title: Mutations of rat surfactant protein A have distinct effects on its glycosylation, secretion, aggregation and degradation.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Haitao
authors.initials: H
authors.lastname: Shen
authors.affiliation: Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Guodong
authors.initials: G
authors.lastname: Fang
authors.affiliation: Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Deng
authors.affiliation: Department of Pathophysiology, The Key Immunopathology Laboratory of Guangdong Province, Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gu
authors.affiliation: Department of Microbiology and Immunology, Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Kangsheng
authors.initials: K
authors.lastname: Li
authors.affiliation: Department of Pediatrics, Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Lian
authors.initials: L
authors.lastname: Ma
authors.affiliation: Department of Pathology, Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, Guangdong Province, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Gu
authors.affiliation: 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.
authors.forename: Yongyu
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense / Q000235:genetics*; D010641:Phenotype; D066329:Protein Aggregates / Q000235:genetics; D011487:Protein Conformation; D059748:Proteolysis; D037662:Pulmonary Surfactant-Associated Protein A / Q000235:genetics* / Q000378:metabolism*; D051381:Rats
chemical_list: D017931:DNA Primers; D066329:Protein Aggregates; D037662:Pulmonary Surfactant-Associated Protein A
keywords: Aggregation; Degradation; Familial pulmonary fibrosis; Glycosylation; Protein secretion; Surfactant protein A