Thom J R, Morris N P
Research Unit, Shriners Hospital for Crippled Children, Portland, Oregon.
J Biol Chem. 1991 Apr 15;266(11):7262-9.
The biosynthesis and proteolytic processing of type XI procollagen was examined using pulse-chase labelling of 17-day embryonic chick sterna in organ culture with [3H]proline. Products of biosynthesis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with and without prior reduction of disulfide bonds. Pro-alpha chains, intermediates, and matrix forms were identified by cyanogen bromide or Staphylococcus aureus V8 protease digestion. The results show that type XI pro-alpha chains assemble into trimeric molecules with interchain disulfide bonds. Proteolytic processing begins at least 40 min after the start of labeling which is later than that of type II procollagen (25 min). This first processing step involves the loss of the domain containing the interchain disulfide bonds which most likely is the carboxyl propeptide. In the case of the pro-alpha 3 chain, this generates the matrix form, m alpha 3, which retains its amino propeptide. For the pro-alpha 1 and pro-alpha 2 chains, this step generates intermediate forms, p alpha 1 and p alpha 2, which undergo a second proteolytic conversion to m alpha 1 and m alpha 2, and yet retain a pepsin-labile domain. The conversion of p alpha 2 to m alpha 2 is largely complete 2 h after labeling. p alpha 1 is converted to m alpha 1 very slowly and is 50% complete after 18 h of chase in organ culture. The apparent proteolytic processing within the amino propeptide, and the differential rate of processing between two chains in the same molecule are unusual and distinguish type XI from collagen types I, II, and III. It is possible that the extremely slow processing of p alpha 1 affects the formation of the heterotypic cartilage collagen fibrils and may be related to the function of type XI collagen.
利用[3H]脯氨酸对器官培养中的17日龄鸡胚胸骨进行脉冲追踪标记，研究了XI型前胶原的生物合成和蛋白水解加工过程。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析生物合成产物，电泳前对二硫键进行还原或不还原。通过溴化氰或金黄色葡萄球菌V8蛋白酶消化鉴定前α链、中间体和基质形式。结果表明，XI型前α链组装成具有链间二硫键的三聚体分子。蛋白水解加工在标记开始后至少40分钟开始，这比II型前胶原（25分钟）要晚。这第一步加工涉及含有链间二硫键的结构域的丢失，该结构域很可能是羧基前肽。就前α3链而言，这产生了基质形式mα3，它保留了其氨基前肽。对于前α1链和前α2链，这一步产生中间体形式pα1和pα2，它们经历第二次蛋白水解转化为mα1和mα2，但仍保留一个对胃蛋白酶敏感的结构域。pα2向mα2的转化在标记后2小时基本完成。pα1向mα1的转化非常缓慢，在器官培养中追踪18小时后完成50%。氨基前肽内明显的蛋白水解加工以及同一分子中两条链之间不同的加工速率是不寻常的，这使XI型与I、II和III型胶原区分开来。pα1的极慢加工可能影响异型软骨胶原纤维的形成，并且可能与XI型胶原的功能有关。
