Sakata N, Sasatomi Y, Ando S, Meng J, Imanaga Y, Uesugi N, Takebayashi S
Second Department of Pathology, School of Medicine, Fukuoka University, 45-1, 7-chome Nanakuma, Jonan-ku, Fukuoka 814-0133, Japan.
Connect Tissue Res. 2000;41(2):117-29. doi: 10.3109/03008200009067664.
Glycoxidative modification of various body proteins, including fibronectin (FN), has been shown to change their structural and functional properties, and be implicated in pathogenesis of diabetic complications. Little is known about the role of secondary structure of glycoxidative FN (gFN) in its domain functions. gFN was prepared by incubation with 25 and 200 mM glucose in 0.2 M sodium phosphate buffer at 37 degrees C on a shaking plate under aerobic and sterile conditions for various time intervals up to 49 days, being defined as gFN25 and gFN200, respectively. Unmodified FN (uFN) was prepared by incubation in 0.2 M sodium phosphate buffer without any glucose at 4 degrees C for 49 days. The extent of glycoxidative modification was examined using a noncompetitive enzyme-linked immunosorbent assay with an antibody against N(epsilon) -(carboxymethyl)lysine (CML), one of the major glycoxidation products. The binding activities of uFN and gFN to collagen, gelatin and heparin were determined by a solid phase enzyme immunoassay or heparin-affinity HPLC. Cell attachment was estimated by the extent of adhesion of FITC-labeled smooth muscle cells to uFN or gFN. Conformational change in gFN was detected by SDS-polyacrylamide gel electrophoresis and spectroscopy (circular dichroism). CML was detected in gFN25 and gFN200 after 49 and 21 days of incubation, respectively. Levels of CML were about six-fold higher in gFN200 than in gFN25 after 49 days. Both gFN25 and gFN200 showed a significant decrease in the ability of binding to collagen and gelatin after 7 days of incubation. The binding activity for heparin was significantly decreased in both gFN25 and gFN200 after one day. Cell attachment activity was reduced to 89% and 76% of the unmodified form in both gFN25 and gFN200 after 49 days, respectively. High molecular weight materials were found in gFN25 and gFN200 after 21 and 7 days, respectively. CD spectrum showed that gFN25 had lost its native conformation after 3 days of incubation, depending upon the concentration and incubation interval of the applied glucose. These in vitro results suggest that the loss of native conformation may reduce the domain functions of gFN, including binding activity to macromolecular ligands and cell attachment, and may play a major role in the pathogenesis of diabetic complications.
包括纤连蛋白（FN）在内的各种人体蛋白质的糖氧化修饰已被证明会改变其结构和功能特性，并与糖尿病并发症的发病机制有关。关于糖氧化修饰的FN（gFN）二级结构在其结构域功能中的作用知之甚少。通过在0.2M磷酸钠缓冲液中，于37℃在有氧和无菌条件下，在摇床上分别与25mM和200mM葡萄糖孵育不同时间间隔直至49天来制备gFN，分别定义为gFN25和gFN200。未修饰的FN（uFN）通过在4℃的0.2M磷酸钠缓冲液中孵育49天，且不含任何葡萄糖来制备。使用针对主要糖氧化产物之一N（ε）-（羧甲基）赖氨酸（CML）的抗体，通过非竞争性酶联免疫吸附测定法检测糖氧化修饰的程度。通过固相酶免疫测定法或肝素亲和高效液相色谱法测定uFN和gFN与胶原蛋白、明胶和肝素的结合活性。通过FITC标记的平滑肌细胞与uFN或gFN的粘附程度来估计细胞附着情况。通过SDS聚丙烯酰胺凝胶电泳和光谱法（圆二色性）检测gFN的构象变化。分别在孵育49天和21天后在gFN25和gFN200中检测到CML。49天后，gFN200中的CML水平比gFN25中的高约六倍。孵育7天后，gFN25和gFN200与胶原蛋白和明胶的结合能力均显著降低。孵育一天后，gFN25和gFN200与肝素的结合活性均显著降低。49天后，gFN25和gFN200中的细胞附着活性分别降至未修饰形式的89%和76%。分别在21天和7天后在gFN25和gFN200中发现高分子量物质。圆二色光谱显示，根据所用葡萄糖的浓度和孵育间隔，gFN25在孵育3天后失去了其天然构象。这些体外实验结果表明，天然构象的丧失可能会降低gFN的结构域功能，包括与大分子配体的结合活性和细胞附着，并可能在糖尿病并发症的发病机制中起主要作用。
