Surfactant Protein SP-A, B, and C: Purification and Biochemical Characterization

Bae, Chong Woo; Choi, Yong Mook; Kang, Joo Hyun; Kim, Kil Lyong; Hahm, Kyung Soo

Abstract

PURPOSE
Several kinds of exogenous pulmonary surfactants (SF), either synthetic or animal- derived, are being used for the replacement therapy in respiratory distress syndrome (RDS) of newborn, especially in premature infants, and improved the neonatal mortality and morbidity. Because synthetic preparations are lack of surfactant protein (SP) and animal-derived preparations cause immunogenecity of heterogenous SP, there have been great necessity for the development of next generation of exogenous SF which made by new technology to produce new type of human SF (contained human synthetic SP). There are two methods to make next generation of SF (mixtures of phospholipids and human synthetic SP) which are using of recombinant SP or synthetic peptides of SP. For the synthesis of SP peptides and production of next generation of SF, at first step, we have isolated SP-A, B, and C from bovine lung SF, and studied the biochemical properties of these proteins.
METHODS
Crude natural surfactant (CNS) and purified natural surfactant (PNS) were isolated from materials which extracted from the bovine lung lavage. The hydrophilic SP-A was purified from PNS by method of modified Hawgood, and hydrophobic SP-B, C were purified by Sephadex LH 60 column chromatography. The purities of the purified SP-A and SP-B, C were assessed by 12% SDS-polyacrylamide gel and tricine buffer SDS-polyacrylamide gel, respectively and the N-terminal amino acid sequences of these proteins were determined using Beckman PI-2090. The polyclonal anti-serum against SP-A was prepared by immunization of the purified SP-A into the mouse and the immunization of the purified SP-A into the mouse and the immunogenecity of SP-A was confirmed by indirect ELISA.
RESULTS
Total 22 gm of CNS, 11 gm of PNS, and 2.5 mg of SP-B and 3.2 mg of SP-C/ 1 gm of CNS, were purified from one bovine both lungs. The molecular weights of SP-A, B, C shown in SDS-polyacrylamide gel were as follows; 28,000-35,000 Da (molecular weight) of SP-A, 15,000-18,000 Da of SP-B, 3,500-5,000 Da of SP-C. The partial N-terminal amino acid sequences of each SPs were; Leu-Glu-His-Asp-Val-Lys- Glu-Val-.... in SP-A, Phe-Pro-Ile-Pro-Ile-Pro-Tyr-.... in SP-B, Leu-Ile-Pro-.... in SP-C, respectively. These results indicated that the amino acid sequences of bovine SPs were different from those of other species, i.e., human, dog and rat, which were reported previously by another investigators and species-specific patterns were shown. The immunogenecity of the purified SP-A was confirmed by the production of polyclonal antibody against mouse. The polyclonal antibody of SP-A could be used for measuring the amount of pulmonary SF in lung lavages. Carbohydrate portion of SP-A was cleaved with N-glycocisidase F. This result suggested that carbohydrate group could be N-glycosylated in some arginine residue of SP-A.
CONCLUSIONS
The SP-A, B, C were purified from bovine lung SF, and N-terminal amino acid sequences of each SP-A, B, C were determined. Further studies were needed for the development and use of next generations of exogenous SF preparation, which based on synthetic SP-peptides, for the treatment of neonatal RDS in the future.