Structural support within the body is provided by extracellular matrices (ECMs). In many instances, connective tissue disorders result from defective ECMs. Since the proteoglycans, collagens and other glycoproteins of the ECM are biosynthetically complex and interact specifically to assemble the ECM, sequential processing which occurs during their movement through specific compartments of the constitutive secretory pathway is critically important for normal ECM maturation and function. The proposed studies are based on the hypothesis that some connective tissue disorders result from errors in the synthesis, processing or trafficking of ECM constituents such as aggrecan, the large aggregating proteoglycan of cartilage. The postulate that chondrodystrophies are the consequence of abnormal changes or defects in aggrecan precursors or the cellular processing machinery and a failure to assemble the normal cartilage ECM will be tested. Studies conducted during the previous funding period demonstrate that the genetic mutation nanomelia is one example of such an aggrecan defect. This premise will be investigated by the transfection of cartilage and non-cartilage cells with genetic constructs of normal and modified aggrecan domains in order to establish requirements for the synthesis, processing and trafficking of aggrecan that reflect inherent properties of the aggrecan molecule itself, and others that may reflect the chondrocyte cytoplasmic environment. In a second approach, human chondrodystrophies such as pseudoachondroplasia will be characterized from the perspective of processing or trafficking errors. Studies of this nature are now feasible, due to recent developments in the culture of human chondrocytes and the availability of patient samples. The analysis of potential processing defects will follow the line of investigation that led to the elucidation of the nanomelia defect. A baseline for aggrecan synthesis, processing and trafficking will be established in normal human chondrocytes. Cases involving potential processing or trafficking errors will be prescreened for low levels of cell-associated alcian blue staining (indicating a proteoglycan defect) and ultrastructural features that suggest deviations from the normal arrangement of intracellular organelles of the secretory pathway (e.g., distended ER). Chondrocytes from patients that exhibit either of these characteristics will be studied further. Expertise gained from the in-depth characterization of aggrecan synthesis, processing and trafficking in normal chondrocytes will be applied to determine the cell biological and biochemical nature of the human defects. Insights gained from these studies will contribute to an understanding of the mechanistic basis of cartilage disease and the requirements for normal ECM formation, and may have broader implications for other connective tissue disorders and the understanding of other proteoglycans, ECM molecules, and membrane-transported proteins in general.
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