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CENTRE FOR NEWFOUNDLAND STUDIES

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TOTAL OF 10 PAGES ONLY

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EFFECTS OF INFLU4MATION AND DBXAHETHASONE ON VARIOUS ASPECTS OF GLYMPRCTEIN BIOmTHESIS

\

A t h e e i s s u b n i t t e d b . ~ the school of Graduate Studies i n p a r t i a l fulfilment of t i e r e k e n t e

f a r the degree of Dootar o f Ph$losyphy

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Department of B-Istry Memorial Un{versity of Newfoundland

s t . b o h n a s, ~evfoundland 1

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nag 1987 ,

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Permiasion. has been granted L'autorieation 4t4 accord4s t o the Nati'onak Library of '

Canada t o microfilm this miarofilmer thesis and- to lena or sell

' Eopiea of the film.

. .

' film. a , - , - .

\ The author (copyright armr) L'auteur (titulairs du droit h i r r e s e r v e d o t h e r d'auteur) ae rbssrvs las ' p u b l i c a t i o n ~ i g h t a , and autrea droita de publicatLon?

.

a h i t h e r t h e t h e s i ~ l nor ni la t h h e

extensive extracts from it e x r r a i t a de

%h,"."-i??: \

may be psinted or othervisql doivent atre' i m p r i d s o u reproduced without h i d h e r , autreaent reproduita aana son written permiselon. .autorieation Lcrite.

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' / .

i i ASSTRACT

' The concentration of acute-phase p r o t e i n s i n plasma i s s i g n i f i c a n t l L increased i n response t o . i a f l a m a t o r y agents.

- Almost a l l acute-phase protein? are glycoproteins in whioh t h e carbohydrate moiety i s a t t a c h e d t o t h e peptide thrpugh an ahparagine nitrogen and lare synthesized

U

dolicfi.pl- s linked intermediates. D@xamethasone, a potent s y n t h e t i c giuoocortiboid, 'has bean shown t o influence t h e biosynthesis

- .

of t h e s e acute-phase glycoproteins i n experiments with

-

a n n a l s and

t m.

This t h e s r a concerna various a s p d a of the+yconylqtion

b -

of acute-phase p r o t e i n s during inflammation and dexamthesone traqtment i n t h e r a t .

I Turpentine-induced l n i l a m a t i o n caused increasqd siialyl and g a l a c t o s y l t r a n n f e r a s e a o t l v i t l e s i n t h e lrver, while i n serum, only t h e s i a l y l t r a n s f e r a g e a c t i v i t y was increased.

The I o m a t i o n of several dolichol-linked l n t o m e d l a t e s ,

.

such as.dolicho1 phosphate mannose, dolichol pyrophosphate N-acetyl chitobiose and do14chol pyrophosphate oligosacchariies was increased In ouitured he$atooytes, a r t h e i r homogenates, isolated iroh inflamed r a t s . Dexamethaeone treatment of ..hepatoqtes from control and inflamed animals a l e 0 caused an increased formation of t h e s e intermediates. The i n a r e a s s i n ' t h e fornation of dolichol-linked i n t e m i a t e s in inflemation was a t t r i b u t e d tp increased endogenous d e l i c h o l phosphate : l e v e l s . I n c o n t r a s t , t h e inoreased 3evele o f t h e intermediates

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iii

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, in dexaiethasone. treatmeirt ware not due to.%= endogenorie ' d o l ~ c h o l phosphate but ware most likely due t o t h e iIpcC1on of glysosyltran6ferases involved in glycopmtei b i ~ ~ t h s s i s .

-

These conclusions were based on the results obtained from the .foIlouing expeyiments: 1) ?stination of the endogenous dolichol phosphate, 2) fornation of d o 9 h o l phosphate

. .

.

mannos's in presence of inoreasing amounts of exogenous

9

, dolichol phosphate, and 3) formation of dolichol-anddolichol phosphate iron mevalonate.

Experiments in hepatocytes with actinonycin D and

-

2t ~ycloheximlde suggested that the lnoraase in dolichol-linked

.

intermediates was dependent on the increased synthesis of glyaosylatahle polypeptides-of the acute-phase proteins.

Nucleotide sugaF pymphoaphatase activities were i n s w e d only in dexamethasone treated hepatocytes, *Hereas, the

---

nucreotrde sugar levels remained unaltered during both

-

inflammation and dexamethssone treatment. The preseht study showing increased synthesis of dolichol-linked intermediates during the biosynthesis of amte-phase proteins in response to inflammation and dexamethasone treatment has provided new

t

*

information on the role of the doli&ol paVIway in 91-rotein synthesis.

