Biophysical characterization of the C-propeptide trimer from human procollagen III reveals a tri-lobed structure.

Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.

Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1.

Recently several cDNAs have been described encoding lysyl oxidase-like proteins. Their deduced amino acid sequences are characterized by a strong similarity in the C-terminal region, corresponding to the lysyl oxidase family catalytic domain, and by marked differences in the N-terminal regions. Different biological functions have been described for lysyl oxidases in addition to their traditionally assumed cross-linking role. To answer the question of whether these different functions are carried out by different lysyl oxidases, purified and active forms of these enzymes are required. At present only the classical form of lysyl oxidase has been purified and characterized. The purpose of this study was to isolate and characterize the lysyl oxidase-like protein. In view of the strong sequence homology with the C-terminal domain of other lysyl oxidases, we chose to purify the protein from bovine aorta using antibodies specific to the N-terminal domain of the proenzyme. We have isolated a 56-kDa protein identified by amino acid sequencing as the bovine lysyl oxidase-like precursor, which is cleaved at the Arg-Arg-Arg sequence at positions 89-91 by a furin-like activity, as revealed after deblocking of the N-terminal residue. The immunopurified protein was largely inactive, but further processing in vitro by bone morphogenetic protein-1 led to an enzyme that was active on elastin and collagen substrates.

Folding and activity of recombinant human procollagen C-proteinase enhancer.

Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains.

Liquid crystalline ordering of procollagen as a determinant of three-dimensional extracellular matrix architecture.

The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged in a tissue-specific manner, as in certain liquid crystals. For example, the continuous twist between fibrils in compact bone osteons resembles a cholesteric mesophase, while in tendon, the regular, planar undulation, or "crimp", is akin to a precholesteric mesophase. Such analogies suggest that liquid crystalline organisation plays a role in the determination of tissue form, but it is hard to see how insoluble fibrils could spontaneously and specifically rearrange in this way. Collagen molecules, in dilute acid solution, are known to form nematic, precholesteric and cholesteric phases, but the relevance to physiological assembly mechanisms is unclear. In vivo, fibrillar collagens are synthesised in soluble precursor form, procollagens, with terminal propeptide extensions. Here, we show, by polarized light microscopy of highly concentrated (5-30 mg/ml) viscous drops, that procollagen molecules in physiological buffer conditions can also develop long-range nematic and precholesteric liquid crystalline ordering extending over 100 microm(2) domains, while remaining in true solution. These observations suggest the novel concept that supra-fibrillar tissue architecture is determined by the ability of soluble precursor molecules to form liquid crystalline arrays, prior to fibril assembly.

Structural requirements for fibromodulin binding to collagen and the control of type I collagen fibrillogenesis--critical roles for disulphide bonding and the C-terminal region.

Fibromodulin belongs to the family of small, leucine-rich proteoglycans which have been reported to interact with collagens and to inhibit type I collagen fibrillogenesis. Decorin and fibromodulin exhibit a noticeable degree of sequence similarity. However, as previously reported [Font, B., Eichenberger, D., Rosenberg, L. M. & van der Rest, M. (1996) Matrix Biol. 15, 341-348] the domains of these molecules implicated in the interactions with type XII and type XIV collagens are different, these being the dermatan sulphate/chondroitin sulphate chain for decorin and the core protein for fibromodulin. At the present time the fibromodulin domains implicated in the interactions with fibrillar collagens remain unknown. In experiments reported here, we have sought to identify the structural requirements for fibromodulin interaction with collagen and for the control of type I collagen fibrillogenesis. Circular dichroism spectra and fibrillogenesis inhibition studies show that fibromodulin structure and its collagen fibrillogenesis control function are strictly dependent on the presence of intact disulphide bridge(s). In addition, we show that the binding of fibromodulin (or fibromodulin-derived fragments) to type I collagen is not necessarily correlated with fibrillogenesis inhibition. To isolate fibromodulin domains, the native proteoglycan was submitted to mild proteolysis. We have isolated an alpha-chymotrypsin-resistant fragment which contains the bulk of the N-terminal and central region of the molecule including the leucine-rich repeats 4 and 6 reported for decorin to be involved in type I collagen binding. This fragment does not bind to type I collagen. Using enzymes with different specificities, a number of large fragments of fibromodulin were obtained, suggesting a compact structure for this molecule which is relatively resistant to proteolysis. None of these N-glycosylated fragments were able to bind to type I collagen in co-sedimentation experiments. Taken together these results suggest that fibromodulin-type I collagen interactions leading to fibrillogenesis inhibition require more than one binding domain. One of these domains could be the C-terminal end of the molecule containing the disulphide loop which is absent in the chymotrypsin-resistant fragment.

Characterization of the interactions of type XII collagen with two small proteoglycans from fetal bovine tendon, decorin and fibromodulin.

