st rsi st

STUDIES Olf RAt c:-UAC'l'In I'ROTltlf z BIHDIIl'CJ'fOlSOLATID RATDPA'rOCY'l'ES A1fD CLZARANCZ J'IOX eIRCUUTION

by

Co CHENG YONCJ YANG, a.sCl.

A thesis submittedtothe School of Craduate St u dies in p.rtial fulfilmentat the requirements

for the deq r e e of Masterof Science

Departme nt ofBi oc hemi stry Memo rial Uni ve rsi tyot Newfoundland

st. John' s , Ne wf ound la nd

1+1

Theauthorhas granted enirrevocablen0n- exclusiveIlcenoe

_ltle

ofCanadatoreproduce,loan,<istri:JUteor sell coples ofhls/hefthesisbyany meansandIn any lannorfoonat,makingthislhesisavai\abIe

to lnterested~: ...

The authorretainsownershipoftheeopyright in hiS/her thesls.Neither the thesisnor substantial extracts fromIt maybeprintedOf otherwise reproduced withouthiSiherper- mission.

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ISBN 0-315-65298-5

ABSTRAC'l'

The clearance mechanism of C-reactive proteins (CRP) ....as examined in this study.~binding assaysus i ng isolatedra t hepatocytes ....ere followed by clearancestudies

~and using perfused rat liver.

It ....as shown that ra t asialo CRP was rapidly cleared from the circulation (half-life about 4min ) , whereas rat and rabbitCRP remained in the circulationformuc h longer periods of tilne (ha lf-li f e 7.Sh for rat CRP).The clearance of rat asialoCRP was shown to be mediated through the hepatic asialoglycoproteinreceptor, andthis clearance was not affected by the phosphorylcholine bindingdomain of rat asialo CRP.Results of the clearance studiesalso suggested that, in addition to liver, lungs might alsobe involvedin the clearance ofCRP.

It was observed thatla r ge amounts ofra t asialoCRP, rat CRP and rabbit CRP bound to isolated rat hepatocytes, and the binding was shown to be mediatedpredomi nantly throughthe phesphorylchollnebinding domain ofth e s e proteins. In addition, rat asble CRP was sho....n to bindto the hepatic asialoqlycoproteinreceptor on the hepatocytes, despite the avidphesphorylchol!ne binding domain-mediated binding of thisprotein. It ....as demonstra.tedthat the binding oflarg e amounts of rat asialoCRP, rat CRP and

11

rabbit CRP to the hepatocytesthroughth e phosphorylcholine bindingdomain resultedfrom a disruptionof the hepatocyte surface.

iii

ACItNOWLEDGEMEN'l'S

I amdeeply grateful to mysup e rv i s o r s, Dr. S. S.

Mookerjeaand Dr. A.Nagpurkar, for the opportunityto carry out this study in their laboratory and for their distin- guished guidance throughout this project. Their advicehas been very inspiringand invaluable . The continued financial support from their grantshas made it pos siblefor me to concentrateon scientific and academic aubjectre , I am especially grateful to Dr. S. Mookerjea for taking every care of me, and to Dr. A. Nagpurkar for critically reading and painstakingly revisingthisthesis.

I am trUly thankfulto Dr.K.H.W. Keough for helpful di s c u s s i on and suggestions. His advice and encouragement havebeen invaluable.

I wish to express my sincere appreciation to membersof BiochemistryDepartmentat Memorial Universityot Newfoundland for creating a pleasant and stimulating atmosphere. I am especially thankful to everyone in our laboratory foral l the help they have given me, and for the friendly, cooperative and pleasant atmospherethey have created.

I wish to thank the School of Graduate Studies in MemorialUn i ve r s i t y of Newfoundland for financial support in the form of a Memorial University Graduate Fellowship.

Thanks are also due to the Heart and Stroke Foundation of

iv

canada and the Medical Research Council of Canada for financial support to th';'s project.

I truly thank my friends for their interest in my proqress.I heartily thank Canadian people tor their generosity and hospitality.They have helped me adapt myself to a totally differentcu l t ur e. lowe many things that I have learned in variousaspects of my life to them.

I am particularlyve ry grateful to my grandmother, my parents and other members of my family for all their love, support and everything they have givenme. Finally, I truthfully thank my motherland. I have all my best wishes to her and dedicate this thesisto her.

TULE OF CONTENTS

Abs t ract Ack nowlegement s Tableof Con t e nts Listof Tables Listof Figures Listof Abbreviations List of ke ywords Chapter 1.INTRODUCTION

se ctionI. OVERVI EWON C-REACTIVEPROTEINS A. DISCOVERYAND DEFINITION OF C-REACTI VEPROTEI NS B. DIS TRI BUTIoN orCRP

C. ISOLATIONAND GENERALPROPERTIESOF CRP 1) . Isolationof CRP

2).Ge ne ralPhy sic ochemicalProperties ofCRP Molecular shape

comp o sit i o n Sta b ilit y 3).Bindingpropert ies ofCRP

Binding of CRP to phosphorylcholine Bindingof CRP to polycations

EM<

Ii Iv vi x xl x11i xv

4).AcutePhase Level sof CRP 10

D. BIOSYNTHESI SAND TURNOVER OF CRP 11

1). Biosynthesis of CRP 11

2). IncreasedCRP synthesi sDuring

Acute PhaseRespo n se 11

3}.Other Sitesof CRPBios ynthesi s 12

4) .1n....YiY2Clearanceof CRP 12

E. PROTEINS RELATEDTOCRP 13

1}. Serum Amyl o idP compo ne nt (SAP) 13 2) . Syrian HamsterFemaleprotein(FP ) 14 3} . OtherPhosphorylchol ine Binding Proteins 14

vi

F. BIOLOGICAL ACTI VI TI ESOF CRP 15 1). Interactionsof CRPwi t h comp lementSystem 15 2). Intera c t io ns of CRP withLymphoid System 17 3). Effectof CRP onthe Activityof Phagocyt ic

Cells 18

4). Effectsof CRPon PlateletFunctions 19 5). Int e r act i o n of CRP with PlasmaLipoproteins 20 6). CRPin DefenceMechanisms against Microbial

Infectionandthe Clearance of Abnormal

Materials from Circulation 22

7). ClinicalApplications of CRP 23

sectionII. GLYCOPROTEIN: ANOUTLINE 25

A. STRUCTUREOF GLYCOPROTEINS 25

1). Compositionof theCarbohydrateMoietyof

Glycoproteins 25

2). Carb'.:lhydrate-PolypeptideLinkages 25 3).Oligos accharides inGlycoprotelns 26

N-linkedoliqosaccharides 26

O-linkedoliqosaccharldes 30

B. BIOSYNTHESISOF GLYCOPROTEINS J1

C. CLEARANCE OF GLYCOPROTEINS 33

1). ClearanceofGlycopro teins throu g h the Hepatic

Asialog lycoproteinReceptor 33

2).Othe r Carbohydrate-dependentClearance

Mecha nisms 36

SectionIII.PURPOSE OFTHE PRESENT STUDY 39

CHAPTER 2 H1I.TERIALS AND METHODS 41

A. MATERIALS 41

1). Ani1na ls 41

2). Reagentsand Chemicals 41

3). Mediumand Buffer 41

vii

B. PREPARATIVE PROCEDURES 42 1).PreparationotSepharose-phenylphosphorylcholine

Affinity Adsorbent 42

2). Preparation of Rat and Rabbit Serum 43 3). Isolation and Purification of Rat and

Rabbit CRP 43

4). preparation ofDesialyla~edProteins 44

5). Radioiodination Procedure 44

C. ANALYTICAL PROCEDURES 45

1).Electrophoresis 45

2). HPLC Analysis 46

D• .I1!...i!IYQ CLEARANCE EXPERIMENTS 47

E. LIVER PERFUSION EXPERIMENTS 49

F. PREPARATION OF HEPATOCYTES 50

G. HEPATOCYTE BINDING ASSAYS 51

Chapter 3 RESULTS 53

SectionI . BINDING OF RAT ASIALO CRP, RAT CRP AND RABBIT CRP TO ISt:lLATED HEPATOCYTES 53 A. PURITY AND IDENTITY OF RAT CRP, RAT ASIALD CRP

AND RABBIT CRP 53

B. CHARACTERIZATION OF 1l'SI-LABELED PROTEINS 54 C. ESTABLISHMENT OF OPTIMUM CONDITIONS FOR

BINDING ASSAYS 60

1). Requirement of eSA to Prevent the Binding of 1l'SI-labeled Proteins to the Tubes 60 2). Determination of Optimum Cell Concentration

and Incubation Time for Binding Assays 60

D. BINDING OF RAT ASIALO CRP, RAT CRP AND RABBIT CRP TO THE HEPATOCYTES AS A FUNCTION OF 12SI_LABELED

PROTEIN CONCENTRATION 65

viii

E. EFFECT OF Ga1NAc AND PHOSPHORYLCHOLINE ON THE BINDING OF RAT ASIALO CRP, RAT CRPANDRABBIT

CRP TO THE HEPATOCYTES 71

F. COMPETITIVE INHIBITION STUDIES ON THE BINDING OF RAT ASIALO CRP.RAT CRP AND ASIALO AGP TO

ISOLATED HEPATOCYTES 71

G. BINDING OF RAT ASIALO CRP TO ISOlATED HEPATOCYTES

WITH DIFFERENT VIABILI.TY 77

Section II. CLEARANCE OF RAT ASIALO CRP. RAT CRP AND RABBIT CRP I..I:L..YI..YQANDIN PERFUSED LIVER 80 A. CLEARANCE OF'25I _ LABELED RAT ASIALO CRP, RAT CRP

