Division of Biomedical Sciences, Faculty of Dentistry, McGill University, 3640 University Street, Montreal, QC, H3A 2B2, Canada.
Amino Acids. 2014 Jun;46(6):1513-26. doi: 10.1007/s00726-014-1719-x. Epub 2014 Mar 19.
Microtubule components α- and β-tubulin undergo a number of posttranslational modifications that modulate their dynamics and cellular functions. These modifications include polyamination and covalent crosslinking by transglutaminase enzymes. We have demonstrated previously that the less dynamic and more stable tubulin form-detyrosinated Glu-tubulin-is found in high molecular weight, oligomeric complexes in bone-forming osteoblasts during differentiation and along with deposition of collagenous extracellular matrix. In this study, we report that oligomeric Glu-tubulin has high nocodazole tolerance, indicating further increased stability. We show that α-tubulin, which gives rise to Glu-tubulin, is a transglutaminase substrate in in vitro assays and that it is crosslinked into oligomers (dimers, trimers and tetramers) by transglutaminase 2 and Factor XIIIA; β-tubulin was not crosslinked by transglutaminase activity. The oligomeric Glu-tubulin was specifically localized to the plasma membrane of osteoblasts as analyzed by subcellular fractionation, cell surface biotinylation experiments and total internal reflection fluorescence (TIRF) microscopy. Glu- and α-tubulin co-localized with cellular Factor XIIIA as analyzed by conventional and TIRF microscopy. The Factor XIIIA-specific substrate peptide bF11 co-localized with α-tubulin and acted as a competitive inhibitor to oligomerization of Glu-tubulin, attenuating its formation in cells. This was associated with significantly decreased type I collagen deposition and decreased secretory activity as measured by synaptotagmin VII levels on the osteoblast plasma membrane. Our results suggest that Glu-tubulin may exist as covalently stabilized form which may be linked to the secretion and elaboration of collagenous extracellular matrix.
微管成分α-和β-微管经历了许多翻译后修饰，这些修饰调节它们的动力学和细胞功能。这些修饰包括多聚胺化和转谷氨酰胺酶的共价交联。我们之前已经证明，在分化过程中形成骨的成骨细胞以及胶原细胞外基质沉积过程中，发现较少动态和更稳定的微管形式——去酪氨酸化的 Glu-微管——存在于高分子量寡聚复合物中。在这项研究中，我们报告说寡聚 Glu-微管对诺考达唑具有高耐受性，这表明其稳定性进一步增加。我们表明，在体外测定中，产生 Glu-微管的α-微管是转谷氨酰胺酶的底物，并且它被转谷氨酰胺酶 2 和因子 XIIIA 交联成寡聚物（二聚体、三聚体和四聚体）；β-微管没有被转谷氨酰胺酶活性交联。通过亚细胞分级分离、细胞表面生物素化实验和全内反射荧光（TIRF）显微镜分析，发现寡聚 Glu-微管特异性定位于成骨细胞的质膜。通过常规和 TIRF 显微镜分析，发现 Glu-和α-微管与细胞因子 XIIIA 共定位。因子 XIIIA 特异性底物肽 bF11 与α-微管共定位，并作为 Glu-微管寡聚化的竞争性抑制剂，在细胞中减弱其形成。这与 I 型胶原沉积减少和突触结合蛋白 VII 水平测量的成骨细胞质膜上的分泌活性降低有关。我们的结果表明，Glu-微管可能以共价稳定的形式存在，这可能与胶原细胞外基质的分泌和修饰有关。
