Identification of 4-Hydroxyproline at the Xaa Position in Collagen by Mass Spectrometry

Abstract

Collagen has a triple helix form, structured by a [-Gly-Xaa-Yaa-] repetition, where Xaa and Yaa are amino acids. This repeating unit can be post-translationally modified by enzymes, where proline is often hydroxylated into hydroxyproline (Hyp). Two Hyp isomers occur in collagen: 4-hydroxyproline (4Hyp, Gly-Xaa-Pro, substrate for 4-prolyl hydroxylase) and 3-hydroxyproline (3Hyp, Gly-Pro-4Hyp, substrate for 3-prolyl hydroxylase). If 4Hyp is lacking at the Yaa position, then Pro at the Xaa position should remain unmodified. Nevertheless, in literature 41 positions have been described where Hyp occurs at the Xaa position (?xHyp) lacking an adjacent 4Hyp. We report four additional positions in liver and colorectal liver metastasis tissue (CRLM). We studied the sequence commonalities between the 45 known positions of ?xHyp. Alanine and glutamine were frequently present adjacent to ?xHyp. We showed that proline, position 584 in COL1A2, had a lower rate of modification in CRLM than in healthy liver. The isomeric identity of ?xHyp, that is, 3- and/or 4Hyp, remains unknown. We present a proof of principle identification of ?xHyp. This identification is based on liquid chromatography retention time differences and mass spectrometry using ETD-HCD fragmentation, complemented by ab initio calculations. Both techniques identify ?xHyp at position 584 in COL1A2 as 4-hydroxyproline (4xHyp).

Collagen analysis with mass spectrometry

“…With the use of MS it proved possible to establish that the PTM was either 3‐hydroxyproline or 4‐hydroxyproline, as shown by Kassel and Biemann () in mussel adhesive proteins. However, van Huizen et al () recently obtained, with MS, the proof of principle that hydroxyproline at the Xaa position (amino acid position 584) in COL1A2 is 4‐hydroxyproline. The authors suggested naming this new PTM “4xHyp.” The “x” differentiates between the Xaa and Yaa position.…”

“…GLH‐3Hyp and GLH‐4Hyp are distinguishable by singly charged fragments at m / z 400 and 454. Reprinted with permission from van Huizen et al () ACS Publications. [Color figure can be viewed at wileyonlinelibrary.com]…”

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

“…Mass spectrometric approaches with protease digestion have been increasingly used for comprehensive identification of prolyl hydroxylation sites in collagen 16 , 17 , 18 , 19 , 20 , 21 , but it is difficult to totally evaluate the degree of the modification with discrimination of the sequence position. Recently, we developed a novel method to quantitatively analyze positional distribution of Pro and Hyp by LC–MS with partial acid hydrolysis [3] .…”

Down-Regulation of Collagen Hydroxylation in Colorectal Liver Metastasis

“…Normally, 4Hyp at the Yaa-position is formed by 4-prolyl hydroxylase, which requires Gly-Xaa-Pro as substrate (2). 4-hydroxyproline rarely occurs at the Xaa positions (4xHyp), neither the enzyme, nor the substrate required for the formation of 4xHyp is known (3). 3Hyp is formed by 3-prolyl hydroxylase, which requires Gly-Pro-4Hyp as substrate (4).…”

“…3Hyp and 4Hyp are involved in triple helix stability and fiber formation, but their specific functions are still not fully understood ( 5 – 7 ). The function of 4xHyp remains unknown ( 3 ). Lysine can be modified into 5-hydroxylysine (5Hyl) by lysyl hydroxylases ( 8 ).…”