Chris SharpCharles Salt Centre

RJ & AH Orthopaedic Hospital

Oswestry

Bone Matrix Biochemistry Bone Matrix Biochemistry UCLUCL--2007 2007

PART 1PART 1Bone BioBone Bio--MarkersMarkers

&&The Mineral Phase, MineralisationThe Mineral Phase, Mineralisation

PART 2

The Organic Phase

FibrillarFibrillar & & MatricellularMatricellular Proteins Proteins of Bone of Bone

Basic Terminology

• amino acids, peptides & proteins

• enzymes, isoenzymes & isoforms

• propepetides & telopeptides

• knock–out “KO” animals

Bone, Muscle & Fat

skeleton comprises skeleton comprises ~ 12% ~ 12%

of body massof body mass

Composition + Structure

Organic 25% 75% Mineral

Structure

Quality

PARTPART 11

Bone BioBone Bio--MarkersMarkers

&&

The Mineral Phase, The Mineral 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

bone formationbone resorption

• clinically meaningful• easy to measure• cheap

BioBio--markers of bone matrix turnovermarkers of bone matrix turnoverProteins 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

analyteanalyte stabilitystabilityinter & intrainter & intra--individual variationindividual variationage, gender & hormone statusage, gender & hormone statusfitness, exercise & dietfitness, exercise & diettime of day, time of year (season)time of day, time of year (season)blood or urine collectionsblood or urine collections

• Analytical variationinter & intrainter & intra--assay variation assay variation (quality of the assay)(quality of the assay)pipettingpipetting skills skills (expertise of the technician)(expertise of the technician)

Composite Material

Organic Phase

type I collagen

Mineral Phasecalcium hydroxyapatite

Impacts on matrix properties:

• matrix stability

Impacts on mineral properties:

• nucleation

• growth

• maturity

Bio-mineralisation Bones & Teeth

• calcium phosphatehydroxyapatite 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” StructuresConodonts 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 PhosphataseOverview

• 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 ALPLe Du et al J Biol Chem 2001

Alkaline Phosphatasesisoenzymes 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 PhosphatasesAnchoring 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/LB Prostate cancer with skeletal metastases, 354 U/L

Magnusson et al. Clin Chem 1998

Origin of BALP isoforms in human cortical and cancellous bone

Sharp et al. CCA 2007 & Magnusson et al. JBMR 1999

B/I B1 B2 B1x mix

BALP isoforms isolated from SaOS-2 cells

Alkaline PhosphatasesFunctions of Bone Alk Phos (BALP)

• Phosphatase activity

– provides Pi for mineralization

– removes pyrophosphate or other inhibitors of mineralization

Alkaline PhosphatasesProposed actions in bone

PPi Pi hydroxyapatite

NTPsother

sources of Pi

other sources

of PPi

NPP1 TNAP

Ca2+

TNAP

ALP removes inhibitors of mineralisation

Hypophosphatasiareduced 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 birth2 Infantile - <6mths, rickets, failure to thrive3 Childhood – premature loss of teeth4 Adult – recurrent, poorly healing fractures5 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

Bone BioBone Bio--markers 1:markers 1: BALPBALPBone 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

y = 44.168e0.0737x

R2 = 0.508

y = 13.604e0.064x

R2 = 0.6

0

200

400

600

800

1000

1200

1400

1600

1800

0.0 10.0 20.0 30.0 40.0 50.0

% skeletal involvement (Coutris Index)

BA

LP is

ofor

m a

ctiv

ity

(U/L

)

♦ B1

□ B2

Tc99-MDP scan

PART 2

The Organic Phase

•• fibrillarfibrillar collagens collagens -- structuralstructural

•• matricellularmatricellular proteins proteins -- biological modulatorsbiological modulators

Gla–Proteins : 1Bone gla-protein (osteocalcin) & Matrix Gla-protein

• vitamin K-dependent γ-carboxylation• characteristic Gla-domains

HOOC GLUtamateCHCH2 + COOH

~HN-CH-CO~

HOOC COOHCHCH2

GLA ~HN-CH-CO~

Gla–Proteins : 3 BGP or Osteocalcin

• 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

Summary

Osteocalcin

25

30

35

20

40

15

10

45

Gla Helix

Asp-Glu HelixNC

- -- - - -

Gla – Proteins : 2Osteocalcin

Hoang QQ et al. Nature 2003; 425:977 structure

interaction with bone mineral

Hoang QQ et al. Nature 2003; 425:977

Gla - Proteins : 5Function 2

OC KO Mice Lee et al. Cell 2007, 130:456-469

insulin secretionβ-cell proliferation

become insulin resistantvisceral fat

“Energy Regulation”

?? FUNCTION : – bone-derived hormone involved in regulation of energy metabolism

Gla - Proteins : 6Function 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 & densityvisceral fat

?? FUNCTION : negative regulator of bone formation –inhibits bone formation

- analogous to MYOSTATIN

Gla - Proteins : 7Comparison of Comparison of GlaGla proteins in boneproteins in bone

Osteocalcin (BGP)

• human gene Chrom 1• bone (5.7kD)• 49 aa, fully processed

(1-49h bone)• 3 Gla sites• binds mineral• regulates growth (?)

