COLLAGEN SUBSTRATE SPECIFICITY OF PROLYL 3-HYDROXLASES Alex W Farnand1,2, Mary Ann E Weis2, Lammy S Kim2, Russell J Fernandes2, David R Eyre2 1. School of Medicine, University of Washington, Seattle, WA, USA.; 2. Orthopaedic Research Laboratories, Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, WA, USA. BACKGROUND RESULTS • By weight, collagens are the most abundant protein in multi- cellular animals. The unique supramolecular structure of 2. RCS-LTC cells express markedly increased levels collagen involves a number of post-translational 1. RT-PCR analysis shows RCS-LTC cells exhibit full expression modifications that are known to be catalyzed by specific of all three P3H enzyme genes; other tissues show reduced or of P3H2 and P3H3 when compared to normal adult enzymes. " lack of expression in one or more P3H genes. rat cartilage • 4-Hydroxyproline (4Hyp), a post-translational modification of the collagen strand, is known to stabilize the collagen triple RCS-LTC cells Rat Cartilage helix. The precise function of 3Hyp, a much more rare A. RCS-LTC Cells B. Adult Normal Rat Cartilage hydroxyproline, is largely unknown, despite its discovery ) **** !"#$%& ##'(& )*+"#& #,%-& #,%.& #,%,& !"#$%& ##'(& )*+"#& #,%-& #,%.& #,%,& T 2.5 over fifty years ago. Ogle et al. J Biol Chem 237 (1962), pp. 3667-3673." C " ! 2.0 ! ( 1.0 •! Mutations in LEPRE1, a gene that encodes prolyl 3- hydroxylase 1 (P3H1)—a protein responsible for 3- n *** o hydroxyproline (3Hyp) formation—was recently found to i s cause a recessive form of severe osteogenesis imperfecta. s Cabral et al. Nat Genet, 39 (2007), pp. 359–365." e r C. Saos-2 Cells D. Human Embryonic Cartilage p x 0.5 •! P3H1 is known to form a trimolecular complex with CRTAP !"#$%& ##'(& )*+"#& #,%-& #,%.& #,%,& !"#$%& ##'(& )*+"#& #,%-& #,%.& #,%,& E (cartilage-associated protein) and cyclophilin B in the e *** v endoplasmic reticulum and is necessary for 3Hyp formation i Vranka et al. J Biol Chem, 279 t at the P986 prolyl residue in collagen. a (2004), pp. 23615-23621." l e R 0.0 •! 3Hyp residues have recently been implicated as having a P3H1 P3H2 P3H3 fundamental role in supramolecular collagen assembly by RCS-LTC cells produce a full complement of the genes assayed for; when compared to other cells and tissues, RCS-LTC cells also produce an extreme abundance of P3H2 (A). Adult Normal Rat Cartilage, similar to the RCS-LTC cells, produces all the genes assayed for; however, expression Quantitative comparison of P3H mRNA expression between the RCS-LTC cells and rat cartilage forming hydrogen bonds between adjacent collagen triple of P3H2 is semi-quantitatively less than the expression seen for P3H1 and P3H2 (B). Saos-2 cells completely lack expression of P3H2 (C). Human yielded similar expression of P3H1, a ~2-fold increase in P3H2 and a ~0.5-fold increase in P3H3 helices. Weis et al. J Biol Chem, 285 (2009), pp. 2580-2590." embryonic cartilage only produces P3H1 to a slight degree and fully lacks expression of P3H3 (D). " expression in the RCS-LTC cells." 3. RCS-LTC collagen showed near complete 3Hyp formation at Pro944. HYPOTHESIS * # * A GFTGLQGLPGPPGPSGDQGTSGPAGPSGPR B Procollagen molecule The respective P3H enzymes—P3H1, P3H2 and P3H3— 3Hyp sites 3Hyp 2+ preferentially target specific prolyl residues in the collagen 100 RCS _1(II) 1376.6 3Hyp A4 A3 A2 A1 fibril for the formation of 3Hyp residues." 80 P944 MS 60 40 N C 1369.12+ 20 Pro P944 0 Relative Abundance Relative 400 600 800 1000 1200 1400 1600 1800 2000 Fibril APPROACH m/z A4 A3 A2 A1 b •!The rat chondrosarcoma cell line, RCS-LTC, lays down an 8 * # * N C abundant matrix of collagen but fails to process this fine GFTGLQGLPGPPGPSGDQGTSGPAGPSGPR y28 y22 y20 y17 y13 y10 y6 filamentous network beyond the stage of polymerized N- y22 2+ procollagen molecules and thus offers a unique way to 100 MSMS 1376 ion 1978.9 80 y222+ study the effects of P3H enzyme activity. 60 b8 989.7 40 774.4 y13 y 2+ y17 y20 y6 10 1168.7 y28 Tandem mass-spectrometric analysis of RCS-LTC cell collagen matrix (Figure A). The relative abundance of the ions shown provides an index of the degree of hydroxylation at 1807.8 •!Using RT-PCR, the relative expression profiles of the P3H 20 570.1 882.4 1274.7 1524.4 Pro944 (top panel, fig. A). As shown, Pro944 is nearly 100% occupied by 3Hyp, which is most similar to vitreous type II collagen (87% occupied). The bottom panel of fig. A 0 shows the MS/MS spectral analysis of the prolyl and suspected 3-hydroxyprolyl versions of the peptide, from which the y ion ladder establishes the position of the added 16 Da enzyme genes and the genes of PPIB and CRTAP—other Abundance Relative A 400 600 800 B 1000 1200 1400 1600C 1800 2000 944 proteins related to the functionality of the P3Hs—were on Pro . Figure B illustrates the potential 3Hyp sites on a procollagen molecule (top) and how axial relationships required for tri-functional intermolecular cross-links form in m/z collagen to produce the collagen fibril (bottom)." compared across RCS-LTC cells, normal-adult rat cartilage, the human osteosarcoma cell line, Saos-2 and in human fetal cartilage. SUMMARY CONCLUSION •!Expression of the three P3H enzyme genes were assayed •!RT-PCR analysis of RCS-LTC cell total RNA showed that the cells express a full complement of the P3H enzyme genes, with The high relative abundance of P3H2 mRNA in RCS-LTC cells, and in normal-adult rat cartilage, via particularly robust expression of P3H2, a phenomenon unmatched by the other tissues investigated." 944 qPCR. •!qPCR analysis of P3H enzyme gene expression in RCS-LTC cells and normal adult rat cartilage showed that the RCS-LTC expression coupled with high occupancy of Pro cells express all three enzymes to a greater extent than does the normal cartilage. Expression of P3H2, in particular, was in the RCS-LTC matrix implicates P3H2 in the •!Collagen produced by the RCS-LTC cells was analyzed for significantly increased in the RCS-LTC cells." formation of 3Hyp at Pro944 supporting evolved 3Hyp formation at previously identified prolyl residues by •!Mass-spectometric analysis of the RCS-LTC collagen showed near complete 3Hyp formation at Pro944, a secondary site substrate preferences for each of the three P3H mass-spectometry. previously found occupied in vitreous type II collagen, but not in cartilage" enzymes. Funded under NCRR Grant TL1 RR 025016 and NIAMS Grant AR 036794 .