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. 2012 Nov 23;287(48):40598-610.
doi: 10.1074/jbc.M112.406850. Epub 2012 Oct 11.

Comprehensive mass spectrometric mapping of the hydroxylated amino acid residues of the α1(V) collagen chain

Chenxi Yang  1 , Arick C Park, Nicholas A Davis, Jason D Russell, Byoungjae Kim, David D Brand, Matthew J Lawrence, Ying Ge, Michael S Westphall, Joshua J Coon, Daniel S Greenspan
Affiliations

Comprehensive mass spectrometric mapping of the hydroxylated amino acid residues of the α1(V) collagen chain

Chenxi Yang et al. J Biol Chem. .

Abstract

Background: α1(V) is an extensively modified collagen chain important in disease.

Results: Comprehensive mapping of α1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets.

Conclusion: The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties.

Significance: Positions, numbers, and occupancy of modified sites can provide insights into α1(V) biological properties. Aberrant expression of the type V collagen α1(V) chain can underlie the connective tissue disorder classic Ehlers-Danlos syndrome, and autoimmune responses against the α1(V) chain are linked to lung transplant rejection and atherosclerosis. The α1(V) collagenous COL1 domain is thought to contain greater numbers of post-translational modifications (PTMs) than do similar domains of other fibrillar collagen chains, PTMs consisting of hydroxylated prolines and lysines, the latter of which can be glycosylated. These types of PTMs can contribute to epitopes that underlie immune responses against collagens, and the high level of PTMs may contribute to the unique biological properties of the α1(V) chain. Here we use high resolution mass spectrometry to map such PTMs in bovine placental α1(V) and human recombinant pro-α1(V) procollagen chains. Findings include the locations of those PTMs that vary and those PTMs that are invariant between these α1(V) chains from widely divergent sources. Notably, an unexpectedly large number of hydroxyproline residues were mapped to the X-positions of Gly-X-Y triplets, contrary to expectations based on previous amino acid analyses of hydrolyzed α1(V) chains from various tissues. We attribute this difference to the ability of tandem mass spectrometry coupled to nanoflow chromatographic separations to detect lower-level PTM combinations with superior sensitivity and specificity. The data are consistent with the presence of a relatively large number of 3-hydroxyproline sites with less than 100% occupancy, suggesting a previously unknown mechanism for the differential modification of α1(V) chain and type V collagen properties.