ANDRABBIT CRP IN RAT 80

8. COMPETITIVE INHIBITION OF THE~CLEARANCE OF RAT ASIALO CRP BY ASIALO AGP, ASIALO-FETUIN

AND AGP 83

C. DISTRIBUTION OF RADIOACl"IVITY IN TISSUES FOLLOWING THE CLEARANCE OF LABELED RAT ASIALO CRP, P.\TCRP

AND RABBITCRP 83

D. HPLC ANALYSIS OF THE RADIOACTIVITY ASSOCIATED WITH

LIVER AND LUNG HOMOGENATES 92

E. CLEARANCE EXPERIMENTS USING PERFUSED LIVER 92

Chapter 4 DISCUSSION 98

A. BINDING OF RAT ASIALO CRP AND RAT CRP TO

ISOLATED HEPATOCYTES 98

8•.IlL.YIYQCLEARANCE OF RAT ASIALO CRP. RAT CRP AND

RABBIT CRP 103

C. CONCLUSION AND FUTURE DIRECTIONS OF RESEARCH 106

REFERENCES 109

LIS'l' OJ'TABLE8

Table 1: Distribu tionotc- reeeefveprote ins Table2: Clini calapplicationsof theme a su rem e nt

of serumCRP 24

Tabl e3: Surfa c eco nt e nt ofasi aloglyc oproteinreceptors

on isolatedhe patocyte 68

Table 4: Rec ove ry of radioa ctiv ityin liv er andlungs 88

LISTOF lIGURES

Fig. 1: Majortypesofcar bohy d r a t e - pe pt i de linkage 27 Fig. 2: Thecommon core of N-linked oligosa c charides 29 Fig. 3: Typiccrl examplesof oligosaccharides in

glycoprote ins 29

Fig. 4: Ashwell'spath"''' ;,' 34

Fig. 5: HPLC profileof is ol a t e d ratCRP 55 Fig. 6: Bindingof rat asidoCRP toaSepharos e-

phenylphosphol.-ylchol ine column 56 Fig . 7: HPLC analysis of mI-labeledrat CRP 57 Fig. 8: Bindingof125I - labe l e d rat CRP toaSe pha r os e -

phenylphosphorylcholine co l umn 59

Fig. 9: Effectof BSA on the binding of 125I-l a be l e d

proteinsto the t....bes 61

Fig. 10 (a &b):di nd i ng of12SI - l a be l e d asialoAGP,

rat asrai.cCRP andrat CRP to isolated hepatocytes asa functionof hepatocyte

concentrations 62

Fig. 11:

Fig. 12 :

Fig. 13:

Fig. 14:

Fig. 15:

Fig. 16 :

Time cours e of the binding of125I - l abell;ld rat CRP and ratas i a l o CRP to isolated hepatocytes

specificBind ingof rat CRP,rat ds-CRP, rabbit CRP, AGP and ds-AGPtois ola t e d hepatocytesas a func t i o n of prote in concent r a tio ns Bindingof12S I-l a be l e d rat asialoCRP andra t CRP to isolatedhepatocytes as a functionof protein concentrations

Effect of GalNAc and phosphoryl choline on the bindingof rat CRP, rat asidoCRP,rabbit CRP and asialoAGP to isolatedhepatocytes Competitive Inhibition of the binding of 125I_labeledasialoAGPto isolatedhe p a t oc yt e s by rat asialo CRP or rat CRP

Effect of GalNAcand phosphorylchol1neon the competitive inhibitionc~the binding of1251_

la bel e d asialoAGP by rat asialoCRP xi

64

66

69

72

75

75

Fig. 17:

Fig. 18:

Fig. 19:

Fig. 20:

Fig. 21:

Fig. 22:

competitive Inhibition of the binding of 1251_

labeled rat asialo CRP and a5ial0 AGP by rat CRPand AGP

Binding of 125I-labeled rat a5ialo CRP to isolated hepatocytes with different viability Clearance of rat asialo CRP, and rat CRP and rabbit CRP from circulation

competitive inhibition of the clearance of 125I - l a be l e d rat a8ialo CRP by a5ial0 AGP Comp'9titive inhibition of the clearance of 12SI_l abeled rat asialo CRP by asiale-fetuin or AGP

Tissue distribution of radioactivity 20 min after the injection of \lSI-labeled rat aslal0 CRP

78

79

81

84

84

87 Fig. 23 (a &b): Time~dependentdistribution of radio-

activity in liver and lungs 89

Fig. 24: Effect of aelalo AGP on the distribution of radioactivity associated with 125I-labeled rat asialo CRP in liver and lungs 91 Fig. 25 (a and b): HPLC elution profiles of the radio-

activity released from liver and lungs

hemogenates 93

Fig. 26: Clearance of rat aaLa Lc CRP, rat CRP and asialo

AGPby perfused liver 96

Fig. 27: Effect of GalNAC, phosphorylcholine and neuraminidase on the clearance of rat as Lake CRP

by perfused liver 97

Fig. 28: Clearance of glycosylated and non-glycDsylated

CRP in liver and lungs 107

oii

LIS'1' OF ABBREVIATIONS

ADP Adenosine diphosphate

AGP a~-ac idglycoprotein(bovine)

Apo Apoprotein

Asialo AGP Desialylated a1-a cidglycoprotein (bov.'.ne) Rat asialoCRP Desialylated rat C-reactive protein Asn As p a r a q ine

BSA Bovine serum albumin

CHBr Cyanogen bromide

CPM Count per minute

CPS c-polysaccharide

CRP C-reactive protein

EDTA Ethylenediamine-tetraaceticacid EGTA Ethyleneglycol-bis-(,8-atli.lnoethyl ether)

NI NI NII NI-t e t r a a c e t i c acid FP

Fuc Gal GalNAc Glc GlcNAc HPLC IL-l IL-6 lodo-Gen

syrian hamster female protein Fucose

Galactose

N-acetylgalactosamine Glucose

N- a c e t y l ql u c os ami n e

High perfor1llance liquid chromatography Interleukin-l

Interleukin-6

1,3I4 , 6-tetrachloro-3a, 6o:-diphenylglycouril

d i i

LOL Low density lipoprotein

Man Mannose

MW Molecular weight

NANA N-acetylneuramlnic acid

NS8 Non-speoific binding

PC (Pwcholine) Phosphorylchol1ne PAF Platelet activating factor PAGE polyacrylamide gel electrophores:is PCBP Phosphorylchol!ne binding protein SAP Serum amyloid P component

so standard deviation

50S Sodium dodecyl sulfate

SEM Standard error

TB Total binding

VLDL Very low density lipoprotein

xiv

LIST OJ' BY WORDS C-reactive protein

Asialoglycoproteinreceptor Hepatocytes

Binding assays Perfusion Clearance Liver Lung

Phosphorylcholinebinding protein Rat

Rabbit

Chap te r 1. INTRODUCTION

C-r e a ctiveprot e i n s (CRP) arepre s e ntina wide ran ge of species, from horseshoecra b (I,imulus pgl yph e mus ) to humanbeing.CRP derives its namefroma phos ph o rylc hollne- mediated reactiv ity with the C-polysaccha rideof the pneumo- cocca l cel l wall (Abernethyand Aver y , 19 41 ;Ma c Leod and Avery, 1941;Volanakis and Kaplan, 1971).Theseproteins have two commoncharacteris ticstha t ha ve been conserved throug hout evolution: i) ape n t amer i c disc-likeconfig u- rat i o n (Osmand et al., 1977) (wi ththeonly exception of horses hoecrab CRP,whichhas a hexa go na l ri ng-s hap e d structure (Robe y andLi u, 19811) , andill a calcium dependentbinding tothe phosphorylchollne ligand(Vola nakis andKaplan , 1971).In addition,the y sha reextensiveamino ac idsequence homology (Oliveiraeta!., 1979; Wanget aI., 19 8 2 ; Pepys et al.,198 4;Ng uy e n etal., 198 6a ). Basedon thei rglyc o s yla t i o n status,CRP canbe divided intotwo cat egories: al Non-gly c os yl a t e d CRP, suchas human and rabbit CRP; andb) GlycosylatedCRP, such as ra t CRP. CRP ar esynt hes ized in the liver by hepatoc yte s (Kushneret al., 198 0 ; Col linset a!., 1984). However, their cl earance pathwayis not known .

Inthe1970^1s,Ashwell di s co v ered tha t , afterth e removal of te rmi n al sial icacids , most mammalian circulatory glyoo-prote i nsare take n upby the li v er via thehep a tic

asialoglycoprotein receptor (Morellet aI., 1971: Ashwell and Harford, 19 82: Ashwell, 1984).Inth eca s e of the CRP family , sinceacme are glycosylatedwhile others arenon - glycosylated, i t isappr o priate to studyi fAshwell's clearancepathwayis applicableto glycosylatedCRP andi t glycosylated and non-glycosylatedCRP are clearedthrough a coemon me c ha n i s m.In addition, i t is important toexamineif the highly conservedphosphorylcholine bindingdomainof CRP would affect thebindingof asialoCRP to the hepatic asialoglycoprotein receptor, andi fthis bindingwould affect theirsUbsequent clearance throughAshwell'5pathway.

