Aristidis S. Charonis

Laminin Alterations After In Vitro Nonenzymatic Glycosylation

Laminin, a basement membrane protein derived from the matrix of the Engelbreth-Holm-Swarm murine tumor, was nonenzymatically glycosylated in vitro in the presence of increasing glucose concentrations. The amount of glucose incorporated per laminin molecule was shown to be proportional to the molarity of glucose used. Nonenzymatic glycosylation resulted in formation of cross-links and alterations of the cruciform shape of laminin molecules; these alterations were dramatic when high concentrations of glucose were used. One of the functions of laminin, the process of self-assembly, was shown to be impaired after in vitro nonenzymatic glycosylation. Glucose incorporation resulted in a dramatic decrease of long-to-long laminin dimers, which normally form during the initial steps of assembly. Furthermore, nonenzymatic glycosylation of laminin reduced its ability to self-associate into complexes larger than dimers, as judged by turbidimetry. The observed decrease of maximal turbidity was proportional to the degree of nonenzymatic glycosylation. Aminoguanidine, which has been suggested to inhibit cross-link formation, was shown to restore to a large extent the shape of laminin, the percentage of long-to-long arm dimers, and the maximal turbidity when included in the mixtures of laminin and glucose. These data suggest that structural and functional alterations of laminin may be primarily due to formation of crosslinks. Such modifications of laminin (along with our basement membrane components) may contribute to the morphological and physiological changes observed in basement membranes under diabetic conditions.

Structural and Functional Changes of Laminin and Type IV Collagen After Nonenzymatic Glycation

Laminin and type IV collagen are two major basement membrane glycoproteins; they are large multidomain macromolecules that are involved in two types of functions. First, they provide the structural framework of all basement membranes, and second, they interact with cell-surface molecules and are key to adhesion, spreading, and proliferation of cells. We summarize experimental evidence that nonenzymatic glucosylation of these two macromolecules in vitro alters their structure, their ability to polymerize, and their ability to promote cell adhesion. Additional studies are needed to document these changes in situ and therefore extend these conclusions to intact basement membranes.

Nonenzymatic glycosylation-induced modifications of intact bovine kidney tubular basement membrane.

We examined structural changes in bovine kidney tubular basement membrane (TBM) following in vitro nonenzymatic glycosylation (NEG). Isolated TBM was incubated for 2 wk at 37 degrees C in the absence of sugar or in the presence of either glucose or ribitol under conditions that minimized degradation and oxidative damage. NEG and crosslink formation in glycated TBM were confirmed by decreased solubility, increased amounts of low mobility material by SDS-PAGE, and increased specific fluorescence compared to controls. Morphological analysis using high resolution, low voltage scanning electron microscopy (LV-SEM) revealed a complex three-dimensional meshwork of interconnecting strands with intervening openings. Glycated TBM underwent distinct morphological changes, including a 58% increase in the amount of image surface area occupied by openings. This was due to an apparent increase in the number of large openings (diameters > 12.5 nm), whereas the number of small openings (diameters < 12.5 nm) remained unchanged. These findings corroborate earlier physiological studies, which established that the loss of glomerular permselectivity seen in patients with diabetic nephropathy is due to the formation of large pores in the kidney filtration barrier of which the BM is a major component. We conclude that NEG and crosslink formation among BM components lead to modifications of BM ultrastructure, which could play a role in loss of barrier function in diabetic microangiopathy and nephropathy.

Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody

Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the binding of the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.

Putrescine: A Novel Inhibitor of Glycosylation-Induced Cross-Links in Laniinin

Objective: To determine whether putrescine, a naturally occuring polyamine, is able to prevent nonenzymatic glycosylation‐induced cross‐linking of basement membrane components. Cross‐linking, leading to the formation of advanced glycosylation end products (AGEs) has been proposed as a major mechanism contributing to structural and functional changes of the vascular wall, thus leading to microangiopathy.

