Accumulating evidence strongly implicates oxidized LDL (Ox-LDL) in the pathogenesis of atherosclerosis. Several receptors have been identified that bind and internalize Ox-LDL, but their relative importance in vivo is unclear. CD36 is an 88-kD transmembrane glycoprotein expressed on monocytes/macrophages, platelets, and microvascular endothelium that has been implicated as a putative receptor for Ox-LDL. We demonstrate that an anti-CD36 monoclonal antibody inhibited 50% of the specific binding and 26% of the specific degradation of Ox-LDL by human monocyte-derived macrophages. To characterize more completely this binding we evaluated interactions between CD36 and Ox-LDL in murine NIH-3T3 cells stably transfected with human CD36 cDNA. Ox-LDL bound to CD36-transfected 3T3 cells in a saturable manner. Specific binding, internalization, and degradation of Ox-LDL were increased fourfold in CD36-transfected cell lines compared with 3T3 cells transfected with vector alone. Binding of Ox-LDL to CD36-transfected 3T3 cells was inhibited by a panel of anti-CD36 antibodies and by soluble CD36 but not by thrombospondin. Specificity of binding was demonstrated by the equivalent binding of LDL and acetylated LDL to control and CD36-transfected 3T3 cells. The epitope or epitopes on Ox-LDL recognized by CD36 are undefined. Two observations suggest that CD36 recognizes a lipid moiety or that the lipid portion of the lipoprotein is essential for apoprotein recognition. The first is that the increased binding of Ox-LDL to CD36-transfected 3T3 cells is abrogated by delipidation of the lipoprotein, and the second is that oleic acid competes for the binding of Ox-LDL to CD36-transfected 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Denaturation of Bordetella pertussis fimbrial preparations by guanidinium hydrochloride (GdnHCl) has been characterized using static light scattering, c.d., fluorescence and antibody recognition. The susceptibility of Fim2 + 3 (a mixed preparation of two fimbrial types) to GdnHCl was found to be highly dependent on pH; as the pH was increased from pH 7.2 to 10.5, the concentration of GdnHCl required to induce 50% denaturation was decreased. At pH 10.5, Fim2 + 3 was denatured by GdnHCl in a three-step pathway comprising: (1) formation of a pre-denaturational intermediate at less than 1.0 M-GdnHCl; (2) dissociation of the fimbrial polymer into subunits between 2 M- and 3.2 M-GdnHCl; and (3) subunit unfolding between 2.8 M- and 3.6 M-GdnHCl. A similar pathway was also found for the denaturation of the individual fimbrial types, Fim2 and Fim3, except that unfolding of either subunit commenced at a lower GdnHCl concentration (2.2 M) than that found for the mixture of fimbriae, Fim2 + 3. The second step in the denaturation pathway, dissociation into subunits, was partially reversible, but the renaturation and reassociation of fully unfolded subunits to form fimbriae-like structures was not achieved. These findings demonstrate that the GdnHCl denaturation of complex polymeric proteins is unlikely to follow a reversible two-state denaturation pathway, and support the involvement of a chaperone-like protein in the folding and assembly of the fimbriae in vivo. Measurement of the ability of anti-fimbrial monoclonal antibodies to recognize intermediates in the denaturation pathway enabled the identification of two types of epitope which were dependent on different aspects of fimbrial tertiary/quaternary structure.
Antibody-binding domains on the major subunits of Bordetella pertussis serotype 2 (Fim2) and 3 fimbriae (Fim3) have been identified using synthetic peptides which were screened for recognition by anti-protein monoclonal antibodies (mAbs). The presence of non-contiguous f imbrial epitopes was demonstrated by both anti-Fim2 and anti-Fim3 mAbs, several of which recognized at least two peptides that were discontinuous in the amino acid sequence of the corresponding subunits. The specificity of one mAb, 51/24, directed against Fim2, was investigated by replacement-set analysis of a 10-residue peptide, and revealed that antibody binding to the peptide was dependent on the sequence NWPQs6 which is non-conserved in Fim3. Furthermore, proline at residue 95 was found to be essential for mAb 51/24 binding. The specific anti-Fim3 mAb, AG3A, was found to recognize the 10-residue carboxy-terminal peptide from both Fim3 and, unexpectedly, from Fim2. This result suggests that mAb AG3A serospecificity a t the protein level is determined by a conf ormational constraint which prevents mAb AG3A binding to the Fim2 C-terminal domain. Several free peptides containing amino acid residues which comprise part of the Fim2 and Fim3 epitopic domains were prepared as immunogens. One of these peptides was immunogenic in the mouse, indicating the location of a T-helper cell epitope within the peptide sequence, and induced a strong anti-peptide antibody response. The other peptides each required immunization as a conjugate with a carrier protein for anti-peptide antibody stimulation. All four anti-peptide antibody preparations only weakly recognized f imbriae-coated ELISA plates. The results of this investigation demonstrate that although short linear peptides can mimic sub-domains of non-contiguous f imbrial epitopes, they are, however, poor candidate antigens for stimulating an anti-f imbrial antibody response.
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