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I would 1fke to O X P ~ P = S my sincere appreciation and deep

' geatitude to ~ r . S.S. Moakerjea for his eupervislon , and

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,

a ' . - .

quihance throughout the course of this work: His continued I enooudagenent and'advice have been very ~u&rtive and in~piring:,

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I am grateful to Dra. J.T. Brosnan and G. Herzbewl for a c t l q as a supervisory committee melnbsre and for their helpfui discussions. I wish to exprese my 'pppreciatign to

, .

them for critically reading my thesis and usefhl euggesti,d.

I would also like to thank Dr. a. Nagpurkar fbe'reading and

helping in writing the thesis.

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J

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4 I would like to thank Dr. 0 . Xomfeld for his generous supply of oligosaccharide standards. I uould\ike to wpress my sincere appreaiation t o ~ a l l newhers of the Biocpemistry

/

Department for creating a very iriendly aoademic ntmoipheie. '

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Many thanks are also due to Mrs. Joan Collins, Mrs. shobhitha- : -

Ratnam and Mr. Uday Saxeda for creating s pleasant and

Ii

comfortable working atmosphere in the lab, w i q spec.ial '

),

tdanka to Mrs. Joah colline for her assistance with the:

'\.

hepatocyte preparations. I appreciate the very efficient assistsnse of nise Carol M"rphy in thb preparation of the '

thrisia. ~ .,

-

i

1 wish to or. ..a. ,ri0h,

Oan,

studies' f~r'~roviding me iinancisl deei&.doe in the form of , Memorial university' Graduate ello ow ship and Bursary. I woula also like to thahk Mebical Remearch council of Canada

b

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and I dedicate t h i s t h e s i s t o them.

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papers published and in preparation.

1. "Key 'role of dolich 1 phosphate in glycaprotein biasynthesis:" S. Mookeqjea, .T. Coolbear P Mohan La1 - sarker: can. J. Bfocham. 6 Cell Biologyql, 1032-40 (1983).

2. "Effect of daxanethasone on mannolipid synthesis by hepat0Eyte.e prepared from control and inflamed rats:"

Mohan sarkar 6 S. Mookerjea: Bimhem. 3. U . 429-36 (1984). - 3 . "Increase of eialyltransferaea activity in the serum and

l i ~ e r ofminilamed rats:" I.H. Praeer, T. Coolbear, Mohen- Sarkae P S. Mookerlea; Biochimica et Bisphyeica A c t a , D.2, 102-105 (1980).

4. "synthesis and eeceetion of serum phosphorylcholine binding peote+n by rat hepatocytes:" Joan Collins Uday saxana, Mohan sarkar, Arun Nagpur$ar and sailen ~ o o i e r j e a ; Blochlmica at Biophysics A c t a . C m , 97-101 (1984).

5. Effect of dexamethasone on the syntheeia of dolichol- linked saccharide and glycoproteina in hepatocytes prepared from control and inflaned rats: Mohan Sarkar P

.

s. Mookerjea; Biochen. J.

w,

675-82 (1985).

6. ,wecove

j

of doiichol-2oligos;;haride in mathanolic

-

,

aqueous phase prepaeed irom rat liver nicrosomes;" Mohan sarkar 6 S. m k e r j e a ; Biochem. J.

m,

913-916 (.1986).

I

7. Regulation of glyooprotjein synthesis by dexamethaeons:

Effects on dollchol-linked sugar intermediates and U nucleotide sugar pyrophbsphataae activity: Mohan sarkae

6 S. Mookerjaa (submitt+).

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v i i

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PAGE

.

...

Table of Concants v i i

. . .

I list of ~ a h l e s

...

x i i

~ i s r of ~ i g u r e s

...

9 L i s t of Abbreviations

...

CHAPTER 2 I~TRODUCTION

- -

,

I 1.1. 1 n f 1 ~ i m a t i o n ...h....

8 1.2. Acpte Phase P r o t e i n s

...

A. Poiyp.ptide s y n t h i k i s

...

;

...

8. S t N E t U r e Bbd ~ y n t h e ~ i b of N-linked 0lig0- s a c c h a r i d e s i n a l v c o n r o t e i n s

...

1. Assembly of t h e - d i d o b a o o h a r f d e chain on d o l i c h o l ~ h o s ~ h a t e

...

1 2. The t r a n d f e r if oliqosaocharide from l i p i d c a r r i e r t o polypeptide

...

3 . Processing of N-linked o l i g o s a

7 .

1.4. Role of t h e Nucleotide s u g a r s

...

28

.

...

1.5. ~l(p0.t

Or

s e c r e t i o n oz Glysoproteins 31

1.6. I n t r o d u c t i o n t 0 P r e s e n t Work

... :... ...

33 -

CHAPTER I 1 MATERIALS AND METHODS

1

2.1. M a t e r i e l s

...

38

...

2.2. Experimental Procedures 39

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viii.*

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' PAOE

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F .I

il iil iii) iv1

Animal and induction of'inflammation

...

Enzyme assays

...

3

... ...