As a necessarybackground for this thesis, an OVQrviQW onC-reactiveproteins and an outline of the structureand clearance pat hwaysof glycoproteins have been provided.

section I.QVE RVnWON C-REACTI YIj'ROIIINS

A.DIScoyERY AND DEFINITIONorC-RUCTIVJjPRQ'l'!XIlB In19 30 ,Til l e t t andFranc i s (193 0)observedtha t sera obtainod frompa t i e nt s during acutefebrile illnesses precipitatedwithan extractof a pneumococcus, whic hwas first designatedas FractionC and subsequentlyas c- polysaccha ride (CPS). The substance responsibletor this reactivitywaste rme d c-precipit i n .It was la t erpr ove n to be a protein not norma l l y presentin thebloodandwas designatedas C- reactiveprotei n (CRP) (Abe r net hy and Avery, 1941)•

SUbsequently ,it was de mons tra tedthat the reaction of CRP towardsCPSis directed to theph o s phorylchol ine residue onCPS (Volanakis andKapl an,19 71 ). Thisbindi ng specificityof human CRPha s been uti lized to defi neCRP homol ogues; inotherspecies.Protein:!:: from the sera of other species which exhibita similar ca 2'- dependent bindingto immobilizedCPs, phosphor ylc holine, or phosphorylcholin e derivativesare also de s i g n ated as CRP.These prote i ns close ly re s e mbl e human CRPin molecularappearance,subu n i t composition and amino acidsequ e nc e (Bach et; a L, , 1971: Pepys ee a1., 1978 a ).

Some membersof the CRP familyha v ebeen referred to by othe r names. For example, li mul u s CRP had been knownas li mul i nbefo r e it was charact erized asame mbe r oftheCRP

family (Robeyand Liu1981). When rat CRP was firstisolated byNagpurkarand Mookerjea, it was termedrat phosphcryl- cho l i ne bind ingprotein (PCBP) onthebasis of itspr op erty of binding tophosphoryl c hol1ne (Na gpur ka r and Mookerj e a , 1981).It was sub s e qu e nt ly sho wnto be ide nt i c a l to rat CRP indepe ndentl yisolatlidbyPontetat al. (1981 ) andde Beer et a1. (198 2a). In thi sthes i s,the termrat CRPisus ed , insteadof rat PCBP.

B. pISTRIBUTION OPCSP

Si nce thediscoveryof CRPinhumans ,CRPhas bee n foundina varietyof species (Table1).CRPhasbeen conserve d throughout theevol u t i o n proces s, from horseshoe cra b s to huma nbeings. Themol ec u l a r app earance, subunit composition, ami n o acid sequ e nc e andbindingspe c i ficity of CRPha vebeen conservedduring this process .

C.ISOLATIONANDPROPERTIES0'CRP 1). Isolationof eRP

CRP was fi r s t isolated in a crystallineformfrom a lipoprotein - richtraction of humanse rum by pre c ipitating with CPS, followedby delipidationandsa l t fractionation (Mccarty , 1947;Woodet a1., 1955) . More recent methodsof purificationinvolve affinity chromatograp hytechniques using immobilizedCPS, phosphorylcholineor phosphoryl- cholineder ivat ives (Os man det a1. , 1975 ; Yo u ng and

Table 1. DI8TR.IBO'I'IONorC-UACTIVB PROTBINS Name Sub u ni t s Glyc o s ylat i on MW

Human CRP! No 117.5kd

Monkey CRP»

Bo v i n eCRP~ No 150 kd

Goat CRP! Yes 120kd

Dog CRP!;!

Rabbit CRPW 120 Jed

Cat CRpi

RatCR~ Yes 125 kd

Mouse CRpl,

,

Chicken CRp!. 21 kd/subuni t

Rainbow

trout CR~ 5? 110 kd

Do q fi s h CRp!l 10 250 kd

Plai c e CRpi 10 Yes 188 kd

~~sucker 10 No? ISOkd

1JJn!.llJ.uiCRFLt 12 Yes 500 kd

lGotschlichand Edel man (1965); ~ileyand Coleman (1970)

!<Maudsleyet a1., (1 9 8 7b ); !!Maud s l e y et aI., (19 8 7 a ) tRobey et aI., (19 84); !Bach et a1.. (1977) ip ep y s an dMaudsley (1987): !!de.Be e r at a1.. (198 2a)

!Na g pur k a r and Mo ok erj e a (1981); loliveiraat aI. , (L980) kBodmerand siboo (L977); lpepy s et al ., (1978 a)

~inkel hakean d Chang (19 8 2 )1 !!Robeyat aI., (1983) .QPepyset al., (19828) ; IlFletcher et 81.. (1981) lIRobey andLiu (1981 )1 rFe r n andez - Mor a n et 81., (1968 )

Williams, 1978 ; Vo lan a ki s at a L, 1978; Nagpurkar and Mookerjaa, 1981). Isolationof CRP from large quantities of se rum, pleural, or peritoneal fluids can beaccomplishedby adsorbing CRP to the affinityresinin the presenc e of calcium and eluti ng with phosphorylc holineor a cal c i um- che lat i ngage nt .

2). G,n.ra1 PhysicoohemicalPropertiesofeRP Molecularsh ape

Ty p i c a lly,CRP compri ses5id e ntica l sUbunits (Ol ive i r a et a1., 1979 ).When examined und er an electronmicroscop e , CRP appearsascyclicpentamersand hasthereforebeen referred toas pe ntraxins (Osmand et al. , 1977) . Hu man, rabbit and rat CRP all have thistypicalconformation. In dogfish (Robe y et a1., 1983), plaice (Pepys et a1., 1982a), and lumpsu cker (Fl e tche r et a1.,1981),CRPis composedof 10 subunits.Thesesubunitseit her formt ....opentameric discs int e r a c t i ng face-to-faceor consistof 5dime rs which aggregatein a pentameric shape.One exc eption tothecommon molecular shape of CRP is horseshoe cr a b CRP, ....hich assumes a hexagonalring-shapedstructure consi s t i ngof 12 subu n i ts (Robey and Liu, 1981).

The subunitsinCRP are usuall yheld together non- cova l e nt l y. Howev e r , inrat CRP,threeof it s five identic a l subunitsare non-covalentlyheldtogether, while the other two are covalently bonded through a disulphidebridge

(Nagpurkar and Hookerjea, 1981; de Beer et al., 1982a).

Compqsit'qn

CRP subunits consist of 205-218 amino acids (Gotschlich and Edelman, 1965; Wang et a1., 1982;Nguyen and suzuki, 19864).The moleCUlar weights of CRP rangefrom 110 to 500 kd (Volanakis et aI., 1978). In some species, such as rat (de Beer et al., 1982a;Nagpurkar and Mookerjea, 1981), goat (Haudsley et aI., 1987a), plaice (Pepys et aI., 1982a), hamster rcee, 1977) and horseshoe crab (Robey and Liu, 1981),CRP subunits contain carbohydrate chains. In the case ot rat CRP,which contains In (wi....)carbohydrate (Nagpurkar and Mookerjea, 1981), it has been shown that each of its sUbunits is glycosylatl,d at position Asn-128 with a complex biantennary oligosaCCharide chain (sambasivam et a L, , 1990).

Inhuman (Gotschlich and Edelman, 1965), rabbit (Robey et aI., 1984), cow (Maudsley at aI., 1987b), and lumpsucker

(Fletcher et aI., 1981), CRP does not contain any carbohydrate. Therefore, based on their glycosylation status, CRP can be divided into two classes: glycosylated CRP and non-glycosylated CRP (Table.1).

The stability of purified CRP is dependent on temperature, pH, and protein concentration (Macintyre, 1988). solutions of CRP at concentrations of 50 JL9'/ml to

1 mg/ml atpH7-8 are st able fo r we eksat 4°C andfor months at _20^tlC.Unde rcondi tionsof lower pH or higher protein concentration, CRPmolecules te ndto aggregate. In alkaline solut ionsor withrepeate d freeze-thawing, di s s o c iat io n of CRP intosubunitsor intermed iateforms may be appreciable.

3).BindingPro pert:llloteRP Binding of eRP to phosphorylcholine

Theligand that CRPbinds be s t isphosphorylcholine (Vol an akisand Kap lan, 19 71) . substitutionof thephosphate monoester groupof phosp hory lcholine with other acidic gr oup s markedly decreases or abolishesthe binding of CRP

(Young and Williams,1978;Gotschlich, 19 8 2). I thas been shown that the interactionof CRP wi thCPS and many other comple x polysaccha ride s ofmicrobial , fungal, or metazoan parasiteorigin is mediatedth rough the phosphorylch oline moietyof thesepo lysaccharides (Toma s z, 1967; Pe r y and Luf f a u , 1979).The higheraffinity for phosphorylcholine compa red with other phosphatemonoesterssuggeststhat CRP re c ognize s both the positive lycharged tri met hy l a mmon i um gro up and the negativelycharged phosphategroup .

Human and ra bbit CRPbi nd5 molecu lesofphos ph o ryl- choline per molecule ofprote in (Li u et aI.,19 8 2) . Dog fi s h CRP bindstwo phos phory lchol i ne moleculesper subunit dimer (Robey et al .,1983).This suggestsone phosphorylchollne bi nd i ng domain on each subu n i t of CRP. oneexceptionis rat

CRP, which bind s thr ee , ratherthan tive, molec u l e s of phosphorylchollne (Na g p u rk a r andMook er j ea, 198 1 ).

Thebi ndi nq of ph ospho rylchol ineby CRPrequires that CRP firstbind sca lcium, usu a lly ina ratioof oneto two Caz..perCRPsubu ni t (Go tsch l ichandEdelma n, 1965 ). The con f ormati onal cha nq'einducedbyCa²⁰makes the phosph o ry l- cho line bind i ng domainofCR Pavai lab le tothe phospho ryl- cho l i ne ligand (Potempa et al., 19 81:'ioungand Wil l iams, 1978)•

The amin oacidseque nc e of CRP has be encomparedwit h the knownseque ncesof phosp horylchollnebindingmyeloma proteins. On thebasis of this comparison , it has been proposedthat thesequ e nc e Phe-Tyr-M et-Glu inCRP maybe partot its phos pho ry lch olinebinding domain(Yo ung and Williams, 1978).

