Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen

Abstract

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet. 10, e1004465). However, the extent of decrease appears to be tissue-and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation.Collagens comprise a large family of structurally related extracellular matrix proteins (1). Among all of the genetic types of collagen identified, fibrillar type I collagen is the most abundant, providing tissues and organs with form and stability. It is a heterotrimeric molecule composed of two ␣1 chains and one ␣2 chain forming a long uninterrupted triple helix with short non-helical domains (telopeptide) at both N and C termini. One of the functionally important characteristics of collagen is its unique, sequential post-translational modifications of Lys residues. The modifications include hydroxylation and monoand diglycosylation of hydroxylysine (Hyl), 3 and oxidative deamination of Lys and Hyl in the N-and C-telopeptides followed by extensive covalent intermolecular cross-linking (2). It is now clear that defective Lys modifications of collagen cause and/or are associated with a broad range of connective tissue disorders, including Ehlers-Danlos syndrome type VIA (3), bronchopulmonary dysplasia (4), Kuskokwim syndrome (5), Bruck syndrome (6, 7), fibrosis (8), disuse osteoporosis (9), and cancer progression (10, 11). Our recent finding showed that a switch of collage...

FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the lysyl hydroxylase 2-driven collagen cross-link switch

“…In contrast, it has been reported that the PPIase activity of FKBP65 is not involved in the modulation of the coacervation and in vitro self-assembly of human tropoelastin3940, indicating that FKBP65 regulates different biologic processes through different mechanisms. Recently, we reported that the PPIase cyclophilin B interacts with all LH1-3 and FKBP65, and that loss of cyclophilin B in mice leads to enhanced LH2 activity generating HLCCs in tendon type 1 collagen41. These together with the current findings strongly indicate an intricate interplay of these ER-resident PPIases with specific LH isoforms, thus, tightly controlling this important post-translational modification42.…”

“…For instance, the specific and dynamic cross-linking pattern in bone/dentin type 1 collagen is important to regulate the process of collagen mineralization45464748. However, the tissue-specific cross-linking pattern cannot be explained solely by the respective LH gene expression41. Likely, these multifaceted post-translational control mechanisms of LH function are, in part, an important contributor to the cell- and tissue-specific collagen cross-linking.…”

“…Immunoprecipitation experiments were performed as described previously41. Briefly, 293F cells were transfected with LH2-3Flag and FKBP65-HA (WT and deletion mutant) plasmids using FuGENE 6 transfection reagent (Promega).…”

P3h3-null and Sc65-null Mice Phenocopy the Collagen Lysine Under-hydroxylation and Cross-linking Abnormality of Ehlers-Danlos Syndrome Type VIA

“…The requirement of CypB for collagen post-translational modification became clear from the effects of PPIB mutations that cause severe osteogenesis imperfecta. They result in loss of prolyl-hydroxylation at Pro-986 (27) and, in CypB knock-out mice, defective lysine hydroxylation in bone (28) and tendon (29) collagens. Furthermore, we know that FKBP65, another peptidyl prolyl isomerase and ER chaperone, can regulate the activity of LH2 the predicted telopeptide lysine hydroxylase (12,30,31).…”

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

“…CypB is an ER-resident peptidyl-prolyl cis-trans isomerase and was the first molecule shown to be associated with LH1 [46]. The absence of CypB or disruption of the interaction with LH1 results in the reduction of lysyl hydroxylation in tendon and skin [31, 32], while the amount of lysyl hydroxylation is increased in bone [47]. Additionally, there is a site-specific effect to the level of lysyl hydroxylation in tendon, skin and bone.…”