Oxford J T, Doege K J, Horton W E, Morris N P
Research Department, Shriners Hospital for Crippled Children, Portland, Oregon 97201.
Exp Cell Res. 1994 Jul;213(1):28-36. doi: 10.1006/excr.1994.1169.
The biosynthesis of type XI and type II collagens was examined using a stable rat chondrocyte cell line established by W. E. Horton et al. (1988, Exp. Cell Res. 178, 457-468.). These cells (IRC; immortalized rat chondrocytes) were created by transformation with a murine retrovirus carrying the v-myc and v-raf oncogenes. They grow in suspension culture as multicellular aggregates and synthesize typical cartilage proteins, aggrecan and link protein. Type II collagen is absent or synthesized at severely reduced levels, as shown by Northern analysis of mRNA. Thus, this cell type represents a unique model in which to study cartilage matrix protein interactions in the absence of type II collagen. A more detailed look at the proteins secreted into the medium by metabolically labeled IRC cells revealed the presence of collagenase-sensitive bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands were identified as the alpha 1, alpha 2, and alpha 3 chains of heterotrimeric type XI collagen by electrophoretic migration after pepsin digestion, by CNBr peptide mapping, and by immunoprecipitation with antibodies to rat alpha 1(XI). mRNA for all three chains was detected by Northern blot analysis. The data indicate that the low level of alpha 1(II) mRNA previously detected in these cells is translated into pro alpha 3(XI) polypeptide chains which are incorporated into molecules of type XI. Under normal culture conditions, homotrimers of type II collagen were not detected. The carboxyl propeptide domain of the fibrillar collagens directs chain selection and molecular assembly of the trimeric molecules. The sequence of the carboxyl propeptide domain from pro alpha 3(XI) of IRC cells was found to be identical to this domain from pro alpha 1(II) of swarm rat chondrosarcoma, supporting previous evidence that pro alpha 3(XI) and pro alpha 1(II) have the same primary structure. When cultured in the presence of 50 mM arginine, IRC cells could be induced to synthesize pro alpha 1(II) chains in excess over pro alpha 1(XI) and pro alpha 2(XI). Only under these conditions were type II collagen molecules detected, suggesting a preferential association of pro alpha 1(II) with the pro alpha 1 and/or pro alpha 2 chains of type XI collagen.
利用W. E. 霍顿等人（1988年，《实验细胞研究》178卷，457 - 468页）建立的稳定大鼠软骨细胞系，对XI型和II型胶原蛋白的生物合成进行了研究。这些细胞（IRC；永生化大鼠软骨细胞）是通过用携带v - myc和v - raf癌基因的鼠逆转录病毒转化而产生的。它们在悬浮培养中以多细胞聚集体的形式生长，并合成典型的软骨蛋白、聚集蛋白聚糖和连接蛋白。如mRNA的Northern分析所示，II型胶原蛋白不存在或合成水平严重降低。因此，这种细胞类型代表了一种独特的模型，可用于在缺乏II型胶原蛋白的情况下研究软骨基质蛋白相互作用。对经代谢标记的IRC细胞分泌到培养基中的蛋白质进行更详细的观察发现，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析时存在胶原酶敏感条带。经胃蛋白酶消化后的电泳迁移、CNBr肽图谱分析以及用抗大鼠α1(XI)抗体进行免疫沉淀，将这些条带鉴定为异源三聚体XI型胶原蛋白的α1、α2和α3链。通过Northern印迹分析检测到了所有三条链的mRNA。数据表明，先前在这些细胞中检测到的低水平α1(II) mRNA被翻译成了前α3(XI)多肽链，这些多肽链被整合到XI型分子中。在正常培养条件下，未检测到II型胶原蛋白的同三聚体。纤维状胶原蛋白的羧基前肽结构域指导三聚体分子的链选择和分子组装。发现IRC细胞的前α3(XI)的羧基前肽结构域序列与群体大鼠软骨肉瘤的前α1(II)的该结构域相同，支持了先前的证据，即前α3(XI)和前α1(II)具有相同的一级结构。当在50 mM精氨酸存在下培养时，IRC细胞可被诱导合成超过前α1(XI)和前α2(XI)的过量前α1(II)链。只有在这些条件下才检测到II型胶原蛋白分子，这表明前α1(II)与XI型胶原蛋白的前α1和/或前α2链优先结合。
