and the sheep antiserum recognize epitopes on the N-terminal half of agrin. By immunohistochemistry on rat renal tissue, we compared distribution of N-terminal agrin with that of C-terminal agrin. The monoclonal antibodies against C-terminal agrin stained almost exclusively the glomerular basement membrane, whereas the anti-N-terminal agrin antibodies recognized all renal basement membranes, including tubular basement membranes. Based on these results, we hypothesize that full-length agrin is predominantly expressed in the glomerular basement membrane, whereas in most other renal basement membranes a truncated isoform of agrin is predominantly found that misses (part of) the C terminus, which might be due to alternative splicing and/or posttranslational processing. The possible significance of this finding is discussed. We determined the specificity of two hamster monoclonal antibodies and a sheep polyclonal antiserum against heparan sulfate proteoglycan isolated from rat glomerular basement membrane. The antibodies were characterized by enzyme-linked immunosorbent assay on various basement membrane components and immunoprecipitation with heparan sulfate proteoglycan with or without heparitinase pre-treatment. These experiments showed that the antibodies specifically recognize approximately 150-, 105-, and 70-kDa core proteins of rat glomerular basement membrane heparan sulfate proteoglycan. Recently, we showed that agrin is a major heparan sulfate proteoglycan in the glomerular basement membrane (Groffen, A. J. A., Ruegg, M. A., Dijkman, H. B. P. M., Van der Velden, T. J., Buskens, C. A., van den Born, J., Assmann, K. J. M., Monnens, L. A. H., Veerkamp, J. H., and van den Heuvel, L. P. W. J. (1998) J. Histochem. Cytochem. 46, 19–27). Therefore, we tested whether our antibodies recognize agrin. To this end, we evaluated staining of Chinese hamster ovary cells transfected with constructs encoding full-length or the C-terminal half of rat agrin by analysis on a fluorescence-activated cell sorter. Both hamster monoclonals and the sheep antiserum clearly stained cells transfected with the construct encoding full-length agrin, whereas wild type cells and cells transfected with the construct encoding the C-terminal part of agrin were not recognized. A panel of previously characterized monoclonals, directed against C-terminal agrin, clearly stained cells transfected with either of the constructs but not wild type cells. This indicates that both hamster monoclonals and the sheep antiserum recognize epitopes on the N-terminal half of agrin. By immunohistochemistry on rat renal tissue, we compared distribution of N-terminal agrin with that of C-terminal agrin. The monoclonal antibodies against C-terminal agrin stained almost exclusively the glomerular basement membrane, whereas the anti-N-terminal agrin antibodies recognized all renal basement membranes, including tubular basement membranes. Based on these results, we hypothesize that full-length agrin is predominantly expressed in the glomerular basement membrane, whereas in most other renal basement membranes a truncated isoform of agrin is predominantly found that misses (part of) the C terminus, which might be due to alternative splicing and/or posttranslational processing. The possible significance of this finding is discussed. Heparan sulfate proteoglycans (HSPGs) 1The abbreviations used are: HSPG, heparan sulfate proteoglycan; HS, heparan sulfate; BM, basement membrane; CHO, Chinese hamster ovary; mAb, monclonal antibody; PBS, phosphate-buffered saline; IF, immunofluorescence; ELISA, enzyme-linked immunosorbent assay; FACS fluorescence-activated cell sorter; PAGE, polyacrylamide gel electrophoresis; MuSK, muscle-specific kinase. are composed of a core protein to which anionic heparan sulfate (HS) side chains are attached. Apart from its presence in basement membranes (BMs), HSPG is found in other extracellular matrices and on many, if not all, cell surfaces (1Gallagher J.T. Curr. Opin. Cell Biol. 1989; 1: 1201-1218Crossref PubMed Scopus (212) Google Scholar, 2David G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (375) Google Scholar, 3Lindahl U. Lidholt K. Spillmann D. Kjellen L. Thromb. Res. 1994; 75: 1-32Abstract Full Text PDF PubMed Scopus (343) Google Scholar). The first extracellular matrix HSPG that was isolated (from mouse Engelbreth-Holm-Swarm sarcoma), characterized, and cloned was perlecan, named after its beads-on-a-string-like appearance in rotary shadowing electron microscopy (4Noonan D.M. Fulle A. Valente P. Cai S. Horigan E. Sasaki M. Yamada Y. Hassell J.R. J. Biol. Chem. 1991; 266: 22347-22939Google Scholar, 5Murdoch A.D. Dodge G.R. Cohen I. Tuan R.S. Iozzo R.V. J. Biol. Chem. 1992; 267: 8544-8557Abstract Full Text PDF PubMed Google Scholar, 6Kallunki P. Tryggvason K. J. Cell Biol. 1992; 116: 559-571Crossref PubMed Scopus (182) Google Scholar). Different isoforms of perlecan have been found, which appear to be the result of alternative splicing (7Joseph S.J. Ford M.D. Barth C. Portbury S. Bartlett P.F. Nurcombe V. Greferath U. Development. 1996; 122: 3443-3452Crossref PubMed Google Scholar, 8Noonan D.M. Hassell J.R. Kidney Int. 1993; 43: 53-60Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Agrin is another extracellular matrix HSPG that is not related to perlecan (9Hagen S.G. Michael A.F. Butkowski R.J. J. Biol. Chem. 1993; 268: 7261-7269Abstract Full Text PDF PubMed Google Scholar, 10Groffen A.J.A. Hop F.W.H. Tryggvason K. Dijkman H.B.P.M. Assmann K.J.M. Veerkamp J.H. Monnens L.A.H. van den Heuvel L.P.W.J. Eur. J. Biochem. 1997; 247: 175-182Crossref PubMed Scopus (32) Google Scholar). Agrin was first isolated from the electric organ of Torpedo (11Godfrey E.W. Nitkin R.M. Wallace B.G. Rubin L.L. McMahan U.J. J. Cell Biol. 1984; 99: 615-627Crossref PubMed Scopus (189) Google Scholar) and was recently identified as an HSPG (12Tsen G. Halfter W. Kroger S. Cole G.J. J. Biol. Chem. 1995; 270: 3392-3399Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). During formation of the neuromuscular junction, motor neurons provide a number of signals that promote the differentiation of the post-synaptic membrane. An early signal is the secretion of specific isoforms of agrin and their incorporation into the basal lamina. Agrin initiates the clustering of nicotinic acetylcholine receptors and other components of the muscle cell surface at the site of contact with axons of motor neurons (13Bowe M.A. Fallon J.R. Annu. Rev. Neurosci. 1995; 18: 443-462Crossref PubMed Scopus (173) Google Scholar, 14Ferns M. Hoch W. Campanelli J.T. Rupp F. Hall Z.W. Scheller R.H. Neuron. 1992; 8: 1079-1086Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 15McMahan U.J. Cold Spring Harbor Symp. Quant. Biol. 1990; 55: 407-418Crossref PubMed Scopus (580) Google Scholar, 16Campanelli J.T. Hoch W. Rupp F. Kreiner T. Scheller R.H. Cell. 1991; 67: 909-916Abstract Full Text PDF PubMed Scopus (106) Google Scholar). The structure of the agrin molecule and the functions of the various domains is extensively studied and presently well understood (17Hoch W. Campanelli J.T. Harrison S. Scheller R.H. EMBO J. 1994; 13: 2814-2821Crossref PubMed Scopus (101) Google Scholar). The C terminus of agrin contains several homology regions with the G-domain of laminin and four epidermal growth factor-like repeats (18Rupp F. Payan D.G. Magill-Solc C. Cowan D.M. Scheller R.H. Neuron. 1991; 6: 811-823Abstract Full Text PDF PubMed Scopus (261) Google Scholar). The binding site of agrin to α-dystroglycan is located on the C terminus as well. Agrin binds to α-dystroglycan via its laminin G repeats (19Hopf C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Abstract Full Text Full Text PDF PubMed Google Scholar). Furthermore, a heparin/HS binding site is located on the C terminus and binding of heparin or HS prevents clustering of the acetylcholine receptors, indicating that HSPGs mediate this activity (20Hopf C. Hoch W. Eur. J. Neurosci. 1997; 9: 1170-1177Crossref PubMed Scopus (29) Google Scholar, 21Hirano Y. Kidokoro Y. J. Neurosci. 