DIFFERENTIAL EXPRESSION OF AGRIN IN RENAL BASEMENT-MEMBRANES AS REVEALED BY DOMAIN-SPECIFIC ANTIBODIES

We determined the specificity of two hamster monoclonal antibodies and a sheep polyclonal antiserum against heparan sulfate proteoglycan iso lated from rat glomerular basement membrane. The antibodies were chara cterized by enzyme-linked immunosorbent assay on various basement memb rane components and immunoprecipitation with heparan sulfate proteogly can with or without heparitinase pre-treatment. These experiments show ed 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 hepar an sulfate proteoglycan in the glomerular basement membrane (Groffen, A. J. A., Ruegg, M. A., Dijkman, H. B. P. M., Van der Velden, T. J., B uskens, C. A., van den Born, J., Assmann, It. 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 re cognize agrin. To this end, we evaluated staining of Chinese hamster o vary cells transfected with constructs encoding full-length or the C-t erminal half of rat agrin by analysis on a fluorescence-activated cell sorter. Both hamster monoclonals and the sheep antiserum clearly stai ned cells transfected with the construct encoding full-length agrin, w hereas wild type cells and cells transfected with the construct encodi ng the C-terminal part of agrin were not recognized. A panel of previo usly characterized monoclonals, directed against C-terminal agrin, cle arly stained cells transfected with either of the constructs but not w ild type cells. This indicates that both hamster monoclonals and the s heep 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 antibod ies against C-terminal agrin stained almost exclusively the glomerular basement membrane, whereas the anti-N-terminal agrin antibodies recog nized all renal basement membranes, including tubular basement membran es. Based on these results, we hypothesize that full-length agrin is p redominantly expressed in the glomerular basement membrane, whereas in most other renal basement membranes a truncated isoform of agrin is p redominantly found that misses (part of) the C terminus, which might b e due to alternative splicing and/or posttranslational processing. The possible significance of this finding is discussed.