Bindi ng ofC;RPtopol y catioDS

In theabsence of ca l c iullion s,CRP bi ndspol yc a t i ons suc h as poly-L-lys l ne andpoly-L-argln ine pol yme rs , ly slne- rich and argin ine- rich hi stone s,mye li nba s i c prote i n, and leucocytecatio nicprotein (SI egeleta1., 1974;Potempaet aI., 1981) . 1.tapp ro priateconcentrat i ons of CRP and ligand, the complexesagg r e gate and pr e c i p i tate.

Inthe absence of phosphorylchollne. calciuminhibits the interaction of CRP andpo l yc a t i o ns.However,in the presenceofphosphorylcholine,calciumpr omotes ratherthan

inhi bitstineee interactions (OlC amel l iet a1.. 19 8 0 ).

4.). llcutePhaseL,velll of eRP

CRP in human (Zell e r , 1986), monkey(Riley and coleman, 1970), rabbit(Winkelhakeand Chang~9 8 2 ) , chicken (Pepys at a1., 1978a ) , and ra i nbow trou t (Wink e lhakaand Chang1982) are typica l acute phasepr ote i ns . Normal sera in these speciescontain only tra c e amountsof CRP. However, during inf lammationor tis!\ue damage. CRP concent:.r:ationsin serum dramatic a l ly inc r e a s e, sometimes up to several thousand-fold over normal values. II) these species, CRP is among the first groupof proteinstha t are synthesizedat a great lyenhanced ra t e during acute phase response, and termi nationof the stimul a t i on is accompanied by there t ur n of their serum conce nt rations tonorma l levels.

CRPin rat (Collinsat aI., 19 8 4 ; de Beer et aI., 198 2a ) is a modera teacu tephase protein . It ispr e s en t in substantial amountin normal ratserum (0.5- 0.6mg/ml).

During acu tephasere a c t i on s, itsserum concentration increasesby abou t two-fold (Collinset aI., 198 4 ) .

CRPin cow (Maudsleyet al. , 1987b), goat (Haudsleyet a1., 1987a),dogfi s h (Robeyet al., 19 B3 ) , plaice(Whiteat al., 1982 ) , and horseshoecrab (RObeyand Li u , 1981 ) are nomal componentsinth e sera. Theydo not undergomajor change in serum concent rationfol lowing an acute stimulus.

10

D. BIOSnrrpSnNIPTURNOVER

0.

Experiments conducted in rabbits (Kushner and Feldman, 19 7 8a ) or with rat hepatocytes (Co ll i n s et aI.,19 8 4 ) have shown th at CRPis synthesized and secretedby the liver . ~

~CRP synthes ishas been demonstrutedusingperfused l iv ers (Kushner etal. , 19 8 0 ). CRPha s been identi f i e d histochemical lyinthe cytoplasmof he pat oc ytesbu t not. in any othertypeof hepaticcel ls ofrabb i t s during inflammation (Kushner and Fel dman,1978a ).

2). Increa s e d CRPsynt h e sisDuring acutePha!lll~

In speciesin whichCRP isan acutephase protein, the peakle v e l of CRP in serum corre lateswell with the se verity of tissuedamage (Kushner et al ., 19 7 8b:Ar o ns en et al., 19 7 2 ). Following anacutestimulUs, in c r e a s e d CRP synthesis starts in the periportal area , andth e n spreadsto involve al lcellsacrosstheliver lobule(Kus h ner and Feldmann, 19 7 8a ). The peak lev e l depe ndson the durationof increased CRP pro d uctionratherthanon the initial rateofincre a s e in synt hesis (Kushneret aI., 1978b). These findi ngs in dicatethat CRP is producedbyprog ressivelyinc r e a s ing number s of he p at oc y t e s after inflammatorystimul us .They also suggest th at a cirC Ulatingmediator(s),which 1s rele a s e d in association ....ithinflammat i on and necrosis, is responsible fo r theinductionofCRP synthesis.Th e r e is

11

evidencethat the circulating me d i ators may be interleukin-1 (IL-I) andinterleukin~li (lL-6) (Moshageet aI.,1988).Both IL-1 and lL-6 havebeen shownto stimulatethe synthes isof CRP by the liver,butIL-6is believedto playa key rolein the process (Mo s haqeet aI., 1988).

3).Other Bites of eREBiosyn tbedl

Although liver is the mainsour c e of serumCRP, it has been shown thatasub s et of peripheral bloodlymphocy t e s are als o able tosy nthes i z eCRPf which apparentlyremainsbound to the su r f a c e of these cells.(Ikuta et aI., 1986; Kuta and 8aum, 198-:).The biological significanceof these findings is not clearat present.

4). 'Invi v oCleara pceof eRP

The half-lifeof human CRPin the circulationofno rmal rabbits is about 4-6 h, and it remainsthesame during the course of acute phase response(Chelladurai et a1., 1983). Invi vo tur nove r studies of CRP havealsobeen carried out in mice and rats (Baltz et a1.,1985). The half-lifeof human eRP in mice and that of rateRP in rats we reals o in therang e of 4-6h. The half-lives ofthe s e CRP were independentof their circulatinglev els andwereno t affectedbyCPS (Baltz at al.,1985).

12

E. PROTEINSRELATED'1'0CHP

1) .

" rum.

Serum amyloid Pcompone nt (SAP) was firs t identifiedas a componentof amyloiddeposits inhumans (Cathc artat a1., 1965).SAP and CRP in humans ar esimilar to each other in the followingaspects: i).They bothbelong to a unique family of protei nscalledpentraxins. Their subunitsare arranged in an annulardisc- likeconfigu rationwithcyclic pentameric symmetry (Osmand et aI., 1977); il). There is about 60,"amino acidsequence homology be'tveenhuman CRP and SAP (Osmand et a1. , 1977; Andersonand Mole, 1982) 1 iii). They bot hhave Ca2'-dependentbinding capa city to cer tai n liga nds (Pepys and Bal tz, 1983;Skinner and Cohen, 1988). novevee, whereasCRP has a strong Ca2'-dependent binding prope rtyto phosphorylcholi ne, SAP binds to agarose, but not to phosphorylcholine (Pepys etaI. , 1977a1Pe py s et a1., 1977b)•

In ot herspecies, serum proteins whLch share",ith human SAP the prope rtyof bindi ng toagarosebut not to phosphorylchol ine hav e also been designatedas SAP {pepyaet a1., 197 8a ).SAP ha s been found in almostall vertebrate species, includingrabbit, rat, mouse, pig, goat,sheep, donke y, human, CO"" mari ne toad, plaice, flounder,and dogfish (Pepys ata1., 19788 ) .

13

2).syr iapHp,t er,. m. h pro~

In syrian hamsters, a CRP analogueis expressedin high levels only in females(cee,1982). Due to its sex-related distribution, th i s proteinis designatedasha mste r female protein(FP). In nOr1llal adul t femal esyrianhamster,FPis present at high serumle v e l s inthe rang'e of0.7- 3.0mg/ml , while in normalmales the level is much le s s (0.01-0.02 mg/ml)•

Like CRPandSAP, FPis composed of five ide ntical subun i tsand exhibits the pentameric structure {cce, 1983).

FPis auni qu e proteinin that it can bind both Phosphoryl- cholineand agarosein thepr e s e nc e of calcium (Coe, 19 8 3 ).

The amino acidsequence ofFPhas691hOMOlogytohumanSAP andsot homologytohuma nCRP (Dowton eta1.,19 80 ).

Althoug hFP binds tophos phory l c hol i ne , it has been shownto beacomponent of hamster amyloiddeposits (cce, 19 8 3 ). Therefo re , FPappearsto beahomologueof bothhuman CRP andSAP.

3).otherPhosp '!:!O ryl oh oHn.BindingPro t e ins Anti-phos p hory lcholi nemyeloma proteins

Anti-phosphorylchollnemyeloma prote ins have phosphorylchollnebinding property. However, un likethe phosphory lchollne bi nd i ng of CRP, their binding of phos phorylchol1neis independent of ca" (Pollet and

14

Edlhoch ,1973).ComparedtoCRP, these proteins showmu c h lessbindingspecificitytor the phosphate moi e tyof the liga n d (You n gand Williams , 197 8).

Pe r f o ri n is apore-forming pro t ein incyt o lyticT- lymphocytes. It hasbeen demons tra t e d thatphosphorylchol1ne acts asa Ca z·-d e p e nde nt receptormolec uleforperfor in (Tsc ho pp eta1. , 1989).

F.BIOLOGICAL ACTIVITIES or CRr

Anumber of biologi c a lpropertiesofCRP havebeen reporte d , ba s edon theirinteractions wit hma c r omol ecul es and wit hvarioustype s of ce l ls . Most of the s eprope rties are dependenton thephosphorylch oline bind ing propertyof CRP.

1). :Interactionsot eRr with complement system The cla s s i c a l comp l e ment pathwa y ca n be activated by CRP- CPS comp lexes ,agg r e ga tedhuma n CRPor bycompl ex e s of CRP with phosphorylcholine-contai ninq or cation-conta ining ligands (Ka p lan andVol an a ki s , 1974 : Cl auset al., 1977a;

Clauset aI. , 197 7bl Richards et a1. , 1979 ).Th e functional and bi olog icaleffects of complementactivatio nge neratedby these complexes includeops on iza t ionand lysis (Horten s e n

15

andGewur z, 197 6a ;Moldet a1.,198 2 ).Phosp horylcholi nehas been showntoinhibit this complementac tivatingactivity of CRP (Kaplanand Volanakis , 1974). The refore, the phospho ryl- choline bindingdomain of CRPhasbeen suggested to be involved inthe complementactivating process.

