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Browse Space| Structure and stability of the triple helix in function and pathology of collagen |
Makareeva, Mertz, DeRidder, Sutter, Leikin; in collaboration with Barnes, Cabral, Marini, McBride
| Type I collagen is a triple-helical protein that forms the stable matrix of bone, skin, and other tissues. Nevertheless, we found that the equilibrium state of collagen as well as its procollagen precursor at body temperature is a random coil rather than a triple helix. Even in a crowded environment mimicking the endoplasmic reticulum (ER), procollagen triple helix folding occurs only below 35°C. Apparently, cells need specialized chaperones to fold procollagen within the ER. Once procollagen is sRecreted from cells, it begins to unfold. Local weakening of its least stable regions and concurrent cleavage of N- and C-terminal propeptides appear to trigger self-assembly of fibers. In fibers, collagen helices are protected from complete unfolding, but local unfolding/refolding of the triple helix still occurs and plays an important physiological role. Over 80 percent of moderately severe to lethal collagen mutations are substitutions of a single glycine in the (Gly-X-Y)n sequence of the triple helix. By disrupting the stability and consequently the folding of the triple helix, the mutations lead to abnormal collagen production, secretion, and fibrillogenesis. Our study revealed that the structural region within which the substitution is located—rather than the type of substituting residue—determines the effect of a glycine substitution on collagen folding and stability. We found evidence for at least six such structural regions within the triple helix. Distinct changes in the folding and structure of the triple helix caused by mutations within different regions seem to correlate with variations in disease phenotype. For instance, we recently established that mutations within the N-terminal (N-anchor) domain lead to a distinct combination of osteogenesis imperfecta and Ehlers-Danlos syndrome. Over the last year, we continued to delineate the structural regions of type I collagen. We expanded the map of changes in the collagen melting temperature (ΔTm) to 36 distinct Gly substitutions. To relate the ΔTm map to peptide-based stability predictions, we proposed a model for extracting the activation energy of local helix unfolding from the reported peptide data. We tested the model and determined its parameters by measuring the hydrogen-deuterium exchange rate for the glycine amino residues involved in interchain hydrogen bonds in collagen. A comparison of the unfolding activation energy (ΔG‡) map and the ΔTm map permitted us to refine the boundaries of the structural regions. The refined regions appear to align with regions important for collagen fibril assembly and ligand binding as reported in the literature. The refined regions also align with distinct OI phenotypes caused by Gly substitutions in the a1(I) chain. The lack of similar correlations in the a2(I) chain corroborates the notion that the loss of helical stability may be less important for OI phenotypes of a2(I) substitutions than initially thought. |
Flor-Cisneros A, Leikin S. Osteogenesis imperfecta. In: Glew RH, Rosenthal MD, eds. Clinical Studies in Medical Biochemistry. Oxford University Press, 2006;30-41.
Makareeva E, Cabral WA, Marini JC, Leikin S. Molecular mechanism of a1(I)-OI/EDS: unfolding of an N-anchor domain at the N-terminal end of the type I collagen triple helix. J Biol Chem 2006;281:6463-70.
