Specific recognition of protein carboxy-terminal sequences by natural IgM antibodies in normal serum.

Our previous study indicated that normal serum contains complement-fixing natural IgM antibodies reacting with a large variety of randomly generated protein carboxy-termini. Here we show that the "carboxy-terminal" IgM (C-IgM) antibodies specifically react with short peptide sequences located immediately at the protein carboxy-terminus. The specificity of C-IgM-peptide interactions is tentatively defined by three to four amino acid residues. All carboxy-terminal peptides in a large peptide library apparently react with C-IgM antibodies. Immobilized synthetic peptides also react with C-IgM antibodies. No interaction of C-IgM antibodies with internal peptide sequences has been observed. C-IgM antibodies are present in germ-free and in athymic adult rats and are absent in newborn rats. The natural ubiquity of protein carboxy-termini in biological structures suggests that C-IgM could play an important role in antigen clearance and presentation to the immune system. From a practical viewpoint, the recognition of carboxy-terminal peptides by complement-fixing C-IgM antibodies has profound implications for the use of peptide- and protein-derivatized delivery vehicles and artificial materials.

The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.

The icosahedral T7 phage (diameter approximately 65 nm) displaying random peptides at the carboxy-terminus of the phage coat proteins was used as a model for drug and gene delivery vehicles containing peptide ligands. We found that displayed peptides were recognized by natural antibodies and induced complement activation. Strikingly, the phage inactivation by complement was peptide-specific that implied the existence of numerous natural antibodies with different peptide specificity. Selection of phage that avoided inactivation by complement allowed the identification of peptides that protected the phage by binding to serum proteins. In rat blood, peptides with carboxy-terminal lysine or arginine residues protected the phage against complement-mediated inactivation by binding C-reactive protein. In human serum, a number of protective peptides with tyrosine residues were selected. The recognition of displayed peptides by natural antibodies appears to represent a universal mechanism for activation of complement at sites that contain identical or homologous proteins with exposed carboxy-termini.

Characterization of N-ethylmaleimide-sensitive thiol groups required for the GTP-dependent fusion of endoplasmic reticulum membranes.

The GTP-dependent fusion activity of endoplasmic reticulum membranes is thought to be required for the structural maintenance and post-mitotic regeneration of the endoplasmic reticulum. This fusion is sensitive to the thiol-alkylating agent N-ethylmaleimide. In many intracellular fusion events N-ethylmaleimide-sensitivity is associated with a homotrimeric ATPase called N-ethylmaleimide-sensitive fusion protein or NSF. The addition of cytosol containing NSF is known to restore fusion activity to N-ethylmaleimide-treated membranes. We found that the inhibition of fusion of rat liver endoplasmic reticulum membranes (microsomes) by N-ethylmaleimide was not reversed by the addition of untreated cytosol. Fusion was also unaffected by treatment with a buffer known to remove NSF from membranes. Accordingly, no membrane-associated NSF was detected by immunoblot analysis. These data suggest that microsome fusion requires an N-ethylmaleimide-sensitive component distinct from NSF. This component was tightly associated with the membranes, so we used a number of chemical probes to characterize it in situ. Its thiol groups did not appear to be part of a GTP-binding site. They showed relatively low reactivity with sodium periodate, which induces the formation of disulphide bonds between proximate thiol groups. The thiols were not protected against N-ethylmaleimide by Zn2+, a potent inhibitor of fusion which is known to efficiently co-ordinate thiol groups. To characterize the topology of the fusion-related thiol groups we used bulky thiol-specific reagents prepared by conjugating BSA or 10 kDa aminodextran to the bifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibition of fusion by these reagents indicated that these thiols are highly exposed on the membranes. This exposure might be important for the function of these groups during GTP-triggered fusion.

Capillary electrophoresis of rat liver microsomes in polymer solutions.

Rat liver microsome components were separated by capillary zone electrophoresis in buffers containing substituted agarose, agarose crosslinked polyacrylamide, polyacrylamide, polyethylene glycol and dextran of various molecular weights. The best resolution of the components was obtained with polymers of 10-21 nm geometric mean radius. Both the crude and the purified preparations of microsomes exhibit a single major peak. In a Tris-borate-EDTA (TBE) buffer, containing polyacrylamide of 5 x 10(6) molecular weight, it has a retardation coefficient, KR, of 0.77 +/- 0.02. Translation of the KR value to geometric mean radii, R, on the basis of the standard curve applicable to polymer solutions, KR vs. R, with polystyrene carboxylate size standards in both dextran and polyacrylamide solution allows one to estimate a value of R as 13-16 nm for the major microsome component. The value is smaller than expected from electron microscopic measurements (100-250 nm), possibly due to the chemical and geometric differences between microsomes and the polystyrene particles used as size standards. The crude preparation also contains a minor component which is smaller and less charged than the major component. Another component, apparently very much larger than the major one, is seen in TBE buffer but not in a potassium-N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) buffer and is therefore thought to be an artifact of interaction with borate. After a short incubation under conditions promoting delayed microsome fusion, i.e. in presence of GTP and Mg++ and in the absence of polyethylene glycol, the electrophoretic pattern changes dramatically: it now exhibits five unretarded, highly mobile and, therefore, presumably large components in addition to the two original retarded components of the microsome and a less highly charged species similar in KR to the smaller of the original two components.

Ca(2+)-mediated interaction between negatively charged and neutral liposomes.

In the present work it is shown that large unilamellar lecithin/cholesterol liposomes are able to sequester small negatively charged liposomes in the presence of divalent cations. Evidence is presented suggesting that the sequestration occurs via the formation of membrane invaginations transformed further into intraliposomal vesicles.