Matrix Gla-protein (MGP)

• human gene Chrom 12 • bone & cartilage (10.6kD)• 84 aa, retains Nt-propep

(1-77h bone)• 5 Gla sites• binds mineral & matrix• inhibits calcification

Gla - Proteins : 8MGP KO MiceLou et al. Nature 1997; 386;78

• smaller

• soft tissue calcification

FUNCTION : inhibits calcification

Arterial network

Bone BioBone Bio--markers 2:markers 2: OsteocalcinOsteocalcinBone Specific – from OSTEOBLASTS

Easily measured – RIA, ELISAReflects bone formation – can be elevated in high

bone turnover states

But: fragments & epitopes

intact

blood

resorption?

Osteopontin & BoneA bridge between bone cells and bone matrix

• osteoblasts• 44kD, 314 residues• present in bone matrix and other cell types • binds integrin αvβ3• dephosphorylated formdoes 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

• structural and functional diversitystructural and functional diversity

•• >1,300 mutations in 23 / 42 >1,300 mutations in 23 / 42 hColhCol genesgenes

Collagens of the ECM : 3Fibrillar Collagens - chain compositonType 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 : 5Post-translational modifications

Intracellular events

• chain association and helix formation

• hydroxylation - Proline - helix stabilityLysine - cross-linking

• glycosylation

Collagens of the ECM : 6Post-translational modifications

Extracellular events

• propeptide cleavage

• lysyl oxidase - forms LysALD - cross-links• monomer assembly

• fibrillogenesis

• stabilisation

Proteoglycans :1~30 extra~30 extra--/ / periperi--cellular PG’scellular PG’s

• tissue organisers• tissue growth & maturation

FunctionsFunctions• protein-protein interactions

• connective tissue assembly• bind growth factors

Proteoglycans :2Small Small LeucineLeucine Rich Proteins Rich Proteins -- SLRPsSLRPs

familyfamily

Proteoglycans :3Decorin & Biglycan

Core protein

CS/DS-GAG

~10 LRRsCys-loopCys-loop

N-linked oligos

Proteoglycans :4

Decorin (PG-II)• 40 / 130 kD• CS chains in bone• wide distribution,

localised with Col I• preOB , OB ,Ocytes• KO = thin skin

Biglycan (PG-I)• 40 / 270 kD• CS chains in bone• pericellular

• osteocytes• KO = osteopenia

Class I Class I -- SLRPsSLRPs

• bind collagen

Proteoglycans :5Proposed interaction of Decorin with

collagen trimer

Weber et al. JBC 1996; 271:31767

• Decorin interacts with the C-terminal region (α1(I)CB6 peptide).

• Some Lys/Hylresidues may be essential for binding.

Both Decorin KO and Biglycan KO mice have abnormal collagen fibres in ECM

biglycan KO mice

• irregular cross-sectional profiles

• reduced bone content

wild type

90nmIozzo, RV. Ann Rev Biochem 1998; 67:609

Proteoglycans : 7

decorin KO mice

• lateral fusion

• fragile skin

Collagen Matrix AssemblyTransport of collagen trimers and initiation of fibrilogenesis

