Molecular mechanisms in basement membrane complications of diabetes. Alterations in heparin, laminin, and type IV collagen association.
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)
Tarsio
J F
JF
Center for Biotechnology, Baylor College of Medicine, Woodlands, Texas.
Reger
L A
LA
Furcht
L T
LT
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism*; D066298:In Vitro Techniques; D007797:Laminin / Q000378:metabolism*; D051379:Mice
D007797:Laminin; D006493:Heparin; D003094:Collagen; D005947:Glucose