BoneBone MatrixMatrix BiochemistryBiochemistry UCLUCL--20072007 Chris Sharp Charles Salt Centre RJ & AH Orthopaedic Hospital Oswestry PARTPART 11 BoneBone BioBio--MarkersMarkers && TheThe MineralMineral Phase,Phase, MineralisationMineralisation PART 2 The Organic Phase FibrillarFibrillar && MatricellularMatricellular ProteinsProteins ofof BoneBone Basic Terminology • amino acids, peptides & proteins • enzymes, isoenzymes & isoforms • propepetides & telopeptides • knock–out “KO” animals Bone, Muscle & Fat skeletonskeleton comprisescomprises ~~ 12%12% ofof bodybody massmass Composition + Structure Organic 25% 75% Mineral Structure Quality PARTPART 11 BoneBone BioBio--MarkersMarkers && TheThe MineralMineral Phase,Phase, MineralisationMineralisation Bone Quality: Bone Mass & Mineral Density Dual-energy x-ray densitometry DXA The Ideal BONE Marker • tissue specificity • molecular specificity • reflect a dynamic physiological process ¾ bone formation ¾ bone resorption • clinically meaningful • easy to measure • cheap BioBio--markersmarkers ofof bonebone matrixmatrix turnoverturnover Proteins made by OSTEOBLASTS: – bone alkaline phosphatase isoforms- BALP –osteocalcin-BGP – intact procollagen propeptides- PINP & PICP Products of OSTEOCLAST activity: – bone acid phosphatase isoforms- BAcP – enzymes used in matrix degradation- cathepsin K – products of collagen breakdown- CTx, ICTP & NTx – collagen crosslinks- Pyr & DPD What can influence a bone marker result ? • Pre-analytical variation ¾¾ analyteanalyte stabilitystability ¾¾ interinter && intraintra--individualindividual variationvariation ¾¾ age,age, gendergender && hormonehormone statusstatus ¾¾ fitness,fitness, exerciseexercise && dietdiet ¾¾ timetime ofof day,day, timetime ofof yearyear (season)(season) ¾¾ bloodblood oror urineurine collectionscollections • Analytical variation ¾¾ interinter && intraintra--assayassay variationvariation (quality(quality ofof thethe assay)assay) ¾¾ pipettingpipetting skillsskills (expertise(expertise ofof thethe technician)technician) Composite Material Mineral Phase Organic Phase calcium hydroxyapatite type I collagen Impacts on matrix Impacts on mineral properties: properties: • matrix stability • nucleation •growth • maturity Bio-mineralisation Bones & Teeth • calcium phosphate hydroxyapatite or “carbonated apatite” (Ca,Sr,Mg,Na,H2O,[*])10 (PO4,HPO4,CO3P2O7)6(OH,F,Cl,H2O,O,[*])2 where [*] represents a lattice defect Ca10(PO4)6(OH)2 Calcium Phosphate “Bony” Structures Conodonts to Bony Fishes Palaeozoic Era 543-248 Myrs Devonian Period 415-360 Myrs BSE-SEM showing “bone mineral density” From Prof Alan Boyde Bone mineral density distribution (BMDD) BMDD in 55 normals OP post-Alendronate Normal (23wt%) Osteomalacia Bone mineral density distribution (BMDD) Optimal Ca2+ distribution in bone mineral with respect to material quality & bone strength Ruffoni et al Bone 2007;40:1308 Bone Mineralisation: A balance of Phosphatases & Pyro-phosphates • Alkaline phosphatase (TNAP) • Nucleoside triphosphate pyrophosphohydrolase (NPP1) • PHOSPHO-1 (phosphatase orphan-1) PPi P + P Alkaline Phosphatase Overview • ecto-enzymes • dimeric • in vitro alkaline pH optimum ~ pH 10 • phosphotransferase, dephosphorylates substrates • widespread tissue distribution • bone isoforms involved in bone mineralisation • most commonly requested analyte in clinical chemistry – bio-marker !! Structure of human placental ALP Le Du et al J Biol Chem 2001 Alkaline Phosphatases isoenzymes and isoforms • Four gene loci = 4 ALP isoenzymes Tissue Non-specific Intestinal Placental Germ Cell Bone ~ 4 isoforms Liver ~ 3 isoforms Kidney ~ ? IEF BALP Alkaline Phosphatases Anchoring of BALP into the cell membrane * Differences between BALP isoforms are due to different glycosylation patterns * Glycosyl-phosphatidylinositol (GPI) anchor * GPI-Phospholipase C and D releases ALP from cell Chromatographic serum profiles A Healthy adult, 174 U/L B Prostate cancer with skeletal metastases, 354 U/L Magnusson et al. Clin Chem 1998 Origin of BALP isoforms in human cortical and cancellous bone BALP isoforms isolated from SaOS-2 cells B/I B1 B2 B1x mix Sharp et al. CCA 2007 & Magnusson et al. JBMR 1999 Alkaline Phosphatases Functions of Bone Alk Phos (BALP) • Phosphatase activity – provides Pi for mineralization – removes pyrophosphate or other inhibitors of mineralization Alkaline Phosphatases Proposed actions in bone TNAP PPi Pi hydroxyapatite P N A P N P T 1 Ca2+ NTPs other other ALP removes inhibitors sources sources of mineralisation of PPi of Pi Hypophosphatasia reduced alkaline phosphatase activity • heritable, rare (about 1/100,000) • low serum ALP activity (hypophosphatasemia) • high serum/urine concentrations of PPi • variable severity of skeletal symptoms • poor skeletal calcification, rachitic