Mannosyltransferasi

...

N-acatylgluaosaminyltransfeeaa~

...

sialyltranaferass

...

Galactosy&tranafera.e

...

succinate: Cytochrome C reductase

...

NADPH - Cyaoohrome C reductaiae

...

B-glu~ur~nidase

...

synthesis ot oliqosaccharide Lipid

...,...

I

Purification of 611$osaooharide lipid b y k E - Cellulree Ch?omatography

...

~na;ysis of Oligo&ccharide Derived from Oiigo- sa~cqaride lipid;

...

1so1ation a&/mntitation or Nucleotide Sugars FL'Om Hepatonftes

! ... .

Biosynthesi and Isolation of [3~]-dolichdl and [3H]-dolickkl phosphate

...

Purificab'ion and Quantitation of [3~]-dolichal

...

and ['H~dolichol phosphate by HPLC raoladon and Wantitstiod of Doliohol arid oolic6ol Phosphate from Rat Liver Homogenatea and Niceoeomes

...

. .

CHAPTER I11 EFFECT OF INFWUlMATION

-

O N S I A L Y L A N D GAWLCTOSYLTRANSPERASE ACTIVITIES IN LIVER

I AND S E R W OF RATS. '-8

J '3.2. Results

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59

...

3 oiscuesion 3!

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PAGE CHAPTER IV EFFECT OF INFLAMlVLTION AND ,DExaMETHASONE ON MRN-P-LmL AND [ G M A C ) I-

. , :,P~;D,","VEBL.","Yp40g;

AND HEPAMCYTES

4.1. Introduction

...

71

, ,

1.2. Resulti

... .:. ...

73- A. Effect of inflammation o n the fornationrof

Man-P-Do1 from GDP- [14c] -marnose in rat liver microsomea

...

%?

8. Effect of ipcreasing amounts of exogenous dolichol phosphate on the fornation of Man-P-ool

.... ...

from G D P - [ ~ ~ c ] mannosa i n niclosmes

.,

74

C. Increqge pi dolichol and dolichol phosphate ooncentration during inflammation

... ;...

78 D. Effeot of inflammation and dexamethasone.on the

incorporation of [14c] Man and [l"] GlcNAc into Man-P-DoL and (GlcNAc]l-Z-P-P-Dol

-

from re.spective nucleotide sugars in hepatocyte hamogenates obtained from control and

inflamed rats

...

81

E. Effect of increasing amount of exogenous dolichbl phosphate on t h e formation of Man-P-

.

Do1 from GDP-[~'c] mennose by hepatocyte homogenate

... :...,.

89 .F. Effect of inflammation and daramethasone on.

the synthesis of [3n]-dalichol and

H HI-

dolichol phosphate from ['HI-mevalonate

...

92

G. Effect Of actinonycin D end ~ycloheximide

.

on the increase op mannosy1 and ~ - a c e t * ~ - glucosaminy tranhferase during inflammation

... ...

and dexemethsone treatment

.:

91

.

H. Studies on the incorporation of 1 1 4 ~ 1 mannose ;

...

info Man-P-~ol iwintact hepatosytes' 102 I. Subcellular distribution of mannosyl-

.

transfarases

...

102

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PAGE

CHAPTER V

.

EP,PECT OF INFLUPUTIO~

AND DEXAMETHASONE ON

.

I THE BIOSYNTXESIS

4

,'

O F DOLICHOL LINKED

.,

OLIGOSbCCHARIDES AND- , G L Y C O P R O T E I N S I N

.

HEPATOCYTES

... ...

115.

.

>

117 the incorporation of [l4c7 mannose from G D P - [ ~ ~ C I nannose into oligosacoharide lipid in

hepatocyte homogenates

...

11'1

~ f f e c t of incraaslng amounts of exogenous dolichol. phosphate on oligoaaccharide

...

lipid synthesis in heparosyte honogcnatas 121'

~ f f e c t e of actinamycin D and cycloherimide pn the oligwacsharida lipid synthesis induced by inflammation and dexamathasone

...

treatment 124

studies on the incorporation of [14c] nannase into [man-14c] oligosaccharid~ lipids and ghycoproteins in intact hepatooytas from '

...

aontral and inflamed rats 127

~ f f e c t of c e l l densit on the [14c1 mannose incorporation into Mag-P-Dol [man- 'C]

oligosacqhqrida lipid and proteins in intact

...

hepatocyte.

...

131

r ~ n a l y s i e of o1igosaocharid.e lipid complex 135

> ,

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isc cuss ion 138

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x i

*

PAGE T~PTER VI EFFECT OF I N F L ~ ~ T I O N

AND DE)ULMETHdSONE ON GDP-MANNOSE AND UPD-

GLCNAc PYROPHOSPHATASE

A C T I V I T I E S I N R A T HEPATOCYTES

Introduction

...

143 !

.

