Heparan sulfate proteoglycans (HSPGs) are essential components of the glomerular basement membrane (GBM) carrying a strong anionic charge. A well-characterized extracellular HSPG is perlecan, ubiquitously expressed in basement membranes. A cDNA construct encoding domains I and I1 of human perlecan was expressed as a fusion protein with glutathione S-transferase. This fusion protein was used to generate monoclonal antibody 95J10. We compared the staining pattern of 95J10 with that of M215, a previously prepared mAb that recognizes HSPG isolated from human GBM. In kidney cortex, the antiperlecan mAb 95J10 showed a strong staining of the mesangium, Bowman's capsule, the tubular basement membrane, and stained the GBM only slightly. In contrast, M215 predominantly stained the GBM in a linear fashion. Immunoelectron microscopy supported these results, showing concentrations of perlecan in some regions of the GBM, whereas the unidentified M215 antigen was homogenously distributed throughout the GBM. In other human tissues, both antibodies also produced a different staining pattern. Furthermore, a polyclonal antiserum recognizing HSPG isolated from the GBM did not recognize perlecan from EHS tumors. These results provide evidence for the presence of another HSPG in the GBM that is immunologically distinct from perlecan. The absence of perlecan splice variants in the kidney suggests that this component is encoded by a different gene than perlecan. Given its marked expression in the GBM, this component could be a determining factor in the maintenance of selective glomerular permeability.Keywords: perlecan; heparan sulfate proteoglycan ; prokaryotic expression; glomerular basement membrane.The glomerular basement membrane (GBM) is a specialized extracellular matrix located at the interface of the glomerular visceral epithelium and the vascular endothelium. The GBM provides a rigid structure that supports the tissue to withstand high local blood pressure, its mechanical strength being enhanced by the highly organized interplay of its biopolymer components [e.g. collagen IV, laminin, nidogen, and heparan sulfate proteoglycan (HSPGs)] [l]. Another crucial function of the GBM is to regulate the passage of proteins depending on their molecular size and charge [2]. The impermeability of the GBM for anionic macromolecules is assigned to the presence of HSPGs anchored within it. The electrostatic charge of these HSPGs can be visualized by electron microscopy using cationic ferritin [2] or heparan sulfate (HS)-specific mAbs [3]. The significance of this mechanism was demonstrated by perfusing rat

The cDNA encoding a human prosome fl-subunit (HSBpros26) was isolated from a lymphoma library using the cDNA of the Xenopus homologue as a probe. The cDNA contains an open reading frame encoding a protein of 233 amino acids and a calculated molecular weight of 25,909. Comparison with interspecies homologues of H SBpros26 from Xenopus (XLB), rat (RN3) and yeast (PRE4) reveals a high degree of identity between thefl-subunits except for the N-terminal end, which is probably cleaved post-translationally. The complete coding sequence of HSBpros26 has been expressed in E. coli. The produced protein of about 27 kDa reacts with the prosomal monoclonal antibody MCP205, kindly provided by Dr. K. Hendil. The molecular weight of the native protein is about 28 kDa indicating that the protein is present as monomers. Finally partially purified HSBpros26 preparations do not contain any proteolytical activity.