Evidence for the Existence of Multiple Heparan Sulfate Proteoglycans in the Human Glomerular Basement Membrane and Mesangial Matrix

Abstract

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

Quantitative Distribution of Glycosaminoglycans in Young and Senile (Cataractous) Anterior Lens Capsules

“…Slow turnover rates of lens capsule material, which provide the opportunity for degradation and posttranslational modi®cation re¯ect a second possibility to be involved in shortening of the GAG chains (Mohan and Spiro, 1991), and thus, to reduce the CCG label intensity. However, quantitative biochemical evaluations of age-related alterations of proteoglycan contents may be superimposed by changes in tissue extractability, due to strong interactions with other components (Groffen et al, 1997). This is a problem which we have overcome using a quantitative cytochemical approach.…”

Antibody Response Against Perlecan and Collagen Types IV and VI in Chronic Renal Allograft Rejection in the Rat

“…34 Perlecan and ␣1(VI)/␣5(IV) collagen are both located on the endothelial side of the GBM, limited to focal accumulations, 31 and to the mesangial matrix. 31,[35][36][37] In addition, type VI collagen is produced by endothelial cells. 38 Together this suggests that initial antibody-mediated GBM damage can occur independent of endothelial cell injury, but may in the long term affect endothelial cell architecture and/or function as observed in patient specimens.…”

“…Perlecan and collagen VI are known to be expressed in most renal BMs, including the TBM. 30,35,46 The induction of antibodies against perlecan or ␣1(VI)/ ␣5(IV) collagen might be the result of the formation of new epitopes exposed in the GBM after injury. Alternatively, antibodies might be induced as a result of (genetic) differences in perlecan or ␣1(VI)/␣5(IV) collagen between donor and recipient.…”

Glomerular basement membrane heparan sulfate in health and disease: A regulator of local complement activation

“…Perlecan is the prominent HSPG in most basement membranes, though in the mature GBM it is confined to the subendothelial side [16]. It consists of a 467 kDa core protein comprising five domains, which bears three heparan sulfate chains within the amino-terminal domain I [17].…”