Yoshikawa K, Takahashi S, Imamura Y, Sado Y, Hayashi T
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153-8902, Japan.
J Biochem. 2001 Jun;129(6):929-36. doi: 10.1093/oxfordjournals.jbchem.a002939.
Our previous report showed that human fetal lung fibroblasts secreted non-disulfide-bonded, non-helical collagenous polypeptides of alpha1(IV) and alpha2(IV) chains depending on culture conditions [Connective Tissue (1999) 31, 161-168]. The secretion of non-helical collagenous polypeptides is unexpected from the current consensus that such polypeptides are not secreted under physiological conditions. The absence of interchain disulfide bonds among alpha1(IV) and alpha2(IV) chains was always correlated with the absence of triple-helical structure of the type IV collagen. The finding corresponds with the fact that the interchain disulfide bonds are formed at or close to the completion of the type IV collagen triple-helix formation. The present report shows that ascorbate is the primary factor for the triple-helix formation of the type IV collagen. When human mesangial cells were cultured with ascorbate, only the triple-helical type IV collagen was secreted. However, when the cells were cultured without ascorbate, the non-helical alpha1(IV) and alpha2(IV) chains were secreted. Relative amounts of the secreted products were unchanged with or without ascorbate, suggesting that ascorbate is required for the step of the triple-helix formation. The ascorbate-dependency of the triple-helix formation of the type IV collagen was observed in all the human cells examined. The non-helical alpha1(IV) chain produced by the ascorbate-free culture contained about 80% less hydroxyproline than the alpha1(IV) chain from the triple-helical type IV collagen. The evidence for the non-association of the non-helical alpha1(IV) and alpha2(IV) chains in the conditioned medium was obtained by an anti-alpha1(IV) antibody-coupled affinity column chromatography for the conditioned medium. Although all the non-helical alpha1(IV) chains were found in the bound fraction, all the non-helical alpha2(IV) chains were recovered in the flow-through fraction. The present findings suggest that ascorbate plays a key role in the trimerization step of three alpha chains and/or in the subsequent triple-helix formation of the type IV collagen.
我们之前的报告显示，根据培养条件，人胎儿肺成纤维细胞会分泌α1(IV)和α2(IV)链的非二硫键结合、非螺旋状胶原多肽[《结缔组织》(1999年)第31卷，第161 - 168页]。从目前关于此类多肽在生理条件下不分泌的共识来看，非螺旋状胶原多肽的分泌是出乎意料的。α1(IV)和α2(IV)链之间不存在链间二硫键总是与IV型胶原三螺旋结构的缺失相关。这一发现与链间二硫键在IV型胶原三螺旋形成接近完成时形成这一事实相符。本报告表明，抗坏血酸是IV型胶原三螺旋形成的主要因素。当人系膜细胞用抗坏血酸培养时，仅分泌三螺旋状的IV型胶原。然而，当细胞在无抗坏血酸条件下培养时，则分泌非螺旋状的α1(IV)和α2(IV)链。无论有无抗坏血酸，分泌产物的相对量均无变化，这表明抗坏血酸是三螺旋形成步骤所必需的。在所有检测的人细胞中均观察到IV型胶原三螺旋形成对抗坏血酸的依赖性。无抗坏血酸培养产生的非螺旋状α1(IV)链所含羟脯氨酸比三螺旋状IV型胶原中的α1(IV)链少约80%。通过对条件培养基进行抗α1(IV)抗体偶联亲和柱层析，获得了条件培养基中非螺旋状α1(IV)和α2(IV)链不缔合的证据。虽然所有非螺旋状α1(IV)链都存在于结合组分中，但所有非螺旋状α2(IV)链都在流穿组分中回收。目前的研究结果表明，抗坏血酸在三条α链的三聚化步骤和/或随后的IV型胶原三螺旋形成中起关键作用。
