Age-related type I collagen modifications reveal tissue-defining differences between ligament and tendon

Abstract

Tendons and ligaments tend to be pooled into a single category as dense elastic bands of collagenous connective tissue. They do have many similar properties, for example both tissues are flexible cords of fibrous tissue that join bone to either muscle or bone. Tendons and ligaments are both prone to degenerate and rupture with only limited capacity to heal, although tendons tend to heal faster than ligaments. Type I collagen constitutes about 80% of the dry weight of tendons and ligaments and is principally responsible for the core strength of each tissue. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including proline and lysine hydroxylation, hydroxylysine glycosylation and covalent cross-linking. The chemistry, placement and quantity of intramolecular and intermolecular cross-links are believed to be key contributors to the tissue-specific variations in material strength and biological properties of collagens. As tendons and ligaments grow and develop, the collagen cross-links are known to chemically mature, strengthen and change in profile. Accordingly, changes in cross-linking and other post-translational modifications are likely associated with tissue development and degeneration. Using mass spectrometry, we have compared tendon and ligaments from fetal and adult bovine knee joints to investigate changes in collagen post-translational properties. Although hydroxylation levels at the type I collagen helical cross-linking lysine residues were similar in all adult tissues, ligaments had significantly higher levels of glycosylation at these sites compared to tendon. Differences in lysine hydroxylation were also found between the tissues at the telopeptide cross-linking sites. Total collagen cross-linking analysis, including mature trivalent cross-links and immature divalent cross-links, revealed unique cross-linking profiles between tendon and ligament tissues. Tendons were found to have a significantly higher frequency of smaller diameter collagen fibrils compared with ligament, which we suspect is functionally associated with the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important to improving the design of orthopedic treatment approaches.

Keywords: ACL, Anterior cruciate ligament; Collagen; Cross-linking; DHLNL, dehydrohydroxylysinonorleucine; HHL, histidinohydroxylysinonorleucine; HHMD, histidinohydroxymerodesmosine; HLNL, hydroxylysinonorleucine; HP, hydroxylysine pyridinoline; LC, liquid chromatography; LCL, lateral collateral ligament; LP, lysine pyridinoline; Ligament; MCL, medial collateral ligament; MS, mass spectrometry; Mass spectrometry; P3H1, prolyl 3-hydroxylase 1; P3H2, prolyl 3-hydroxylase 2; PCL, posterior cruciate ligament; Post-translational modifications; QT, quadriceps tendon; Tendon.

Fig. 1

SDS-PAGE reveals a decrease in collagen extractability in adult tendons and ligaments compared to fetal tissue. Acid labile aldimine cross-links are broken with mild acetic acid treatment, allowing native type I collagen monomers to be extracted from the tissue. Collagen was more acid extractable from fetal tissues (A) than adult tissues (B). Anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), lateral collateral ligament (LCL), medial collateral ligament (MCL), quadriceps tendon (QT). SDS-PAGE sample loads were normalized to the dry weight of lyophilized tissues. The reduction in collagen extractability from adult tissues is consistent with an increase in mature collagen cross-links with development.

Fig. 2

Type I collagen from ligaments exhibits age-dependent 3-hydroxyproline profile. LC-MS profiles of in-gel trypsin digests of the collagen α1(I) chain from fetal and adult bovine ACL. (A) MS profile of fetal bovine ACL shows only partial 3-hydroxylation (~8%) at the α1(I) C-terminal GPP motif; (B) MS profile at the same site of adult bovine ACL shows a hydroxylation like bovine tendon 3-hydroxylation (~60%). The trypsin digested peptide is shown with P^# indicating 3Hyp, P* indicating 4Hyp and K* indicating Hyl. See Table 1 for more details.

Fig. 3

Post-translational variances in linear cross-linking α1(I)K87 and α2(I)87 from type I collagens. (A, B) LC-MS profiles of in-gel trypsin digests of the collagen α1(I) chain from fetal and adult bovine tendon. (A) Fetal bovine tendon shows complete glycosylation of α1(I)K87 (613.3³⁺). (B) The α1(I)K87 residue from adult bovine tendon is hydroxylated but not subsequently glycosylated (505.2³⁺). The trypsin-digested peptide is shown with P* indicating 4Hyp, M* indicating oxidized methionine. Summary of modifications on lysine 87 from (C) α1(I) and (D) α2(I) chains of type I collagen. Lysine modifications include unmodified lysine (Lys), hydroxylysine (Hyl), galactosyl-hydroxylysine (G-Hyl) and glucosylgalactosyl-hydroxylysine (GG-Hyl). The percentages were determined based on the ratios between the ion-current yields of each post-translational variant as previously described. See Table 2 for more details.

Fig. 4

Increased hydroxylation of the linear C-telopeptide cross-linking lysine residue in adult ligaments. The C-telopeptide cross-linking lysine of type I collagen is a useful predictor of cross-linking quality across tissues. Percent hydroxylation is calculated from tryptic peptides using mass spectrometry (n = 3). Note that lysine hydroxylation in adult QT is significantly less than adult ligaments; * p < 0.05 by t-test assuming unequal variance.

Fig. 5

Ligament type I collagen has elevated N-telopeptide cross-linking lysine hydroxylation compared to tendon. LC-MS profiles of collagenase-digested adult ACL (A), Fetal ACL (B), Adult QT (C) and Fetal QT (D). (A) Adult ligaments had the highest level of hydroxylysine (~1015²⁺) at the N-telopeptide cross-linking site of all tissues tested. A small population of peptide containing hydroxylysine + oxidized methionine (~1023²⁺) was observed in adult tissues.

Fig. 6

Increased pyridinoline cross-linking with increased tissue maturity. Concentration of hydroxylysine pyridinoline cross-linking residues in fetal and adult bovine tendon and ligaments expressed as moles/mole of collagen (n = 3). (HP, hydroxylysine pyridinoline). None of the tissues contained measurable levels of LP (lysine pyridinoline). *p < 0.05 by t-test assuming unequal variance.

Fig. 7

Tissue pyrrole content determined from Ehrlich reagent color change. Bovine tissues were minced and incubated in Ehrlich’s reagent for 5 min. No color change was detected in fetal tendon (n/d). The color reactions (n = 2) were measured using online color detection software.

Fig. 8

Transmission electron microscopy of collagen fibrils from bovine ligament and tendon. Representative transmission electron microscopy images showing slight distinctions between collagen fibrils between ligament (ACL) and tendon (QT) (A). Scale bar is 100 nm. Collagen fibril diameter plot illustrates bimodal distribution in both tissues (B). Fibril density, mean fibril area and fibril area fraction graphs illustrate several subtle distinctions between the tissues (C).