Molecular Mechanisms in Basement Membrane Complications of Diabetes: Alterations in Heparin, Laminin, and Type IV Collagen Association

Abstract

We report alterations in the ligand-binding properties of laminin, the major noncollagenous protein of basement membranes, resulting from nonenzymatic glycosylation in vitro. Mouse laminin was incubated in vitro with 500 mM glucose, and the level of nonenzymatically glycosylated amino acids increased 2-, 6.2-, and 12-fold after incubation for 1, 3, and 12 days, respectively. Ligand binding assays conducted in solution with varying concentrations of [3H]heparin and a constant amount of control or nonenzymatically glycosylated laminin showed a reduction in heparin binding proportional to laminin glycosylation. An analysis of the stoichiometry of [3H]heparin binding to control and nonenzymatically glycosylated laminin at saturating levels of heparin was performed. The results indicated that the total number of heparin binding sites on laminin decreased 4.7- and 14.7-fold due to nonenzymatic glycosylation of laminin for 1 and 3 days, respectively. [3H]heparin binding to 12-day nonenzymatically glycosylated laminin was abolished. Scatchard analyses of the binding data for heparin and laminin gave a dissociation constant (Kd) of 3.4 X 10(-8) M. The data for nonenzymatic glycosylation of laminin for 1 day in vitro resulted in a biphasic heparin binding curve. Both high- and low-affinity binding was observed (Kd values of 3.3 x 10(-8) and 2.6 x 10(-7) M, respectively). Similar high- and low-affinity binding sites were seen on 3-day nonenzymatically glycosylated laminin (Kd values of 2.1 x 10(-8) and 3.9 x 10(-7) M, respectively). [3H]heparin binding at a fixed concentration to a constant amount of control or 12-day nonenzymatically glycosylated laminin and type IV collagen was also studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel urinary biomarkers in pre-diabetic nephropathy

“…Studies have shown that prevalence of microalbuminuria is near 10 % in pre-diabetes and 25 % in diabetes [26]. Chronic exposure to abnormal glycemic levels cause oxidative stress, glycation of membrane proteins, and hyperfiltration resulting in microalbuminuria [27,28]. However, it is not very sensitive and specific marker of nephropathy.…”

Microalbuminuria in a Middle-Aged Workforce: Effect of hyperglycemia and ethnicity

“…Therefore, perhaps the most likely single explanation for the increased permeability of glomerular basement membranes to plasma proteins in early NIDDM and its relationship with hyperglycemia is some biochemical mechanism. Increased glycation of laminin and type IV collagen components of the glomerular matrix cause decreased incorporation of heparan sulfate proteoglycan into complexes of fibronectin and collagen in the glomerular basement membrane and extracellular matrix (30). The anionic sites of heparan sulfate proteoglycan impart strong electronegativity to the glomerular basement membrane, and it is postulated that this charge effect helps keep the surface hydrated and prevents the filtration of negatively charged plasma protein such as albumin (31).…”

Microalbuminuria: Implications for micro- and macrovascular disease

“…In poorly regulated diabetic patients, the degree of glycosylation of collagen IV and fibronectin is much higher than in control subjects (69). However, nonenzymatic glycosylation of collagen IV and fibronectin will decrease the affinity to HS-PG (69). This might lead to a greater turnover of HS-PG in poorly regulated diabetic patients (70) and reduced density of HS-PG in extracellular matrix.…”

“…In poorly regulated diabetic patients, the degree of glycosylation of collagen IV and fibronectin is much higher than in control subjects (69). However, nonenzymatic glycosylation of collagen IV and fibronectin will decrease the affinity to HS-PG (69).…”