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. 2021 May 7:10:100067.
doi: 10.1016/j.mbplus.2021.100067. eCollection 2021 Jun.

In-depth correlation analysis demonstrates that 4-hydroxyproline at the Yaa position of Gly-Xaa-Yaa repeats dominantly stabilizes collagen triple helix

Yuki Taga  1 , Keisuke Tanaka  1 , Shunji Hattori  1 , Kazunori Mizuno  1
Affiliations

In-depth correlation analysis demonstrates that 4-hydroxyproline at the Yaa position of Gly-Xaa-Yaa repeats dominantly stabilizes collagen triple helix

Yuki Taga et al. Matrix Biol Plus. .

Abstract

There is a general consensus that collagen stability is largely maintained by Pro and its major hydroxylated form, 4-hydroxyproline (4Hyp). However, positional difference in their stabilizing effect at the Xaa or Yaa position of collagenous Gly-Xaa-Yaa sequences has remained inconclusive. Here, we position-specifically evaluated the correlation of imino acid contents to denaturation temperature (Td) of collagen among various vertebrate and invertebrate species, using a recently developed LC-MS methodology. 4Hyp at the Yaa position showed the highest positive correlation with Td, followed by Pro at the Xaa position, which was even further increased by excluding invertebrates. We confirmed that Gly-Pro-4Hyp liberated after bacterial collagenase digestion was highly positively correlated with Td. Furthermore, other tripeptides with Yaa position 4Hyp also had comparable positive correlation, excepting negative correlation of Gly-Gly-4Hyp, while tripeptides with Xaa position Pro did not. These data provide evidence that 4Hyp dominantly contributes to thermal stability of collagen depending on its sequence position, especially in vertebrates.

Keywords: APDS, 3-aminopyridyl-N-hydroxysuccinimidyl carbamate; Collagen; Denaturation temperature; Hydroxyproline; Hyp, hydroxyproline; MRM, multiple reaction monitoring; Td, denaturation temperature; Thermal stability.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Correlation analysis between total imino acid contents and Td in collagens from various species. Correlation with Td was analyzed for total contents of (A) Pro, (B) Hyp, and (C) Pro + Hyp. Pearson’s correlation coefficient (r) and statistical significance (p) are shown. Values in parentheses are calculated by excluding invertebrates.
Fig. 2
Fig. 2
Correlation analysis between position-specific imino acid contents and Td in collagens from various species. Correlation with Td was analyzed for (A and C) 4Hyp, (B) 3Hyp, and (D and E) Pro at the (A, B, and D) Xaa or (C and E) Yaa positions. Pearson’s correlation coefficient (r) and statistical significance (p) are shown. Values in parentheses are calculated by excluding invertebrates.
Fig. 3
Fig. 3
Correlation analysis between amounts of collagenase-liberated tripeptides and Td in collagens from various species. Correlation with Td was analyzed for (A) Gly-Pro-4Hyp, (B) all Gly-Xaa-4Hyp tripeptides (Gly-Ala-4Hyp + Gly-Glu-4Hyp + Gly-Leu-4Hyp), (C) all Gly-Pro-Yaa tripeptides (Gly-Pro-Ala + Gly-Pro-Arg + Gly-Pro-Gln), (D) all Gly-4Hyp-Yaa tripeptides (Gly-4Hyp-Ala + Gly-4Hyp-Gly), and (E) Gly-Gly-4Hyp. Pearson’s correlation coefficient (r) and statistical significance (p) are shown. Values in parentheses are calculated by excluding invertebrates.
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