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

Abstract

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.collagen cross-linking | lysyl hydroxylase | FKBP65 | Bruck syndrome | fibrosis C ollagen I is an essential component of the extracellular matrix (ECM) of tissues such as bone and skin, and is involved in a wide variety of biological processes. A deregulated synthesis of collagen type I results in pathologies ranging from severe bone and skin anomalies to fibrosis (1-5). Hydroxylation of specific lysine (Lys) residues into 5-hydroxylysine (Hyl) is performed by lysyl hydroxylases (LHs), also known as the procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) family. Collagens deposited in the ECM are stabilized by the formation of intermolecular crosslinks by members of the lysyl oxidase family, LOX and LOXL (6). Two collagen cross-linking pathways have been identified, the allysine route and the hydroxyallysine route (7). In the allysine route, a telopeptide Lys is oxidized by lysyl oxidases into an aldehyde; in the hydroxyallysine route, this occurs with a telopeptide Hyl. In turn, the reactive aldehydes interact with the Lys or Hyl residues in the helical part of collagen to form difunctional and finally trifunctional cross-links (8-10). The trifunctional cross-links derived from the hydroxyallysine route are referred to as pyridinolines. Collagens cross-linked by means of pyridinolines are difficult to degrade (11-13).The LH family consists of three individual members, designated LH1, LH2, and LH3. Hydroxylation of Lys present...

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

“…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).…”

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

“…During the preparation of this manuscript, two groups independently reported that FKBP65 does indeed interact with LH2 by forming a protein complex2132. One of these studies21 showed that FKBP65 interacts with LH2, but not LH1 or LH3, and is critical for the dimerization and function of LH2.…”

“…One of these studies21 showed that FKBP65 interacts with LH2, but not LH1 or LH3, and is critical for the dimerization and function of LH2. This explains why helical Lys hydroxylation is not significantly affected in bone type 1 collagen in Bruck syndrome 1 and 2 as reported previously153033 and in our study (Fig.…”

“…Very recently, Gjaltema et al . reported that FKBP65 forms a complex with LH2, but not LH1 or LH3, and promotes LH2 dimerization, which is crucial for LH2 enzymatic activity21. However, the molecular mechanism by which FKBP65 regulates LH2 activity and its functional outcomes, i.e.…”

Genetic causes and mechanisms of Osteogenesis Imperfecta

“…Due to the similarities in phenotypic features, cellular localization and the lack of intrinsic lysyl-hydroxylase function of FKBP65, it was speculated that FKBP65 and LH2 may need to form a complex to exert enzymatic function [2,12]. Indeed, a recent study has shown that physical interaction between FKBP65 and LH2 promotes dimerization and activation of LH2 in cell culture [64]. This interaction was specific to LH2 as FKBP65 did not bind with either LH1 or LH3, corroborating the observation that loss of Fkbp10 does not reduce helical lysyl hydroxylation [63,64].…”