Zhou F Y, Kan M, Owens R T, McKeehan W L, Thompson J A, Linhardt R J, Höök M
Department of Biochemistry and Biophysics, Texas A & M University, Houston 77030, USA.
Eur J Cell Biol. 1997 May;73(1):71-80.
Heparin and related molecules have been identified as important participants in fibroblast growth factor (FGF) signaling although the mechanisms of action remain unclear. We have used heparin oligosaccharides to examine steps in the signaling process which could be affected by the polysaccharide. Immobilized FGF-1 and FGF-2 bound all sizes of oligosaccharides tested, ranging from tetrasaccharide to decasaccharide, at physiological salt concentration. Each group of oligosaccharide was eluted from the FGF affinity columns in several peaks, and larger oligosaccharides showed higher apparent affinity for the immobilized growth factors compared to the shorter ones. Heparin hexasaccharides were the smallest fragments providing complete protection of FGF-1 and FGF-2 against trypsin digestion. Tetrasaccharides, however, were able to provide partial protection. The requirement of heparin for ligand-receptor interaction was evaluated in receptor binding assays using Sf9 insect cells engineered to overexpress different recombinant FGF receptor (FGFR) species including FGFR1 beta, FGFR1 alpha or FGFR4 at the cell surface. In these assays hexasaccharides were the smallest fragments capable of stimulating FGF-receptor interaction. Over the range of concentrations examined, neither hexasaccharides nor octasaccharides were able to stimulate receptor binding to the level attained by intact heparin. In fact, these oligosaccharides interfered with the ability of intact heparin in promoting FGF-receptor binding. The presence of both stimulatory and inhibitory activities in hexasaccharide and octasaccharide populations could be attributed to structural heterogeneity within the oligosaccharide preparations. However, similar observations were obtained with "highly-sulfated" structurally homogeneous preparations of hexasaccharide and octasaccharide, although these molecules generally had greater stimulatory and less inhibitory activity than their structurally heterogeneous counterparts. Hexasaccharides were found to be the smallest fragments able to potentiate the FGF-1-induced 3T3 cell proliferation while their effect on FGF-2 signaling was less clear. These observations suggest that heparin can modulate FGF-signaling at several stages with different end results.
肝素及相关分子已被确认为成纤维细胞生长因子（FGF）信号传导中的重要参与者，尽管其作用机制尚不清楚。我们已使用肝素寡糖来研究信号传导过程中可能受多糖影响的步骤。在生理盐浓度下，固定化的FGF-1和FGF-2能结合所测试的各种大小的寡糖，范围从四糖到十糖。每组寡糖从FGF亲和柱上以多个峰洗脱，与较短的寡糖相比，较大的寡糖对固定化生长因子表现出更高的表观亲和力。肝素六糖是能为FGF-1和FGF-2提供完全抗胰蛋白酶消化保护的最小片段。然而，四糖能够提供部分保护。在受体结合试验中，使用经工程改造在细胞表面过表达不同重组FGF受体（FGFR）种类（包括FGFR1β、FGFR1α或FGFR4）的Sf9昆虫细胞来评估肝素对配体-受体相互作用的需求。在这些试验中，六糖是能够刺激FGF-受体相互作用的最小片段。在所研究的浓度范围内，六糖和八糖均无法将受体结合刺激到完整肝素所达到的水平。事实上，这些寡糖会干扰完整肝素促进FGF-受体结合的能力。六糖和八糖群体中同时存在刺激和抑制活性可能归因于寡糖制剂中的结构异质性。然而，对于六糖和八糖的“高度硫酸化”结构均一的制剂也得到了类似的观察结果，尽管这些分子通常比其结构异质的对应物具有更大的刺激活性和更小的抑制活性。已发现六糖是能够增强FGF-1诱导的3T3细胞增殖的最小片段，而它们对FGF-2信号传导的影响则不太明确。这些观察结果表明，肝素可在多个阶段调节FGF信号传导，并产生不同的最终结果。
