Epitope mapping for monoclonal antibodies identifies functional domains of pulmonary surfactant protein A that interact with lipids.

Y Kuroki,F X Mccormack,Y Ogasawara,R J Mason,D R Voelker

doi:10.1016/s0021-9258(18)43951-8

Abstract

Pulmonary surfactant protein A (SP-A) contains 4 domains: a disulfide forming amino terminus, a collagen-like region, a neck region, and a carbohydrate recognition region. The protein binds the lipids dipalmitoylphosphatidylcholine and galactosylceramide and induces aggregation of phospholipid vesicles. SP-A also inhibits lipid secretion and enhances the uptake of phospholipid by alveolar type II cells. Previously described monoclonal antibody 1D6 blocks the inhibitory effect of SP-A on lipid secretion by type II cells, but antibody 6E3 has no effect. In the present study we mapped the epitopes for monoclonal antibodies 1D6 and 6E3 by enzyme-linked immunoassay of recombinant proteins expressed using the baculovirus system, and investigated the domain that is responsible for the SP-A interactions with lipid. Monoclonal antibody 1D6 bound to mutant SP-As in which the neck portion of the molecule was deleted or substituted with that of mannose-binding protein A, but 6E3 failed to bind to these mutants. In contrast, 1D6 did not bind to a chimera in which the carbohydrate recognition domain (CRD) was substituted with that of surfactant protein D (SP-D). In addition, 1D6 failed to recognize antigen in cells infected with the recombinant virus directing the synthesis of a Cys204-Cys218 (small disulfide loop) deletion within the CRD. Antibody 1D6 completely blocked the binding of SP-A to dipalmitoylphosphatidylcholine and galactosylceramide and liposome aggregation. By comparison, 6E3 failed to completely attenuate the interactions of SP-A with lipids. However, both 6E3 and 1D6 blocked the uptake of lipid by type II cells that is caused by SP-A. From these data, we conclude that: 1) the epitope for antibody 6E3 is located at the neck domain of SP-A and that for antibody 1D6 is at the small loop region in the CRD; 2) the CRD is essential for the SP-A functions of lipid binding, liposome aggregation, the inhibitory effect on lipid secretion, and the augmentation of lipid uptake by type II cells, and these activities are largely attributable to amino acid residues within the steric inhibitory footprint of 1D6 bound to the small disulfide loop region; and 3) the neck domain of SP-A may also be involved in the process of SP-A-mediated uptake of phospholipids by alveolar type II cells.

Highlights

From The Lord and Taylor Laboratory for Lung Biochemistry, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 and the Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262
We conclude that: 1)the epitope for antibody 6E3 is located at the neck domainof SP-Aand that for antibody 1D6 is at the small loop regionin the carbohydrate recognitiondomain (CRD); 2) the CRD is essential for the SP-Afunctions of lipid binding, liposome aggregation, the inhibitory effect on lipid secretion, and the augmentation of lipid responsible for the biophysical properties of surfactant, Surfactant proteinA (SP-A) may play a n important role in phospholipid homeostasis in the alveolar space
For protein expression,the recomantibody (6E3)failed to alter theinhibitory effect of this protein [1].The purpose of this study was to map epitopes for monoclonal antibodies that exhibited different effectsupon SP-A functions using recombinant proteins expressed in the baculobinant virus stock was used to infect Sf9 cells a t a multiplicity of 2-5 at 28 "C for 1h, and the infective media was removed and the cells were incubated in Excel1400 media.Media was harvested and the recombinant SP-A and the chimeras of SP-Aand surfactant protein D (SP-D) werepurified by affmitychromatographyon mannose-Sepharose6B byadsorption in the virus system

Summary

Present address

Critical Care Medicine, 231 Bethesda Ave., Cincinnati,OH 45267. Tel.: palmitoylphosphatidylcholine;GalCer, galactosylceramide; CRD, car-. For protein expression,the recomantibody (6E3)failed to alter theinhibitory effect of this protein [1].The purpose of this study was to map epitopes for monoclonal antibodies that exhibited different effectsupon SP-A functions using recombinant proteins expressed in the baculobinant virus stock was used to infect Sf9 cells a t a multiplicity of 2-5 at 28 "C for 1h, and the infective media was removed and the cells were incubated in Excel1400 media.Media was harvested and the recombinant SP-A and the chimeras of SP-Aand SP-D werepurified by affmitychromatographyon mannose-Sepharose6B byadsorption in the virus system This approach has enabled us to map domains presence of 1-5 m~ CaCl, and elution with 2-10 m~ EDTA as described involved in lipid binding, liposome aggregation, and lipid up- previously [29].For the purification of the deletion mutants of SP-Aand take by alveolar type I1 cells. The chimera with SP-A and MBP-A, a protein A-Sepharose CL-4B column covalently coupledwith monoclonal antibody 1D6 wasused, since

EXPERIMENTAL PROCEDURES

RESULTS

Findings

DISCUSSION