Ithas beend",monstrated that huma n CRP selectively de pos i ts on damaged tissue andnecrotic cells, and binds to membranes whichhav e beenst ru c t urall y altered (Volanakis an d wirtz, 1979;Narkates and Volanakis, 198 2; Vola na ki s and Narkates, 1981).The binding of rat CRP to multilamell a r phosphatidylcholinellposomeshas beenshown to require the disruption ofth eli pos ome struc tu re bythe incorporation of ly s ophosph a t i d ylchol i ne (Nagpurkar et21.1.,1983). The binding isspecific toward s the polar phosphorylcholinehe a d groups on the surfaceofth e liposomes. Similarresults....ere obta i nedfromth e binding studies of human CRP to unilamel larliposomes (Volanakisand Wirtz, 1979). More importa ntly, SUbsequentto thebi nd i ng of CRP to damagedor al te redcellmembranes, the complementpath....ay was shownto be activated (Volanakisand Wirtz, 1979 ). It has therefore b'''2nsuggested tha t CRPrecogni z e sda ma g e d cells~ , and by SUbsequently activatingth e complement path....ay, generates the chemotactic and opsonic activities requiredto promotephagocy tosis,leading eventuallyto the resolution andre pa i r ofth ele s i o n (Volanakis , 1982).

16

2).InteractioD'oteRr YUh LymphoidBy't."

A possible interaction between CRP and lymphoid cells was first suggested in 1937 by Abernethy and Francis when they reported a delayed skin reaction to CPS in patients with elevated serum levels of CRP (Abernethy and Francis, 1937).This suggestion wassuppo r t e d by the observation of Wood et al (1953). They found that, while immunizing rabbits, animals which produced CRP in response to the injection of antigen had higher titer of specificantibody against the antigen than those which failed to produce CRP.

In addition, Williams et at. (198 0, 1982) reported the presence of CRP on the surface of 1.0-35\ of peripheral lymphocytes of patients with rheumatic fever, poststreptococcal cholera, or acute streptococcal infections.

A number of ~experiments have provided more direct evidenceof the interaction of CRP with T- lymphocytes. Fluorescein-labeled CRP was shown to bind to peripharal blood lyrophocytes (Oishi et at., 1973).The binding of CRP to lymphocytes could be inhibited by phosphorylcholine or by pre-treatment of the cells with phosphl)llpase C (Hornung, 1972). Mortensen et at. (1975) observed that purified human CRP inhibited rosette formation, proliferative responses to soluble antigens and to all agenetic cells in mixed lymphocyteculture.Further experiments demonstrated a selective binding of human CRP to

17

antigen-stimulated lymphocytes (Croft et a1., 1976).The amountsof migration inhibitory factorand macrophage chemotacticfactor synthesized byantig e n-st i mul a ted and mitogen-stimulated lymphocytesdecreased when the lymphocyteswerecul t ured in the presenceof CRP(Mor te n se n et a!., 19 77).

3).ErtectotCRPonthe Actiyity ofPhagocytic C.lls In19 51,WOOd.reportedthatmoc1e s t co ncentration sof purified humanCRP or acute phase se r a with low levelsof CRP markedly st i mulat e d themi g r a tionof human polymorpho- nuclearleu c oc ytes , whereas high concentrationsof humanCRP inh ibitedthismi gration (Wood, 19 51). Hokama et a1. showed that the rate and degree of neutrophilphagocytosisof st r a i ns ofst r e ptococ ci pneumoni aeand serrati aMarcescens and of carbonyl ironspherules inwhol eblood were increased by the addition of human CRP (in the rangeof betwe e n10 and 30 JLg/ml ) (Hak a maet aI., 19 62) .Subse que nt l y, Ganrot and Kindmark (19 69 ) and Kindmark (1971) demon strated the phaqocytos is-promotingeffect of CRP inse r um- f ree~ aGsay systemsusing awi dera nge of bacterial sp eci e s , and succeededin reco veringCRP from bacterial su r f a c es

(Kindmark,1972). Inadd i t i o n, Mortensenet a1. (197 6) observed thatthe numberof monocytes ingesting CPS- c oa t ed erythrocytes was reduced from 82' to 13' when CRP was removed fr om the erythroc ytesthat had reacted with CRP and

,.

complement.Re-addi t ionof CRPrestoredthe inges tionto 76%, indicatinga requirementof CRP intri g ge ri ng ph a goc y t os is•

Recently, ra t CRP has been shown to occur as a membrane-associatedproteincons titutive lyexpressed on liver macrophages (Kupffer cells) (Kempkaet 211., 1990) . It functions as a galactose-specificreceptor for the binding of particulateligands.This membrane bound rat CRP has been shownto be identical to serum rat CRP and could be functio nallyreplaced by purified rat CRP.

4). EffeClt_ of CRPon Platel e t Punc tions

cevu e e and Fiedel were thefir s t to demonstratethat human CRP inhibits platelet aggre ga tio nsti mul at ed by ADP, epinephrine,or thr ombin (Fiede l and Gewurz, 1976a1Fiedel and Gewurz, 19 7 6b ). Thei r re s ul t..s suggested thatth e inhibitoryeffect of human CRP on plateletactivities might be due to aninterferencewith prostaglandinmeta bolism

(Fiedel et 211., 1977) . It was afterwa rdsshown that a low molecu lar weight factor in CRPprepa ra tionswas essential in th i s inhibitoryeffect of human eRP (Fiedel et 211., 198221).

Subsequent ly, rat CRP was shownto inhibit platelet aggre g a t i on induced by plate let -activatingfactor (PAF) in a dose-dependent manner (Nagpurkar et 211., 1988 ).Rat plate l e tsare normal ly re fra cto ry to aggregation by PAF.

Howeve r ,when ra bb i t antiserumagains t rat CRP was addedto

19

rat platelet-rich plasma,aggregationof rat plateletsby PAr was observed . In addition, rat CRP also inhibitedPAF- ind uc e d aggregationof humanplateletsin a dose-dependent manner.It was suggested that the highco n c e nt r a t i o n of rat CRP present innormal rat serum could be the cause ofthe refractoryresponse of rat platelets to PAF. Li ke rat CRP, rabbit andhumanCRPha v e also been shown toinh ib i t plateletaggregationinduc e d by PAF (Vigo , 1985 : Kilpatrick andVirella, 1985) .

Using an HPLC-g el filt r a tion technique, rat CRP has bee n shown to bind to PAr (Randell et al., 1990a).The formationofthe ratCRP- PAF complex was calciumdependent and couldbe inhibited byphosphorylcholine , suggestingthe involvementof the phosphorylcholinebinding domain of rat CRP. The binding of CRP to PAF has beenus e d to eKplainthe inhibitoryeff ectof CRPon PAF-i nducedaggregationof platelets (Randel let al.I 1990a).

Rat CRP has been shown to bind to platelets (Rand e ll et a1., 1990b).It has been suggested that the binding of CRP to the surface phospholipids of plate letsthroughits phosphorylcholine binding domainmay contributeto its inhibitionof platelet activities (Fiedel et a1., 1976c:

Randell et a1.,1990b) .

5). Jp tar. crUoA ot CRP with PlasmaLipop rot eins

Using immobilizedrat CRPonSepharose 4B, it was shown

20

thatrat CRPselectivelyboundape Ba nd E containing lipoproteins (Saxenaet aI., 1987a). Huma n andra bb i t CRP have beenshownto havesi mila r reactivitywith apo B containing lipoprote ins(de Beer et aI.,1982b; Rowe et a1., 1984a). This binding of lipopro tei ns was ca lciumdependent and the bound lipoprot e in s were eluted by phos ph orylc holine . Therefore, it presumablyinvolvesthe phosphorylcholine binding domain of CRP.

In rabbits, some or al l of the CRP in acute phase serum ex i s t s as a complexwith eitherlow densitylipo pr o t eins (LDL) or very low density li p opro t e i ns (VLDL) (Pontet et al., 1979; Cabana et a1.,1982).The formatio nof these complexesdependson the concentrationof apo B containing lipoproteins in the serum(Roweeta1.I 19 84a ). Inhumans, CRP co-existsin the circul a tion wi t hpla s mali p i ds or lipopro teins, but does notbindto them or formcomp le xes to any appreciableexte nt (de Beer et a1.I 198 2b ). However, in the serum of patientswithtypeIII hyperlipoproteinemia, whichcontains the abnormallipoproteinP-VLDL, the formationof CRP- lipop roteincomplexeswas demonstrated (Rowe et a1.I 19 84b ).

Investigationson the ef fectofrat CRP onthe binding of humanLOLto LDLre ce pt ors onli v er cel l membranes fr om est r a di o l - trea t e drats showed that ra t CRP inhibited the binding of LDL to thesere c e pt ors (saxena et a1.I 19 8 6). It was shown that this inhibitoryeffect ofrat CRP could be

21

due to the tomation er a LDL-rat CRP complex, rather than the binding ot" rat<:RPto theLDLreceptors (Saxena et al., 1986)•

I1I\lllobilized ratCRP hasbeen tested for its effectiveness to bind plasmaVLOLandLDLusing a extracorporealplasmapheretic system with rabbits (Saxena et aI., 1990). Resultsshowed that sepharose-rat CRP columns bound somecirculating plasma lipoproteins and most (>90%) of the bound lipoprotein fraction containedVLDLand LDL.

The interaction between CRP- and apo a-containing lipoproteins may have important.in..-.YJ.ygfunctional effects.

By binding to damaged cell membranes and then selectively interacting with apo a-containing lipoproteins, CRP may contribute to the specific localization of lipoprotein particles required for active cellular metabolism or processes of repair (Pepys et a1., 1985).This may be relevant to the pathogenesis of atherosclerosis .

