Different splice variants of cartilage alpha1(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.

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 Wharton's 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.

Processing of type XI collagen. Determination of the matrix forms of the alpha1(XI) chain.

Type XI collagen is mainly found as a minor constituent in type II-containing fibrils and presents a alpha1(XI)alpha2(XI)alpha3(XI) stoichiometry. This molecule was shown to be partially processed in its intact tissue form. Moreover, alternative splicing has been demonstrated in the variable region of the N-terminal domain of alpha1(XI) and alpha2(XI) chains. In this work, the processing of a major intact form of alpha1(XI) from matrix laid down by chick chondrocytes in culture was identified using N-terminal sequencing and antibodies to synthetic peptides corresponding to the N-terminal propeptide cDNA-derived sequence. The results show that the fully processed form of alpha1(XI) begins at Gln254 of the N-terminal propeptide, seven residues before the end of the proline/arginine-rich protein region encoded by exon I (Zhidkova, N. I., Justice, S. K., and Mayne, R. (1995) J. Biol. Chem. 270, 9486-9493). This sequence is immediately followed by a sequence encoded by exon III. The processing takes place at an Ala-Gln sequence that corresponds to a consensus sequence for procollagen N-proteinase. The antibody raised against a sequence located within the region corresponding to exon IV (anti-P8) fails to recognize this fully processed form of the alpha1(XI) chain. It recognizes, however, two minor bands of high molecular mass. These results suggest that a major cartilage form of alpha1(XI) is the product of alternative splicing in which sequences encoded by both exons II and IV are skipped. The presence of a highly acidic subdomain encoded by exon III at the N terminus of the major form of the alpha1(XI) chain, as predicted by these data, provides potential sites for interaction of collagen XI with other molecules.

Diversity in the processing events at the N-terminus of type-V collagen.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Synteny between the loci for a novel FACIT-like collagen locus (D6S228E) and alpha 1 (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.

Localization on the mitochondrial F1 ATPase alpha subunit of an epitope masked in the membrane-bound enzyme using a monoclonal antibody and synthetic peptides.

The epitope of the monoclonal antibody 20D6 was localized by N-terminal sequencing of the smallest immunoreactive peptides obtained after CNBr and trypsin cleavage of the F1 alpha subunit of the mitochondrial ATPase/ATP synthase. Immunochemical analysis of overlapping synthetic octapeptides, covering the immunoreactive peptide sequence, has defined the seven-amino-acid sequence recognized by 20D6 as 84EGDIVKR90. The binding of 20D6 was lost after substituting either I87 by K or S, or R90 by C or A as it occurs in the alpha subunit sequence of Escherichia coli or chloroplast ATPase, respectively. This explained the lack of immunoreactivity of 20D6 to these species and indicated the importance of charged as well as hydrophobic residues in the epitope. Immunochemical analysis of synthetic peptides by polyclonal anti-F1 antisera showed that this region is highly immunodominant. In a competitive ELISA, the monoclonal antibody bound with similar affinity to F1 in the presence and absence of substrate as well as to cold dissociated F1, indicating that the epitope was located on the surface of the alpha subunit and not buried between F1 subunits. The lack of binding of 20D6 when F1 is bound to the membrane showed that the epitope exposed at the surface of purified soluble F1 became masked after binding to the membrane. This suggests that it is located at the interface between F1 and the membrane.

Inhibition of mitochondrial F1-ATPase activity by an anti-alpha subunit monoclonal antibody which modifies interactions between catalytic and regulatory sites.