Tubulointerstitial nephritis antigen (TIN-ag) is expressed in distinct segments of the developing human nephron

Tubulointerstitial nephritis antigen (TIN-ag) is a 58 kDa glycoprotein restricted within the kidney to basement membranes underlying the epithelium of Bowmans capsule and proximal and distal tubules. Autoantibody formation against this component has been described in association with primary immune-mediated tubulointerstital nephritis, membranous nephropathy and anti-glomerular basement membrane nephritis. In the present report, the ontogeny of this protein was studied in human fetal kidney tissue by immunohistochemical analysis of immature and developing nephrons using a panel of monoclonal and polyclonal antibodies. TIN-ag is first detected in basement membranes underlying the epithelium of Bowmans capsule of early capillary loop stage glomeruli and the primitive proximal tubule. No detectable expression is observed in the basement membranes of the branching ureteric bud, nephrogenic vesicle, or comma shape and s-shape stages of nephrogenic development. Increased staining of the proximal tubular basement membrane is associated with outgrowth of the primitive tubule from the urinary pole of the developing glomerulus. In more mature fetal tubules, TIN-ag expression closely resembles that of previously reported observations in mature tissue where it is present in high amounts in the basement membranes of proximal tubules, and to a lesser extent in Bowmans capsule and distal tubules. Our results suggest that TIN-ag expression is developmentally regulated in a precise spatial and temporal pattern throughout nephrogenesis.

Effects of Nonenzymatic Glycation on Molecular Interactions of Basement Membrane Molecules

Nonenzymatic glycation of proteins is considered one of the major pathogenetic processes responsible for diabetic microangiopathy. The basement membrane of the microvasculature is thought to undergo important structural and functional alterations in diabetes mellitus. In this chapter, the structural characteristics of the macromolecular components of the basement membrane are briefly described; the focus is on the intrinsic basement membrane components that are thought to be the building blocks for this structure. It should be emphasized that the current list of well-characterized basement membrane macromolecules is a partial one, and certainly other components will soon be added.

A melanoma cell surface laminin binding protein with apparent Mr 90,000.

Laminin promotes adhesion of various cell types via multiple interactions with cell surface components. We have used a laminin domain involved in adhesion of melanoma cells, peptide F9 (Charonis et al., J. Cell Biol. 107:1253 [( 1988]), to examine its specific interaction with cell surface components. Cells were surface labeled, solubilized, and the cell surface associated macromolecules were purified via laminin and F9 affinity columns. We have observed that a macromolecule with apparent molecular weight 90,000 interacts with laminin and peptide F9. This macromolecule does not change electrophoretic mobility upon reduction, cannot be removed from the cell surface by high salt treatment and partitions in the detergent phase of Triton X-114. These results suggest that this macromolecule is associated with melanoma cell surfaces and may be involved in their interaction with laminin.

Glycosylation-induced Modifications of Intact Basement Membrane

We examined structural changes in bovine kidney tubular basement membrane (TBM) following In vitro nonenzymatic glycosylation (NEG). Isolated TBM was incubated for 2 wks at 37° C in the absence of sugar or in the presence of either glucose or ribitol under conditions which minimized degradation and oxidative damage. NEG and crosslink formation in glycated TBM were confirmed by increased specific fluorescence compared to controls. Morphological analysis using high resolution, low voltage scanning electron microscopy (LV-SEM) revealed a complex three-dimensional meshwork of interconnecting strands with intervening openings. Glycated TBM underwent distinct morphological changes including a 58% increase in the amount of image surface area occupied by openings. This was due to an apparent increase in the number of large openings (diameters >12.5nm), whereas the number of small openings (diameters <12.5nm) remained unchanged. These findings corroborate earlier physiological studies which established that the loss of glomerular permselectivity seen in patients with diabetic nephropathy is due to the formation of large pores in the kidney filtration barrier of which the BM is a major component. We conclude that NEG and crosslink formation among BM components lead to modifications of BM ultrastructure which could play a role in loss of barrier function in diabetic microangiopathy and nephropathy.

Tubular Basement Membrane Autoantibodies

Publisher Summary This chapter focuses on tubular basement membrane autoantibodies. Antibodies to kidney tubular basement membrane (TBM) are present in tubulointerstitial nephritis (TIN), a common disease leading to renal insufficiency. The antibodies are usually detected in the clinical setting by direct immunofluorescence during evaluation of renal biopsy specimens. A sensitive radioimmunoassay was used in studies of TBM antibodies in patients with anti-GBM nephritis. The common renal disease associations with anti-TBM disease include membranous glomerulonephritis and antiglomerular basement membrane disease. Anti-TBM occurs in membranous glomerulonephritis or in drug-induced interstitial nephritis in male patients.