Effect of the ‐Gly‐3(S)‐hydroxyprolyl‐4(R)‐hydroxyprolyl‐ tripeptide unit on the stability of collagen model peptides

Abstract

The collagen triple helix is probably the most abundant protein motif in the human body. It comprises three left-handed polyproline II-like helices with a Gly-Xaa-Yaa repeat. These form a right-handed super helix with a one-residue stagger [1,2]. The collagen triple helix has many unique properties. One of them is the requirement for many post-translational modifications to produce the final tissue form of the molecule [3]. In vertebrate collagens, most of the Pro residues in the Yaa position of the -Gly-Xaa-Yaarepeat sequence are nearly completely 4-hydroxylated to 4(R)-hydroxyproline [4(R)Hyp] by the enzyme prolyl 4-hydroxylase (EC 1.14.11.2). This modification in the Yaa position is strongly related to the stability of the collagen triple helix. Prolyl 4-hydroxylation also occurs in the Xaa position in invertebrates. In addition to prolyl 4(R)-hydroxylation, a small numbers of proline residues are modified to 3(S)-hydroxyproline [3(S)Hyp] [4,5] in many types of vertebrate collagens, such as types I, II, III, IV, V and X. Invertebrate collagens also contain 3(S)Hyp, for example interstitial and cuticle collagens of annelids [6], crab sub-cuticular In order to evaluate the role of 3(S)-hydroxyproline [3(S)-Hyp] in the triple-helical structure, we produced a series of model peptides with nine tripeptide units including 0-9 3(S)-hydroxyproline residues. The sequences are H-(Gly-Pro-4(R)Hyp) l -(Gly-3(S)Hyp-4(R)Hyp) m -(Gly-Pro-4(R)Hyp) n -OH, where (l, m, n) = (9, 0, 0), (4, 1, 4), (3, 2, 4), (3,3,3), (1, 7, 1) and (0, 9, 0). All peptides showed triple-helical CD spectra at room temperature and thermal transition curves. Sedimentation equilibrium analysis showed that peptide H-(Gly-3(S)Hyp-4(R)Hyp) 9 -OH is a trimer. Differential scanning calorimetry showed that replacement of Pro residues with 3(S)Hyp residues decreased the transition enthalpy, and the transition temperature increases by 4.5°C from 52.0°C for the peptide with no 3(S)Hyp residues to 56.5°C for the peptide with nine 3(S)Hyp residues. The refolding kinetics of peptides H-(Gly-3(S)Hyp-4(R)Hyp) 9 -OH, H-(Gly-Pro-4(R)Hyp) 9 -OH and H-(Gly-4(R)Hyp-4(R)Hyp) 9 -OH were compared, and the apparent reaction orders of refolding at 10°C were n = 1.5, 1.3 and 1.2, respectively. Replacement of Pro with 3(S)Hyp or 4(R)Hyp has little effect on the refolding kinetics. This result suggests that the refolding kinetics of collagen model peptides are influenced mainly by the residue in the Yaa position of the -Gly-Xaa-Yaa-repeated sequence. The experiments indicate that replacement of a Pro residue by a 3(S)Hyp residue in the Xaa position of the -Gly-Xaa-4(R)Hyp-repeat of collagen model peptides increases the stability, mainly due to entropic factors.Abbreviations CRTAP, cartilage-associated protein; DSC, differential scanning calorimetry; Hyp, hydroxyproline; P3H1, prolyl 3-hydroxylase 1.

Prolyl 3-Hydroxylase 1 Null Mice Display Abnormalities in Fibrillar Collagen-rich Tissues Such as Tendons, Skin, and Bones

“…3-Hydroxyproline occurs at a single site in the alpha I chain of type I collagen, at proline 986 (13). The in vivo function of a single 3-hydroxyproline residue in the triple helical domain of fibrillar collagen is still unclear, however, it was recently shown that, the conversion of a Pro to 3(S)-Hyp residue in the Xaa position of a Gly-Xaa-Yaa synthetic peptide slightly increases the stability of the triple helical structure (27). Recent observations on the existence of additional conserved 3-hydroxyproline sites, sequence motif, and spacing indicate a role for 3-hydroxyproline in the ordered self assembly of collagen supramolecular structures (13).…”

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

“…Functionally, 4-Hyp residues have well characterized effects in stabilizing triple helices (16), whereas Hyl residues are important to the formation of stable covalent cross-links between collagen chains (17), and Hyl glycosylation may be important in assembly and secretion of at least some collagens (18). The function of 3-Hyp residues is unclear, with conflicting reports on small stabilizing (19) or destabilizing (20) effects on triple helix stability. However, loss of 3-Hyp residues in collagens can have catastrophic phenotypic consequences (21), implying an important biological function(s).…”

Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta

“…Hypomorphic CRTAP mutations lead to partial loss of 3-hydroxyproline (3Hyp) in fibrillar collagen, overmodification of other residues and result in recessive OI type VII, which clinically overlaps with dominant forms 2 . The physiological function of 3Hyp is incompletely understood, but biochemical and genetic studies suggest that it is involved in collagen-protein interactions and required for normal bone mineralization 6–7 .…”