Plate 32 Rheumatoid arthritis. Three-dimensional representation of HLA class II molecule containing the antigenic processed peptide in the antigen-binding groove (light blue stretch, top of the figure). The five amino acid sequence QR/RRAA ('shared epitope') is common among all HLA alleles correlated to rheumatoid arthritis. (Kindly provided by A La Cava and S Albani, University of Carolina, USA, with permission.)
Plate 33 (A) Secretory component (the polymeric Ig receptor). Paired immunofluorescence staining for IgA and SC in tissue section from human nasal mucosa; the same field is shown after incubation with 'red' anti-IgA + 'green' anti-SC, (i) (red filtration) and (ii) (double exposure). IgA alone is present in immunocytes and throughout the stroma; SC alone is present in the Golgi zones (arrows) adjacent to nuclei of glandular acinar cells; both IgA and SC (mixed color) are present basolaterally on acinar cells and apically in their cytoplasm.
Plate 34 (B) Paired immunofluorescence staining for IgA and total SC, or for unoccupied and bound SC, in tissue sections of human colonic mucosa. (i) left part (red filtration) and right part (double exposure), comparable fields in adjacent sections of gland incubated with 'red' anti-SC and with 'red' anti-IgA + 'green' anti-SC, respectively. Prominent SC-containing granules are present in the Golgi zones, whereas the rest of the cytoplasm and the basolateral aspects of columnar epithelial cells are positive for both SC and IgA (mixed color). Goblet cells are devoid of both markers. (ii) (red filtration), (iii) (left part, double exposure), and (iv) (green filtration), same field in a section of gland incubated with 'red' anti-I determinant (specific for unoccupied SC) + 'green' anti-A determinant (accessible on free as well as bound SC). The two antigenic determinants show distinct differential distribution with a relative dominance of I in the Golgi zones, and of A in the remaining cytoplasm and weakly also basolaterally. Right part of (iii) (double exposure) shows a comparable field after prolonged exposure time for red (anti-I) emission. Note that neither of the two anti-SC reagents produced fluorescence of elements in the lamina propria.
Plate 35 (C) (i) Paired immunofluorescence staining to demonstrate in vitro SC affinity to IgA immunocytes in section of human colonic mucosa preincubated with free SC followed by 'red' anti-SC + 'green' anti-IgA. Note in double exposure (center) that most IgA cells show varying degree of mixed color as evidence for binding of SC to cytoplasmic polymeric IgA (pIgA), whereas a few cells (probably pure monomer producers) lack SC-binding and are therefore only green. The purely red cells in the double exposure are SC-binding IgM immunocytes. The columnar epithelial cells of the glands at top and bottom contain innate SC and transport pIgA; they therefore show mixed cytoplasmic color with the exception of the Golgi zones which contain SC but no IgA. (ii) Similar SC-affinity test on section of human salivary gland. Double exposure (center) shows various tints of yellow in IgA immunocytes located between acini A and duct D on the figure; in several immunocytes the yellow color is restricted to areas close to the nucleus (arrow), suggesting accumulation of pIgA. One immunocyte (at the bottom) is purely green, suggesting production of only monomeric IgA. Acini and duct are faintly double-stained for unoccupied SC and translocated pIgA. (Kindly provided by P Brandtzaeg, F-E Johansen, P Krajci and IB Natvig, University of Oslo, Norway, with permission.)
Plate 36 (A) Anti-lysozyme binding site Surface complementarity between the antigen, hen egg-white lysozyme and the antibody HyHEL-5 binding site. In this cross-reaction through the antigen-antibody complex, 5 A thick, centers of atoms are connected by sticks and atomic volumes are shown as Conolly dot surfaces. Atom color-coding; oxygen, red; nitrogen, blue; sulfur, yellow; carbon in lysozyme, white; carbon in VL domain, magenta; carbon in VH domain, brown. The intermolecular interface, approximately traced by a line dividing the antigen atoms (lower part) from those of the antibody (upper part), consists of tightly packed regions, and occasional loosely packed regions, 'cavities'. The intermolecular cavities, for example the one delimited by lysozyme residue Arg 68 and the antibody residues L 91, H 33 and H 35 (blue arrow, on the top), are not any larger, however, than the occasional intramolecular cavities (white arrow, on the bottom). Crystallographic coordinates by Sheriff S et al. (1987), Proceedings of the National Academy of Science of the USA 84: 8075-8079, with permission.
Plate 37 (B) Electrostatic fields at the surface of the HyHEL-5 antibody binding site. This monoclonal antibody is specific to lysozyme, a protein with an exceptionally high positive charge. Using the crystallographic coordinates of the HyHEL-5 VH and VL domains (Sheriff et al, (1987) Proceedings of the National Academy of Science of the USA 84: 8075-8079, with permission.) molecular surface envelope was generated by the program GRASP (A Nicholls, Columbia University 1991) and was color-coded by the electrostatic field produced by all the formally charged residues in the molecule: red, negative potential; blue, positive potential; color intensity proportional to the strength of the field). At the center of the binding site there is a deep, electronegative cavity 'below' which is a protruding side-chain of the Arg residue L93, with a weaker, positive, electrostatic field. In the HyHEL-5-lysozyme complex, the central negatively charged cavity becomes occupied by the two positively charged lysozyme side-chains, Arg 45 and Arg 68. The positive field generated by the antibody residue Arg L93 gives rise to a dipole, close to the antibody surface, perhaps helping to preorient the antigen at its approach to the binding site.
Plate 38 (C) Comparison of anti-digoxin and anti-lysozyme binding sites. In these 'elevation plots', points of the molecular surfaces, generated by the program GRASP, have been color-coded according to its distance normal to a three-dimensional envelope of the molecule. The envelope was defined, and constructed, as a smooth, curved plane connecting the 'highest' peaks of the molecular surface. The deepest protrusions on the surface of the molecule are shown in blue, the most elevated points of the surface are shown in red. The binding site of the 26-10 antibody directed against the hapten digoxin is a deep, narrow well (high 'global' curvature); the binding sites of the anti-lysozyme antibodies D1.3 (crystallographic coordinates by Bhat TN et al, (1990) Nature 347: 483-486, HyHEL-5 and HyHEL-10 (coordinates by Padlan EA et al, (1989) Proceedings of the National Academy of Science of the USA 86: 5938-5942) are all larger and more shallow (low 'global' curvature). The D1.3 binding site contains a smaller, deep 'local' hole in its surface (complementary to the lysozyme residue Gin 121), the HyHEL-5 binding site contains a similar but shallower hole (complementary to Arg '68,). The HyHEL-10 elevation plot shows virtually no local depressions. (Kindly provided by J Novotny and M Davis, Bristol-Myers Squibb Research Institute, USA with permission.)
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