162 Collagen II R992C mutation leads to unfolded protein response Hye Jin Chung, Andrzej Steplewski, Deborah Jensen, Katarzyna Gawron, Andrzej Fertala Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, United States Single amino acid substitutions in collagen II are associated with spondyloepiphyseal dysplasia. These mutations cause alterations of the structure of individual collagen molecules, their fibrillar assembly and affect the ability of collagen molecules to properly interact with extracellular matrix components. Recently we demonstrated that certain mutations not only affect the structure of mutant molecules but also induce intracellular accumulation of mutant proteins which can lead to apoptosis. Here we analyzed the effects of R992C on collagen II thermostability, the presence of intramolecular disulfide bonds and cell response to the presence of the mutant molecules. We demonstrated that R992C alters the melting profile of the collagen triple helix, introduces atypical disulfide bonds, decreases the rate of secretion of mutant molecules and increases intracellular accumulation. Expression of mutant collagen II in a chondrocytic cell line demonstrated that these cells undergo apoptosis in response to the presence of mutant collagen II. Biochemical and microscopic assays have shown that apoptotic changes were associated with processes typical of the unfolded protein response in cells expressing R992C. Overall our study suggests that mutations in collagen II associated with atypical unfolding of the triple-helical domain of collagen may trigger a cascade of intracellular events cumulating in apoptosis. We postulate that the described changes are part of the pathomechanism of heritable disorders of cartilage caused by mutations in collagen II. doi:10.1016/j.matbio.2008.09.377 163 P16 and P19; mineralization-related Echinoderm phosphoproteins Keith Alvares, Saryu Dixit, Elizabeth Lux, Arthur Veis Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University. Chicago, IL. 60611, United States Acidic proteins of similar character have been implicated in mineral deposition in vertebrate bones and teeth and invertebrate tissues such as shells, spicules and teeth. To explore the relationships between the vertebrate and invertebrate proteins, we have focused on the mineral- related proteins of the sea urchin teeth L. varigatus, which have a rich cellular component, and a magnesium-rich calcite mineral phase. The mature mineralized portion of 100 teeth was solubilized in 0.6N HCl, after an initial 6.0 M Gdn-HCl extraction. The extracted proteins were separated by SDS polyacrylamide gel electrophoresis, and by passage over a CHT ceramic hydroxyapatite column, separating distinct apatite binding and non-binding fractions. The most prominent component in each fraction was purified, digested with trypsin, fractionated by HPLC and peptide peaks sequenced by automated Edman degradation. The peptides shared homol- ogy with predicted proteins P16 and P19, previously described from the sea urchin spicules by mRNA isolation. The full-length protein sequences were cloned from our L. varigatus tooth cDNA library. P19, a Glu-rich intracellular protein, did not bind to 45 Ca or apatite while P16 bound to 45 Ca and hydroxyapatite and had a transmembrane sequence with a dentin phosphophoryn-like Ser-Asp-rich domain in the extracellular space. Both proteins were phosphorylated in vivo. Although both proteins were occluded in the mineral phase they appear to have distinct functions relative to the mineralization process. doi:10.1016/j.matbio.2008.09.378 164 Distinct OI phenotype caused by COL1 C-proteinase site mutations A.M. Barnes a , K. Lindahl b , M. Whyte c , T. Hefferan d , C.-J. Rubin b , A. Kindmark b , W. McAlister c , S. Mumm c , Ö. Ljunggren b , J.C. Marini a a BEMB, NICHD/NIH, Bethesda, MD, United States b Deparment of Endo, Med Sci, Uppsala Univ, Sweden c Shriner's Hosp for Children, St. Louis, MO, United States d Department of Orthop, Mayo Clinic, Rochester, MN, United States Osteogenesis imperfecta (OI) is often caused by mutations in the type I collagen genes. Mutations in the type I procollagen C-propeptide cleavage site are of interest because they disrupt a processing step. We identified two children with mild OI who had cleavage site mutations in COL1A1 (P1: α1(I) D1041N) or COL1A2 (P2: α2(I)A1029T). P1 DEXA z-score and pQCT vBMD were +3, contrasting with radiographs demonstrating osteopenia and os-in- os vertebrae, and histomorphometry revealing increased bone remodeling, without a mineralization defect or signs of osteosclerosis. P2 had a DEXA z-score of 0, gracile long bones with radiographic osteopenia, and decreased BV/TV and increased BFR without a mineralization defect on histomorpho- metry. Steady-state collagen electrophoresis showed slight backstreaking of α1(I) and α2(I) in cell layers of both probands, with a slight baseline delay in P1. Chain incorporation was normal in P1 and slightly delayed in P2. Pericellular processing of P1 was delayed, with increases in both pCα1 and proα2, while P2 had increased pCα2 and proα2 and normal processing kinetics. Together with an adult with an α1(I)A1040T substitution (Int Conn Tis 82S1: CC01), our cases suggest that defects in proα1(I) processing lead to high childhood BMD, with signs of osteopetrosis occurring subsequently. Proα1(I) cleavage appears crucial to C-propeptide processing, while defective proα2(I) cleavage occurs after α1(I) processing. doi:10.1016/j.matbio.2008.09.379 165 Molecular packing structure of native type II collagen Olga Antipova a,b , Raul Barrea a,b , Joseph P. Orgel a,b a Illinois Institute of Technology, Chicago IL, United States b BioCAT, APS, Argonne National Lab, IL, United States The naturally crystalline arrangement of collagen molecules in fibrils found in some tissues, allows the use of fiber diffraction methods for structural characterization. This alternative biophysical method has the potential to give structural information about collagen type II with minimum interference from sample preparation and may give the opportunity to produce relatively detailed three-dimensional visualization of the fibrils sub-structure. Towards this end, experiments with Multiple Isomorphous Replacement (MIR) were carried out to so that a one- dimensional electron density map of native collagen structure may be determined. Several experiments were performed at the BioCAT facility at Argonne National Laboratory with variations of: sample holder designs, sample preparation procedures, heavy atoms for MIR, temperatures and setups for small and medium angle diffraction. Some more optimum combinations of these produced data of resolution 15 Å or better in the axial direction. This study revealed that the parameters of collagen type II fibrils from lamprey notochord are very similar if not the same as collagen type II fibrils in mammalian tissues: 30 nm in diameter, axial periodicity of 67 nm, amino acid distribution is the same. Analysis of the one dimensional electron density map showed that the telopeptides, which are crucial for fibrillogenesis and organization of collagen type II tissues, have a very specific folded conformation, reminiscent of that seen in the C-telopeptide of type I collagen. This type of structural information is essential for understanding the mechanisms of development and disease pathologies. doi:10.1016/j.matbio.2008.09.380 Abstracts S49