Results

...

145 Increase of nucleotide sugar pyrophosphatas*

a c t i v i t y by deramethasone

...

145 Mechanism of dexanathasone eff c t on the

inmease of nucleotide sugar pyrophosphatafe a c t i v i t i e s

...

i b l ind ~ T P dn

Determination o f ~ n u c ~ o t i d a su&r &ola l p

...

hepatocyte culture

Discussion

...

162

-

.

CHAPTER VII DISNSSI~*

Role of dolichol phosphate i n glycoprotain s y n t h e s i s

...

169

Role of dolichol linkedglycosyltransierase a c t i v i t i e s i n regulating N-linked glyco-

protein lynthesis

...

170

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7.3. Role of nucleotide sugars i n N-linked glyco- p r o t e i n synthsgis

...

172

7 . 4 . Role of the expression and. t r a n s l a t i o n of m A s encoding N-linked glycoproteine i n glycoprotein eynthesis as r e l a t e d t o t h e e f f e c t s of deaaaethasone and inflammation on d a l i c h o l phosphate depenaent g l y c o s y l -

t r a n s f e r a s e s

..(. ...

175 7 . 5 . Conclusions

...

176

.

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xiii

Y'

List of Tables PAGE

Table 1. Effect of injection of Eroton oil into rats

... ,

5

Table 2. Phyeioal and chemical propereies of typical Bate-phase protein-

...

8

Table 3. Effect of turpentine induced i n f l h a t i o n on dolichol and dolichol phosphate

~ ~ n ~ e n t ~ a t i o n s in rat liver homogenatee

.

and microsomes

...

8 0

9

Table 4. Effeot of dexamethasone on the synthesis of

...

Man-P-~ol by rat hepatocytea 88

Table 5. Effect of exogenous dolichol phosphate on the

-

incorporation of [ldcl-mannose from G D P - L ~ ~ I mannose into Man-P-Do1 in hepatocyte

...

homogenates 91

Tabla 6. Effects of dexamethasone and actinom =in D on '-

+ the fornetion of Wan-P-Do1 and ( ~ l c ~ ~ c ) l - ~ - P-P-Dcl in hepatocytes obtained from

control ra'ts

...

98

Table 7. Effects of dexamethasone and actinomyoin D on the fornation of Man-P-Do1 and ( G ~ O N A ~ ) ~ - ~ P-P-Do1 activitied in'hepatocytea obtained from inflamed rate

... .'. ..

99

L

Table 8. Effects of dexamethasone and cyoloheliimide on the fomtion'of Man-P-W1 and ( G ~ C N A O ) ~ + - P-P-Do1 in hepatocytei obtained f m m

w n t r o l rats

...

LOO

Table 9. Effeots of dexamethasone and ~yslohoximid&

-

on the fornation of ManiPDol and ( G ~ E N A C ) ~ - ~ -P-P-Do1 in hepatocytes obtained from

inflamed rats

...

lo1

Tabla 10. Effeo of dexamethaaone on the incorporation of [lfc) mannohe into Pan-P-Dol. in rat hepataeytee

...

104

*able 11. Subcellular localization of mannosyl-.

transferasas and other marker enzymes

...

106

E

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' Xi"

PAGE T a b l e 1 2 . E f f e c f Of exogenous d o f a c h o l phosphare on

? h e i n c o r p o r a t i o w of 6 C l mannose ~ n + o o l i g o s a c c h a r i d e l i p i d

...

122 T a M e 13. E f f e c t of actinomycin D on t h e . d e x a m r t h a e o n e

induced i n c r e a s e of o i ~ g o e a c c h a r i d e l i p i d , s y n t h e s i s i n h e p a r o c p e a o b t a r n e d Erorn

...

c o n t r o l r a t s 125

T a b l e 1 4 . E f f e c t of acrinomycin D on dcxane*hasone and inflamhlation i n c e d increases of o l i g o s a c c h a r i d e lip%orma+.,ion i n h e p a t o c y t e s o b r a i n e d from i n f l a m e d

rare'

...

126

.

T a b l e 15. E f E s c t of c y c l o h e x i m i d a on t h e dexamethasqne induced i n c r e a e e of o l i g o a a c c h a r i d e l i p i d L e y n t h e s i a i n , h e p a t o c y t e s o b r a i n e d f r o m

...

c o n t r o l r a t s 1 2 0

T a b l e 1.6. E f f e c t of c y c l o h e x i m i d r on t h e i n E l a m a r i o n

., , .

and deramet.haason induced i n c r e a i e of a l i g o s a c c h a r i d e l i p i d s y n t h e e i s i n hepat.ocy$.es obt-ained from i n f l a m e d

rats

...

129

able 1 7 . E f f e p f o f dexametnasane on *.he i n c o r p o r a t i o n

,

of C Cl mannose i n f o o l i g o s a c c h a r i d e l i p i d qnd c e l l u l a r p r o t e i n s

...

