A Novel 3-Hydroxyproline (3Hyp)-rich Motif Marks the Triple-helical C Terminus of Tendon Type I Collagen

Abstract

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...

Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

“…The spatial arrangement of these sites, which are staggered by a D-period, suggested a fundamental role for these modifications in the supramolecular assembly by forming hydrogen bonds between adjacent collagen triple helices (20,21). In rat tail tendon, a 3-Hyp-rich motif was found at the C-terminal end of the triple helix (22).…”

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

“…They also predicted the tissue-specific occurrence of 3-Hyp residues within Gly-Pro-Pro repeats at the COOH termini of the COL1 domains of ␣1(V) and other fibrillar collagen chains, based on mapping of such residues to Gly-Pro-Pro repeats at the COL1 COOH termini of ␣1(I) and ␣2(I) chains from tendon but not from skin or bone (30). Here, we confirm the presence of X-position Hyp residues at sites 434, 665, and 695 and in all three COL1 COOH-terminal Gly-Pro-Pro repeats (sites 1004, 1007, and 1010) in bovine placenta ␣1(V) chains (supplemental Spectra 1), whereas human recombinant pro-␣1(V) chains contained an X-position Hyp residue only at site 434.…”

“…Recent analyses of collagenous proteins by MS relied heavily on a priori biological knowledge to assess prolyl hydroxylation (i.e. PTM assignment based upon hydroxylation motifs) (30,32) rather than PTM assignment based upon localizing fragments from MS n spectra. We report comprehensive mapping of all PTMs involving hydroxylated residues on bovine placenta ␣1(V) and human recombinant pro-␣1(V) collagen chains and provide manually verified mass spectral evidence for each modified site.…”

Murine tendon function is adversely affected by aggrecan accumulation due to the knockout of ADAMTS5

“…17 Chondroitin Sulfate/Dermatan Sulfate (CS/DS) and Hyaluronan (HA) Contents GAGs were solubilized and prepared for FACE analyses. 18 Briefly, four FDLs were combined and digested with proteinase or 0.1 M NaOH, GAGs desalted by MicroCon3 filtration, digested with chondroitinase ABC prior to fluorotagging with 2-aminoacridone/sodium cyanoborohydride followed by electrophoretic separation of disaccharide products.…”