Abstract
We have shown recently that glycosylation of threonine in the peptide Ac-(Gly-Pro-Thr)(10)-NH(2) with beta-d-galactose induces the formation of a collagen triple helix, whereas the nonglycosylated peptide does not. In this report, we present evidence that a collagen triple helix can also be formed in the Ac-(Gly-Pro-Thr)(10)-NH(2) peptide, if the proline (Pro) in the Xaa position is replaced with 4-trans-hydroxyproline (Hyp). Furthermore, replacement of Pro with Hyp in the sequence Ac-(Gly-Pro-Thr(beta-d-Gal))(10)-NH(2) increases the T(m) of the triple helix by 15.7 degrees C. It is generally believed that Hyp in the Xaa position destabilizes the triple helix because (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) form stable triple helices but the peptide (Hyp-Pro-Gly)(10) does not. Our data suggest that the destabilizing effect of Hyp relative to Pro in the Xaa position is only true in the case of (Hyp-Pro-Gly)(10). Increasing concentrations of galactose in the solvent stabilize the triple helix of Ac-(Gly-Hyp-Thr)(10)-NH(2) but to a much lesser extent than that achieved by covalently linked galactose. The data explain some of the forces governing the stability of the annelid/vestimentiferan cuticle collagens.
Highlights
The observation of di- and trisaccharides of galactose is consistent with the carbohydrate compositions of the cuticle collagens of Lumbricus terrestris and Nereis virens
For the R. pachyptila cuticle collagen, which has a low content of Pro/Hyp residues (ϳ5%), it was suggested that glycosylation of Thr with galactose must compensate for the presence of Hyp in the Xaa position to achieve the high observed thermal stability [3]
We have recently shown that substitution of Thr by Thr-O(-D-galactose) in the peptide Ac-(Gly-Pro-Thr)10-NH2 induces the formation of a collagen triple helix, with the latter peptide having essentially a random coil structure in aqueous solution [8]
Summary
The observation of di- and trisaccharides of galactose is consistent with the carbohydrate compositions of the cuticle collagens of Lumbricus terrestris (earthworm) and Nereis virens (clamworm). In vertebrates and most invertebrates, Xaa is often proline and Yaa is often 4-trans-hydroxyproline, and the thermal stability of a triple helix from a particular species increases as the total content of these imino acids increases [1]. Even though the Hyp content of the L. terrestris cuticle collagen is high (16 –18%), the thermal stability is low (Tm ϭ 22 °C) [7].
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