Murata Y, Kuroki Y, Akino T
Department of Biochemistry, Sapporo Medical College, Japan.
Biochem J. 1993 Apr 1;291 ( Pt 1)(Pt 1):71-6. doi: 10.1042/bj2910071.
Surfactant protein A (SP-A), with a reduced denatured molecular mass of 26-38 kDa, is characterized by a collagen-like sequence in the N-terminal half of the protein. This protein forms an oligomeric structure which is dependent upon this collagenous domain. SP-A has been demonstrated to function as an inhibitor of phospholipid secretion by primary cultures of alveolar type II cells via a cell surface receptor for the protein. However, the receptor-binding domain of SP-A has not been identified. The purpose of the present study was to investigate the role of the C-terminal domain of SP-A in binding to type II cells and regulation of phospholipid secretion. A monoclonal antibody to human SP-A, whose epitope was localized at the C-terminal domain of the protein, abolished the inhibitory activity of human SP-A on lipid secretion by type II cells, and attenuated the ability of human SP-A to compete with 125I-(rat SP-A) for receptor binding. SP-A was then digested with collagenase and the collagenase-resistant fragment (CRF), which is the C-terminal domain of SP-A (thus lacking the N-terminal domain), was isolated. Gel filtration chromatography revealed that CRF exists as a monomer in solution containing Ca2+. CRF had the ability to inhibit phospholipid secretion, although at a higher concentration than for SP-A, and was also able to compete with 125I-(rat SP-A) for binding to type II cells. A direct binding study showed that CRF bound to type II cells in a concentration-dependent manner. The present study demonstrates that the non-collagenous, C-terminal, domain of SP-A is responsible for the protein's inhibitory effect on lipid secretion and its binding to type II cells.
表面活性蛋白A（SP-A），其变性分子量降低至26 - 38 kDa，其特征在于该蛋白N端的一半具有类似胶原蛋白的序列。这种蛋白形成一种寡聚结构，该结构依赖于这个胶原结构域。已经证明SP-A通过该蛋白的细胞表面受体作为肺泡II型细胞原代培养物中磷脂分泌的抑制剂。然而，SP-A的受体结合结构域尚未确定。本研究的目的是研究SP-A C端结构域在与II型细胞结合及调节磷脂分泌中的作用。一种针对人SP-A的单克隆抗体，其表位定位于该蛋白的C端结构域，消除了人SP-A对II型细胞脂质分泌的抑制活性，并减弱了人SP-A与125I-（大鼠SP-A）竞争受体结合的能力。然后用胶原酶消化SP-A，并分离出抗胶原酶片段（CRF），它是SP-A的C端结构域（因此缺少N端结构域）。凝胶过滤色谱显示CRF在含有Ca2+的溶液中以单体形式存在。CRF具有抑制磷脂分泌的能力，尽管其浓度高于SP-A，并且也能够与125I-（大鼠SP-A）竞争与II型细胞的结合。直接结合研究表明CRF以浓度依赖的方式与II型细胞结合。本研究表明，SP-A的非胶原C端结构域负责该蛋白对脂质分泌的抑制作用及其与II型细胞的结合。
