Actin depolymerizing activity in serum can be attributed to the two proteins brevin and vitamin D binding protein (DBP). To investigate their mechanisms of action, we used a number of techniques, including procedures involving the fluorescent pyrene-labeled actin probe, to compare the interaction of the two proteins with G- and F-actin in vitro. With a fluorescence enhancement assay, we determined that brevin forms a 1:2 complex and DBP forms a 1:1 complex with pyrene-G-actin. We also found that both proteins reduce the viscosity of F-actin measured with high-shear and low-shear viscometers, with brevin effective at much lower concentrations than DBP. In polymerization experiments, brevin inhibits filament elongation at substoichiometric levels by inhibiting monomer addition at the barbed end but can also accelerate polymerization by nucleating assembly of filaments which grow from the pointed end. DBP does not nucleate filament assembly and inhibits filament elongation at either end only at near-stoichiometric levels. Brevin, but not DBP, accelerates disassembly of filaments diluted into a depolymerizing medium. This is consistent with the capability of brevin to sever preformed filaments associated with erythrocyte membranes and to increase the number of filament ends as estimated by a cytochalasin binding assay. In steady-state experiments involving the use of pyrene-actin, brevin produces only a small increase in the apparent monomer concentration when the critical concentrations at the two ends of the filaments are the same (i.e., in 0.1 M KCl). However, when the critical concentration at the pointed end is higher than that at the barbed end (i.e., in 2 mM MgCl2), low molar ratios of brevin sharply increase the monomer concentration to the critical concentration of the pointed end. This allows substoichiometric amounts of brevin to completely depolymerize filaments when the total actin concentration is at or below that of the pointed end. In contrast to brevin, DBP increases the amount of nonfilamentous actin in a stoichiometric and dose-dependent manner regardless of the nature of the salt in the medium. We conclude from this study that brevin is similar in its mechanism of action to other proteins known to bind to the barbed end of filaments and that DBP is related in its action to proteins that complex monomers and prevent them from participating in the polymerization process.

Myelin proteolipid protein (PLP), the major protein of mammalian CNS myelin, is a member of the proteolipid gene family (pgf). It is an evolutionarily conserved polytopic integral membrane protein and a potential autoantigen in multiple sclerosis (MS). To analyze antibody recognition of PLP epitopes in situ, monoclonal antibodies (mAbs) specific for different regions of human PLP (50–69, 100–123, 139–151, 178–191, 200–219, 264–276) were generated and used to immunostain CNS tissues of representative vertebrates. mAbs to each region recognized whole human PLP on Western blots; the anti‐100–123 mAb did not recognize DM‐20, the PLP isoform that lacks residues 116–150. All of the mAbs stained fixed, permeabilized oligodendrocytes and mammalian and avian CNS tissue myelin. Most of the mAbs also stained amphibian, teleost, and elasmobranch CNS myelin despite greater diversity of their pgf myelin protein sequences. Myelin staining was observed when there was at least 40% identity of the mAb epitope and known pgf myelin proteins of the same or related species. The pgf myelin proteins of teleosts and elasmobranchs lack 116–150; the anti‐100–123 mAb did not stain their myelin. In addition to myelin, the anti‐178–191 mAb stained many neurons in all species; other mAbs stained distinct neuron subpopulations in different species. Neuronal staining was observed when there was at least approximately 30% identity of the PLP mAb epitope and known pgf neuronal proteins of the same or related species. Thus, anti‐human PLP epitope mAbs simultaneously recognize CNS myelin and neurons even without extensive sequence identity. Widespread anti‐PLP mAb recognition of neurons suggests a novel potential pathophysiologic mechanism in MS patients, i.e., that anti‐PLP antibodies associated with demyelination might simultaneously recognize pgf epitopes in neurons, thereby affecting their functions. © 2006 Wiley‐Liss, Inc.

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We coupled a monoclonal anti-human IgD to the gp350 gylcoprotein of Epstein-Barr virus, which has been shown to bind to the complement receptor 2 (CR2), and compared its B cell stimulatory ability to that of anti-Ig and to a multivalent anti-Ig-dextran conjugate. The anti-Ig-gp350 conjugate stimulated higher levels of human B cell proliferation in vitro than did anti-Ig or anti-Ig conjugated to control viral protein, comparable to the proliferation stimulated by the multivalent anti-Ig-dextran. This enhanced proliferation was dependent on binding of the conjugate to CR2, inasmuch as an anti-CD2 antibody blocked the enhanced proliferative response. This enhanced proliferative response was associated with prolonged elevations of intracellular ionized calcium, which was comparable to the response stimulated by anti-Ig-dextran. These findings suggest the use of gp350 as a carrier molecule for weakly immunogenic peptides or antigens which, when bound to gp350, would enhance B cell clonal expansion and activation of antigen-specific B cells.