I*

I Table 18. Efgect. of c e l l d e n s i t y and dexamethaeone o n [ c l @annose i n c o r p o r a t i o n i n t o Man-P-D8l

...

o l i + o s a c c h a r i d e l i p i d and p r o t e i n s 134 T a b l e 1 9 . E f f e c t of a c t i n o m y s l n D and c y c l o h e x i r n i d e

on der&?lethasdn? i s d u c e d s t i m u l a t i o n of n u c l e o l d e s u g a r pyrophoPphafaae

...

a c r l v i t y i n h e p a t o c y r e c u l t u r e 152 T a b p 20. ~ i b e t - i c p a r a m e t e r s of n u o l e o t i d e s u g a r

pyrophoopharase a c t l v i r y i n c o n t r o l and dexamethasone t r e a t e d h e p a r o y r t e s

...

155

T a b l e 21. E f f e c t o f d i n e r F a p r o p r p q a n o 1 and ATP on the i n c o r p o r a t i a n of C C l mannose i n t o - a l i g o s a c c h a r i d e l i p i d by membranes

d e r i v e d from c o n t r o l a n d dexaaerhaeone

t r e a t - e d h e p a r o c y t e s

...

161

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,

'73

. .

X V

-

PAGE -.

Table 2 2 . L e v e l s of n u c l e o r i d e sugars in/

heparocytes

. . . . . . . . . . . . . . . . . . . . . . .

163 . .

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x v i , .

' -2 PAGE

~ 1 s t o f Figures

F i g u r e I. schematic d i a g r a m of i n n a m a t o r y

.

p r o c e s s

...

2

F i g u r e 4 .

,

~ i g u r e 5.

F i g u r e 6.

sequence o r e v e n t s i n the a c u t e - p h a s e p r o t e i n r e s p o n s e

...

5

schematic diagram f o r c o - t r a n s l a t i o n a l

.

p r o t e i n t r a n s l o c a t i o n acroae tqe rough end.oplasnio r e t i c u l u m membrane

...

1 3

yre

s t r u c t u r e Of alrparagine-linked o1igo- -

a o o h a r i d e c h a i n s

...

1 6

.truEtu.e of ~ - g l ; c o a i d i a a l l y l i n k e d o l i g o e a c c h a r i d e

...

1 7

s t r u c t u r e of d o l i c h o l phosphate

...

1 9

ids synthetic ...

pathways oe d o l i c h o l

...

phosphate 2 1 ,

s t r u c t u r e of g l u c o s e c o n t a i n i n g o1ig.-

;~~~~?.!f~~".~~.~~!~~!?~.!h."~!. .

..?23

The do!lEhol C y c l e

... .-..

2 4

~ i G u r a $ 0 . B i o e q n t h a t i c pathways or f o r n a t i o n of nucl-otide s u g a r s

...

3 0 F i g u r e 11. b c h m a t i c diagram on t h e l i v e r , p e r f u e i n

. ... ... .

system

.:. j.

$< 4 0

p i g i r e 1 2 . p sol at ion o e d o l i s h o ~ l i n k e d sugar

... / ...

4 6

~ i g u ; e 1 3 . n r f e c t of'ine~amnation on e i a l y l t r a n ~ i / r a s e

~ c t i v i t y i n r a t l i v e r homogenate

...

6 0

~ i g u r e 14. ~ f i e c t o f inflammation on s i e l y l t r a n a e r a s e

...

a c t i v i t y i n r a t l i v e microsones 61

fi

~ i g u r s 15. ~ f f e c t o f ' inflammation on s i a l y l t r a n ( e r a s e

... ....

- -

a c t i v i t y i n t h e r a t sem

.:.;. 1 .

6 2

n g u r e 16. ~ i i s c t of inflammation on g a l a c t o a y t r a n s -

/ ....

f e r a s e a c t i v i t y i n r a t l i v e r hmog a t e 6 4

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I w i i

% PAGI

r i g u r e 1 7 . ~ f f e c t f a r a s a a c t i v i t y . i n r a t l i v e r miorosome qf inflammation on g a l a c t o s y l t r a n s -

....

. 6 5

p i g u r e 1 8 . ~ f c e c t € e r a s e a c t i v i t y i n t h e t a t asrum of inflammation on g a l p c t o a y l t r a n s -

...

. 6 6 F i g u r e

p.

Formation of 1 1 4 ~ 1 Man-P-Dol&s a f u n c t i o n

...

of t i n e i 5

F i g u r e 2 0 . ~ ~ c c o r p o r a t i o n o f [ 1 4 c ] nnose i n t o Man-P-Do1 as a f u n c t i o n o f GDP-[14CI mannose

~ o n c e n t r a t z o n

... m...