deformities, fractures, early tooth loss Hypophosphatasia – mutations in TNALP gene that impact on enzyme function Clinical forms: 1 Perinatal – die in utero or shortly after birth 2 Infantile - <6mths, rickets, failure to thrive 3 Childhood – premature loss of teeth 4 Adult – recurrent, poorly healing fractures 5 Odonto HPP – loss of deciduous teeth <3yrs, dental but not skeletal problems Diagnosis: • plasma pyridoxal phosphate increased • plasma ALP decreased No established treatment Micro-CT images of upper tibae from WT & TNALP-/- mice Anderson et al. Am J Pathol 2004; 164:841 BoneBone BioBio--markersmarkers 1:1: BALPBALP Bone Specific ALP isoforms – from OSTEOBLASTS Reflects bone formation – elevated in high bone turnover states Easily measured – RIA, ELISA or enzyme activity BALP isoforms in Paget’s Disease of Bone Coutris Index vs BALP-B1 & B2 Activities 1800 1600 y t 1400 i 0.064x v i ♦ B1 y = 13.604e t 1200 c R2 = 0.6 a 1) 000 m L 0.0737x / y = 44.168e or B2 (U 800 □ 2 of R = 0.508 s i 600 LP 400 A B 200 0 0.0 10.0 20.0 30.0 40.0 50.0 99 Tc -MDP scan % skeletal involvement (Coutris Index) PART 2 The Organic Phase •• fibrillarfibrillar collagenscollagens -- structuralstructural •• matricellularmatricellular proteinsproteins -- biologicalbiological modulatorsmodulators Gla–Proteins : 1 Bone gla-protein (osteocalcin) & Matrix Gla-protein HOOC GLUtamate CH CH2 + COOH ~HN-CH-CO~ HOOC COOH CH CH2 GLA ~HN-CH-CO~ • vitamin K-dependent γ-carboxylation • characteristic Gla-domains Gla–Proteins : 3 BGP or Osteocalcin Summary • conserved across spieces • specific to osteoblasts & bones/teeth • contains up to 3 Gla sites • binds metal ions, Ca2+, Mg2+ etc. and bone mineral • homology with blood clotting factors • various forms in serum can reflect bone formation and resorption Osteocalcin C Asp-Glu Helix N 35 45 30 40 10 15 20 25 -- -- -- Gla Helix Gla – Proteins : 2 Osteocalcin Hoang QQ et al. Nature 2003; 425:977 structure interaction with bone mineral Hoang QQ et al. Nature 2003; 425:977 Gla - Proteins : 5 Function 2 OC KO Mice Lee et al. Cell 2007, 130:456-469 insulin secretion β-cell proliferation become insulin resistant visceral fat “Energy Regulation” ?? FUNCTION : – bone-derived hormone involved in regulation of energy metabolism Gla - Proteins : 6 Function 1 OC KO Mice Ducy et al. Nature 1996; 238;448 • deletion of OG1 and OG2 from mOC locus • serum OC wt 362, -/- 0 ng/ml • KO (-/-) normal at birth - by 6 months, cancellous & cortical BFR, cortical thickness & density visceral fat ?? FUNCTION : negative regulator of bone formation – inhibits bone formation - analogous to MYOSTATIN Gla - Proteins : 7 ComparisonComparison ofof GlaGla proteinsproteins inin bonebone Osteocalcin (BGP) Matrix Gla-protein (MGP) • human gene Chrom 1 • human gene Chrom 12 • bone (5.7kD) • bone & cartilage (10.6kD) • 49 aa, fully processed • 84 aa, retains Nt-propep (1-49h bone) (1-77h bone) • 3 Gla sites • 5 Gla sites • binds mineral • binds mineral & matrix • regulates growth (?) • inhibits calcification Gla - Proteins : 8 MGP KO Mice Lou et al. Nature 1997; 386;78 • smaller • soft tissue calcification Arterial network FUNCTION : inhibits calcification BoneBone BioBio--markersmarkers 2:2: OsteocalcinOsteocalcin Bone Specific – from OSTEOBLASTS Easily measured – RIA, ELISA Reflects bone formation – can be elevated in high bone turnover states But: fragments & epitopes intact blood resorption? Osteopontin & Bone A bridge between bone cells and bone matrix • osteoblasts • 44kD, 314 residues • present in bone matrix and other cell types • binds integrin αvβ3 • dephosphorylated form does not bind O’clasts • OPN inhibits mineralisation Collagens • 30% protein mass of body • maintain structure of tissues • cell adhesion • wound healing • pathology Collagens of the ECM : 1 • 27 different types • 42 genetically distinct α-chains • structuralstructural andand functionalfunctional diversitydiversity •• >1,300>1,300 mutationsmutations inin 2323 // 4242 hColhCol genesgenes Collagens of the ECM : 3 Fibrillar Collagens - chain compositon Type I α1[α1(I)3] two forms α2[α1(I)]2 α2(I) Type II α1[α1(II)]3 two forms Type III α1 [α1(III)]3 Structure of Type I procollagen & collagen Collagens of the ECM : 5 Post-translational modifications Intracellular events • chain association and helix formation • hydroxylation - Proline - helix stability Lysine - cross-linking • glycosylation Collagens of the ECM : 6 Post-translational modifications Extracellular events • propeptide cleavage • lysyl oxidase - forms LysALD - cross-links • monomer assembly • fibrillogenesis • stabilisation Proteoglycans :1 ~30~30 extraextra--// periperi--cellularcellular PG’sPG’s • tissue organisers • tissue growth & maturation FunctionsFunctions • protein-protein interactions