Yang Wenbing, Shen Haitao, Fang Guodong, Li Hui, Li Lan, Deng Fang, Gu Wei, Li Kangsheng, Ma Lian, Gu Jiang, Wang Yongyu
Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China.
Department of Pathophysiology, The Key Immunopathology Laboratory of Guangdong Province, Shantou University Medical College, Shantou, Guangdong Province, China.
Life Sci. 2014 Nov 11;117(2):47-55. doi: 10.1016/j.lfs.2014.09.006. Epub 2014 Sep 19.
Surfactant protein A (SP-A) plays critical roles in the innate immune system and surfactant homeostasis of the lung. Mutations in SP-A2 of the carbohydrate recognition domain (CRD) impair its glycosylation and are associated with pulmonary fibrosis in humans. We aim to examine how mutations in SP-A that impair its glycosylation affect its biological properties and lead to disease.
We generated rat SP-A constructs with two types of mutations that impair its glycosylation: N-glycosylation site mutations (N21T, N207S and N21T/N207S) and disease-associated CRD mutations (G231V, F198S). We transfected these constructs into Chinese hamster ovary (CHO)-K1 cells and assessed biochemical differences in cellular and secreted wild-type and mutant SP-As by western blot, immunofluorescence, and sensitivity to enzymatic digestion.
Mutations of the CRD completely impaired SP-A secretion, whereas mutations of N-glycosylation sites had little effect. Both types of mutations formed nonidet p-40 (NP-40) insoluble aggregates, but the aggregates only from CRD mutations could be partially rescued by a chemical chaperone, 4-phenylbutyrate acid (4-PBA). The majority of CRD mutant SP-A was retained in the endoplasmic reticulum. Moreover, both types of mutations reduced SP-A stability, with CRD mutant SP-A being more sensitive to chymotrypsin digestion. Both types of soluble mutant SP-A could be degraded by the proteasome pathway, while insoluble aggregates could be additionally degraded by the lysosomal pathway.
Our data provide evidence that the differential glycosylation of SP-A may play distinct roles in SP-A secretion, aggregation and degradation which may contribute to familial pulmonary fibrosis caused by SP-A2 mutations.
表面活性蛋白A（SP-A）在肺部固有免疫系统和表面活性物质稳态中发挥关键作用。碳水化合物识别结构域（CRD）的SP-A2突变会损害其糖基化，并与人类肺纤维化相关。我们旨在研究损害SP-A糖基化的突变如何影响其生物学特性并导致疾病。
我们构建了具有两种损害其糖基化的突变类型的大鼠SP-A构建体：N-糖基化位点突变（N21T、N207S和N21T/N207S）以及与疾病相关的CRD突变（G231V、F198S）。我们将这些构建体转染到中国仓鼠卵巢（CHO）-K1细胞中，并通过蛋白质印迹、免疫荧光以及对酶消化的敏感性来评估细胞内和分泌的野生型及突变型SP-A的生化差异。
CRD突变完全损害了SP-A的分泌，而N-糖基化位点突变影响较小。两种类型的突变均形成了不溶于非离子去污剂P-40（NP-40）的聚集体，但只有CRD突变形成的聚集体可被化学伴侣4-苯基丁酸（4-PBA）部分挽救。大多数CRD突变型SP-A保留在内质网中。此外，两种类型的突变均降低了SP-A的稳定性，其中CRD突变型SP-A对胰凝乳蛋白酶消化更敏感。两种类型的可溶性突变型SP-A均可通过蛋白酶体途径降解，而不溶性聚集体还可通过溶酶体途径降解。
我们的数据表明，SP-A的差异糖基化可能在SP-A的分泌、聚集和降解中发挥不同作用，这可能导致由SP-A2突变引起的家族性肺纤维化。