Interactions of collagen fibrils with other matrix proteins DeRidder, Sutter, Makareeva, Leikin; in collaboration with Forlino, Marini, Nagase, Tenni, Visse
| Interactions of collagen with ligands, particularly other extracellular matrix proteins and proteoglycans, are believed to be an important factor in connective tissue diseases. In tissue, ligands interact primarily with collagen fibrils rather than with triple helical monomers. We recently developed a new confocal microscopy assay with differential fluorescent labeling that allows us to visualize and quantitatively measure binding of various matrix proteins to individual collagen fibrils under physiological conditions. We found that MMP1 (matrix metalloprotease 1) attacks damaged and poorly assembled rat tail-tendon collagen fibers via preferential binding to microfibrils that become more exposed at fiber defects. Given that MMP1 is responsible for remodeling type I collagen fibers, such preferential binding may be physiologically important. In another set of experiments, we demonstrated high reproducibility of the confocal microscopy assay for measurement of recombinant decorin binding to human collagen fibrils. Over the last year, we focused on competitive binding experiments between fluorescently labeled and unlabeled decorin. The resultant measurements revealed an enhancement of the binding by fluorescent labels and demonstrated that the enhancement depends on the number of attached fluorophores. We therefore developed a protocol for characterization of and correction for the fluorophore effects. We are currently conducting systematic measurements of interactions between collagen fibers and matrix proteins potentially important in OI and other connective tissue disorders. |
Type I collagen homotrimers and their role in osteoporosis, cancer, OI, and EDS; Han, Makareeva, DeRidder, Sutter, Leikin in collaboration with Byers, McBride, Pace
| Normal type I collagen is a heterotrimer of one a2(I) and two a1(I) chains; formation of a1(I) homotrimers, on the other hand, has been implicated in several cases of OI and EDS and observed in cultures of breast cancer cells. It has also been suggested that type I homotrimers play at least some role in age-related osteoporosis. A polymorphism in an SP1 promoter region resulting in increased production of the a1(I) chain was recently linked to increased predisposition to osteoporosis in the general population. It was proposed that such predisposition might be caused by a1(I) homotrimers comprising about 10 to 15 percent of all newly synthesized type I collagen in affected individuals. Despite their potential importance, the molecular mechanisms of pathology associated with the homotrimers remain unclear. Our previous studies revealed increased thermal stability, altered domain structure, and reduced intermolecular attraction resulting in higher solubility and abnormal fibrillogenesis of murine a1(I) homotrimers. Last year, we characterized in more detail human homotrimers produced by dermal fibroblasts from a patient with Ehlers-Danlos syndrome caused by the lack of the a2(I) chain synthesis. Our most important discovery was a reduced rate of homotrimer cleavage by tissue collagenases (rhMMP-1 and rhMMP-13)—about 10 to 20 percent of that seen in heterotrimeric type I collagen. The resulting cleavage rate misbalance may have important implications for remodeling tissues with mixed-collagen composition, e.g., heterotypic fibrils containing type I homo- and heterotrimers or type I homotrimers and type III collagen. More detailed examination suggested that the initial MMP binding to collagen is not affected by the lack of the a2(I) chain but that the chain is essential for the next step of triple helix unwinding and opening that precedes cleavage. We believe that the abnormal homotrimer cleavage rate may play an important role in various pathologies associated with homotrimer synthesis. We maintain that better understanding of the underlying molecular mechanism may help in the development of new strategies for treatments. |
Translational studies of patients with novel/unusual OI mutations; Makareeva, DeRidder, Sutter, Leikin; in collaboration with Barnes, Cabral, Marini
| To gain further insight into molecular mechanisms of bone pathology associated with abnormal folding, function, and interactions of collagens, we continued our collaboration with clinical researchers in the NICHD's Bone and Extracellular Matrix Branch to characterize the pathology in patients with unusual OI mutations and/or phenotypes. Our goal was to find and characterize molecular abnormalities of collagen from these patients. Previously, such studies revealed, for example, the molecular mechanism of OI/EDS caused by mutations within the N-anchor domain of the triple helix. More recently, we suggested a possible molecular origin of pathology in patients with a familial R888C substitution in the a1(I) chain and an unusual combination of OI and EDS symptoms. We completed a study of post-translational modification, thermal stability, and folding of collagen and procollagen from patients with severe/lethal skeletal disorders reminiscent of osteogenesis imperfecta caused by null mutations in cartilage-associated protein (CRTAP) or prolyl-3-hydrohylase (P3H1), not by mutations in collagen. The CRTAP and P3H1 form a tight three-protein complex in ER, which also involves cyclophilin B. Disruption of the complex significantly delays type I procollagen folding, resulting in overhydroxylation and overglycosylation of Lys residues, which is also observed in many OI cases. P3H1 is responsible for 3-hydroxylation of Pro986 in the a1(I) chain, which CRTAP or P3H1 null mutations prevent. However, our measurements suggested that the lack of the Pro986 hydroxylation may not be the primary cause of the delayed procollagen folding and may not be the underlying cause of the severe/lethal symptoms. Instead, we believe that the CRTAP and P3H1 may be essential for retaining cyclophilin B within ER and that the prolyl isomerase activity of cyclophilin B is essential for procollagen folding. Several research groups are testing such a hypothesis. |
Barnes AM, Chang W, Morello R, Cabral WA, Weis M, Eyre DR, Leikin S, Makareeva E, Kuznetsova N, Uveges TE, Ashok A, Flor AW, Mulvihill JJ, Wilson PL, Sundaram UT, Lee B, Marini JC. Deficiency of cartilage-associated protein in recessive lethal osteogenesis imperfecta. N Engl J Med 2006;355:2757-64.