1989; 9: 1555-1561Crossref PubMed Google Scholar, 22Wallace B.G. J. Neurosci. 1990; 10: 3576-3582Crossref PubMed Google Scholar). Most alternative splicing sites have been identified in the C terminus, three on rat agrin called x, y, and z and two on chick and Torpedo agrin called A and B, corresponding to rat y and z. This alternative splicing affects the activity of agrin to cluster acetylcholine receptors. The most active forms only occur in neural tissue, whereas the isoforms from non-neural tissue are less active (14Ferns M. Hoch W. Campanelli J.T. Rupp F. Hall Z.W. Scheller R.H. Neuron. 1992; 8: 1079-1086Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 23Godfrey E.W. Exp. Cell Res. 1991; 195: 99-109Crossref PubMed Scopus (58) Google Scholar, 24Hoch W. Ferns M. Campanelli J.T. Hall Z.W. Scheller R.H. Neuron. 1993; 11: 479-490Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 25Ferns M.J. Campanelli J.T. Hoch W. Scheller R.H. Hall Z. Neuron. 1993; 11: 491-502Abstract Full Text PDF PubMed Scopus (279) Google Scholar). This suggests that splicing is regulated in a tissue-specific manner. The N-terminus of agrin contains nine domains homologous to the Kazal family of protease inhibitors and several laminin III homology regions (18Rupp F. Payan D.G. Magill-Solc C. Cowan D.M. Scheller R.H. Neuron. 1991; 6: 811-823Abstract Full Text PDF PubMed Scopus (261) Google Scholar). On the N-terminal half of agrin, several attachment sites for HS side chains are found. Up until now, only one alternative splicing site has been found in the N-terminus. The insert on the N-terminal half is required for binding to the extracellular matrix which is mediated by laminin (26Denzer A.J. Gesemann M. Schumacher B. Ruegg M.A. J. Cell Biol. 1995; 131: 1547-1560Crossref PubMed Scopus (110) Google Scholar, 27Tsen G. Napier A. Halfter W. Cole G.J. J. Biol. Chem. 1995; 270: 15934-15937Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 28Denzer A.J. Brandenberger R. Gesemann M. Chiquet M. Ruegg M.A. J. Cell Biol. 1997; 137: 671-683Crossref PubMed Scopus (138) Google Scholar). Expression of agrin is not restricted to neurons. Agrin mRNA can be detected in a wide range of different tissues (24Hoch W. Ferns M. Campanelli J.T. Hall Z.W. Scheller R.H. Neuron. 1993; 11: 479-490Abstract Full Text PDF PubMed Scopus (180) Google Scholar), and the agrin protein has been demonstrated to be a basal lamina component in many tissues including kidney during early development (29Godfrey E.W. Dietz M.E. Morstad A.L. Wallskog P.A. Yorde D.E. J. Cell Biol. 1988; 106: 1263-1272Crossref PubMed Scopus (52) Google Scholar). Recently, we have shown the presence of agrin in the glomerular basement membrane (glomerular BM) in adult human kidney (30Groffen A.J.A. Ruegg M.A. Dijkman H.B.P.M. van der Velden T.J. Buskens C.A. van den Born J. Assmann K.J.M. Monnens L.A.H. Veerkamp J.H. van den Heuvel L.P.W.J. J. Histochem. Cytochem. 1998; 46: 19-27Crossref PubMed Scopus (148) Google Scholar). In this study, we raised and characterized two hamster monoclonal antibodies (mAbs) and a sheep polyclonal antiserum against rat glomerular BM HSPG. The specificity of the mAbs is studied on Chinese hamster ovary (CHO) cells that are transfected with constructs coding for full-length agrin or the C-terminal half of agrin. These studies showed that these antibodies recognize epitopes on the N-terminal half of agrin. The staining on rat kidney tissue of these mAbs is compared with that of different mAbs against well-defined epitopes on the C terminus of agrin. These experiments suggest that most renal BMs except the glomerular BM express a truncated isoform of agrin that lacks (part of) the C-terminal half due to alternative splicing and/or posttranslational processing. We speculate on the possible involvement of the C terminus of agrin in glomerular function. For immunization, rat glomerular HSPG was isolated from rat glomeruli (31van den Born J. van den Heuvel L.P.W.J. Bakker M.A.H. Veerkamp J.H. Assmann K.J.M. Berden J.H.M. Kidney Int. 1992; 41: 115-123Abstract Full