in the vicinity of the cell

I

fibronectin

fibronectin scaffold &integrin nucleation centre

cell

I

II I

I I

I

integrins

α11β1

α2β1

I I

Velling et al JBC 2002;277:37377

small PG’s

Collagen Crosslinking

Pyridiniumcrosslink

helix

telo

telo

Collagen Stabilisation

STA ⇓B ⇓IL ⇓ITY

• non-crosslinked

• divalent

• trivalent

Collagen Matrix Stabilisation• dependent on 2 enzymes that act on

lysine residues:-

• lysyl hydroxylase- 3 isoenzymes

• lysyl oxidase- 5 isoenzymes

Collagen Matrix Stabilisation• lysyl hydroxylase

- helix & telopeptide acting isoforms- tissue specific

-NH-CH-C=O COOH -NH-CH-C=O COOHCH2 CO CH2 CH2

CH2 + CH2 CH2 + CH2

CH2 CH2 O2 HCOH COOHH2CNH2 COOH CO2 H2CNH2

Lysine αKG HyLys Succ

Asc / Fe++

EC 1.14.11.4

Collagen Matrix Stabilisation• lysyl oxidase

- Cu2+ - dependent amine oxidase- forms Lys/HylALD in ¼-stagger collagen

-NH-CH-C=O Cu++ -NH-CH-C=O[CH2]4 +O2 [CH2]3 + NH3 + H2O-NH3+ Pydx Phos HC=O

Lysine / HyLys Lysylald / HyLysald

EC 1.4.3.13

Collagen cross-linking

ProcollagenHelical–Lys Hydrox

Telopeptide-Lys HydroxHylALD

pathwayLysALD

pathwayHylALD

bonebonetelo-Hyl

ketoketo--iminesimines

pyridiniumpyridinium & & pyrrolicpyrrolic crosslinkscrosslinks

LYSYL OXIDASE

divalent XL

trivalent XL

skin, tendontelo-Lys

aldiminesaldimines

histidinehistidine adductsadducts

HHLHHL

Pyridinoline Pyrrole

Known Trivalent Collagen Crosslinks

Crosslinking at the Ct-telopeptide

Di-valent

Ct-telo

InterInter--molecularmolecular

((telotelo –– helix)helix)reduced keto-imine

DHLNL

Review of Ct-telo structuresnon-crosslinked

Ct-telo

α2teloα1teloα1telo

IntraIntra--molecularmolecular

((telotelo –– telotelo))~40% Pyr

Trivalent

Ct-telo

Collagen Matrix Stabilisation

Overview• condensations of Lys or Hyl• Nt- and Ct-telopeptides• inter- and intra-molecular• temporal sequence• type and extent of X-linking influenced by

lysyl hydroxylaseBailey AJ. Amino Acids 1991;1:293-306

Collagen genes, mutations & diseases

• COL1A1, COL1A2 OI, EDS VIIA & VIIB

• COL2A1 chondrodysplasias &

osteoarthrosis

• COL3A1 EDS IV

Collagen knock-outs 1Human models

osteogenesis imperfecta (OI)

• mutations cause dysfunctional or reduced amounts of collagen

Spectrum of severitylethal > severe > moderate > mildType II Types III & IV Type I

Osteogenesis Imperfecta

Bullough’s Orthopaedic Pathology 3rd Ed.

Collagen knock-outs 2Animal model

• osteogenesis imperfecta mouse (OIM)

• natural deletion of COLIA2 gene

• only homotrimer type I collagen present

Defects (?)

Collagen knock-outs 3Characteristics of the OI Mouse

• smaller (25% lighter)

• generalised osteopenia

• decreased bone strength

• fractures

• progressively deforming

Collagen knock-outs 4Bone matrix of the OI Mouse

• type I collagen homotrimer [α1(I)3] • disorganised collagen matrix

• altered mineral crystal size & composition

Important role of α2(I)-collagen chain in maintaining bone quality

Bone Matrix Changes in Osteoarthritis: 1

Quality = composition & structureBone Structure: histomorphometry

Fazzalari Group, Truong et al Arth Res & Ther 2006; 8:R188

Bone Matrix Changes in Osteoarthritis: 2

Quality = composition & structure

Matrix Composition: gene expression (mRNA)

Normalised to GAPDH• ↑ALP• ↑OCN• ↑OPN

• ↑COL1A1/COL1A2

Fazzalari Group, Truong et al Arth Res & Ther 2006; 8:R188

Biomarkers 3: collagenBiomarkers 3: collagenPINP PICP

(PINP) Amino propeptide Carboxyl propeptide (PICP)

Helical domain

(Nt-telo) Amino telopeptide Carboxyl telopeptide (Ct-telo)

Biomarkers 4: type I collagen turnoverBiomarkers 4: type I collagen turnoverlocation marker Type I collagen

formationType I collagen

breakdown

α1(I).α1(I)

ICTP α/β CTx-I

helical domain peptides

hydroxyprolinehelical peptide

NSNS

collagen cross-links

trivalent divalent

NS NS NS

propeptides intact PINP PINP-Col1

trivalently XLtelopeptides INTP

NTx-I

divalently XL

telopeptides α1(I).α2(I)

SummaryGlycoproteins – Alkaline phosphatases,

BALP isoforms & role in mineralisation

Matricellular proteins

Gla-proteins – BGP (OCN) & MGP, possible roles in bone & soft tissues

Osteopontin – OPN, role in mineralisation

Collagen – Type I, structure, assembly & cross-linking

Proteoglycans – Decorin & Biglycan, roles in collagen fibril assembly & organisation