A monoclonal antibody, 7B3, specific to the alpha subunit of the mitochondrial ATPase-ATP synthase inhibited the rate of ATP hydrolysis by either soluble F1 or electron transport particles up to a maximum of 75%. However, 7B3 did not modify the rate of ITP hydrolysis. In addition, the apparent Km for MgATP extrapolated at high ATP concentrations had the same value in the absence as in the presence of 7B3. The antibody did not change the inactivation rate of F1-ATPase induced by dicyclohexylcarbodiimide or 4-chloro-7-nitro-2,1,3-benzoxadiazole. These observations indicate that 7B3 did not directly interfere with the catalytic sites of ATP or ITP hydrolysis. On the contrary, 7B3 modified the interaction between nucleotide sites and therefore the regulation of the rate of ATP hydrolysis. Indeed, 7B3 changed into a positive cooperativity the negative cooperativity observed when measuring the rate of ATP hydrolysis as a function of ATP concentration. 7B3 also increased the binding of ADP to F1. 7B3 prevented the rapid phase of inactivation of F1 by 5'-p-fluorosulfonylbenzoyladenosine. This phase has been correlated to the binding of 5'-p-fluorosulfonylbenzoyladenosine to regulatory sites (Di Pietro, A., Godinot, C., Martin, J. C., and Gautheron, D. C. (1979) Biochemistry 18, 1738-1745). The inhibition of ATP hydrolysis is concomitant with the binding of 1 mol of IgG or of 2 mol of Fab fragments per mol of F1. However, by further increasing the ratio Fab/F1, only 1 mol of Fab remained bound to F1 without change in inhibition of ATPase activity. All these experiments strongly support the suggestion that F1 conformational changes occurring upon binding of 7B3 to alpha subunit induce a modification of interactions between nucleotide sites. This modification would be consecutive to a change in the normal interaction between the alpha and beta subunits which is required to observe an active rate of ATP hydrolysis or synthesis. In conclusion, the use of this monoclonal antibody demonstrates for the first time in mammalian F1 the role of the conformation of the alpha subunit in the regulation of the ATPase activity.

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

To test whether ATP synthesis could occur via a mechanism of rotational catalysis in which the alpha and beta 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-alpha 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 ant-mouse IgG). It is concluded that the rotation of the alpha subunits is extremely unlikely to play an essential role in the mechanism of ATP synthesis.

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

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 beta-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-beta monoclonal antibodies bound more easily to the beta-subunit than to active F1, and recognized equally active F1 and F1 integrated in the membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F1 and better exposed in the isolated beta-subunit. In addition, unfolding F1 by urea strongly increased the binding of one anti-beta monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the beta-subunit. One anti-alpha 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 alpha-subunit buried inside F1 integrated in the membrane, while 7 B3 binds to a domain of the alpha-subunit well exposed at the surface of the inner face of the mitochondrial membrane.

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.

Monoclonal antibodies to mitochondrial F1-ATPase and oligomycin sensitivity conferring protein (OSCP). Tools for recognition of well conserved and essential antigenic sites.

The preparation of anti-OSCP monoclonal antibodies is described for the first time. One of these antibodies prevents the activating effect of OSCP in reconstitution experiments. These antibodies and antibodies previously obtained against the alpha- and beta-subunits of pig heart mitochondrial F1-ATPase have been used to look for well conserved epitopes in various species. One anti-beta antibody can recognize all species tested while the anti-OSCP antibodies only recognize the pig or beef enzyme. The above anti-beta antibody inhibits ATP synthesis without modifying the rate of ATP hydrolysis. This antibody also prevents the ADP-induced hysteretic inhibition of F1-ATPase.

Characterization of monoclonal antibodies against mitochondrial F1-ATPase.

Four stable murine hybridoma clones producing homogeneous antibodies against pig heart mitochondrial F1-ATPase have been established. All antibodies exhibit specific binding to F1-ATPase. Three of them interact with the beta subunit and one binds strongly to the alpha and barely to the beta subunit. All of them exhibit linear Scatchard plots and high binding affinities, with dissociation constants between 4.7 X 10(-8) M and 6.5 X 10(-10) M. The minimal number of moles of IgG bound at saturation per mole of immobilized F1-ATPase is 2.2 for the anti-alpha-subunit antibody and 2.5 for one anti-beta-subunit antibody. This suggests that more than two copies of the alpha and beta subunits are present in the enzyme. Two antibodies seem to recognize F1-ATPase equally before or after denaturation with sodium dodecyl sulfate. The two other antibodies exhibited a much higher affinity for the nondissociated enzyme, indicating that they are very sensitive to a specific conformation of the enzyme.