Decoding the comprehensive substrate-specificity and evidence of altered site-specific collagen prolyl-3-hydroxylation, lysyl-hydroxylation, and lysyl O-glycosylation in P4ha1 and P4ha2 deleted mutant mice

Question

1. How does deletion of prolyl 4-hydroxylases affect collagen deposition in mouse skin?

2. How many PTM sites on Col1a1, Col1a2, and Col3a1 chains were identified in wild-type mice skin?

3. What is the most common motif for 4-hydroxyproline in collagen 1?

4. What is the site-specificity of P4ha1 and P4ha2 in fibrillar collagens?

Accepted Answer

Deletion of prolyl 4-hydroxylases adversely affects collagen deposition in the extracellular matrix (ECM) of mouse skin. The relative label-free quantitation of collagen 1 chains (Col1a1 and Col1a2) revealed a significant decrease in collagen amount in mice with complete deletion of P4ha2. Partial deletion of P4ha1 and P4ha2 also resulted in lower levels of collagen 1. Complete deletion of P4ha2 led to the lowest amount of collagen 1 chains deposited in the skin, indicating a potential decrease in collagen 1 biosynthesis. Additionally, deletion of P4ha2 resulted in an elevation of about 34% in the level of Col3a1 compared to wild-type mice.

Accepted Answer

In our analysis, we identified a total of 421 site-specific collagen PTMs on Col1a1, Col1a2, and Col3a1 chains extracted from wild-type mice skin. Col1a1 was found to be the most modified, while Col1a2 was the least modified among the three fibrillar collagen chains. These PTMs include 160 site-specific PTMs on Col1a1, 124 PTMs on Col1a2, and 137 PTMs on Col3a1. The PTMs include 106 4-hydroxyproline sites, 89 4-hydroxyproline sites on Col1a2, 112 4-hydroxyproline sites on Col3a1, 12 5-hydroxylysine sites on Col3a1, and 1 glucosyl-galactosyl-hydroxylysine site on Col3a1. These PTMs play a crucial role in the structure and function of collagen in the extracellular matrix (ECM) of wild-type mice skin.

Accepted Answer

The most common motif for 4-hydroxyproline in collagen 1 is the -Pro-Pro-Gly-motif. In the proteomics analysis, a total of 35 4-hydroxyproline sites on this motif were identified. Other motifs with detected 4-hydroxyproline sites include -Leu-Pro-Gly-motif (12 sites), -Arg-Pro-Gly-motif (5 sites), -Ser-Pro-Gly-motif (11 sites), -Gln-Pro-Gly-motif (2 sites), and -Val-Pro-Gly-motif (2 sites). Additionally, 1 site each was detected on the -Gly-Pro-Gly-motif, -Lys-Pro-Gly-, -Met-Pro-Gly-, -Asn-Pro-Gly-motif, and -Thr-Pro-Gly-motif. These findings validate the initial findings on collagen prolyl 4hydroxylase motif-specific activity.

Accepted Answer

The site-specificity of P4ha1 and P4ha2 in fibrillar collagens has not been fully explored. However, studies have shown that P3H1 has three specific sites in collagen I, which are majorly modified by P3H1. Similarly, P3H2 has site-specificity in collagen IV. To identify the specific sites of P4ha1 and P4ha2 in mice skin fibrillar collagen chains, researchers quantified the 4-HyP occupancy levels in wild-type and different C-P4h deletion mutants. This research aims to understand the site-specificity of P4ha1 and P4ha2 in fibrillar collagens, which could provide valuable insights into collagen modification and its implications in various biological processes.

Accepted Answer

P4ha1 is the predominant collagen modifying enzyme, ubiquitously expressed in all tissues. It plays a crucial role in the site-specificity of 4-hydroxylation on fibrillar collagen chains. The complete deletion of P4ha1 results in embryonically lethal mice, while a partial deletion retains the enzyme's activity. Quantitative analysis revealed that 23 sites on fibrillar collagen chains (Col1a1, Col1a2, and Col3a1) are fully (>=99%) 4-hydroxylated in wild-type and P4ha1 (+/-) and P4ha2 mutants. These sites are considered P4ha1-specific, highlighting the enzyme's importance in maintaining collagen structural stability and functioning in the skin extracellular matrix (ECM).

