Wenbing Yang1, Haitao Shen1, Guodong Fang1, Hui Li1, Lan Li1, Fang Deng1, Wei Gu2, Kangsheng Li3, Lian Ma4, Jiang Gu1, Yongyu Wang5. 1. Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China. 2. Department of Pathophysiology, The Key Immunopathology Laboratory of Guangdong Province, Shantou University Medical College, Shantou, Guangdong Province, China. 3. Department of Microbiology and Immunology, Shantou University Medical College, Shantou, Guangdong Province, China. 4. Department of Pediatrics, Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, China. 5. Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China. Electronic address: yywangut@163.com.
Abstract
AIMS: 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. MAIN METHODS: 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. KEY FINDINGS: 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. SIGNIFICANCE: 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.
AIMS: 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. MAIN METHODS: We generated ratSP-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. KEY FINDINGS: 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. SIGNIFICANCE: 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.