6). eRrin Derenoe ",cbagh;, agaig8t Inflction and th, Chlrano. of' l.bDormal "at,rialsfromciroulation

The phosphoryl choline binding properly of CRP enables i t to recognize and bind a wide range of either microorganisms or their degradation products. It has been suggested that CRP may reduce the toxicity of the microorganisms by simply binding to them.Besides, secondary processes such as complement activation and phagocytosis may

22

alsocontributeto pro tectionagainst infection (Yotheret al. , 1982).ThisroleotCRPhas been demonst r a tedbyan experiment in whichhuma n CRPwas shown to protectmice from le t ha l streptococcuspneumoniaeinfection (Moldetar., 1981a). Thisresult supports the conceptth a t CRP may actin thisway both ince r t ai n primitivean i mals , which havenot evolved spe c i f i cant i bod y mechanisms,and in moredeveloped animalsat anearlysta ge of infection, before the production of specificantibodiesget under way.

studieson the interaction between CRPand certain abnormal materialsna v e also providedevidencethat supp ort the rol eof CRPin defense mechanisms (Pe py s , 1981). Human CRP hasbe e n showntobindto chroma tininthe presence of calcium(Robey eta1., 198 4 ).It has also beenshow nthat human CRP mediates the solubili za tio nofch r omat i n by complement (Robey et a1. , 1985).Inaddition, CRP from rat and horseshoecrab have been found tobi nd mercurybothin

~and ~,andit ha s beensuggested that CRP may act as a scavenge r for toxi c metals (Agr a wal and Bhattacharya, 198 9 ).

7).Clini call\ppHcaUonsofCRP

Duringsevereinfections,CRP concentrationinserum incre a s e s ,and the actua l deg r e e of increase usua lly corresponds with these v erityof th e infec t i on (Sabe land Hanson , 1974; Mood l e y, 1981 ).Basedon these obs e rva tio n s ,

23

measur emen t ot serumCRPhasbee nus ed in clinical diagnosis, especiallyindi s t i ng u i s h i ng bacterial intections tromviral ones,and in thema na g eme nt ot a widera nge of clini c al condit ions (Table2).

Table 2.cU.Di cal 1pplicatioll.s of tbeMe.s uremell.totSerwaCRP

screening tor organic di s e a s e

Monito ringthe extent and activityot disease Inf e c t i o n

Infl a mma t i on Ma lig na ncy Necrosis Detectionof infect ions

(Taken fromPepys and.Baltz (1983».

24

sectionrz, GLYCOPROTUNtNfO ~

Glycopr o t einsarepro t e ins to whicholigosaccharides are cova lent lyattached. It is wellknown tha t the carbohydra techainson glycopro teinsplayimportantroles in theirbiologic al functio nsand me t a bol ism.The cle a r a nc e of glycosylatedCRPhas been examinedin this study.Abrief outli ne onthe structura l featuresand carb ohy d rate- de pe nde nt clearancemechanism of glycoproteinsis therefore providedinth i s section .

A. STRUCTUREor GLYCOPROTE INS

1). composition oftheCarbohydrate Moiety of alycopr01;eins Although at lea s t 200 dif:fere nt mon os a cchari de s have been discoveredinna t ure,only 11are knownto occur in glycop roteins (Sharon and Lis, 1981 ). Mostof the s e monosaccha rides ar e hexoseeor the i r simple deriva t i ve s, such as N-acetylhexosami nesand uronic acids. In addi tio n, manyan ima l glycoprote i nsconta i nmore compl e x mo no - sacchar ides ,e.g. , th e sialicacids. Inglycoproteins , the s e monos a c chari de s exist as residu es inoligosa ccha r i de chains.

The red uci nggroups at th e end ofthe s e ollgosac c harides are invo lve d inthe carbohyd rate-pept ide linkag e.

2).C'.lrboh y4rab-Polypepti4ll....Li..nmn

The car bohydratecha i ns on glyc o proteins areclass i f ied

25

according to their linkage to the polypeptide chains (Kornfeld and Kornfeld, 1980).A carbohydrate chain is termed an N-linked or asparagine-linkedoligosaccharide i f i tis attached to the amide group of an asparagine side chain (Fig.1). On the other hand, if a carbohydrate chain is attached to a hydroxyl group in the side chain of serine, threonine, hydroxylysine or hydroxyproline, it is called an a-linked oligosaccharide (Fig.1).

In most glycoproteins that have been examined so far, the carbohydrate chains seem to be preferentially attached to peptide sequences forming ,a-turns (Rademacher at al., 1988).Where N-linked oliqosaccharldes are attached, the amino acid sequence next to the glycosylated asparagine is always asparaginyl-X-serine/threonine (X can be any amino acid except glycine). In glycoprotelns where more than one N-llnked oligosaccharide is present, several amino acids separate the sites of attachment of the carbohydrate chains.

In contrast, a-linked oligosaccharldes may be attached to neighbouring amino acid residues.

3).oligonccbaric!esinGlycopr0t.ein, N-linked oligosaccharides

The N-linked oligosaccharides are by far the most conncn ones found in glycoproteins (Kornfeld and Kornfeld, 1985).A conaon feature of N-linked oligosaccharldes is that they have a common inner core, consisting of a branched

26

Fig . 1. tla j or Types of C arbo hydrate-peptide Linkage

1. N -glycosidic linkage

GlcNAc - -- Asparagi ne

2. a-g lycosidic linkage

CK. O K 0

r ):

_ o~ ° 'v--

-ox-o

H~:c.. <:..

GalNAc- - - Th rlS e r

27

penta saccharide: trillIan nosyl-d i-N-a c e t ylgI uc os a mine [HanJ (GlCNAC)2) (Fig.2).

nc.

1I. .. Cl• •

>

a4apu4 from8Jluoa.IUMI UI (1111)

The periphera l mannoseresidues are usually substituted. Due to these substitutions, th r ee majortypes of N-linkedoligosaccha ridesare formed:thehigh-maDnose type (oligomannosid1c type), the complex type, and the hybrid type of oligosaccharides (Kornfeld and Kornfeld, 1985). Typica l examples of thethreetype s of N-linked oligosaccharides areshownin Fig.:1.

The high-mannoseoligosaccharidestypically have two to s Ix additionalmannese re s i due s linkedto tnepenta - saccharide co re,Whereasthecompl e x oligosaccharides containadi v ers e groupof additional sugarsto the core structure.The hybrid oligosaccharides ar e so named because they have feat uresof bothhigh-mannossandcomplex

2.

K- Gl"eo.ld1o

B:J.cb-DlUUlOI IOUcOlAocharl4e1

y-y...

y y) .- .-.-

y-Y

. - l>-e

~., -r

. - l>

. - A-e-y

H1tlr14 cUicl aoo!lartdl'

:~y, T

/y- .-.-

L:1- e-Y

O- Qlycolldla

. -0-

l>-O - ^lAUfr'MM&lJOOpro~

n.L.u~I'bn1Zl.llroopro~1n.I

z.r. !::J..W. O.a.lIlM, ^ftIlU,

....""' ... UI (1..1)

ollgosaccharldes .

The common pentasaccha rideof N-linkedoligosaccharides maybemodified both by the addi tio n of extra sugar re s i d ue s thatelongate the outer chains an d by the additionof extra br a nc he s to theama nn os e residuesof the core structure.

The outerbranchesof the ollqosaccharidesare also often referredto ali antennaeof the ol1qosaccharides.The ma j o rit yof N-linkedol iqosa cch a ridesco n tain two, thre e, or fou rant ennae(Paz-Parenteet al., 1982 ; Yamashitaet al., 198 3 :Franc o i s-Ge r a r d et aL ,19 8 3 ) .

The high-mannos 8oliqosacchar ide s oc cur inani mals , higherplan t s , yeast, and fungus glyco prote i ns. Onthe ot he r ha nd, complexoligosaccharid e sappear to be confi ne dto anima ls. In mammals , no mor e thansix mannose resi du e s at tached to the core structu re ha ve been found. But more prl1ltltiveorganisms,suc h as yeast.may contain much lal:ger high-Illannoseoligosaccharides (Schachter, 1984).

O- linked 01iqQS1lcsbu'des

Incontrastto N-llnkedoligosaccharides. tev general i za tions can be made abou t a- linkedoligosac charides.

Thecar boh yd ratestructuresvary frOIDa singlegalac tose residue (e.g., in colla gen) tobranc he d Ol!qos acchari de s of up to16 to 18 monosac c ha r i d e res iduBs (e.g. in soluble bl ood group sUbs tances ). Perhapsthe on lycommon feature in the structure of a-linke d oliqosa c ch a r i d e s is theGalNAc

'0

attached toth e hyd r oxylgroupof se rineor threoni ne in manyO-linked oligosaccha rides (Fi g. 3).

B. BIQBXNTHEBIBQPGLYCOPRQTEI NS

The polypeptidechainsof glycoproteins aresynt he s ize d in thesame way as non- g l yc os y l a t e d proteins.They are assembled on membra ne -boundribosomeson rough endoplasmic reticu l um, under directcontrol of the geneticcode.The initial glycosylationoccurseitherwhilethe polypeptide is still nasc e nt; on the ribosome or shortly aftertranslation ha s been completed.

Unlike thepolypeptide chains,carbohydra te cha i ns in glyc o pr o t e i ns are not prima ry gene products. They are synthesized by enzymesknownas glycosyl tr an s f era s e s

(Rademacher et a1., 1988). These transferasesarebound to th e metnbranes ofthe endoplasmicre tic ul um or the Golgi apparatus.They act in a veryspecificorder, without using a template, but. rather by recog n i z i ng the actual structure of the growing oligosaccharideasthe appropriatesubstrate.

