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. 2011 Mar 11;286(10):7732-7736.
doi: 10.1074/jbc.C110.195768. Epub 2011 Jan 14.

A novel 3-hydroxyproline (3Hyp)-rich motif marks the triple-helical C terminus of tendon type I collagen

David R Eyre  1 , MaryAnn Weis  2 , David M Hudson  2 , Jiann-Jiu Wu  2 , Lammy Kim  2
Affiliations

A novel 3-hydroxyproline (3Hyp)-rich motif marks the triple-helical C terminus of tendon type I collagen

David R Eyre et al. J Biol Chem. .

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 α1(I) and α2(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 α1(I) and α2(I) chains but only one 3Hyp residue in skin α1(I) and none in α2(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 tissue-specific manner.

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Figures

FIGURE 1.
FIGURE 1.
Tandem mass spectrometry of tryptic peptides from the α1(I) chain reveals a 3Hyp repeat in the C-terminal sequence from the triple-helical domain of tendon but not skin or bone type I collagen. a, the three collagen preparations were run on SDS-PAGE for in-gel trypsin digestion of the excised α1(I) chains. Also identified in a are the principal β dimer components common to all three tissues and a slower β11 band prominent in extracts of skin collagen (see “Discussion”). b, the parent ion ladders given by the C-terminal tryptic peptide from the α-chain of each tissue. These full scan mass spectra are taken from the tryptic peptide LC/MS profile of each α-chain scrolling across an elution window that combines all post-translational variants of this peptide. c, the MS/MS fragmentation spectrum of the parent ion (1876.32+) of the tendon peptide with three additional hydroxyls on X position prolines. The sequence is shown with b and y ion breakages that establish the proline residues bearing the additional 16-dalton masses. P#, 3Hyp; P*, 4Hyp.
FIGURE 2.
FIGURE 2.
Tandem mass spectrometry of tryptic peptides from the α2(I) chain reveals a similar 3Hyp ladder to that in α1(I) for tendon collagen but not skin or bone. The α2(I) chains were resolved as shown in Fig. 1, cut out, and subjected to in-gel trypsin digestion and LC/MS analysis. a, the parent ion ladder from the C-terminal tryptic peptide of rat tail tendon α2-chain is shown, and below it is the MS/MS fragmentation profile from the ion bearing three additional 16-dalton masses on Y position prolines. The position of predicted 3Hyp residues was determined from the b and y ion masses as indicated for this variant and also for the other variant parent ions in the ladder. The sequence determined matches that in the Ensembl rat genomic database (ENSRNOT00000016423). b, MS results from the α2(I) chain of mature bovine Achilles tendon show a similar +16-dalton series of molecular ions for this peptide. The bovine Achilles α1(I) chain gave a similar ladder (data not shown). P#, 3Hyp; P*, 4Hyp.
FIGURE 3.
FIGURE 3.
Molecular location of the novel 3Hyp repeat at the C terminus of the triple-helix in relation to known 3Hyp sites. The axial positioning of the three classes of 3Hyp that can be defined by their sequence context is shown in the upper procollagen molecule. How these are positioned relative to each other in a fibril of D-staggered molecules is shown below. Sequences of the C-terminal GPP repeat domain for all clade A and clade B collagen α-chains from the human genome (Ensembl and NCBI genomic databases) are aligned for comparison and to put the rat sequences studied here in a broader context.
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