Mahnaz Moradi-Améli

Synteny between the loci for a novel FACIT-like collagen locus (D6S228E) and α1(IX) collagen (COL9A1) on 6q12–q14 in humans

A 1.8-kb cDNA encoding portion of a novel collagenous chain was isolated from a human rhabdomyosarcoma cell line by cross-hybridization using a chicken type V collagen probe. Sequence analysis suggests that this chain belongs to the recently discovered group of collagens, termed the FACIT class of macromolecules. This cDNA was used to locate the corresponding gene (D6S228E) to chromosome 6, notably at position 6q12-q14. Interestingly, within this region of human chromosome 6 residues the alpha 1 (IX) collagen gene (COL9A1), a member of the FACIT group.

Microfibrillar composition of umbilical cord matrix: Characterization of fibrillin, collagen VI and intact collagen V

Ultrastructural studies made on human umbilical cord revealed that the striated collagen fibrils of the Whartons jelly matrix are mixed with many microfibrillar structures. Microfibrils were found with a tubular cross-section of 10-12 nm diameter and were organized as beaded filaments characteristic of fibrillin-rich microfibrils. Beads had an average diameter of 25 nm and were spaced at about 50-80 nm. This ultrastructural observation was confirmed by indirect immunofluorescent staining of the jelly matrix using monoclonal antibody to fibrillin. Another constituent of the microfibrillar network was present as typical 100-nm periodic filaments of type VI collagen. Indirect immunofluorescent staining using antibodies to collagen VI showed for the first time that this collagen appeared to be distributed largely in the jelly matrix. In addition, other microfibrils with no specific banding pattern were observed. These microfibrils may constitute an organization of type V collagen different from the one which is generally assembled in heterotypic fibrils with collagen I. Among the latter heterotypic fibrils, type V collagen was studied using an anti-peptide antibody to the most N-terminal non-collagenous region of its alpha 2(V) chain. This antibody recognized a filamentous mesh decorating the bundles of collagen fibrils by immunofluorescent staining. This indicates that at least this part of alpha 2(V) chain may be accessible to the antibody at the surface of the fibrils.

Common topology within a non-collagenous domain of several different collagen types

The secondary structure of a conserved non-collagenous module in alpha 1(V), alpha 1(XI), alpha 1(IX), alpha 1(XII), alpha 1(XIV) and alpha 1(XVI) collagen chains and in proline- and arginine-rich protein was analyzed using different algorithms. The results predict that a common anti-parallel beta-sheet structure composed of nine consensus beta-strands is present in these non-collagenous modules. A model for the packing of these beta-sheets is proposed which suggests that the predicted beta-sheet structure may be involved in molecular recognition functions.

Stoichiometry of the oligomycin-sensitivity-conferring protein (OSCP) in the mitochondrial F0F1-ATPase determined by an immunoelectrotransfer blot technique

The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the alpha and beta subunits of F1-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with 125I-labelled purified monoclonal antibodies specific to various peptides. The 125I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H2O2 and alpha-naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F1.

Different splice variants of cartilage α1(XI) collagen chain undergo uniform amino-terminal processing

Collagen XI is found mainly as a component of cartilage fibrils. Among the different transcripts identified by RT-PCR for the alpha1 (XI) chain, the major tissue form has been reported to be the splicing product of exons I, III and V. In this study, two other splice isoforms of the alpha1(XI) chain were identified using N-terminal sequencing. Like the major alpha1(XI) chain, the fully processed isoforms begin at Gln254 within the N-terminal domain encoded by exon I. This sequence is followed by sequences encoded by exon IIA or III. An anti-peptide antibody allowed the identification of the exon IV encoded sequence within both isoforms. Therefore, these isoforms of the alpha1(XI) chain correspond to the splicing of exons I, IIA, III, IV and V or of exons I, III, IV and V, thus presenting larger acidic sequences than the major form. They could mediate strong ionic interactions within the cartilage matrix.

The rotation of the α subunit of F1 relative to minor subunits is not involved in ATP synthesis. Evidence given by using an anti-α subunit monoclonal antibody

Abstract To test whether ATP synthesis could occur via a mechanism of rotational catalysis in which the α and β subunits of F1 would rotate with respect to the minor subunits, we have measured the rate of ATP synthesis after binding various masses of antibodies to F1. If the rotation was an essential feature of the mechanism, the rate of ATP synthesis should be inhibited either completely or proportionately to the load carried by F1. Bivalent immunoglobulins (IgG) or monovalent Fab fragments of an anti-α monoclonal antibody (7B3) were bound to F1 present in electron-transport particles in a ratio of 2 Fab or 2 IgG per F1. This binding similarly inhibited the rate of ATP synthesis by a maximum of about 50%. When anti-mouse immunoglobulins were added to the F1-7B3 (IgG) complex, no significant change in the rate of inhibition was observed. In conclusion, the rate of ATP synthesis was the same when F1 was loaded with 100 kDa (2 Fab), 300 kDa (2 IgG, 7B3) or 900 kDa (2 IgG + 4 anti-mouse IgG). It is concluded that the rotation of the α subunits is extremely unlikely to play an essential role in the mechanism of ATP synthesis.

[73] Monoclonal antibodies to F1-ATPase subunits as probes of structure, conformation, and functions of isolated or membrane-bound F1

Publisher Summary This chapter describes the preparation of monoclonal antibodies (McAb) against pig heart mitochondrial F 1 -ATPase subunits and their use to determine the stoichiometry, the conformations, and the functions of the isolated or membrane-integrated enzyme. The antibodies able to recognize sequences of only a few amino acids serve as tools to define structure-function relationships in enzymes. Conventional antisera are complex mixtures of antibodies of different classes with different affinities for various antigenic determinants; therefore, they cannot provide precise information on the domains or sub-domains of proteins. Because of the complexity of the immune response, no conventional antiserum is exactly like another even if both come from the same species. In contrast, monoclonal antibodies are attractive reagents as once the cell line is established, it provides a permanent source of an antibody. In addition, as monoclonal antibodies recognize a single antigenic determinant, they are suitable as specific probes of protein assembly.

Availability to monoclonal antibodies of antigenic sites of the α and β subunits in active, denatured or membrane-bound mitochondrial F1-ATPase

Abstract The binding of five monoclonal antibodies to mitochondrial F1-ATPase has been studied. Competition experiments between monoclonal antibodies demonstrate that these antibodies recognize four different antigenic sites and provide information on the proximity of these sites. The accessibility of the epitopes has been compared for F1 integrated in the mitochondrial membrane, for purified β-subunit and for purified F1 maintained in its active form by the presence of nucleotides or inactivated either by dilution in the absence of ATP or by urea treatment. The three anti-β monoclonal antibodies bound more easily to the β-subunit than to active F1, and recognized equally active F1 and F1 integrated in membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F1 and better exposed in the isolated β-subunit. In addition, unfolding F1 by urea strongly increased the binding of one anti-β monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the β-subunit. One anti-α monoclonal antibody (20 D6) bound poorly to F1 integrated in the membrane, while the other (7 B3) had a higher affinity for F1 integrated in the membrane than for soluble F1. Therefore, 20 D6 recognizes an epitope of the α-subunit buried inside F1 integrated in the membrane, while 7 B3 binds to a domain of the α-subunit well exposed at the surface of the inner face of the mitochondrial membrane.