Laminin Alterations After In Vitro Nonenzymatic Glycosylation ARISTIDIS S. CHARONIS, LORREL A. REGER, JAY E. DEGE, KOKKONA KOUZI-KOLIAKOS, LEO T. FURCHT, ROBERT M. WOHLHUETER, AND EFFIE C. TSILIBARY 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 cross- links. 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. Diabetes 39:807-14,1990 N onenzymatic glycosylation is the binding of glu- cose to proteins, primarily to the e-amino groups of lysine residues, without the mediation of any enzymes (1). Two stages of this reaction can be distinguished: a reversible one, where a Schiff-base product and eventually an Amadori product are formed, and an ir- reversible one, where advanced glycosylation end products are formed by the cross-linking of two glycosylated e-amino groups of two lysine residues (2). It has recently been suggested that aminoguanidine can block the forma- tion of cross-linked end products without any major inter- ference with the direct binding of glucose to the free amino groups (3). Nonenzymatic glycosylation normally occurs at very slow rates in long-lived proteins. In diabetic conditions, hyper- glycemia greatly accelerates this reaction. Many proteins have been observed to undergo nonenzymatic glycosylation in diabetes, e.g., hemoglobin (4,5), albumin (6), low-density lipoproteins (7), erythrocyte membrane proteins (8), lens crystallins (9), and glomerular basement membrane colla- gen (10). This structural modification may lead to altered physicochemical properties. It has been reported that gly- cosylated hemoglobin has a lower affinity for oxygen (11), glycosylated albumin has a decreased ability to bind to bil- irubin and c/s-parinaric acid (12), and glycosylated lens crystallins may aggregate and cause opalescence (9). Such functional changes may be the result of either a direct in- corporation of glucose onto lysine residues (12) or subse- quent cross-linking between modified and unmodified lysine residues (2,9). In general, proteins with long half-lives are more suscep- tible to severe structural and functional alterations because of nonenzymatic glycosylation. Therefore, macromolecular components of basement membranes that turn over very slowly are primary targets for such modifications. In the past, we have presented evidence that nonenzymatic glycosyla- tion of the main noncollagenous NC1 domain of type IV collagen, a major glycoprotein of basement membranes, in- From the Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota. Address correspondence and reprint requests to Aristidis S. Charonis, De- partment of Laboratory Medicine and Pathology, University of Minnesota Med- ical School, Box 609 UMHC, 420 Delaware Street, SE, Minneapolis, MN 55455. Received for publication 9 October 1989 and accepted in revised form 13 March 1990. DIABETES, VOL. 39, JULY 1990 807 Downloaded from http://diabetesjournals.org/diabetes/article-pdf/39/7/807/357766/39-7-807.pdf by guest on 04 November 2022