Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2 Rutger A. F. Gjaltemaa,b,1, Miesje M. van der Stoela, Miriam Boersemaa,2, and Ruud A. Banka,1,2 aMatrix Accumulation, Tissue Repair, and Inflammation Research Group, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; and bEpigenetic Editing Research Group, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands Edited by Darwin J. Prockop, Texas A&M Health Science Center, Temple, TX, and approved May 16, 2016 (received for review January 7, 2016) Collagens are subjected to extensive posttranslational modifications, telopeptides of collagen, where such a sequence motif is not present such as lysine hydroxylation. Bruck syndrome (BS) is a connective (16–18). LH2 consists of two splice variants, LH2A and LH2B (19), tissue disorder characterized at the molecular level by a loss of with LH2B containing an extra exon known as 13A. Although both telopeptide lysine hydroxylation, resulting in reduced collagen isoforms contain the same dioxygenase catalytic domain responsible pyridinoline cross-linking. BS results from mutations in the genes for hydroxylation, only LH2B is thought to be involved in pyridinoline coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isom- cross-linking by displaying selective hydroxylase activity toward erase (PPIase) FKBP65. Given that the immunophilin FKBP65 does the telopeptide Lys of collagen (18, 20), whereas the biological not exhibit LH activity, it is likely that LH2 activity is somehow de- function and specificity of LH2A is unknown. LHs display activity pendent on FKPB65. In this report, we provide insights regarding only when present in a dimer (21–23). Homodimers of LH1, the interplay between LH2 and FKBP65. We found that FKBP65 LH2, and LH3 are likely the rule, but notable amounts of the forms complexes with LH2 splice variants LH2A and LH2B but not heterodimer LH1/LH3 can be formed as well (24). with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 Mutations in PLOD2 are linked to the development of Bruck did not affect complex formation. Both depletion of FKBP65 and syndrome type 2 (BS2) (MIM 609220) (25–28), a heritable auto- inhibition of FKBP65 PPIase activity reduced the dimeric (active) somal recessive bone disease characterized by congenital contrac- form of LH2 but did not affect the binding of monomeric (inactive) tures with pterygia, early onset of bone fractures, postnatal short LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B stature, and severe limb deformity and progressive scoliosis. In cannot form heterodimers with each other but are able to form addition, mutations in FKBP10 result in BS type 1 (MIM 259450) heterodimers with LH1 and LH3. Collectively, our results indicate (27, 29–34) and in osteogenesis imperfecta and Kuskokwin syn- that FKBP65 is linked to pyridinoline cross-linking by specifically drome (35–38).AsisthecaseforPLOD2 mutations, FKBP10 mediating the dimerization of LH2. Moreover, FKBP65 does not in- mutations result in a dramatic underhydroxylation of collagen teract with LH1 and LH3, explaining why in BS triple-helical hydrox- telopeptide Lys and a subsequent decrease in the pyridinoline cross- ylysines are not affected. Our results provide a mechanistic link linking of collagen type I in bone (26, 32, 39). FKBP65, encoded by between FKBP65 and the loss of pyridinolines and may hold the FKBP10, belongs to the family of immunophilins, a class of proteins key to future treatments for diseases related to collagen cross-linking that exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity that anomalies, such as fibrosis and cancer. can be targeted by immunosuppressive drugs, such as FK506 (tacrolimus) and rapamycin (40, 41). Despite the high number of collagen cross‐linking | lysyl hydroxylase | FKBP65 | Bruck syndrome | prolyl residues in collagen, the PPIase activity of FKBP65 toward fibrosis Significance ollagen I is an essential component of the extracellular matrix (ECM) of tissues such as bone and skin, and is involved in a C Collagens are structural extracellular matrix proteins that pro- wide variety of biological processes. A deregulated synthesis of vide mechanical support to tissues. To gain stability, collagens collagen type I results in pathologies ranging from severe bone can form pyridinoline cross-links via enzymatically formed inter- and skin anomalies to fibrosis (1–5). Hydroxylation of specific mediates initiated by lysyl hydroxylase (LH) 2. Individuals with lysine (Lys) residues into 5-hydroxylysine (Hyl) is performed by mutations in the gene encoding LH2 share highly overlapping lysyl hydroxylases (LHs), also known as the procollagen-lysine, traits with individuals with mutations in the gene encoding the 2-oxoglutarate 5-dioxygenase (PLOD) family. Collagens deposited immunophilin FKBP65 that shows no LH activity. We found