West Leigh, Govindraj Prasanthi, Koob Thomas J, Hassell John R
Center for Research in Skeletal Development and Pediatric Orthopaedics, Shriners Hospitals for Children-Tampa, 12502 Pine Drive, Tampa, Florida 33612, USA.
J Orthop Res. 2006 Jun;24(6):1317-26. doi: 10.1002/jor.20160.
Perlecan is a heparan sulfate proteoglycan present in the growth plate and essential for endochondral ossification. We evaluated the synthesis and structure of perlecan in the different zones of the growth plate. The growth plates from fetal bovine ribs were isolated and sequentially sliced into 1-mm sections containing the hypertrophic zone, lower proliferative zone, upper proliferative zone, intermediate zone, and resting zone, respectively. The slices were then either incubated in culture medium with 35SO4 to measure total sulfated proteoglycan synthesis and perlecan synthesis, extracted for perlecan core protein analysis by Western blot, or extracted for perlecan isolation and subsequent characterization of glycosaminoglycan size and disaccharide composition. 35SO4 incorporation into perlecan was three-fourfold higher in the proliferating/hypertrophic zone than the resting zone. Western blot showed perlecan content was greatest in the lower and upper proliferating zones and that a perlecan fragment lacking portions of the N- and C-terminal domains containing heparan sulfate was also present in all zones. Purified perlecan from the hypertrophic/lower proliferative zone had larger chondroitin sulfate chains and a different composition of CS and HS disaccharides than the perlecan isolated from the resting zone. These results indicate perlecan deposition is increased and is turned over during proliferation to be replaced by a perlecan with a different sulfation pattern.
基底膜聚糖是一种硫酸乙酰肝素蛋白聚糖，存在于生长板中，对软骨内骨化至关重要。我们评估了生长板不同区域中基底膜聚糖的合成和结构。分离出胎牛肋骨的生长板，并依次切成1毫米厚的切片，分别包含肥大区、下增殖区、上增殖区、中间区和静止区。然后将切片在含有35SO4的培养基中孵育，以测量总硫酸化蛋白聚糖的合成和基底膜聚糖的合成，提取用于通过蛋白质印迹法分析基底膜聚糖核心蛋白，或提取用于基底膜聚糖的分离以及随后对糖胺聚糖大小和二糖组成的表征。与静止区相比，增殖/肥大区中35SO4掺入基底膜聚糖的量高出三到四倍。蛋白质印迹显示，基底膜聚糖含量在下增殖区和上增殖区最高，并且在所有区域中还存在一种缺乏包含硫酸乙酰肝素的N端和C端结构域部分的基底膜聚糖片段。从肥大/下增殖区纯化的基底膜聚糖具有更大的硫酸软骨素链，并且与从静止区分离的基底膜聚糖相比，其硫酸软骨素和硫酸乙酰肝素二糖的组成不同。这些结果表明，基底膜聚糖的沉积增加，并且在增殖过程中发生周转，被具有不同硫酸化模式的基底膜聚糖所取代。