Cabral WA, Chang W, Barnes AM, Weis M, Scott MA, Leikin S, Makareeva E, Kuznetsova NV, Rosenbaum KN, Tifft CJ, Bulas DI, Kozma C, Smith PA, Eyre DR, Marini JC. Prolyl 3-hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling lethal/severe osteogenesis imperfecta. Nat Genet 2007;39:359-65.
Cabral WA, Makareeva E, Colige A, Letocha AD, Ty JM, Yeowell HN, Pals G, Leikin S, Marini JC. Mutations near amino end of a1(I) collagen cause combined OI/EDS by interference with N-propeptide processing. J Biol Chem 2005;280:19259-69.
Cabral WA, Makareeva E, Letocha AD, Scribanu N, Fertala A, Steplewski A, Keene DR, Persikov AV, Leikin S, Marini JC. Y-position cysteine substitution in type I collagen (alpha1(I) R888C/p.R1066C) is associated with osteogenesis imperfecta/Ehlers-Danlos syndrome phenotype. Hum Mutat 2007;28:396-405.
| Insights into OI mechanisms from murine models |
Mertz, Han, Leikin; in collaboration with Forlino, Marini, McBride, Uveges
| Murine OI models offer unique opportunities for more systematic studies of the molecular mechanism of pathology in animals with the same mutation but different genetic background, age, sex, and so forth. We currently work with all three existing murine models: the oim mouse with nonfunctional a2(I) chains, the Brtl IV mouse with a knockin G349C substitution in the a1(I) chain, and a new model with a knockin G610C substitution in the a2(I) chain. Our previous studies revealed several changes in the physical and chemical properties of collagen from oim and G610C animals, including altered thermal stability (higher in oim and lower in G610C) and abnormal collagen-collagen interactions. However, the Brtl IV animals, with no such abnormalities in type I collagen, exhibit more severe bone phenotype. The most important molecular abnormality that we discovered in the latter animals is selective retention and intracellular degradation of molecules with a single mutant chain. The retention of the mutant collagen within ER produces visible swelling of ER cisternae, potentially resulting in ER stress and malfunction of collagen-producing cells, including osteoblasts. To improve our understanding of how such retention might result in bone failure in the Brtl IV animals, we continued to characterize the structure and composition of the animals' bone by infra-red microspectroscopy and reflection microscopy. It appears that non-lamellar layers produced during initial, faster growth of the long bones in mutant animals display abnormal collagen and mineral organization, which might be responsible for bone brittleness. These findings appear consistent with (1) the idea of more severe cellular malfunction associated with faster collagen production and (2) the observed gradual normalization of bone strength in post-pubertal animals. However, more studies are needed to verify such a hypothesis. |
Forlino A, Kuznetsova NV, Marini JC, Leikin S. Selective retention and degradation of molecules with a single mutant alpha1(I) chain in the Brtl IV mouse model of OI. Matrix Biol 2007;26:604-14.
