Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro 986 in A-clade chains ␣1(I), ␣1(II), and ␣2(V). Two partially modified sites (A2 and A3) were found at Pro 944 in ␣1(II) and ␣2(V) and Pro 707 in ␣2(I) and ␣2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro 470 in ␣2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian ␣1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.Collagens are the most abundant and ubiquitous proteins in multi-cellular animals. It is well established that 4-hydroxyproline (4Hyp) 2 residues stabilize the collagen triple helix through water-bridged intramolecular hydrogen bonding (1). However, the function of the much less abundant 3-hydroxyproline (3Hyp), although discovered 50 years ago, is unknown (2). Only 1-2 residues of 3Hyp occur per chain in collagen types I and II and 3-6 residues occur per chain in collagen types V and XI. The content is highest in type IV collagens of basement membranes in which 10% of the total hydroxyproline can be 3Hyp (3).Specific prolyl 3-hydroxylases (P3Hs) are responsible for 3Hyp synthesis. Three different genes encoding P3H1, P3H2, and P3H3 are present in the human genome, which show tissue specificity in their expression (4, 5). Substrate proline residues occur in a prerequisite sequence -Pro-4Hyp-Gly. The ␣1(I) chain has only one established 3Hyp site at Pro 986 in a motif conserved across vertebrate species (human GLPGPIGPPGPR) a close variant of which also occurs in type II collagen (human GIPGPIGPPGPR).Renewed interest in 3Hyp was recently sparked by the discovery that a recessive form of osteogenesis imperfecta (OI) is caused by mutations in CRTAP. This gene encodes a protein (cartilage-associated protein) that is bound to P3H1 and cyclophilin B in the endoplasmic reticulum and is requi...

As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes.

Because of its unique physical and chemical properties, rat tail tendon collagen has long been favored for crystallographic and biochemical studies of fibril structure. In studies of the distribution of 3-hydroxyproline in type I collagen of rat bone, skin, and tail tendon by mass spectrometry, the repeating sequences of Gly-Pro-Pro (GPP) triplets at the C terminus of ␣1(I) and ␣2(I) chains were shown to be heavily 3-hydroxylated in tendon but not in skin and bone. By isolating the tryptic peptides and subjecting them to Edman sequence analysis, the presence of repeating 3-hydroxyprolines in consecutive GPP triplets adjacent to 4-hydroxyproline was confirmed as a unique feature of the tendon collagen. A 1960s study by Piez et al. (Piez, K. A., Eigner, E. A., and Lewis, M. S. (1963) Biochemistry 2, 58 -66) in which they compared the amino acid compositions of rat skin and tail tendon type I collagen chains indeed showed 3-4 residues of 3Hyp in tendon ␣1(I) and ␣2(I) chains but only one 3Hyp residue in skin ␣1(I) and none in ␣2(I). The present work therefore confirms this difference and localizes the additional 3Hyp to the GPP repeat at the C terminus of the triple-helix. We speculate on the significance in terms of a potential function in contributing to the unique assembly mechanism and molecular packing in tendon collagen fibrils and on mechanisms that could regulate 3-hydroxylation at this novel substrate site in a tissuespecific manner.Prolyl 3-hydroxylation, a long recognized quantitatively minor post-translational modification of collagen (1), has received much attention in the last few years after gene mutations affecting its formation were found to cause recessive forms of osteogenesis imperfecta (2-5). A single primary site of 3-hydroxyproline (3Hyp) 2 is present in normal collagen ␣1(I) and ␣1(II) chains at Pro-986 of the triple-helix (6, 7) but is not hydroxylated in the tissues of mice and humans with recessive osteogenesis imperfecta caused by mutations in CRTAP or LEPRE1 (the gene encoding P3H1) (2-5). The LEPRE1 gene encodes P3H1, which is one of three prolyl 3-hydroxylases (P3H1, P3H2, and P3H3) in the mammalian genome. CRTAP encodes a protein that is homologous to the N-terminal half of P3H1, which it associates with P3H1 together with cyclophilin B to form the functional enzyme complex required for Pro-986 3-hydroxylation of unfolded collagen chains in the endoplasmic reticulum (8).We recently identified several other sites of prolyl 3-hydroxylation in fibrillar collagen chains including Pro-707 in ␣2(I) and ␣2(V), Pro-944 in ␣1(II) and ␣2(V), Pro-470 in ␣2(V), and Pro-434, Pro-665, and Pro-692 in ␣1(V), ␣1(XI), and ␣2(XI) but a lack of any 3-hydroxyproline in the mammalian ␣1(III) chain (7). The D-periodic spacing between three of these additional sites in clade A collagen chains (␣1(I), ␣2(I), ␣1(II), and ␣2(V)) and between two in clade B (␣1(V), ␣1(XI), and ␣2(XI)) suggested a role in fibril formation. In pursuing this concept further, we investigated the potential for differences in 3-hydrox...