Mark J. Hackett, Graham N. George, Ingrid J. Pickering, and B. Frank Eames

(ACS) – Proteoglycans (PGs) are heavily glycosylated proteins that play a critical role in both development and disease. During development of the vertebrate skeleton, for example, abundant PG secretion in the extracellular matrix provides function to both cartilage and bone.1 Defects in PG synthesis and homeostasis can lead to skeletal disease. A main feature of the joint disease osteoarthritis is loss of PGs in cartilage matrix.2 PG function is regulated by sulfation, during which sulfates are added to specific sugar residues as esters. Sulfation of PGs is important for at least two biological roles of cartilage matrix that are relevant to defects in osteoarthritis patients: tissue integrity and growth factor signaling.2,3 The amount of PG sulfation in cartilage matrix is thought to be directly proportional to how much water it absorbs, which determines the compression-resistant strength of cartilage.4 In addition to this structural role, PG sulfation influences binding affinities (and thus signaling activity) of growth factors and is required for normal growth factor signaling in cartilage matrix.5,6 In fact, many of the studies of the signaling role of cartilage PGs originate from genetic perturbations to PG sulfation pathways.7−12 Despite the importance of sulfated PGs, methods for their visualization are quite limited. Histological stains, such as Alcian blue or Safranin O, can reflect PG sulfation; however, the mechanisms through which the dye binds are unclear and can be nonspecific.13 Moreover, although antibodies have been generated against sulfated PGs, these may be too specific as an antibody might sense only one isomer of a particular sulfated PG.14 We present herein methods for direct visualization of sulfur chemical forms, and specifically sulfate esters, in situ within developing cartilage matrix through the use of chemically specific X-ray fluorescence imaging (XFI).15

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