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Figures

FIGURE 1.
FIGURE 1.
Hydroxylated amino acid residues and saccharide attachments of the bovine placenta α1(V) collagen chain. Sequence coverage was 94%, with identification and mapping of 106 Y-position Hyp, 22 Gly-Hyp-Hyp X-position Hyp, 1 Gly-Hyp-Ala, 1 Gly-Hyp-Val, 3 Hyl, and 34 Glc-Gal-Hyl residues. Red, hydroxylated, non-glycosylated residues; green, Glc-Gal-Hyl residues; underlined, hydroxylated/glycosylated residues mapped in previous studies (4, 32). Sequences not identified in the course of MS analysis are in light blue. The seven COL1 amino acid residues that differ between human and bovine are orange. *, 12 Y-position Pro residues hydroxylated in human or bovine α1(V) COL1 domain but not the other. ^, Lys residue hydroxylated in human recombinant pro-α1(V) but not in bovine placenta α1(V). #, Hyl residues identified by Wu et al. (4) as being involved in interchain covalent cross-links. !, Hyp residues detected in the X-position of Gly-X-Y triplets lacking a Y-position Hyp. Amino acid residues are numbered from 1 to 1014, with residue number 1 being the first amino acid and residue number 1014 being the final amino acid residue of the COL1 triple helical domain.
FIGURE 2.
FIGURE 2.
X-position Hyp residues in Gly-X-Val and Gly-X-Ala triplets. Peptides were prepared by trypsin digestion of bovine placenta α1 (V) collagen chains. Mass error from the expected product ion monoisotopic mass is typically less than 5 ppm. y14+4OH fragment ion and internal fragment ions (PGPV-28 + 2OH, PGPV+2OH, and PGPVGA+2OH) confirm that one Hyp is in the Gly-X-Val triplet in the upper spectrum (A). y11+OH, y12+OH, and y14+2OH fragment ions confirm that one Hyp is in the Gly-X-Ala triplet in the lower spectrum (B). OH, one hydroxylation.
FIGURE 3.
FIGURE 3.
Hydroxylated amino acid residues and glycosylated Hyl residues of recombinant human pro-α1(V) collagen chains produced in 293-EBNA human embryonic kidney cells. Sequence coverage was 90% of the entire pro-α1(V) chain and 96% of the COL1 domain. Within the COL1 domain 98 Y-position Hyp, 9 Gly-Hyp-Hyp X-position Hyp, 1 Gly-Hyp-Val, 1 Gly-Hyp-Ala, 1 Gly-Hyp-Thr, 7-Hyl, and 23 Glc-Gal-Hyl residues were identified. Red, hydroxylated, non-glycosylated residues; green, Glc-Gal-Hyl residues; dark blue, Gal-Hyl residue; purple, residue found as both Glc-Gal-Hyl and Gal-Hyl. Underlined, hydroxylated/glycosylated residues mapped in previous studies (4, 32). Sequences not identified in the course of MS analysis are in light blue. The seven COL1 amino acid residues that differ between human and bovine are orange. *, 12 Y-position Pro residues hydroxylated in human or bovine α1(V) COL1 domain but not the other. #, Hyl residues identified by Wu et al. (4) as being involved in interchain covalent cross-links. ^, Lys residues hydroxylated in bovine placenta α1(V) but not in human recombinant pro-α1(V). A vertical arrow marks the site of proteolytic removal of the signal peptide, as previously determined by Edman degradation NH2-terminal amino acid sequencing (40) and as confirmed by MS analysis in the present study. The brackets indicates limits of the small COL2 hypothetical triple helical domain, in which interruptions of Gly-X-Y repeats are underlined. Amino acid residues NH2-terminal to the COL1 domain are numbered 1 to 558, starting with the initial Met residue of the signal peptide. Amino acid residues of the COL1 domain are numbered 1 to 1014, for easy comparison to the similarly numbered residues of the bovine α1(V) COL1 domain in Fig. 1. Amino acids of the COOH-propeptide are shown for the sake of completeness, but are not numbered, due to lack of identified hydroxylated residues.
FIGURE 4.
FIGURE 4.
Expression of P3H1, P3H2, P3H3, and prolyl 3-hydroxylation complex components CRTAP and peptidyl prolyl cis-trans isomerase B (PPIB) in 293-HEK cells. Expression of P3H1, P3H2, P2H3, CRTAP, and GAPDH expression was ascertained by RT-PCR analysis of RNA from MB436 cells (top panel), a cell line previously shown to be positive for expression of all three P3Hs (43), and from 293-HEK cells (bottom panel). Positions of size markers, given in base pairs (bp), are shown to the left of each panel.
FIGURE 5.
FIGURE 5.
A chimeric MS2 spectrum revealing a pair of isomeric differently modified α1(V) chain peptides that co-eluted and co-fragmented. Peptides were generated by AspN cleavage of bovine placenta α1(V) chains. MS1 in the insert was obtained by averaging MS1 across the elution window. MS2 revealed that the two separate cluster peaks in MS1 belong to peptides with identical primary sequence but to differing degrees of hydroxylation. Peptides with a total of three hydroxylations contain Hyp693, Hyp696, and Hyp705 (MS2 for peptides with a total of 3 hydroxylation in the MS1 of the inset is not shown here). MS2 for peptides with a total of four hydroxylations (shown here) reveals that the extra Hyp can be either Hyp692 or Hyp695.
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