Text PDF PubMed Scopus (212) Google Scholar). Proteoglycans were isolated by guanidine extraction, dialysis against urea, DEAE ion-exchange chromatography, chondroitinase ABC digestion (Sigma) and by fast protein liquid Q-Sepharose chromatography. For immunoprecipitation, HSPG was isolated from rat glomerular BM obtained by a detergent procedure (32van den Heuvel L.P.W.J. van den Born J. van de Velden T.J.A.M. Veerkamp J.H. Monnens L.A.H. Schroder C.H. Berden J.H.M. Biochem. J. 1989; 264: 457-465Crossref PubMed Scopus (54) Google Scholar), from which proteoglycans were isolated as described (31van den Born J. van den Heuvel L.P.W.J. Bakker M.A.H. Veerkamp J.H. Assmann K.J.M. Berden J.H.M. Kidney Int. 1992; 41: 115-123Abstract Full Text PDF PubMed Scopus (212) Google Scholar). All procedures were performed at 4 °C in the presence of 10 mm sodium EDTA, 1 mm phenylmethylsulfonyl fluoride, 5 mmbenzamidine HCl, and 10 mm N-ethylmaleimide. Male Chinese hamsters were immunized with rat glomerular HSPG according to a previously described protocol (31van den Born J. van den Heuvel L.P.W.J. Bakker M.A.H. Veerkamp J.H. Assmann K.J.M. Berden J.H.M. Kidney Int. 1992; 41: 115-123Abstract Full Text PDF PubMed Scopus (212) Google Scholar). Hybridomas were screened in indirect immunofluorescence (IF) on rat kidney cryostat sections (see below) for a staining pattern comparable with the staining of previously described mAbs against human glomerular BM HSPG, which primarily stain the glomerular BM in a bright linear pattern and the tubular BMs to a lesser extent (33van den Born J. van den Heuvel L.P.W.J. Bakker M.A.H. Veerkamp J.H. Assmann K.J.M. Berden J.H.M. J. Histochem. Cytochem. 1994; 42: 89-102Crossref PubMed Scopus (64) Google Scholar). Two hybridomas were found to meet these criteria, called MI-90 and MI-91, and were subcloned twice by limiting dilution so that all subclones displayed the same reactivity pattern. From these two clones, ascites production was elicited in nude mice. A sheep was immunized intracutaneously with rat glomerular HSPG according to a previously described protocol (32van den Heuvel L.P.W.J. van den Born J. van de Velden T.J.A.M. Veerkamp J.H. Monnens L.A.H. Schroder C.H. Berden J.H.M. Biochem. J. 1989; 264: 457-465Crossref PubMed Scopus (54) Google Scholar), and after the last booster the sheep was bled and the serum (called GR-14) was collected. The reactivity of the antibodies with various BM components was determined in ELISA as described (34Raats C.J.I. Bakker M.A.H. van den Born J. Berden J.H.M. J. Biol. Chem. 1997; 272: 26734-26741Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Rat glomerular BM HSPG, human glomerular BM HSPG (isolated as described, Ref. 32van den Heuvel L.P.W.J. van den Born J. van de Velden T.J.A.M. Veerkamp J.H. Monnens L.A.H. Schroder C.H. Berden J.H.M. Biochem. J. 1989; 264: 457-465Crossref PubMed Scopus (54) Google Scholar), rat L2-laminin, mouse fibronectin, mouse collagen type IV (all from Sigma; all at 1 μg/well), and HS from bovine kidney (Seikagaku Kogyo, Tokyo, Japan; 5 μg/well) were coated in PBS. Primary antibodies MI-90 and MI-91 were diluted 1:100, and GR-14 was diluted 1:1000. The following peroxidase-conjugated secondary antibodies in a dilution of 1:500 were used: rabbit anti-hamster IgG (Nordic, Tilburg, The Netherlands) for mAb MI-90, goat anti-hamster IgG (Cappel-Organon Teknika, Turnhout, Belgium) for mAb MI-91, and rabbit anti-sheep Ig (Cappel) for GR-14. Rat glomerular BM HSPG was labeled with 125I according to the chloramine T-method (35Greenwood F.C. Hunter W.M. Glover J.S. Biochem. J. 1963; 89: 114-123Crossref PubMed Scopus (6742) Google Scholar). Part of the sample was treated with heparitinase (EC 4.2.2.8; from Sigma) 1 unit/ml in 10 mm HEPES, pH 7.0, containing 2 mm CaCl2 for 6 h at 43 °C. Radiolabeled HSPG was pre-absorbed with protein G-Sepharose CL-4B to remove eventual radiolabeled IgG. Normal hamster IgG and culture supernatants of mAbs MI-90 and MI-91 were incubated with protein G-Sepharose, to which goat