McCormack F X, Stewart J, Voelker D R, Damodarasamy M
Division of Pulmonary/Critical Care Medicine, University of Cincinnati College of Medicine, Ohio, USA.
Biochemistry. 1997 Nov 11;36(45):13963-71. doi: 10.1021/bi970745q.
The carbohydrate recognition domain (CRD) of surfactant protein A (SP-A) is critical for the modulation of surfactant secretion from isolated type II cells and for the Ca(2+)-dependent aggregation of surfactant liposomes, but the domains of SP-A that mediate lipid binding have not been precisely mapped. To determine the role of the CRD in lipid interactions and other functions, the conserved amino acids of the putative Ca2+ and carbohydrate binding site (Glu195, Glu202, Asn214, Asp215) were substituted with alanine. The wild-type recombinant protein, SP-Ahyp, and mutant SP-As SP-Ahyp,E195A, SP-Ahyp,E202A, SP-Ahyp,N214A, and SP-Ahyp,D215A, were expressed in insect cells using baculovirus vectors and compared functionally. The Ca(2+)-dependent binding and aggregation of liposomes at pH 7.0 by SP-Ahyp,N214A were comparable to SP-Ahyp, but these activities were blocked in SP-Ahyp,E195A, SP-Ahyp,E202A, and SP-Ahyp,D215A. In contrast, the SP-Ahyp,D215A but not the other mutant proteins induced the Ca(2+)-independent aggregation of phospholipid liposomes at pH 4.0. The mutant recombinant proteins did not compete with 125I-labeled rat SP-A for high-affinity receptor occupancy on isolated type II cells and were much less potent than SP-Ahyp as regulators of surfactant secretion and uptake from type II cells. We conclude that (1) lipid binding and pH-dependent liposome aggregation are mediated by the CRD of SP-A, (2) distinct but overlapping domains within the CRD are required for pH- and Ca(2+)-dependent liposome aggregation, and (3) conserved acidic and polar residues of the carbohydrate binding site of SP-A are essential for interactions with type II cells.
表面活性蛋白A（SP-A）的碳水化合物识别结构域（CRD）对于调节分离的II型细胞的表面活性剂分泌以及表面活性剂脂质体的钙（Ca2+）依赖性聚集至关重要，但介导脂质结合的SP-A结构域尚未精确确定。为了确定CRD在脂质相互作用和其他功能中的作用，将假定的Ca2+和碳水化合物结合位点（Glu195、Glu202、Asn214、Asp215）的保守氨基酸替换为丙氨酸。使用杆状病毒载体在昆虫细胞中表达野生型重组蛋白SP-Ahyp以及突变体SP-A，即SP-Ahyp,E195A、SP-Ahyp,E202A、SP-Ahyp,N214A和SP-Ahyp,D215A，并进行功能比较。SP-Ahyp,N214A在pH 7.0时对脂质体的Ca2+依赖性结合和聚集与SP-Ahyp相当，但这些活性在SP-Ahyp,E195A、SP-Ahyp,E202A和SP-Ahyp,D215A中被阻断。相反，SP-Ahyp,D215A而非其他突变蛋白在pH 4.0时诱导磷脂脂质体的Ca2+非依赖性聚集。突变重组蛋白在分离的II型细胞上不能与125I标记的大鼠SP-A竞争高亲和力受体占据，并且作为II型细胞表面活性剂分泌和摄取的调节剂，其效力远低于SP-Ahyp。我们得出结论：（1）脂质结合和pH依赖性脂质体聚集由SP-A的CRD介导；（2）CRD内不同但重叠的结构域是pH和Ca2+依赖性脂质体聚集所必需的；（3）SP-A碳水化合物结合位点的保守酸性和极性残基对于与II型细胞的相互作用至关重要。