that in the ECM are stabilized by the formation of intermolecular cross- FKBP65 is necessary for the dimerization of LH2, which is required links by members of the lysyl oxidase family, LOX and LOXL (6). for activity of LH2. Collagen cross-linking plays important roles in Two collagen cross-linking pathways have been identified, the bone diseases as well as in such pathologies as cancer and fi- allysine route and the hydroxyallysine route (7). In the allysine brosis. Our study has elucidated a mechanism of how to interfere route, a telopeptide Lys is oxidized by lysyl oxidases into an al- in a specific type of collagen cross-linking and can help advance dehyde; in the hydroxyallysine route, this occurs with a telo- the design of new treatments toward these pathologies. peptide Hyl. In turn, the reactive aldehydes interact with the Lys or Hyl residues in the helical part of collagen to form difunc- Author contributions: R.A.F.G., M.B., and R.A.B. designed research; R.A.F.G., M.M.v.d.S., and tional and finally trifunctional cross-links (8–10). The trifunc- M.B. performed research; R.A.F.G., M.M.v.d.S., M.B., and R.A.B. analyzed data; and R.A.F.G., tional cross-links derived from the hydroxyallysine route are M.B., and R.A.B. wrote the paper. referred to as pyridinolines. Collagens cross-linked by means of The authors declare no conflict of interest. pyridinolines are difficult to degrade (11–13). This article is a PNAS Direct Submission. The LH family consists of three individual members, designated 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. LH1, LH2, and LH3. Hydroxylation of Lys present in the Gly-X-Lys 2M.B. and R.A.B. contributed equally to this work. sequence in the helical domain of collagen is carried out by LH1 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and LH3 (14, 15), whereas LH2 hydroxylates Lys residues in the 1073/pnas.1600074113/-/DCSupplemental. 7142–7147 | PNAS | June 28, 2016 | vol. 113 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1600074113 Downloaded by guest on September 26, 2021 displayed only a fraction of the signal. This suggests that endog- enous LH2 and FKBP65 together form a complex. Moreover, in human fibrotic kidney tissue, PLA confirmed the interaction of LH2 and FKBP65 (Fig. 1C), underscoring the in vivo relevance of the LH2/FKBP65 complex. Because when using commercially available LH2 antibodies we could not distinguish whether LH2A, LH2B, or both splice variants can form a complex with FKBP65, we performed coimmunopreci- pitation (co-IP) on HEK-293T cells that overexpressed combina- tions of FLAG-tagged LH2A or LH2B with FKBP65. Pulldown on LH2A-FLAG and LH2B-FLAG both resulted in co-IP of FKBP65, whereas no co-IP was observed under the control conditions (Fig. 2A). These findings indicate that both LH2 splice variants (LH2A and LH2B) can bind FKBP65, and that this interaction is not de- pendent on the extra exon 13A in LH2B. LH1 and LH3 Do Not Form a Complex with FKBP65. Mutations of FKBP65 in Bruck syndrome affects the lysyl hydroxylation only of the telopeptides, not of the triple helix (32, 39). After finding that FKPB65 complexes with LH2, we wondered whether FKBP65 also interacts with LH1 and LH3, the LHs involved in the hydroxylation of Lys in the helical part of collagen. Therefore, we cotransfected Fig. 1. Endoplasmic reticulum-localized LH2 and FKBP65 exhibit physical in- HEK-293T cells with LH1-His or LH3-His together with FKBP65 teractions. (A) Immunofluorescent detection of LH2, FKBP65, aSMA, and and investigated complex formation by means of co-IP. Even α β B procollagen-I 1 in NHDFs with or without TGF 1 treatment for 2 d. ( )Im- though overexpression of both proteins was achieved, neither LH1 munofluorescent detection of LH2 and FKBP65 in NHDFs transfected with nor LH3 showed significant interaction with FKBP65, with only esiRNA against PLOD2 or control (RLUC), followed by treatment with or without TGFβ1for2d.(C) Fluorescent proximity ligation assay for LH2 and a very faint band of FKBP65 observed after LH1 and LH3 IP FKBP65 in NHDFs transfected with esiRNA against PLOD2 or control (RLUC), (Fig. 2B). followed by treatment with or without TGFβ1for2d.(D, Upper)Immuno- histochemistry for LH2 and FKBP65 on serial sections from fibrotic explanted Mutations in the Catalytic Domain of LH2 and FKBP65 Do Not Reduce human renal allograft tissue. (D, Lower) PLA on identical serial sections for LH2 Complex Formation. To examine whether the catalytic activity of and FKBP65 or IgG isotypes as a control. The arrow indicates colocalization of LH2 affects complex formation withFKBP65,wegeneratedamu- LH2 and FKBP65 in the serial sections. (Scale bars: white, 100 μm; black, 50 μm.) tation of the conserved residue of LH2A (R728K) and its corre- sponding residue in LH2B (R749K) to ablate the catalytic activity of LH2 (Fig.