anti-hamster IgG (Cappel) was immobilized. Normal mouse IgM (MOPC-104E; Bionetics, Kensington, MD) and mouse anti-HS side chain mAb JM-403 (31van den Born J. van den Heuvel L.P.W.J. Bakker M.A.H. Veerkamp J.H. Assmann K.J.M. Berden J.H.M. Kidney Int. 1992; 41: 115-123Abstract Full Text PDF PubMed Scopus (212) Google Scholar,36van den Born J. Gunnarsson K. Bakker M.A.H. Kjellen L. Kusche-Gullberg M. Maccarana M. Berden J.H.M. Lindahl U. J. Biol. Chem. 1995; 270: 31303-31309Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) were incubated with protein G-Sepharose to which goat anti-mouse IgM (Cappel) was immobilized. Normal sheep IgG and sheep antiserum GR-14 were directly coupled to protein G-Sepharose. After washing with PBS-Tween, the immobilized antibodies were incubated overnight at 4 °C with radiolabeled rat glomerular BM HSPG (with or without heparitinase pretreatment). After washing in PBS-Tween, sample buffer containing 5% β-mercaptoethanol was added, the sample was boiled for 3 min and centrifuged, and the supernatant was subjected to polyacrylamide gel electrophoresis on a 7% homogenous gel. The gel was dried and used for autoradiography for 3 days at −70 °C. To investigate the reactivity of our antibodies with agrin, we used CHO-cells, stably transfected with constructs encoding full-length rat agrin or the C-terminal half of agrin (construct N1, amino acids 1137–1940) (17Hoch W. Campanelli J.T. Harrison S. Scheller R.H. EMBO J. 1994; 13: 2814-2821Crossref PubMed Scopus (101) Google Scholar). The cells were cultured as described (17Hoch W. Campanelli J.T. Harrison S. Scheller R.H. EMBO J. 1994; 13: 2814-2821Crossref PubMed Scopus (101) Google Scholar) and continuously selected for the presence of the construct by Geneticin (G418, 500 μg/ml; Life Technologies, Inc., Paisly, Scotland). Untransfected CHO cells were used as a negative control. On cytospins of CHO cells, indirect IF was performed with anti-HSPG mAbs MI-90, MI-91 antiserum GR-14, and anti-agrin mAb Agr-131 (17Hoch W. Campanelli J.T. Harrison S. Scheller R.H. EMBO J. 1994; 13: 2814-2821Crossref PubMed Scopus (101) Google Scholar) (Table I) as described for the kidney cryostat sections (see below). For analysis by fluorescence-activated cell sorter (FACS), samples of 100 μl of cell suspension containing 5 × 106 cells were permeabilized and fixed with 1 ml of PermeaFix (Ortho Diagnostic, Beerse, The Netherlands) according to the instructions of the manufacturer. The cells were incubated with 100 μl of primary antibody (Table I) for 30 min at 0 °C. The cells were washed and incubated with fluorescein isothiocyanate-conjugated secondary antibody for 30 min at 0 °C. The cells were washed twice and kept in 1% paraformaldehyde until analysis on a Coulter Epics XL flow cytometer (Coulter Corp., Hialeah, FL). Mean fluorescence was determined on a scale between 0 and 1024, and values were corrected for staining by the secondary antibody alone.Table IPrimary and secondary antibodies used in indirect immunofluorescencePrimary antibodyAntigenDilutionSecondary antibodyDilutionSourceMI-90Rat glom HSPG1:400Goat anti-hamster IgG1:50Jackson, West Grove, PAMI-91Rat glom HSPG1:800Goat anti-hamster IgG1:200Cappel-Organon Teknika, Turnhout, BelgiumGR-14Rat glom HSPG1:800Rabbit anti-sheep IgG1:100Southern, Birmingham, ALAgr-30, −33, −131, −137, −435Rat agrin1:1000Sheep anti-mouse IgG F(ab)21:500Organon Teknika, Turnhout, Belgium Open table in a new tab Indirect IF was performed as described previously (34Raats C.J.I. Bakker M.A.H. van den Born J. Berden J.H.M. J. Biol. Chem. 1997; 272: 26734-26741Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) on 2 μm cryostat sections of rat kidney. To evaluate the specificity of the hamster and sheep antibodies for the HSPG core protein, pretreatment of rat kidney cryostat sections was performed by incubating nonfixed sections with 0.25 units/ml heparitinase in 10 mm HEPES, pH 7.0, containing 2 mm CaCl2 for 30 min at 37 °C. The reactivity of hamster mAbs MI-90 and MI-91 and sheep antiserum GR-14 in