Age-Related Type I Collagen Modifications Reveal Tissue-Defining Differences Between Ligament and Tendon

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, however; healing outcomes for tendons are often faster than ligament injuries. 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 propose is a consequence of the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important for designing improved orthopedic treatment approaches.

Re-evaluation of lysyl hydroxylation in the collagen triple helix: lysyl hydroxylase 1 and prolyl 3-hydroxylase 3 have site-differential and collagen type-dependent roles in lysine hydroxylation

Collagen is the most abundant protein in humans and is heavily post-translationally modified. Its biosynthesis is very complex and requires three different types of hydroxylation (two for proline and one for lysine) that are generated in the rough endoplasmic reticulum (rER). These processes involve many enzymes and chaperones which were collectively termed the molecular ensemble for collagen biosynthesis. However, the function of some of the proteins in this molecular ensemble is controversial. While prolyl 3-hydroxylase 1 and 2 (P3H1, P3H2) are bona fide collagen prolyl 3-hydroxylases, the function of prolyl 3-hydroxylase 3 (P3H3) is less clear. A recent study of P3H3 null mice demonstrated that this enzyme had no activity as prolyl 3-hydroxylase but may instead act as a chaperone for lysyl hydroxylase 1 (LH1). LH1 is required to generate hydroxylysine for crosslinking within collagen triple helical sequences. If P3H3 is a LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should have similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared lysyl hydroxylation in type I and V collagen from P3H3 and LH1 null mice. Our results indicate LH1 plays a global role for lysyl hydroxylation in triple helical domain of type I collagen while P3H3 is indeed involved in lysyl hydroxylation particularly at crosslink formation sites but is not required for all lysyl hydroxylation sites in type I collagen triple helix. Furthermore, although type V collagen from LH1 null mice surprisingly contained as much hydroxylysine as type V collagen from wild type, the amount of hydroxylysine in type V collagen was clearly suppressed in P3H3 null mice. In summary, our study suggests that P3H3 and LH1 likely have two distinct mechanisms to distinguish crosslink formation sites from other sites in type I collagen and to recognize different collagen types in the rER.Author summaryCollagen is one of the most heavily post-translationally modified proteins in the human body and its post-translational modifications provide biological functions to collagen molecules. In collagen post-translational modifications, crosslink formation on a collagen triple helix adds important biomechanical properties to the collagen fibrils and is mediated by hydroxylation of very specific lysine residues. LH1 and P3H3 show the similar role in lysine hydroxylation for specific residues at crosslink formation sites of type I collagen. Conversely, they have very distinct rules in lysine hydroxylation at other residues in type I collagen triple helix. Furthermore, they demonstrate preferential recognition and modification of different collagen types. Our findings provide a better understanding of the individual functions of LH1 and P3H3 in the rER and also offer new directions for the mechanism of lysyl hydroxylation followed by crosslink formation in different tissues and collagens.

Discovery of Lysine Hydroxylases in the Clavaminic Acid Synthase-Like Superfamily for Efficient Hydroxylysine Bioproduction

ABSTRACT Hydroxylation via C—H bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available l-lysine with l-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two l-lysine 3S-hydroxylases and four l-lysine 4R-hydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereoselective hydroxylation into either C-3 or C-4 positions of l-lysine, yielding (2S,3S)-3-hydroxylysine and (2S,4R)-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant l-lysine hydroxylase-expressing Escherichia coli cells were used as a biocatalyst for l-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2S,3S)-3-hydroxylysine was produced from 600 mM l-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2S,4R)-4-hydroxylysine was produced from 300 mM l-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies. IMPORTANCE The present study identified six l-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of l-lysine hydroxylases was relatively narrow, we found that (2S,3S)-3-hydroxylysine was hydroxylated by 4R-hydroxylase and (2S,5R)-5-hydroxylysine was hydroxylated by both 3S- and 4R-hydroxylases. Moreover, the l-arginine hydroxylase VioC also hydroxylated l-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of l-lysine into (2S,3S)-3-hydroxylysine and (2S,4R)-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic l-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.

Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer.

Distinct Characteristics of Mandibular Bone Collagen Relative to Long Bone Collagen: Relevance to Clinical Dentistry

Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw.