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Proteolysis of recombinant fragment of human surfactant protein D

Presented at the Neonatal Society 2007 Summer Meeting (programme).

Duvoix A1, Mackay R-M2, Henderson N2, Postle A2, Clark H1,2

1 MRC Immunochemistry Unit, Biochemistry Dept, University of Oxford, Oxford, UK
2 School of Medicine, University of Southampton, Southampton, UK

Background: Surfactant Protein D (SP-D) acts in the first line immune defence of the lungs but is absent in premature infants, 40% of whom develop neonatal chronic lung disease (CLD). Moreover, inducing SP-D expression with Dexamethasone improves pulmonary status of those infants and low levels of SP-D have been linked to CLD (1). This implies that an early recombinant SP-D therapy could attenuate the inflammatory processes in CLD. Our team has created a recombinant fragment of the human (rfh) SP-D that includes the hydrophobic neck and the CRD and seems to reverse the symptoms of the SP-D knock-out mice (2).

Aim: In this paper we study the in vitro effect of different proteolytic enzymes usually found in inflamed lungs, such as Neutrophil elastase, Cathepsin G and Protease 3 as well as Pseudomonas elastase (Pseudomonas aeruginosa is a common infectious agent in lungs), on rfhSP-D.

Methods: We performed SDS-PAGE gels followed by staining with Coomassie blue or Western blots with anti-human SP-D antibody. We also performed ELISA using the same antibody.

Results: We show that all 4 enzymes used cleave rfhSP-D at 37ºC and that all the cleavages are inhibited by physiological concentration of calcium. We also determine that anti-SP-D ELISA does not generate a good picture of the cleavages obtained previously and thus cannot be used as a good “diagnostic” tool.

Conclusion: rfhSP-D is sensitive to cleavage by enzymes likely to be present in inflamed and infected lungs, although physiological concentrations of calcium inhibit those cleavages.

Undertaken in accordance with UK ethical guidelines.

1. Beresford MW, Shaw NJ. Pediatr Res. 2003 Apr;53(4):663-70.
2. Clark H, Palaniyar N, Hawgood S, Reid KB. Ann N Y Acad Sci. 2003 Dec;1010:113-6.

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