In addition to the major collagens, such as type I or type II, connective tissues contain a number of less abundant collagens and proteoglycans, whose association contributes to the different properties of the tissues. Type XII and type XIV collagens have been described in soft connective tissues, and type XIV collagen has been shown to interact specifically with decorin through its glycosaminoglycan chain (Font et al., J. Biol. Chem. 268, 25015-25018, 1993). Interactions between these collagens and the small proteoglycans have been characterized further by studying the binding of type XII collagen to decorin by solid phase assays. Our results show a saturable binding of the proteoglycan through its glycosaminoglycan chain to type XII collagen, which does not seem to involve the large non-collagenous NC3 domain of the molecule. This interaction is strongly inhibited by heparin. Furthermore, we report that another small proteoglycan, fibromodulin, isolated from tendon under non-denaturing conditions, is able to bind to type XII collagen. This interaction has been characterized and, unlike that observed with decorin, type XII collagen-fibromodulin interaction seems to take place with the core protein of the proteoglycan. In addition, we report that type XII-type I collagen interactions are not necessarily mediated by decorin as previously suggested.

Binding of collagen XIV with the dermatan sulfate side chain of decorin.

As an approach to elucidate the role of collagen XIV, which is still unclear, molecules exhibiting affinity for this collagen have been sought in connective tissue. Extracts from fetal bovine tendon were resolved by gel electrophoresis and electrophoretically transferred to nitrocellulose. The blot was overlaid with native collagen XIV and the collagen XIV-binding molecules revealed by immunodecoration with a monoclonal antitype XIV collagen antibody. This experimental approach allowed us to reveal in tendon extracts a diffuse band, with an apparent molecular mass of approximately 100 kDa, that binds collagen XIV. This molecule was also found associated with the fractions containing partially purified type XIV collagen. This 100-kDa molecule was sensitive to chondroitinase ABC and, after chondroitinase digestion, yielded a core protein of about 48 kDa. N-terminal sequence analysis of the proteoglycan after blotting allowed us to identify it as decorin. By solid phase assays we have studied this newly described association between decorin and type XIV collagen and shown that it is a saturable process. In addition, preliminary determination of the domains of the two molecules involved in the association has been performed. The possible role of these interactions is discussed.

Purification and characterization of native type XIV collagen.

A new molecule, type XIV collagen, with domains homologous to type IX and XII collagens has been recently discovered in pepsin extracts of fetal bovine tissues (Dublet, B., and van der Rest, M. (1991) J. Biol. Chem. 266, 6853-6858). In the present study, we describe the purification and the characterization of the intact native form of this newly discovered collagen. By using only two chromatographic steps we were able to obtain pure type XIV collagen. Furthermore, minor modifications of the protocol allowed us to perform the simultaneous large scale purification of type XII and type XIV collagens from the same tissue. Intact type XIV collagen migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as two bands of 220 and 290 kDa (reducing conditions). After collagenase treatment, a single band of 190 kDa is observed, which represents the large non-collagenous domain of the molecule (NC3). Rotary shadowing electron micrographs of intact type XIV collagen show a cross-shaped structure formed by a thin tail attached through a central globule to three identical "fingers." These properties are similar to those previously described for intact chicken type XII collagen (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156), but the two molecules are different gene products and have charge and glycosylation differences. Finally, we show that the three chains of purified type XIV collagen have an apparent molecular mass of approximately 220 kDa and are not cross-linked to each other by bonds other than disulfide bridges. The same observation was made for type XII collagen. In both cases, the 290-kDa migrating band in SDS-PAGE is due to incomplete denaturation in electrophoresis sample buffer in the absence of urea.

Immunological determination of the oligomeric form of mitochondrial creatine kinase in situ.

Whereas factors governing the interconversion of the two oligomeric forms of mitochondrial creatine kinase are relatively well known, few informations are yet available on the actual form in situ. Antibodies against purified pig and rabbit heart mitochondrial creatine kinase were obtained. The former exhibits a marked specificity for the dimer while the second reacts with both dimer and octamer. They allowed to demonstrate that no dimer can be detected in mitochondria and that CKm occurs naturally exclusively as an octamer. We present arguments that the larger part, if not the totality, of the octamer is membrane-bound rather than soluble in the intermembrane space. However, these findings do not refute the previously proposed models for the regulation of CKm activity in the mitochondrion but urge to envisage a more complex one.

Isoelectric point heterogeneity of the two oligomeric forms of heart mitochondrial creatine kinase.

Rabbit heart mitochondrial creatine kinase has been recently shown to exist in two oligomeric forms: a dimer and an octamer, the latter being the form associated with the inner mitochondrial membrane [(1988) Biochem.Biophys. Res. Commun. 153,1310.]. We report here on the determination of the isoelectric points (pI) of the two purified forms by thin layer isoelectric focusing. The pI of the dimer is 8.2 and that of the octamer is 8.8; the former is higher by more than one pH unit than that of the cytoplasmic form MM-CK. It is proposed that the higher pI of the octamer is responsible for its binding to the inner membrane.

The radiation inactivation method provides evidence that membrane-bound mitochondrial creatine kinase is an oligomer.

