鲐鱼软骨糖胺聚糖的硫酸软骨素的结构及其与生长因子的相互作用

为了开发具有药用价值的硫酸软骨素(chondroitin sulfate,CS)的新资源,从鲐鱼(Pneumatophorus japonicus Houttuyn)软骨蛋白聚糖(proteoglycan,PG)制备了糖胺聚糖(glycosaminoglycan,GAG),用酶降解和阴离子交换HPLC法测定了GAG的CS的组成及其含量,用凝胶层析法测定了GAG的分子量,用表面等离子体谐振(surface plasmon resonance,SPR)法测定了其与多效生长因子(PTN)、中期因子(MK)和肝细胞生长因子(HGF)相互作用的动力学参数结合速率常数(ka)、解离速率常数(kd)和平衡解离常数(KD)。结果显示,鲐鱼软骨GAG含量约为651μg/mgPG或1.09μmol/mgPG(按照二糖单位计算),主要含CS(1.03μmol/mgPG,按照二糖单位计算),CS酶解产生的主要二糖单位是ΔDi-6S(38.8%)和ΔDi-4S(46.3%),有少量的ΔDi-0S(8.4%)和ΔDi-diSD(6.5%)。GAG(CS)的分子量为78kD。GAG(CS)与生长因子的相互作用的动力学参数ka((mol/L)-1.s-1)、kd(s-1)和KD(nmol/L)分别为(2.77±0.17)×105、(7.74±1.56)×10-5和(0.28±0.06)(MK),(1.05±0.22)×104、(4.16±0.80)×10-3和(417±131.3)(PTN),(7.04±0.94)×105、(7.84±2.82)×10-3和(11.1±3.80)(HGF)。该GAG同MK、HGF和PTN有高的或较高的亲和性,暗示鲐鱼软骨GAG的CS有可能通过调节生长因子的信号转导途径而对某些疾病发挥治疗作用,具有潜在进一步药用开发价值。

Chondroitin Sulfate in the Preparation of Glycosaminoglycan from Mackerel (Pneumatophorus japonicus Houttuyn) Nasal Cartilage and Its Interaction with Growth Factors

To search for new resource of chondroitin sulfate(CS) for therapeutics,glycosaminoglycan(GAG) was isolated from nasal proteoglycan(PG) of mackerel(Pneumatophorus japonicus Houttuyn).The disaccharide composition of CS in the GAG preparation was determined by anion-exchange HPLC after digestion with chondroitinase.The molecular size of GAG was determined by gel filtration,and the interaction of GAG with growth factors was analyzed to determine the kinetic parameter ka(association rate constant)、 kd(dissociation rate constant) and KD(equilibrium dissociation constant) through surface plasmon resonance(SPR) analysis.The content of GAG was 1.09 μmol/mg PG(as disaccharide unites) or 651 μg/mg PG.CS is the major ingredient of the GAG preparation(1.03 μmol/mg PG).Unsaturated disaccharide units produced from CS were ΔDi-6S(38.8%),ΔDi-4S(46.3%),ΔDi-0S(8.4%) and ΔDi-diSD(6.5%).Molecular size of GAG(CS) was approximately 78 kD.The kinetic parameter ka((mol/L)-1·s-1),kd(s-1) and kD(nmol/L) of GAG(CS) with the growth factors were(2.77±0.17)×105,(7.74±1.56)×10-5 and(0.28±0.06) for midkine(MK);(1.05±0.22)×104,(4.16±0.80)×10-3 and(417±131.3) for pleiotrophin(PTN);and(7.04±0.94)×105,(7.84±2.82)×10-3 and(11.1±3.80) for hepatocyte growth factor(HGF),respectively.High affinity with MK and HGF and higher affinity with PTN was observed,suggesting that the CS chains in mackerel nasal cartilage may regulate growth factor signaling and have a potential for therapeutic applications to some diseases.