7 6

-

F i g u r e 2 1 . The p r o g r e s s i o n of Nan-P-Do1 p r o d u e t i o n as f u n c t i o n o f t i m e of inflammation

...

7 7

F i g u r e 2 2 . E f f e o t of i n c r e a s i n g amount of exogenous d o l i c h o l p h o s p h a t e on l n c o r p o r a t l o n of [14c]

.annose i n t o Nan-P-Do1

...

79

~ i g u r e 2 3 . I n c o r p o r a t i o n o f [ 1 4 ~ 1 ' ~ a n from 0 ~ p - 1 ~ ~ 1

.

mannose i n t o Man-Pool i n h e p a t o c y t e

*

homogenatel

...

8 2

'PL- 2 4 . I n c o r p o r a t i o n of [ l l c ] G ~ O N A C from U D P - ~ 1 4 ~ 1 GlcNac i n t o ( G l ~ N l l c ) ~ - ~ - P - P - D o l i n h e p a t o c y t e homogenates

...

8 3

p i s u r e 25. I n s o m o r a t i o n of [ ~ ( c I nan from G D P - [ ~ ~ c ] mannose i n t o Nan-P-Do1 i n h e p a t o s y t e homogenates

I as a f u n c t i o n o f amount of homogenate

p r o t e i n

...

8 5

dexamathasone o n t h e i n c o r p o r a t i o n of [ l a c ] man from G D P - [ ~ ~ c ] mannose i n t o

...

Nap-P-Do1 i n h e p a t o e y t e homogenates 8 6 F i g u r e 27. E f f e c t of dexamathasone on t h e i n & r p o r a t i o n d

of ~ 1 4 ~ 1 mannose from G D P - C ~ ~ C ] mannose i n t o Ran-P-Ool i n h e p a t o c y t e h m o g e n a t e e

...

8 7

Figure 28.

~.'"n:gp~~~;::~:~~:g:,IMa:h;::~:;P:~14cl

mannose i n t o Man-P-Do1 i n h e p a t o c y t e p i g l l r e 23. E f f e t of d e r a n e t h a s o n e on t h e i n c o r p o r a t i o n

of CSH] < e v a l o n a t e i n t o ['HI-dolichol i n

(23)

F i g u r e 30. E f f e c t of dexalnethdsone on t h e i n c o r p o r a t i o n of ( 3 ~ 1 mevaianate i n t o

HI

d o l i o h o l phosphate i n h e p a t o c y t e s

...

96 F i g u r e 31. E f f e c t of dexamethaaane on t h e I n c o r p o r a t i o n

of [ 1 4 c ] mannore fro. [ 1 4 c l mannose i n t o an-P-~ol i n * i n t a c t h e p a t o c y t e s

...

r..

...

103 F ~ q u r e 32. E f f e c t of dexamethasone on t h e r e t e o f

o l i g o s a c e h a r i d s l i p i d s y n t h e e i a by

... .

hepatooyte homoganates from o o n t r o l r a t s 118 ' F l g u r e 33. E f f e c t of dexamethasone on t h e r a t e o f o l i g o -

saccharide l l p i d s y n t h e s i s by h e p a t o c y t e homogenates from i n f l a m e d r a t s

...

120

.

F i g u r e 34. E f f e c t s or exogenous d o l i d o l phosphate on

.

t h e i n c o r p o r a t i o n of [lac] l a n n e s e i n t o o l i g o s a c c h a r i d e l i p i d

...

123

F i g u r e 35.

.

e f f e c t of dexanethaeone i n o o r p o r a t i o n i n t o o l i g o s s o o h a r i d a l i p i d i n on [I4cl mannome t

-

i n t a t t h e p a t o s y t e s 130

,

...

~ i g u r e 36. E f f e c t of d e x m e t h a e o n e on [ l a c ] mannose - - - i n c o r p o r a t i o n i n t o c e l l u l a r p r o t e i n i n t a c t h e p a t o c y t e s

... .; ...

i n 1 3 2 . -

~ i g u r r 3,. Chronatoqrsphy of o l l g o s a c c h b r i d s d e r i v e d fro. o l i q o s a c c h a r i d e complex i s o l a t e d

...

from h s p a c o c y t e honoqenatoe 136 F i g u r e 38. Chromatography of o l i g o e a c c h a r i d a d e r i v e d

from o l i g o s a c c h a r i d e l i p i d complex r e p a r e d Lf z n s u b a t i o n of h s p a t o c y t e s w i t h 9 1 laannose

...

- 137

~ i g u r e 39. E f f e c t of dexamethasone oh n u c l e o t i d e s u g a r pymphosphatase a c t i v i t i e s as a f u n c t i o n o f assay time

...

146

Figure 40. E r f e c t of dexamethasone on n u c l e o t i d e s u g a r pyrophosphatase a c t i v i t i e s as a f u n c t i o n o f protein c o n c e n t r a t i o n i n t h q e s s a y

...~.

