Malemud C J, Goldberg V M, Moskowitz R W, Getzy L L, Papay R S, Norby D P
Biochem J. 1982 Aug 15;206(2):329-41. doi: 10.1042/bj2060329.
Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [(35)S]sulphate or [(14)C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [(35)S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [(35)S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60-80% of the [(35)S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (K(av.)) of the proteoglycan monomer on Sepharose CL-2B was 0.28-0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2-D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [(35)S]sulphate and [(14)C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60-70%), with 9-11% keratan sulphate in the monomer proteoglycan; (3) K(av.) values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH(4)-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.
在体外特定培养条件下，研究了手术关节置换时从骨关节炎股骨头获取的人骨软骨赘（骨赘）中蛋白聚糖的生物合成。骨赘主要存在于两个解剖位置，边缘和关节上。将切碎的组织切片在[(35)S]硫酸盐或[(14)C]葡萄糖胺存在下孵育。骨赘将两种标记的前体掺入蛋白聚糖中，随后通过CsCl等密度梯度超速离心和Sepharose CL - 2B柱色谱对其进行表征。用0.5m - 氯化胍提取的物质在离心后，A1组分中[(35)S]硫酸盐占78.1%。在缔合条件下，该A1组分中只有23.0%的[(35)S]硫酸盐在Sepharose CL - 2B的空体积中洗脱。组织4m - 氯化胍提取物中约60 - 80%的[(35)S]硫酸盐与单体蛋白聚糖（D组1分）相关。蛋白聚糖单体在Sepharose CL - 2B上的平均分配系数（K(av.)）为0.28 - 0.33。约12.4%的这种单体在体外与高分子量透明质酸形成稳定聚集体。对较低浮力密度组分（D2 - D4组）进行Sepharose CL - 2B柱色谱分析，结果表明其在Sepharose CL - 2B上的洗脱图谱与D1组显著不同，表明新合成的蛋白聚糖具有多分散性。对糖胺聚糖的组成和链大小分析显示如下：(1) [(35)S]硫酸盐和[(14)C]葡萄糖胺在DEAE - 纤维素上的0.5m - LiCl组分中优先洗脱；(2) 主要的硫酸化糖胺聚糖是硫酸软骨素6 - 硫酸盐（60 - 70%），单体蛋白聚糖中硫酸角质素占9 - 11%；(3) 木瓜蛋白酶消化和NaBH(4)处理的D1单体在Sephadex G - 200上的K(av.)值为0.38，在Sepharose CL - 6B上为0.48。合成的糖胺聚糖与内源性糖胺聚糖的比较表明类型相似。这些研究表明，人骨赘在体外合成硫酸化蛋白聚糖，其某些特征与成熟人关节软骨相似，特别是在蛋白聚糖单体大小和硫酸软骨素6 - 硫酸盐占优势方面。它们与关节软骨的主要区别在于缺乏大量硫酸角质素以及与透明质酸单体相互作用相关的聚集特性。
