The carbohydrate recognition domain of surfactant protein A mediates binding to the major surface glycoprotein of Pneumocystis carinii.
Pneumocystis carinii is a common cause of life-threatening pneumonia in immunodeficient patients. Pulmonary surfactant protein A (SP-A), an alveolar glycoprotein containing collagen-like and carbohydrate recognition domains (CRD), binds P. carinii and enhances adherence to alveolar macrophages. In this study, we examined the structural basis of the interaction between SP-A and the major surface glycoprotein of P. carinii (MSG). Rat SP-A bound to purified rat P. carinii-derived MSG in a saturable and calcium-dependent manner, which was partially reversible by coincubation with excess monosaccharides, or pretreatment of MSG with N-glycanase. Mutant recombinant SP-As with neutral amino acid substitutions for the predicted calcium- and carbohydrate-coordinating residues of the CRD were synthesized in insect cells using baculovirus vectors and tested for binding to MSG. Substitutions of negatively charged (Glu195, Glu202, and Asp215) and polar residues (Asn214) of the CRD with alanine but not substitution of the Arg197 with glycine reduced the binding of SP-A to mannose-Sepharose beads and to MSG. Deletion of the N-linked oligosaccharides from SP-A by mutagenesis of the consensus sequences for glycosylation had no effect on binding. We conclude that the CRD mediates the binding of SP-A to oligosaccharides attached to MSG.
McCormack
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Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA. frank.mccormack@uc.edu
Festa
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Andrews
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Linke
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Walzer
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D000818:Animals; D001665:Binding Sites; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D011010:Pneumocystis / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000302:isolation & purification / Q000378:metabolism*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism
D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins; C077556:GPA protein, Pneumocystis carinii sp.