147

(24)

\

x i x

i PAGE

-.I"

F i g u r a 4 1 E f f e o t o f dewamethaeone o n n u o l e o t i d e s u g a r p y r o p h o s p h a t a s e a c t i v i t i e s for 1 2 h i n h e p a t o c y t e homogenates f r o m c o n t r o l

r a t 6

...

148

F i g u r e 4 2 E f f e c t o f dexamethasone o n n u s l e o t i d e s u g a r p y r o p h o r p h a t a s e activities for 1 2 h in - h e p a t o c y t e homogenate%from' infkimed

.

q a t r

...

150

F i g u r e 4 1 . ~ c l e o t i d e e u q a r p y r o p n o s p h a t a s e a c t i v i t y

$,

m i X t u r e ~ o f h o m q e n a t e from c o r l t r o l and dexanethasona t r e s t e d h e p a t o c y t e a

...

154

F i g u r e 4 4 . f f e c t o f dexanethasone o n N-acetylglucos-

I

minyl t t a n s f e r a s e a c t i v i t y e i t h e r

-

i n t h e

...

re88nEe or absence o f dimeroaptopropanol

n d ATP 157

F i g u r e 45. f f e o t of daxalnethasone o n a r s n n o a y l t r a n s f e r a s e e t i v i t y e i t h e r i n t h e presence or a b s e n c e

...

f dimerosptopropanol and aTP r . . 160

-

\

(25)

PAGE L i e of m b r e v i a t i o n s

ATP

.

Adenosine 5 ' - t r i p h o s p h s t e

ASN A r p a r a g i n e

CNP c y t i d i n e 5

'

-monophosphate

CTP Cytidine 5 ' - t r i p h o e p h a s e

cn C e n t i m e t r e

, -

14c Carbon 1 4

c i & r i e

DEAE Diethylamino%thyl

Dol-P DOlichol monophoaphate

Dol-P-P Bolichol lpyrophoaphate

dpm DisintegAation p a r minute

DNA ~ e o x y r i b d n u ~ l e i c a c i d

EDTA srhylened/iaminetetracetste

-

Fm-6-P muctose-6-phosphate

N E FUEOBB

~ a l G a l a c t o ~ e

GERL ~ o l g i - e n d o p l s s m i c r e t i c u l u m lysosome

GlcN Glucosamine

C ~ O N A C N-acetylglucosamina

GalNAc I-L N - a c e t y l g i t l a c t o s a ~ i n e

Gal-1-P Galactose-1-phosphate

I GkC Glucose

010-6-P Gluooee-6-phosphate

Glc-P-Do1 ~ l i ~ h o l monophoaphate glucose

GICNAE-P-P-~01 D o l i c h o l p y r o p h o e p 4 a t e N- a ~ e t y l g l u c o ~ a r n i n e

(GlcNAC) 2-P-P-DOl W l i c b l pymphoaphate N-lchitobh

GDP Gvanoslne 5'-diphosphate

9 G r a v i t a t i o n a l f o r c e u n i t

W Gram

h !lour

HSP Hepatocyte stimulating f a o t o r

(26)

IL-1 d Km 9 m N A IES n l m 9 nM n Man Man-6-P A 0 1

/

.in.

. '

m,

Man-P-DO1 NADPH Nemc mc1 PA RER

RNA \

S e r SRP SA T h r T r i s

UDP

'

w .

V/V

YmBx

-

W/V

x x i

-

PAGE

I n t e r l e u k i n - 1

~ i c h a e l i d c o n s t a n t

Messenger ribonucleic a c i d m C i A l i n v i e , 2-(N-morpholino) ethanesdlphonic a c i d Millilitre

M i l l i g r a m M i l l i n o l a r Molar

Mannose L

Hannoee-6-Phosphata

. * /

,

.

Hil1;nole

Minute

.

M i l l i m e t e r

Dolichol monophosphate mannwe P.'

m e a d nictin@de adenine dinmlechide p h o s p h a t e

N-acetylneuraminic a c i d Nan0 mole

-

l o g or hydrogen i o n c o n c e n t r a t i o n Rough e n d o p l a a m i c r e t i c u l u m R i b o n u e l e i c a ~ i d s e r i n e

Siymal r e ~ o q n i t i o n p a r t ~ c l e S i a l i o . a i d

T h r e a n i n e

T r i s (hydroxymethyl) aminonethane u r i d i n e 5 ' d i p h o s p h a t e U l t r a v i o l e t volume/volume Maximum v e l o s i t y ueight/volume Microgram

~ i c r o l i t r e

(27)

I x x i i

PAGE

V M Mioronolar

uci ., Microcurie ' .

*.d

.rS- .

