Milev P, Maurel P, Chiba A, Mevissen M, Popp S, Yamaguchi Y, Margolis R K, Margolis R U
Department of Pharmacology, New York University Medical Center, New York, New York 10016, USA.
Biochem Biophys Res Commun. 1998 Jun 18;247(2):207-12. doi: 10.1006/bbrc.1998.8759.
We have used a slot-blot radioimmunoassay to quantitate the levels of hyaluronan-binding chondroitin sulfate proteoglycans in developing rat brain from embryonic day 14 (E 14) to eight months postnatal. Recombinant nonhomologous regions of the core proteins were used for immunization to obtain polyclonal antibodies specific for aggrecan, the alpha and beta domains of versican mRNA splice variants, and N- and C-terminal portions of neurocan, while brevican was quantitated using a specific monoclonal antibody. The concentration of aggrecan increased steadily during brain development up to 5 months of age, when it reached a level that was 18-fold higher than at E14. Alternatively spliced versican isoforms containing the alpha domain of the glycosaminoglycan attachment region were present at a relatively low level during the late embryonic and early postnatal period, decreased by approximately 50% between 1 and 2 weeks postnatal, and then increased steadily in concentration to reach a maximum at 100 days that was 7-fold that present at 10 days postnatal. In contrast to these results, versican isoforms containing the beta domain more than doubled in concentration between E14 and birth, after which they decreased by greater than 90% to reach a low "mature" level that remained unchanged between 2 and 8 months. The N- and C-terminal portions of neurocan (produced by a developmentally-regulated proteolytic cleavage in the middle of its chondroitin sulfate attachment region) both increased in embryonic brain during development, reached a peak in the early postnatal period, and then declined thereafter. As in the case of aggrecan, only traces of brevican were detected in embryonic brain and its concentration increased steadily after birth to reach an adult level that was approximately 14-fold higher than that present in neonatal brain. These striking and distinctive changes in the concentrations of the different members of this family of structurally related proteoglycans in developing brain, including changes in opposite directions for versican mRNA splice variants, indicate that the individual proteoglycans and their isoforms probably serve unique functions during nervous tissue histogenesis.
我们采用斑点印迹放射免疫分析法，对胚胎期第14天（E14）至出生后8个月的发育中大鼠脑内透明质酸结合硫酸软骨素蛋白聚糖的水平进行定量分析。利用核心蛋白的重组非同源区域进行免疫，以获得对聚集蛋白聚糖、多功能蛋白聚糖mRNA剪接变体的α和β结构域、神经蛋白聚糖的N端和C端部分具有特异性的多克隆抗体，而短蛋白聚糖则使用特异性单克隆抗体进行定量分析。聚集蛋白聚糖的浓度在脑发育至5月龄时稳步增加，此时达到的水平比E14时高18倍。含有糖胺聚糖附着区域α结构域的可变剪接多功能蛋白聚糖异构体在胚胎后期和出生后早期水平相对较低，在出生后1至2周间浓度下降约50%，然后浓度稳步增加，在出生后100天时达到最高值，是出生后10天时的7倍。与这些结果相反，含有β结构域的多功能蛋白聚糖异构体在E14至出生间浓度增加了一倍多，此后下降超过90%，达到低水平的“成熟”状态，在2至8个月间保持不变。神经蛋白聚糖的N端和C端部分（由其硫酸软骨素附着区域中部发育调控的蛋白水解切割产生）在胚胎脑发育过程中均增加，在出生后早期达到峰值，然后随后下降。与聚集蛋白聚糖的情况一样，在胚胎脑中仅检测到痕量的短蛋白聚糖，其浓度在出生后稳步增加，达到成年水平，比新生脑内的水平高约14倍。发育中脑内这种结构相关蛋白聚糖家族不同成员浓度的显著且独特变化，包括多功能蛋白聚糖mRNA剪接变体的相反方向变化，表明各个蛋白聚糖及其异构体在神经组织组织发生过程中可能发挥独特功能。