Thistype of synthesisis notve ry accurateandthe r e f o r e re s ul t s in mlcrohete rogenei tyin the carbohydratemoietyof glycop rot eins. j.' o r example,o:l-acid glycoprotein (AGP) from human -erunhas at le a s t 19 variant carbohydrate structures at the five at tachmen tsite s alongthe polypeptidechain.

For O-linkedollgosaccharides, the suga rsare addedone ata timetothe polypepti d e chal ns.Usua llyGalNAcisadded

31

first, which is elongated by a variable nUmber of additional sugar residues, ranging from just a few to 10 or more (Ruoslahti, 1988).

Unlike the synthesis of a-linked oligosaccharides, the initial step in the synthesis of N-linked o1igosaccharides cannot be accomplished by the addition of individualsugars to the polypeptide chains. Rather, it requires the transfer

~of a single speciesof oligosaccharide, containing 3 Glc, 9 Han and 2 GlcNAc residues, from dolichol pyro- phosphate oligosaccharide to an asparagine residue in the polypeptide chains (Kornfeld and Kornfeld, 1985). The oligosaccharides are almost always transferred to growing polypeptide chains, ensuring maximum access to the target asparagine residues.

Once initial glycosylationhas occurred within the rough endoplasmic reticUlum, the oligosaccharideis trillU'Ded immediately. The trimming process continues as the glyco- protein is transported to Golqi apparatus. The diversity of the oligosaccharide structures on mature glycoproteins results mainly from this extensive modification of the originaloligosaccharide (Berger et a1., 1982;Kornfeld, 1982; Schachter et a1., 1983).

C. CLEARANCE OF QLYCOPRQTEINS

Proteins are degraded in a controlled manner during normal growth and differentiation (Rechsteiner et a1. 1987).

32

In addition to featuressuch as conformation of the pOl ype pt i d e,the carbohydra temoietyhas been shown to play an important roleinre g ul a t i ng the catabolismot glycoproteins. Several carbohydrate-dependent clearance pathways for glycoproteins hav e been discovered.

1) . Clearanceof glVcopr otei ns tbrp ug btb . HepaU"

Asia1oqlycoproteinRlpe ptp r

In thell:lt e 60Is , Ashwelland Morel l discovered that the clearancerate of ceruloplasmin, a copper tr a ns p or t i ng glycoproteinin serum, wasdramaticallyenha ncedwhen its te rmi na l sialic acids were removed by neuraminidase treatment (Ashwell and Harf or d , 1982). The as LeLo- ceru loplasminwas rapidlyta ken upanddegradedin the liver. Similar reSUltswere obtaine dwit hothe r mammalian serumglycopr ot e i n s . Detailedinvestigatio ns of this phenomenon revealed a receptor for asialog lycoprotelnson the su rfaceof hepat ocy tes (the hepaticasialog lycoprotein receptor). Thisrecep tor re cogni ze s the term i na l galactose residuesof a variety of asia loglycoproteinsandmediates the upta ke of asialoglycoprote'i ns intoLy sos e n e e for hydrolysis. ThUS, a clearancepathwayfor mammalian cirCUlating qlycoproteins ha sbe en established , whichis often re f erred to as Ashwel l 'spathway (Fig. 4).

Thehepaticasialoglycoproteinreceptorhasbe e n isolatedand purified (Schwartz and Ashwell, 1984). nue to

33

NANA

I

Gal I GlcNAc Coresuga

I

8 I ·

Gal I GlcNAc Coresu

I

~~.~i~_~~~~

8 !

glycop-' l oln~/ ••laloglyc oprolein

•'7'""HrpotW GriaIo,lVcopntrin _ _-"'..._ _ NlllJlfor

degradation

1

Fig.4.A8hweU'. pathway

its le ct i n-li ke specitic bin dingto galactose and the file t that thebind ing can be spe c ificallyinhi bite d byGa lNAc, this re c e ptor isal s o called he patic Ga l/Ga l NACspec i fi c lect in. It isthe~irs tanim allectindiscove red and characterized. Sim ilar to plantlecti na,th is le c t inal so agglutinatesneur a min idase- t reatedcellsand is mitog e ni c for lYlllphoc ytes .Th e s e prope rtiesimpl ymult i-v ale nc yof bind ingsitesonone functio nal le ctin mo lecu le.

The bind i ngandup t a ke of asialoq l yc o prote ins into hep atocytesals ose em to relyon lIIulti pIe intera c tio n s of the asialoglyc oproteinswiththe hepatic aaialoglycoprotoin receptor (Baenzigerand Fiete, 1980 ).Asia logl y c op r o t e i ns or purif ied asialoqlycopept i de s....it hmu lt iplete rm inal galactoseresidue s are boundwi thhighaffinitytothis re ce pt nr and are rapidly taken up into the cel L Asialo- glycoproteinswi th twoorlesstem ina l ga l acto seresidu e s usu a lly bindpoorly.Thu sasialo AGP,whIchhas.anytri- or tetraante nnaryN-linked oligos a c cha rides , is an exce lle nt substrate forthis receptor , where a sserum as1a lo- tran s f errinwithbia ntennaryoligosaccharldes isapoor substra t e (Ha t to n et a1.. 1979 ).

Sialic acids havebeen shownto playa dual rolein this clearancepathway (Hudginet a1..1974) . On one hand, the removal of terminal si alic acidsfr omgl y c c protei ns isa pr e -re quirement fortheirbindingto thehepat icasLako-

35

glyc oprot einrec epto r. On the other hand , the pres e nceof sialic ac i d s onthe receptor isnece s s arytoke epth e receptorbiolog i c all yfun c tional.Ne u r amin i da s e treataoent of the hepatocytesor the purifiedhepati cas ialogly c oprote in rece p t or re sultedin compl e te inhi b i t i on of the interacti on between asi a loglyc o pro te ins and thehepatic receptor. The removal ofsialic ac id s fromthecar bohy d ra tech ai ns ofthe hepatiorec eptor induc e s se l f-ag g reg a tio n thr ou gh inte r - actionwith 8xpos e d ga lac t os eterminalresidue s, and impai r s itsbinding ofasi a 1og1ycopr o tei ns .

2).Other Carbobydrat_- da p_ndentcl ••rancaMechanhms Clearance sys t ems inwh i c hsug a rs otherthan gala ctos e serveas determinantshavebeenide ntified. Besides hep ato- cyees, non- pa r en c h yma l cells in theliver, particularl y Kupffer cells,also pl a y animpor t a nt role inthe cl earance of gl y c opr o t e i ns .

After AGP has been successi velytreate d wi t h neurami nidaseand,a-galactosidasetoexpo s eits GleNAc residues, the modified prote i n israpidly taken up by the reticuloendothelialsyste min thelive r.Hanna s e-terminat ed gl yc o p r ot e i ns are cleared in a simil ar way (stahl et a L,, 1976). The re fo r e , thelecti n on the surfaceof Kupffercell s bindsglyeoproteinscarrying ei t he r terminalGlcNAc or Man residues.

36

An ana logous lect i nis alsowide ly di s t r ibute don phagocyticcells,e.g. alveol armacrophages.Th e funct i on of theabove mentioned le ct i ns may inc lude the clearance of agedor partiallydegr a dedserum glycoproteins fr omthe cir cul a t i o n , andthede s t ructi on of microorgan isms, such as yeasts , whichar e rich in ol igomannosidicchains.The ingestion of yeastsby alveolarmacrophages, for examplo, is inhibitedby mannosean dglycoproteinswith terminal mannose residues (Warr, 1980 ).

Unlikemammalian serum glycoproteins, norm al circu- lat i ngglycopro teins inavian species usually po s s e s s exposedgalactoseresidues. Obvious ly, therefore,clearance via thehepatic Gal/GaINACspecific lect in cann otexistin these species. It was found that in these species a cle a r a nc e system whic h re c ogn i ze ste rmi na l GICNAcresidues of glycoproteinsopera tes onthe surfaceofth e hepatocytes (Re g oecz i et aI., 1975).Theelegant workofRegoeczi and his co-wo rkers showed that ttle half-li f e of avian glyco - prote ins wa s dramatica l ly de c r e a s e d after beingtr e atedwi t h ,a- g a l a c t os i da s e to exposetheir penUltimateGlcNAc re s i du e s

(Regoecziet a1.I 1975).

I thas been found that the car bohydratebinding domain of several cl on e d anima l le c t in s sharesomeCOml1lon fe a t ur e s inthe i r ami noacid sequences(Drickame r, 1988). Homo logous carbohydratebindingdo mains have alsobeenfound inothe r

37

ani mal lec t i n s.By searchinq th e known prote i n sequencestor thecarbo~ydrate~indin9'motit , addi t iona l animal le~tins havebeendiscovered,includinqpUlmona rysurfac tant pr o tei n A,proteoqlycancorG proteins, andlymphocytere cep t or tor the Fc portionotIq E(Dr i c kamer, 1988).

3.

SectionIII.PORP08I!orTR£ PRESENTnODY

As describedearlie r inthi s chapter, CRPinvestiga ted so far are al l synthesizedmain lyin thelive r.However, the ir catabolic path way is not clear.Basedon Ashwel l's discovery,th ehepa ticasialog lycoprotei n re c eptor is a potent ialsite for the clearance ofmamma l iancircu lato ry glycoproteins . Rat CRP, being a serumglycop rote i n , offe red us anoppor t unit y to study itscleara nceby Ashwe l l 's pathway.