ELISA with different BM components is shown in Fig. 1. For MI-90, a clear reactivity was observed with both rat glomerular BM HSPG and human glomerular BM HSPG. For MI-91, a reactivity was observed with rat glomerular BM HSPG, but not with human glomerular BM HSPG. Neither of the mAbs bound to rat L2-laminin, mouse fibronectin, mouse collagen IV, or HS. Antiserum GR-14 bound to rat glomerular BM HSPG, human glomerular BM HSPG, and to a lesser extent to rat L2-laminin, but not to the other BM components. After immunoprecipitation of intact, 125I-labeled rat glomerular BM HSPG with hamster mAbs MI-90, MI-91, and sheep antiserum GR-14, gel electrophoresis and autoradiography revealed a broad smear with a molecular mass larger than 220 kDa, which might have resulted from different core proteins and/or different extent of glycosylation (Fig. 2, lanes 2, 3, and7). This is in agreement with previous findings (32van den Heuvel L.P.W.J. van den Born J. van de Velden T.J.A.M. Veerkamp J.H. Monnens L.A.H. Schroder C.H. Berden J.H.M. Biochem. J. 1989; 264: 457-465Crossref PubMed Scopus (54) Google Scholar). The same band is seen with the mouse anti-HS side chain mAb JM-403 (lane 5) (33van den Born J. van den Heuvel L.P.W.J. Bakker M.A.H. Veerkamp J.H. Assmann K.J.M. Berden J.H.M. J. Histochem. Cytochem. 1994; 42: 89-102Crossref PubMed Scopus (64) Google Scholar). To assess the molecular mass of the HSPG core protein(s), HSPG was treated before immunoprecipitation with heparitinase to degrade the HS glycosaminoglycan side chains. This resulted in a major band of ∼170 kDa and several bands with molecular masses of ∼105 and 70 kDa, which were equal for MI-90, MI-91, and GR-14 (lanes 9, 10, and 14). mAb JM-403 directed against HS side chain, however, is negative after treatment with heparitinase, as expected (lane 12). Non-relevant hamster IgG (lanes 1 and 8), mouse IgM (lanes 4 and 11), and normal sheep IgG (lanes 6 and 13) with the same Ig concentration served as negative controls. These results indicate that the mAbs MI-90 and MI-91 and the sheep antiserum GR-14 are directed against the core protein(s) of an HSPG present in rat glomerular BM. To evaluate recognition of rat agrin by our antibodies, indirect IF was performed on CHO cells transfected with constructs encoding full-length agrin (37Campanelli J.T. Roberds S.L. Campbell K.P. Scheller R.H. Cell. 1994; 77: 663-674Abstract Full Text PDF PubMed Scopus (340) Google Scholar) or the C-terminal half of agrin (N1) (25Ferns M.J. Campanelli J.T. Hoch W. Scheller R.H. Hall Z. Neuron. 1993; 11: 491-502Abstract Full Text PDF PubMed Scopus (279) Google Scholar). Binding of antibodies was evaluated either microscopically on cytospins or by FACS analysis. First, microscopical evaluation of cytospins showed that both hamster anti-HSPG mAbs MI-90 and MI-91 and sheep anti-HSPG GR-14 recognized full-length agrin as expressed by CHO cells transfected with full-length agrin (Fig. 3, A-C), whereas they did not bind to the C-terminal half of agrin as expressed by cells transfected with construct N1 (Fig. 3, E-G, TableII). Neither did they bind to wild type CHO cells. The positive control anti-agrin mAb Agr-131 bound to both full-length agrin and to the C-terminal half of agrin as expressed by cells transfected with full-length agrin or construct N1, respectively (Fig. 3, D and H, Table II) To confirm this crucial conclusion, we also performed fluorescence analysis by FACS and quantified mean fluorescence intensity. As shown in Table II, full-length agrin expressed by CHO cells is recognized by both hamster mAbs and the sheep antiserum. These antibodies, however, were negative on N1-transfected cells and on wild type CHO cells. Anti-agrin mAb Agr-131, however, recognized both full-length and C-terminal agrin. These results indicate that hamster mAbs MI-90 and MI-91 and the sheep antiserum GR-14 recognize epitopes on the N-terminal half of agrin.Table IIStaining of agrin-expressing CHO cells by MI-90, MI-91, GR-14, and Agr-131AntibodyWild typeFull-length agrinC-terminal