Lyophilized suspensions of rabbit heart mitochondria have been irradiated with varying doses of gamma rays. Mitochondrial creatine kinase activity was inactivated exponentially with a radiation inactivation size of 352 or 377 kDa depending upon the initial medium. These values are in good agreement with the molecular mass previously deduced from by permeation experiments: 357 kDa. This is the first direct evidence showing that the native form of mitochondrial creatine kinase is associated to the inner membrane as an oligomer, very likely an octamer.

Interaction of creatine kinase and hexokinase with the mitochondrial membranes, and self-association of creatine kinase: crosslinking studies.

Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes. The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers. Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase. Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu++-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization. In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.

Only one of the two interconvertible forms of mitochondrial creatine kinase binds to heart mitoplasts.

When analyzed by cellulose acetate electrophoresis, solubilized pig or rabbit heart mitochondrial creatine kinase is shown to exist under two distinct forms. The less cathodic one (form 1) is a dimer and the other having a higher cathodic mobility (form 2) has a molecular weight of about 350,000. The latter form can be converted into the former by incubation at alkaline pH or when the enzyme forms a reactive or an abortive complex with its substrates. This conversion is a reversible phenomenon and is not due to proteolysis. When rabbit heart mitoplasts are treated with the creatine kinase releasing agents, the enzyme is always solubilized as its form 2 and conversion to form 1, when it occurs, always take place after solubilization. Form 2 is also the only form which can be bound to pig or rabbit mitoplasts. Thus form 2 may be the actual form associated with heart mitochondria in vivo.

Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts.

This paper demonstrates that the mitochondrial isoenzyme of creatine kinase (CKm) can be solubilized from rabbit heart mitochondria, the outer membrane of which has been removed or at least broken by a digitonin treatment or a short hypotonic exposure, but which has retained an important part of the capacity to phosphorylate ADP. Phosphate, ADP, or ATP, at concentrations which are used to study oxidative phosphorylation and creatine phosphate synthesis, solubilize CKm; the same is true with MgCl2 and KCl. The effect of adenine nucleotides does not seem to be due to their interaction with the adenine nucleotide translocase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that CKm is the main protein released in the described conditions; however, it does not amount to more than 1% of the total protein content of the mitoplasts. When the apparent Km for ATP of CKm was estimated by measuring creatine phosphate synthesis, the values obtained using water-treated mitochondria (0.21 mM) were slightly higher than those of intact mitochondria (0.12 mM) but the difference was not significant. In the former preparation 77% of CKm was in a soluble state. If we can extrapolate these results to intact mitochondria and suppose that in this case a fraction of CKm is also soluble in the intermembrane space, this does not support the theory of functional association between CKm and the adenine nucleotide translocase.

Effects of SH group reagents on creatine kinase interaction with the mitochondrial membrane.

Solubilization of the specific mitochondrial isoenzyme of creatine kinase (CKm) from rabbit heart mitochondria by treatment with SH group reagents has been studied. From the various compounds tested only the negatively charged organomercurials are able to induce an extensive solubilization of the enzyme. This effect is fully reversible since the solubilized enzyme readily reassociates with the membrane when the bound organomercurial is removed by treatment of the homogenate by an excess of dithiothreitol. Solubilization by negatively charged organomercurials can be partly prevented by pretreatment of mitochondria with either disulfide or uncharged organomercurials. No clear-cut relationship has been pointed out when the amount of SH titrated by various reagents has been compared with the extent of CKm solubilization. More detailed studies with para-chloromercuribenzoate (pCMB) show that extensive CKm solubilization (about 75%) occurs for pCMB concentration as low as 25 microM, whereas pronounced inhibition of the enzyme is observed only for concentrations greater than 200 microM. By cross-reassociation of enzyme solubilized either by para-hydroxymercuribenzoate (pHMB) or by 20 mM sodium phosphate (NaPi) with mitochondria depleted of CKm by pHMB or by NaPi treatment, SH groups whose titration impedes CKm reassociation with the mitochondrial membrane have been tentatively located on the enzyme. Thus, negatively charged organomercurials, could induce a reversible conformational modification of the enzyme which is no longer able to bind on the inner mitochondrial membrane. Furthermore, our results show that the binding of an excess of mitochondrial CK, which has been previously reported, could reflect unspecific binding since it occurs only on mitoplasts incubated in very hypotonic medium, but not in isotonic medium.

Carnitine metabolism in early stages of Duchenne muscular dystrophy.

Muscle carnitine deficiency was found in 12 children affected with Duchenne muscular dystrophy (DMD), the diagnosis being made at a preclinical stage or at the beginning of the clinical symptoms. Enzymatic activities related to fatty acid transport and carnitine metabolism were studied in these patients and normal subjects: palmitoyl carnitine transferase was increased, palmitoyl carnitine hydrolase was not found in the muscle, palmitoyl coenzyme A synthetase was normal and palmitoyl coenzyme A hydrolase was increased.