Since not all CRP are glycosylated , it is appropriate to determinei fglycosylatedCRPandnon-glycosyl ated CRP follow differentcle a r anc epathwa ys. CRP bindsto ph os ph orylch o l i ne, and thecel l surfaceis abunda nt with phosphory1c ho1 ineresidues.It isthe r e t o r ere l e va nt to examinei fthe phosphory1chol lnebindi ngpro pertyof CRP wou l d affect thebinding of asia loCRPtoth e hepatic asialog;J.ycoprotelnreceptor and theauba e qu e nt; clearance of theseproteinsthroughthisre c ep t o r .

Inthepre s e nt study, thebi ndi ng of rat asia l oCRP , rat CRP and rabbitCRP to Isolatedhepatocytes has been examined.The clearanceof theseprote i ns ha s been investigated~and alsousingperfus e drat live r.

competitive inhibition experime nts hav e been carriedout.1n ill1:2and~betwe en ra t asialoCRPan d asialoAGP . The

39

effect of GalNAc and phosphorylcho lineon th e bi nd i ng of rat asialoCRP hasbeen studied , ra t and rabbitCRP to isolated hepatocytesand on th e clearanceofthe s e proteinsby perfused li v er .

40

CHAPTER 2 MATESUL8ANJ)MEtHOps

A.IllInl\1.M&

1). An i mal s Male Sprague-Oawleyrats (body weight 250-300 g) and malewhiteNew Zealand rabbits (b o d y weight 1.5-2.5 k.g) ver'e purchased from CharlesRiver CanadaInc.,La Praire, PQ and were fed chow ad libitum.

2).Reaq8n~!!IaM chem i cal. waymouth'BHB752/1medium(IX) was from Gibco laboratories, USA. Nal2S₁(reductant free, 2mci in 4-5~l0.1MNaOH) solution was fromDu Pont Canada Inc. Cyanogen bromide (CNBr) activatedSepharose 4B was from Pharmacia. lodo-Gen (1 ,3 ,4,6-tetrachlorO-30:,60:-

diphenylglycouril) was from Pierce. Neuraminidasefrom .!a....

perfringens (EC 3.2 .1.18),col lagenasefrom£l..:- hfstg l y ti c u m, typeIV (EC 3.4.24.3), O:l-acidglycoprotein (BOvine), Sephadex G-25- 150(bead size 50-150JL)and all other chemicals werefrom Sigma Chemical Company.

3). Medium andBuffer ~Waymouth'sMB752/1 medIum plus 0.2\ (w/v) bovi n e serum albumin(BSA) (pH 7.4). Hanks ' buffer' 0.137M NaCI, 26 DIM NaHCO" 0. 34 rnMNa2HPo"

0.44mMKHzPO" 0.5mMEGTA, 0.8mMMgSO,' 7H20, 5. 4llIM KCI (pH 7.4). Kreb's -bicarhonate /B SA' 118mHNaCI, 23.8 mM NaHeo" 0.95mMKHzPO" 1.2mMMgSO,~7H20, 4.8 mMKCl, 2.9 mM

41

CaCl z·2HZO, 0.25lBSA (pH 7.4) . Trigbufter · 5mMTris-HCI butter (pH 7.5)containing0.1 5 M NaCl and2mMca z-.

B. PRlpnauYlPROCEDURES

1). pr e p ar a t i o pot seDba rose -pb eDy lphospborylcholine Affinitylld s o r be n t The affinity ad s orbe nt for th e isolation and puri fic ationof ratand rabbitCRPwa s prepare das described by Nagp u r ka r andHookerjea (1981).Typically, 4- ni t r op h en y l - p h os p h o ry l c h o line (0.69, 1.19 nmol) inmeth anol (50ml) was red ucedwith Hz at 1 atm us ing 0.29 of5%

palladium on charcoalas catalystfor 2 h at room temperature. The reactionmi xture....as filteredandthe filtrate ev apor ated und e r reduced pres sure. The re sUlt ing4- aminophenylphospho ryl choline....asimme dia t e ly diss olved in 100ml of 0.1 NNaRCo) buffer (pH 8.3) containing 0.5M NaCI and ....as added to 15 g of CNBr-a c t ivatedSepharose 48 hi.ch had beenpreviously....ashedwith 1roMHCI. The mi x t u re as gently mixed for 4 h at room temperature , filteredand then washed alternati velywith 0.1M NaHCD) buffer (pH 8.3) containing 0.5M NaCl and0.1 M CH)COONabuffer (pH 4.2) cont ai n i ng 0.5K NaCI. The Sepharose-phenylphosphol.ylcholi ne was treated overnight at 4°C with 200 ml of 1 M ethanolamine (pH 9.0) and finallyre-sus pended in 5\lIMTris-Hel (pH 7.B) and st ored at0- 4°C.

42

2).Prlparationof R.t and Rabbit Ser. Rats were anaesthetized l ightlywit hethe r, the abdomencut openand blood obta inedfrom the abdominal aorta.'l:he blood was al lowedtocl o t for 1 hour at roomtemperaturefol lowedby another hour at 4°C. It was thencentrifuged for 5 minat 1,000 Kg in a bench top centrifuge. The ser umwas taken out and used immod i a t e l y orstore d at_20°C until use.

Inflammationwas induced inrabbi tsby subcuta neousl y injecting turpe nti ne (0.5ml/kgbodyweight) in the thighs. Blood was obtained after 48 hby cardi acpuncturefrom rabbitsunder ar.aesthesiainducedby intraperitonea l injectionof sodium phenobarbital (1ml/ kq body weight). Serum was preparedas described for rats .

3). Isolationand Purification ot Rat and RabbiteRP Rat CRPandra bbi t CRP wereisolatedandpurifiedfrom no rmal rat serum and inflamed ra bb it serum, re s pective l y , according to theprocedureof NagpurJcarand MooJcerjea (19B1) . Sepharose- phenylphosphorylcholineaffinityadso rbe ntwas packed ina colum n(20 x 2 em)and equilibrated with5- 10 be d vol ume ofTris buffer at a flow ra t e of 1 ml/min .Serum

«40 ml) was brough t to a concentrationof5tnMwit hrespect to Tris-HCI , 2 111M with re s p e c t to ca Zo, and applied on the colum n. The column was washed extens ivelywith Tris buffer unt ilabsorbanceat 280 nm ofth e eluen t wa s le s s than 0.02. CRP was then elutedwith 8roMphosphor y lcholineinTria

"

buffe r.After dia lys i ng exhaustivelyagainstTris buffer(4 cha nges),the proteinsol ut ionwas concentratedby ultra- filtra tion ,and aliquotswere st o red at _20^Ge. Pr otein con cen trationwasmeasuredby the method of Lowry et al.

(1951) us ing eSA as st a ndard.The pur ityof CRPwaschecked by el e c t r ophore sisand highperformance liquid

ch ro matog ra phy (HPLC).Pr e paratio ns whic h gave a si ngle band on electropho res i sor asingl e peak on HPLC were used for furt he r studi e s.

4).'np.nUopof Millo Pfottip!!! Ra t CRP (10 mg) was des!alylatedby inCUb a ting with0. 5 inte r na tiona l ac t ivity unitsof neuraminidasein10ml ofTris buffe r at 37°Cfor 3 h. The incubation mixt ur e wasthenapp l ied to a sepharo s e - pheny1pho sph orylchol ine atf i nit ycolumn (2em x3!5 em).The columnwas wa s h e dextens i ve ly withTris buffer.Ratasi a lo eRPboun d tothe colUlllnwas el uted withBmMphospho ryl- cho line in Tri s buf f e r. AGP and fetuinwere desialylated by treatingwith0.05MHZS04 at so-c for60min, followingthe procedure of spiro (1964). The amount ofsialic aci d released dur i ng inc uba t i o nandsia lic acidremainingon the prote inwas determinedusingthe method of Warren (1959). Typically,desialylation re s u l t e d inthe removal of >90",of thesi a l ic acid fromrateRP and AGP.

5). RacHoiodinaUonProc.dur81 Rat CRP, rat as ialoCRP, ra bbit CRP, AGP, and asia l0AGP were iodinatedwith Iodo-Gen

44

iodinationre a ge nt as the catalyst followingthe manufacture rIs procedure (Pierce) . Briefly, 2mCi of carrier free Na125I was diluted to60~lwith0.1N NaOH.rcdo-c e n coated tube swcrerinsed withTris buffer. Proteinsolution in Trisbuffer was the nadded , to a ratio of 10 I-Ig protein/I-Iglodo-Gen, followed by500~CiNa¹2SI in15~1of 0.1N NaOH.The reaction mixturewas le f t at room temperature for 10 - 1 5min withoccasional shaking,and then applied to a G-25column(20 ml bed volume) . whichhad been previouslyequilibrated with Trisbutfer followed by0.5ml of BSA(1mg/ml in Tr isbu f f e r ).The la bele d protein was collected from the void volume and dialysedagainst Tris buffer. Sample s were aliquotedand storedat

-20°C. Thespecificra dioa c t i v i t y was in th era nge of 1.0- 1.5x10 6CPM//Jgprotein andgreater tha n96\of the rad i oa c t ivi ty was precipitableby ice-coldtr i c hlo r oa c eti c acid (S\)-phosphotungsticacid(n).

C. MfALYTI CAL PROCEDQRg0

1). Electropho r esis Pr oteinsampleswere analyzedby either nativeor 50S polyacrylamidegel electrophoresis (Native- PAGE, and SDS-PAGE)usingthe separationun it of a Phastsystem (Pharmacia) anda-25\gradientgels (Pha s t Ge l , Pharmacia). After electrophoresis, th e gels were stained with CoomassieR 350 dye (Pha rmac i a ) us i ngthe devQlopment unit of the PhastSystem.

45