half agrinCytospinFACSCytospinFACSCytospinFACSMI-90−0+++306−0MI-91−7+++412−0GR-14±54++178±63Agr-131±51+++326++163Cytospins or FACS of CHO cells transfected with constructs coding for either full-length agrin or the C-terminal half of agrin or wild type CHO cells stained with hamster mAbs MI-90, MI-91, sheep antiserum GR-14, or anti-agrin mAb Agr-131. Staining on cytospin preparations is quantified as follows: −, negative; +, weak; ++, moderate; +++, strong. Staining intensity on the FACS is quantified as mean fluorescence intensity (on a scale from 0 to 1024). Results from one representative out of four experiments are shown. Open table in a new tab Cytospins or FACS of CHO cells transfected with constructs coding for either full-length agrin or the C-terminal half of agrin or wild type CHO cells stained with hamster mAbs MI-90, MI-91, sheep antiserum GR-14, or anti-agrin mAb Agr-131. Staining on cytospin preparations is quantified as follows: −, negative; +, weak; ++, moderate; +++, strong. Staining intensity on the FACS is quantified as mean fluorescence intensity (on a scale from 0 to 1024). Results from one representative out of four experiments are shown. In indirect IF, mAbs MI-90 and MI-91 stain most BMs in a linear way, the glomerular BM with the highest intensity (Fig. 4,A and B). This staining pattern closely resembles the staining of previously developed mAbs directed against human glomerular BM HSPG (38van den Born J. van den Heuvel L.P.W.J. Bakker M.A.H. Veerkamp J.H. Assmann K.J.M. Weening J.J. Berden J.H.M. Kidney Int. 1993; 43: 454-463Abstract Full Text PDF PubMed Scopus (146) Google Scholar). Only mAb MI-91 weakly stained Bowman's capsule and BMs of smooth muscle cells in arteries and arterioles. Both mAbs stained BMs of endothelial cells in blood vessels but not the mesangial matrix and peritubular capillaries. (Fig. 4, A andB) mAb MI-90 also stained human kidney cryostat sections, and mAb MI-91 also stained mouse kidney cryostat sections. (TableIII). The polyclonal antiserum GR-14 stained the glomerular BM, all tubular BMs, and BMs of endothelial cells in a bright linear way but not the mesangial matrix nor the peritubular capillaries. Bowman's capsule, and the smooth muscle cells in arteries and arterioles were stained weakly (Fig. 4 C). This staining pattern is similar to that of a previously described polyclonal antibody directed to human glomerular BM HSPG (32van den Heuvel L.P.W.J. van den Born J. van de Velden T.J.A.M. Veerkamp J.H. Monnens L.A.H. Schroder C.H. Berden J.H.M. Biochem. J. 1989; 264: 457-465Crossref PubMed Scopus (54) Google Scholar). GR-14 also stained human and mouse kidney cryostat sections. (Table III) The staining pattern of MI-90, MI-91 and GR-14 could not be prevented by pre-treatment of the sections with heparitinase, indicating that the antibodies are directed against the core protein of HSPG. mAb JM-403 directed against the HS side chain, however, was negative after treatment with heparitinase, as expected. This confirms our data from the immunoprecipitation with heparitinase-pretreated glomerular BM HSPG (Fig. 2, lanes 8–14).Table IIIKidney staining of agrin by MI-90, MI-91, GR-14, and Agr-131AntibodyRatHumanMouseGBMTBMMMBCPTCVSMMI-90++++/++−−−−+−MI-91++++/++−±−±−+GR-14++++/++−±−±++Agr-131+++−/±−−−−+NDIndirect immunofluorescence of MI-90, MI-91, GR-14, or Agr-131 on rat, human, and mouse renal tissue. GBM, glomerular basement membrane; TBM, tubular basement membrane; MM, mesangial matrix; BC, Bowman's capsule; PTC, peritubular capillaries; VSM, vascular smooth muscle; ND, not determined. Open table in a new tab Indirect immunofluorescence of MI-90, MI-91, GR-14, or Agr-131 on rat, human, and" @default.
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• W2157299631 date "1998-07-01" @default.
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• W2157299631 title "Differential Expression of Agrin in Renal Basement Membranes As Revealed by Domain-specific Antibodies" @default.
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