Accepted Answer

Promiscuous sites on fibrillar collagen chains are specific locations where different isoforms of prolyl-4-hydroxylases (P4ha1, P4ha2, and P4ha3) can catalyze the 4-hydroxylation reaction. These sites allow for cross-reactivity between the isoforms, enabling them to perform the same reaction. In total, four 4-HyP sites were identified on fibrillar collagen chains, where catalysis can occur by both P4ha1 and P4ha2 isoforms. The level of P4ha3 is usually lower in wild-type tissues, but it can be elevated upon partial deletion of P4ha1 and P4ha2 in mice. The site-specific activity of P4ha3 upon P4ha1 and P4ha2 deletion has not been documented yet. Site-specific quantitation of 4-hydroxyproline occupancy in mice skin fibrillar collagen chains revealed decreased occupancy on eight sites upon partial deletion of P4ha1 or P4ha2, but the levels were either similar to wild-type or slightly elevated upon complete deletion of P4ha2. Four sites showed decreased 4-hydroxyproline occupancy upon partial deletion of P4ha1 or P4ha2, but elevated levels in P4ha1+/+; P4ha2-/- and P4ha1+/-; P4ha2-/- mutants. These promiscuous sites are important for understanding the cross-reactivity and compensatory mechanisms between the different P4ha isoforms.

Accepted Answer

The deletion of P4ha1 and P4ha2 significantly increases the 3-hydroxyproline occupancy on Col1a1 P874 and Col1a2 P803. In wild-type mice skin, Col1a1 P874 and Col1a2 P803 had 5.7% and 13.55% occupancy respectively. However, these sites were not detected in mice skin previously by Pokidysheva et.al. in 2013. Partial deletion of P4ha1 and full deletion of P4ha2 resulted in increased 3-hydroxyproline occupancy on these sites. Complete deletion of P4ha2 also enhanced site-specific prolyl 3hydroxylation on classical sites of P3h1 on collagen 1. On-site Col1a1 P1153, there was a significant decrease in 3-hydroxyproline occupancy in P4ha1+/-; P4ha2+/- mice, but an increase in complete deletion mutants of P4ha2 mice.

Accepted Answer

The correlation analysis shows that as the 4-hydroxylation occupancy of 4-HyP 1154 decreases in P4ha1 +/-; P4ha2 +/- mice, the occupancy of the 3-hydroxylation site on P1153 also decreases. However, in the double allele deletion mutant of P4ha2, the 3-hydroxylation level increases. This suggests that the presence of 4-hydroxylation on the Yaa position facilitates the recruitment of 3-hydroxylation in the Xaa position in the repetitive sequence in collagen I chains. Therefore, 4-HyP occupancy directly affects the 3-HyP site on collagen 1 alpha 1.

Accepted Answer

The study shows that partial deletion of P4ha1 and full deletion of P4ha2 significantly increase the hydroxylysine level in Col1a2 N-terminal helical cross-linking site K183. Similarly, an elevation in hydroxylysine level is observed in non-cross-linking Col1a1 K731 and Col1a2 K315 sites. The occupancy level of galactosyl-hydroxylysine is also elevated upon partial deletion of P4ha1 and complete deletion of P4ha2. These findings suggest that prolyl 4-hydroxylases may crosstalk with the hydroxylation levels of helical cross-linking lysine sites and non-cross-linking lysine sites in collagen 1.

Accepted Answer

Collagen-modifying enzymes play a crucial role in collagen biosynthesis by heavily modifying newly translated collagen chains in a site-specific manner. These enzymes are responsible for the post-translational modifications of collagen, which are essential for the proper formation and function of collagen in the extracellular matrix (ECM). The modified collagen chains then form a triple helix structure, which is transported to the ECM. In the ECM, terminal proteinases cleave the propeptides of the collagen triple helix, allowing for cross-linking and the formation of collagen fibre assembly. This process is vital for the structural integrity and mechanical properties of tissues, as collagen is a major component of the ECM. Overall, collagen-modifying enzymes ensure the proper biosynthesis and deposition of collagen in the ECM, contributing to the maintenance of tissue structure and function.

Decoding the comprehensive substrate-specificity and evidence of altered site-specific collagen prolyl-3-hydroxylation, lysyl-hydroxylation, and lysyl O-glycosylation in P4ha1 and P4ha2 deleted mutant mice

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Most frequently asked questions

1. How does deletion of prolyl 4-hydroxylases affect collagen deposition in mouse skin?

2. How many PTM sites on Col1a1, Col1a2, and Col3a1 chains were identified in wild-type mice skin?

3. What is the most common motif for 4-hydroxyproline in collagen 1?

4. What is the site-specificity of P4ha1 and P4ha2 in fibrillar collagens?

5. What is the significance of P4ha1 in fibrillar collagens?

6. What are promiscuous sites on fibrillar collagen chains?

7. How does P4ha1 and P4ha2 deletion affect 3-hydroxylation occupancy on Col1a1 and Col1a2?

8. How does 4-HyP occupancy affect 3-HyP site on collagen 1 alpha 1?

9. How do P4ha1 and P4ha2 deletions affect lysyl-hydroxylation in collagen?

10. What is the role of collagen-modifying enzymes in collagen biosynthesis?

References

Skyline: an open source document editor for creating and analyzing targeted proteomics experiments

The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.

Epithelial-mesenchymal transition and its implications for fibrosis.

Collagens—structure, function, and biosynthesis

The Collagen Family

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