| High-definition imaging infrared micro-spectroscopy of cartilage in mice with normal and impaired diastrophic dysplasia sulfate transporter |
Mertz; in collaboration with Forlino, Lupi, Rossi
| We recently developed high-definition infrared micro-spectroscopy for label-free imaging of thin, solvated tissue sections with significantly increased chemical resolution and spectral reproducibility. This new technique resolves different glucosaminoglycan (GAG) types and the extent of their sulfation and distinguishes between collagen and other proteins. During the last year, we applied the new technology to study a mouse model of diastrophic dysplasia based on a knockedin homozygous mutation in SLC26A2 sulfate/chloride antiporter. We measured quantitative, 5 µm-resolution distributions of major extracellular matrix components across femur head cartilage of wild-type (WT) and mutant newborn mice. We found that the extent of GAG sulfation increased toward the femur head center both in the mutant and WT. In the mutant, the GAGs were 2.3-fold undersulfated at the articular surface but nearly normal in the head center. This normalization may be caused by faster degradation of undersulfated GAGs, increased intracellular sulfate as a result of GAG catabolism, or a slower rate of GAG synthesis. The mutation also affected the concentrations and spatial distributions of other extracellular matrix components. Sugar groups were nearly uniformly distributed in WT but depleted near the articular surface in the mutant. Concentration of non-collagenous proteins was 1.8-fold lower in the mutant and gradually decreased (1.5-fold) from the articular surface toward the femur head center in both genotypes. Collagen concentration in WT also gradually decreased (2-fold) toward the femur head center but remained nearly uniform across the mutant's femur head, probably because of delayed development. At the articular surface, collagen concentration in the mutant was about 1.5-fold lower than in WT. The lower densities of collagen, GAGs, and sugar sulfate may be responsible for lowering mutant cartilage elasticity and increasing its permeability to synovial enzymes, thereby contributing to the observed cartilage degradation in diastrophic dysplasia. |
Mertz EL. Anomalous microscopic dielectric response of dipolar solvents and water. J Phys Chem A 2005;109:44-56.
United States Patent
Patent pending: Flow-through, inlet-gas-temperature-controlled, solvent-resistant, thermal-expansion compensated cell for light spectroscopy. Mertz EL, Sullivan JV.
| DNA interactions; new insights from classical diffraction patterns |
Leikin; in collaboration with Baldwin, Brooks, Kornyshev, Lee, Seddon, Wynveen
| Interactions between DNA play an important role in packaging genetic material inside cells and viruses and in many other fundamentally important biological processes. Earlier, we found that these interactions are intimately related to the finer details of the molecular structure of DNA, particularly to the helical nature of its surface charge pattern. Our theory of these interactions suggested explanations for the observed counterion specificity of DNA condensation, DNA overwinding from 10.5 base pairs (bp) per helical turn in solution to 10.0 bp per turn in aggregates, nontrivial cholesteric pitch behavior upon compression of DNA aggregates, subsequent transition from the cholesteric to hexagonal (hexatic) phase, and several quasi-crystalline phases of even more densely packed DNA aggregates. One of the most important and yet controversial predictions based on our theory was that electrostatic interactions might contribute to sequence homology recognition and pairing of intact DNA double helices observed before genetic recombination. Last year, we completed the first set of experiments probing the recognition between intact, duplex DNA in vitro. We imaged a mixture of two fluorescently tagged, double-helical DNA molecules with identical nucleotide composition and length (50 percent GC; 294 bp) but different sequences. In electrolytic solution at minor osmotic stress, the DNAs formed discrete liquid-crystalline aggregates (spherulites). We observed spontaneous segregation of the two types of DNA within each spherulite, which, consistent with our predictions, revealed that nucleotide sequence recognition occurred between double helices separated by water in the absence of proteins. |
Kornyshev AA, Lee DJ, Leikin S, Wynveen A. Structure and interactions of biological helices. Rev Mod Phys 2007;79:943-96.
Kornyshev AA, Lee DJ, Leikin S, Wynveen A, Zimmerman SB. Direct observation of azimuthal correlations between DNA in hydrated aggregates. Phys Rev Lett 2005;95:148102.
1 Brown University Medical School, Providence , RI