Characterization of opticin and evidence of stable dimerization in solution
Characterization of opticin and evidence of stable dimerization in solution
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DOI：
10.1074/jbc.m303117200
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发表时间：
2003-11-14
影响因子：
4.8
通讯作者：
Bishop, PN
中科院分区：
文献类型：
--
作者：
Le Goff, MM;Hindson, VJ;Bishop, PN
视神经肽是细胞外基质富含亮氨酸的小重复蛋白（SLRP）家族的III类成员，最初在眼睛中与玻璃体液的胶原纤维相关。重组和组织提取形式的牛视光蛋白进行生物化学和生物物理特性。SDS-PAGE后，两种形式产生的主要组分是45-52 kDa之间的宽带。有证据表明两阶段加工，此外，蛋白水解裂解产物类似于25 kDa。圆二色谱的去卷积显示β-折叠（41%），β-转角（21%）和α-螺旋（10%），热变性实验显示中点为47 ℃的转变。用光散射和分析超离心法测定的重均分子量表明，opticin在溶液中以类似于90 kDa的稳定二聚体形式存在，其可以通过用2.5 M盐酸胍变性或在SDS-聚丙烯酰胺电泳过程中解离成单体。Opticin仍然是一个二聚体去除后的氨基末端区域的O-唾液酸糖蛋白内肽酶消化，表明二聚体的形成是由富含亮氨酸的重复介导的。二聚化可能具有许多功能性后果，包括二价配体相互作用。
Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein (SLRP) family that was initially identified in the eye in association with the collagen fibrils of the vitreous humor. Recombinant and tissue-extracted forms of bovine opticin were subjected to biochemical and biophysical characterization. Following SDS-PAGE the predominant component produced by both forms was a broad band between 45-52 kDa. There was evidence for two-stage processing and, additionally, a proteolytic cleavage product of similar to25 kDa. Deconvolution of circular dichroism spectra revealed beta-sheet (41%), beta-turn (21%), and alpha-helix (10%), and thermal denaturation experiments showed a transition with a midpoint of 47degreesC. Weight-averaged molecular mass measurements using both light scattering and analytical ultracentrifugation demonstrated that opticin exists in solution as a stable dimer of similar to90 kDa, which can be dissociated into a monomer by denaturation with 2.5 M guanidine hydrochloride or during SDS-polyacrylamide electrophoresis. Opticin remains a dimer after removal of the amino-terminal region by O-sialoglycoprotein endopeptidase digestion, suggesting that dimer formation is mediated by the leucine-rich repeats. Dimerization could have a number of functional consequences, including divalent ligand interactions.