Crystal structure of the N-terminal NC4 domain of collagen IX, a zinc binding member of the laminin-neurexin-sex hormone binding globulin (LNS) domain family.
ABSTRACT Collagen IX, located on the surface of collagen fibrils, is crucial for cartilage integrity and stability. The N-terminal NC4 domain of the alpha1(IX) chain is probably important in this because it interacts with various macromolecules such as proteoglycans and cartilage oligomeric matrix protein. At least 17 distinct collagen polypeptides carry an NC4-like unit near their N terminus, but this report, describing the crystal structure of NC4 at 1.8-A resolution, represents the first atomic level structure for these domains. The structure is similar to previously characterized laminin-neurexin-sex hormone binding globulin (LNS) structures, dominated by an antiparallel beta-sheet sandwich. In addition, a zinc ion was found in a position similar to that of the metal binding site of other LNS domains. A partial backbone NMR assignment of NC4 was obtained and utilized in NMR titration studies to investigate the zinc binding in solution state and to quantitate the affinity of metal binding. The K(d) of 11.5 mM suggests a regulatory rather than a structural role for zinc in solution. NMR titration with a heparin tetrasaccharide revealed the presence of a secondary binding site for heparin on NC4, showing structural and functional conservation with thrombospondin-1, but a markedly reduced affinity for the ligand. Also the overall arrangement of the N and C termini of NC4 resembles most closely the N-terminal domain of thrombospondin-1, distinguishing the two from the majority of the published LNS structures.
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ABSTRACT: Although the pericellular matrix (PCM) plays a central role in the communication between chondrocytes and extracellular matrix, its composition is largely unknown. In this study, the PCM was investigated with a proteomic approach using chondrons, which are enzymatically isolated constructs including the chondrocyte and its surrounding PCM. Chondrons and chondrocytes alone were isolated from human articular cartilage. Proteins extracted from chondrons and chondrocytes were used for two-dimensional electrophoresis. Protein spots were quantitatively compared between chondron and chondrocyte gels. Cellular proteins, which had similar density between chondron and chondrocyte gels, did not proceed for analysis. Since chondrons only differ from chondrocytes in association of the PCM, protein spots in the chondron gels that had higher quantity than that in the chondrocyte gels were selected as candidates of the PCM components and processed for mass spectrometry. Among 15 identified peptides, several were fragments of the three type VI collagen chains (α-1, α-2, and α-3). Other identified PCM proteins included triosephosphate isomerase, transforming growth factor-β induced protein, peroxiredoxin-4, ADAM (A disintegrin and metalloproteinases) 28, and latent-transforming growth factor beta-binding protein-2. These PCM components were verified with immunohisto(cyto)chemistry for localization in the PCM region of articular cartilage. The abundance of type VI collagen in the PCM emphasizes its importance to the microenvironment of chondrocytes. Several proteins were localized in the PCM of chondrocytes for the first time and that warrants further investigation for their functions in cartilage biology.Histochemie 06/2011; 136(2):153-62. · 2.61 Impact Factor
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ABSTRACT: Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families. The C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains. Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.BMC Evolutionary Biology 12/2008; 8:316. · 3.29 Impact Factor
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ABSTRACT: Cartilage plays an essential role during skeletal development within the growth plate and in articular joint function. Interactions between the collagen fibrils and other extracellular matrix molecules maintain structural integrity of cartilage, orchestrate complex dynamic events during embryonic development, and help to regulate fibrillogenesis. To increase our understanding of these events, affinity chromatography and liquid chromatography/tandem mass spectrometry were used to identify proteins that interact with the collagen fibril surface via the amino terminal domain of collagen α1(XI) a protein domain that is displayed at the surface of heterotypic collagen fibrils of cartilage. Proteins extracted from fetal bovine cartilage using homogenization in high ionic strength buffer were selected based on affinity for the amino terminal noncollagenous domain of collagen α1(XI). MS was used to determine the amino acid sequence of tryptic fragments for protein identification. Extracellular matrix molecules and cellular proteins that were identified as interacting with the amino terminal domain of collagen α1(XI) directly or indirectly, included proteoglycans, collagens, and matricellular molecules, some of which also play a role in fibrillogenesis, while others are known to function in the maintenance of tissue integrity. Characterization of these molecular interactions will provide a more thorough understanding of how the extracellular matrix molecules of cartilage interact and what role collagen XI plays in the process of fibrillogenesis and maintenance of tissue integrity. Such information will aid tissue engineering and cartilage regeneration efforts to treat cartilage tissue damage and degeneration.Proteomics 12/2011; 11(24):4660-76. · 4.43 Impact Factor