Antibody Imprint of a Membrane Protein Surface

Abstract

Structural features of the integral membrane protein flavocytochrome b (Cyt b) were discovered using an antibody "imprint" of the Cyt b surface. Amino acid sequences were selected from a random nonapeptide phage-display library by their affinity for the monoclonal antibody 44.1 binding site, which recognizes the native conformation of the p22 phox subunit of Cyt b. Transferred nuclear Overhauser effect spectroscopy and rotating frame Overhauser effect spectroscopy NMR were used to study the antibody-bound conformation of a synthetic peptide derived from phage-displayed sequences. The NMR data supported the phagedisplay analysis suggesting the existence of a complex epitope and allowed the modeling of the close spatial proximity of the epitope components 29 TAGRF 33 and 183 PQVNPI 188 from discontinuous regions of p22 phox . Although these regions are separated by two putative membrane-spanning domains and are 150 residues apart in the sequence, they appear to combine to form a complex epitope on the cytosolic surface of the transmembrane protein. NMR constraints, measured from the antibody-bound conformation of a composite peptide mimetic of the Cyt b epitope, and one constraint inferred from the phage-display results, were used to demonstrate the close proximity of these two regions. This information provides a low resolution view of the tertiary structure of the native discontinuous epitope on the Cyt b surface. Given additional antibodies, such imprint analysis has the potential for producing structural constraints to help support molecular modeling of this and other low abundance or noncrystallizable proteins.Human phagocyte flavocytochrome b (Cyt b) 1 is an electron transferase that directs metabolic electrons across the plasma membrane to reduce molecular oxygen and form superoxide (O 2 . ). The production of superoxide by phagocytes, which involves modulation of Cyt b structure, is essential for antimicrobial host defense (1), plays a central role in inflammatory tissue injury (2), and may play a more general role in cellular regulation (3). Individuals with a defective Cyt b have insufficient superoxide production, suffer from chronic granulomatous disease (4), and sustain repeated life-threatening infections (5). Despite its cardinal role in many inflammatory processes and possibly in growth regulation, few experimental methods have been able to provide information on the structure of Cyt b. Therefore, we have developed an alternative method to describe topological features of the Cyt b surface. Because antibodies and their cognate antigens form complementary surfaces of interaction (6), we have sought structural information about Cyt b from an antibody binding site, specific for the Cyt b surface. We recently reported mapping of monoclonal antibody epitopes and functional sites of Cyt b, using random sequence phage-display peptide libraries (7;8). mAb 44.1 recognizes Cyt b in situ in permeabilized human neutrophils and thus contains information about the three-dimensional structure of the protein sur...

Functional Epitope on Human Neutrophil Flavocytochrome b558

“…Human neutrophil Cyt b (30 ng per lane) was partially purified by heparin-agarose affinity chromatography prepared as described (10), or by immunoaffinity purification of Cyt b using mAb 44.1 (8). Following separation of proteins by SDS-PAGE and transfer to nitrocellulose, the blot was probed with 13 nM (2 g/ml) of the primary Abs NL7, 54.1, or 7D5.…”

“…Tertiary structural elements of native Cyt b have further been determined by this method, revealing discontinuous regions of protein that are contained within a single epitope (8,9). In the current report, we show that both p-iodonitrotetrazolium violet (INT) and cytochrome c reduction are inhibited by mAb NL7, which binds 498 EKDVITGLK 506 , a cytosolic segment of gp91 phox .…”

Immunofluorescence and Confocal Microscopy of Neutrophils

“…5 We routinely use the following antibodies for immunofluorescence microscopy of human neutrophils [6][7][8][9][10]. To detect NADPH oxidase components, antibodies specific for p22 phox (mAb 44.1) and gp91 phox (mAb 54.1) [20,21] are now available from Santa Cruz Biotechnology (Santa Cruz, CA); mAb 7D5 detects an epitope of gp91 phox present in mature flavocytochrome b 558 [22] and can be used on intact PMNs to detect protein upregulation at the cell surface [7]; a rabbit mAb specific for human p40 phox (Epitomics, Burlingame, CA); an antibody that specifically detects active Rac (NewEast Biosciences, Malvern, PA); and rabbit antisera from William Nauseef (University of Iowa) specific for p47 phox and p67 phox [6]. Mouse mAbs specific for human CD63, lamp-1, and CD11b are obtained from the University of Iowa Developmental Studies Hybridoma Bank.…”

Phage Display Epitope Mapping of Human Neutrophil Flavocytochromeb 558

“…Determination of structural and functional aspects of epitopes bound by specific antibodies can provide information about the protein against which the antibody is directed (11). Antipeptide and antisubunit polyclonal antibodies against regions of Cyt b have been used to locate the corresponding epitopes (12,13) and information derived from identification of epitope mimetics has led to the description of anti-Cyt b "antibody imprints" (14). We continue to elucidate the structure of Cyt b domains recognized by monoclonal antibodies to better define its transmembrane topology and gain insight into its functional organization.…”

“…Rinsing and blocking were performed as described (14), except a different blocking buffer (Hanks' balanced salt buffer with 10 mM HEPES, pH 7.4, 0.5% bovine serum albumin) was used. Cyt b was probed for 1 h at 25°C with 80 l of the indicated mAb at a concentration of 3 g/ml.…”