Non‐enzymatic glycation of type I collagen diminishes collagen–proteoglycan binding and weakens cell adhesion

Abstract

Non-enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans1(PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell-collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG-collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content -effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, had similar structures and comparable diameter distributions. B-cells expressing the cell surface heparan sulfate PG syndecan-1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein-collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG-collagen interactions, thereby disrupting matrix integrity and cell-collagen interactions, adhesion, and migration.1 The abbreviations used are: proteoglycans (PG); keratan sulfate (KS); heparan sulfate (HS); dermatan sulfate (DS); chondroitin sulfate (CS); glycosaminoglycan (GAG); affinity coelectrophoresis (ACE); circular dichroism (CD); advanced glycation end products (AGE); cyanogen bromide (CB); 3-(N-morpholino)-2hydroxypropanesulfaonate (MOPSO) *Address correspondence to: James D. San Antonio, Department of Medicine and the Cardeza Foundation for Hematologic Research, Thomas Jefferson University, 809 Curtis, 1015 Walnut St., Philadelphia, PA 19107-5099, Ph: 215-955-6121, Fax: 215-923-3836 [Linsenmayer, 1991;Prockop and Kivirikko, 1995] for review). It is synthesized as a soluble, procollagen form containing two α1, and one α2 chains, each of about 1000 amino acids. Upon secretion from the cell the propeptides are cleaved, and the collagen monomer is assembled into the fibril, proposed to consist of aggregates of microfibrils, or 5-mer bundles of overlapping monomers. Type I collagen fibrils contribute to the integrity and function of many tissues via interactions with other collagens, PGs, and growth and differentiation factors [DiLullo et al., 2002]. In aging, type I collagen b...

Interstitial Fluid and Lymph Formation and Transport: Physiological Regulation and Roles in Inflammation and Cancer

“…Collagen is a protein polyampholyte with both positively and negatively charged groups that self-compensate at pH 7.4. Although charge may vary with the microenvironment (161), collagen I fibrils generally possess a slightly positive charge (pI ϳ8.0) at physiological pH (267,384), estimated to ϩ14 mol/mol in reconstituted collagen (236), while collagen II has a zero net charge density in cartilage (485), leaving a slight net positive charge on the molecule (160,267). Accordingly, since collagen has almost no net charge, it behaves as a tissue element with effectively no areas with high local charge (i.e., it does not support an electron cloud and has no Debye length) (502).…”

Lumican is required for neutrophil extravasation following corneal injury and wound healing

“…Tissue culture chamber slides (Labtec, Naperville, IL) and cell culture inserts (3 mm pore size, BD Biosciences) were pre-coated with lumican purified from bovine aorta (non-glycanated) (Funderburgh et al, 1991) or keratan sulfate proteoglycan (KSPG) (glycanated) from bovine corneas (Reigle et al, 2008) at 10 mg/ml for 60 minutes at 37°C in a humidified atmosphere containing 95% air, 5% CO 2 . For neutralizing antibody studies, 24-well culture plates (Iwaki, Tokyo, Japan) and cell culture inserts (FluoroBlok TM Insert System, 3 mm pore size, BD Falcon TM , San Jose, CA) coated with type 1 collagen, were coated with GST-Lum 17-338 or GST at 10 mg/ml in PBS for 60 minutes at 37°C in a humidified atmosphere containing 95% air and 5% CO 2 .…”

“…Tissue culture chamber slides (Labtec, Naperville, IL) were pre-coated with lumican purified from bovine aorta (Funderburgh et al, 1991) (non-glycanated) or keratan sulfate proteoglycan (KSPG) (glycanated) from bovine corneas (Reigle et al, 2008) as described previously. After washing in PBS, the slides were blocked with 1% BSA for 60 minutes at 37°C in a humidified atmosphere containing 95% air, 5% CO 2 .…”

Advanced glycation end-products: Mechanics of aged collagen from molecule to tissue

“…On one hand, they alter molecular recognition [16] by modifying the biochemistry of specific protein binding sites [17]. On the other hand, AGEs crosslinks such as glucosepane are known to alter the mechanical properties of load bearing proteins such as collagen [18,19] , leading to stiffer tissues and decreased viscoelasticity [20][21][22].…”