,

(28)

1

I CHAPTER I

1.1. L m u W a u Q N ,

u i cr e r n o n s e t o Inflammation. t h e a c u t e i n f l a n m t o n '

, Y'

I n f l a m a t i a n i n insmale nay be caused by various f a c t o r s , which i n o l u d e : l o c a l i n j e c t i o n o f . i n f l a m n b t o r y a g e n t s (I, 2 ) . t h e r m a l or n e c h a n l c a l i n j u r y (3)

,

major surgery ( I ) , b a c t e r i a l

_

i n f e o t l o n ar e n d o t p x i n i n j e c t i o n ( 5 . 6 ) . and n e o p l a s t i c growth i

( 5 8 7 ) .

.

I'

.

J

The. l o o a l r e a c t i o n b f t i s s u e t o i n j u r y or i n f e c t i o n i s

-

t e m e d s c u t e inflarmaation, and t h e s y s t e m i c and m e t a b o l i c changer; t h a t oocur d u r i n g i n f l e m p t i o n a r e te-ed, t h e acute-phase r e s p o n s e 1 8 ) . The l o c a l a n d systemio r e a c t i o n s t h a t o c c u r d u r i n g a c u t e and c h r o n i c inflammation have been d e s c r i b e d by GLenn S

a.,

(91 i n a scheme shown i n f i g . 1.

-

(29)

2

.The "lnelemnatory P r o c e s s a 4 DAMAGING AGENTS Antigen-antibody r e a c t i o n s C h e n i ~ a l - p h y s i ~ 8 X l irritants

,,, /

;g;:ial i n f a c t i o n O t h e r s

LOCAL REACTIONS SYSTEMIC REACTION

( A c u b Phase Response) Venular d i l a t i o n I n c r e a s e d body t e m p e r a t u r e

Slow v e n u l a r f l o w P a i n

I n c r e a s e d blood v i n c o s i $ y

.

Granulocytoeie and ' E n d o t h e l i a l l e a k a g e , lymbhocytosis

B ~ . y t h r o s t l s i e ' I n o r e a s e d fibrinogen ! , P l a t e l e t a g g r e g a t i o n I n ~ r e a a e d C-reactive '

Thrombus f o r m a t i o n ( 2 ) - p r o t e i n

F i b x i n a c c u m u l a t i o n

-

I n w a s e d c- ard 8 - g l d m I e

Neutrbphil and lymphocyte Increased 02-glycqaotelns

a c c u m u l a t i o n Decreased lbulnin

P h a g a c y t o r i s of i r r i t a n t Decrkased &tjm *ran t

a n d damaged t i s s u e s I n c r e a s e d serum copper Leukocyte and p l a t e l e t I n c r e a s e d muaoproteins .

.

breakdown .Increased g l y c o p r o t e i n s

Increased l a c t a t e I n o r e a s e d . p i t u i t a r y a n d ;

dehydragenase a d r e n a l f u n c t i o n

I n c r e a s e d gamma-globulin

> , ,

PRDl'ECTIVE AND INHIBIlWW

LEAKY MEMBRANES

Fig. 1. s c h e m a t i c diagram of i n i l a n m a t o r y p r o c e s s . The numbers i n p a r e n t h a s i s i n d i c a t e t h e o r d e r i n which t h e e v e n t s a r e b e l i e v e d t o occur. Reproduoed from Glenn st 81.

, ( 9 ) .

7

(30)

, .

3

~ l e n n & 41 (9) have suggested that'tbe systemic reaction is induced by the loeal reaction, which includes phenomena suoh as venular dilatilon, endothelial leakage and oedena, platelet aggregatioh, fibrin famatian, leucocyte :accumulation, release of lysosomal enzymes from laucocytes and tiasues, fa&ation and release of small nolecular weight mediators (histamine. 5-hydroxytryptamine, kinins), .mesenchymal oell proliferation and others.

syatemic response includes fever, pain, leucocytoris, increased level of'acute-phase proteins: increased function of the pituitary-adrana; system and decrqayied level of serum iron. Vsrioul forms of inflamation are associated with an increased pameability of lysosoma~ Fenbranes and release of acid hydrolases (10.11). The %ease o Y y s o s e m a l e n z y i ~ e s

...

has been referred to by Reisamann (12) as the oqtidal c o b o n pathway" in inflmmation.

.

Fig. 1 shows that the systemic response is accompanied ' b

by various metabolic, humoral and physiologic alterations.

. <

p ever is oonsideeed a part of the systemic re-a to different types of stimuli, which pr ides pertinentgr0-h conditions for invasiva m i c r ~ ~ r g a n i s m s 13).

So

Fever causes increased

.

utilization of nkrients to meet the, elevated energy requirements of body cells: Increased iluooneogenesis and glycogenolysis, elevatlon.of amino acid degradatio".vith

. .

accelerated ureogenesis and anmoniogenasis, increseOQ

. , .

a

catabolism ,of somati0 proteins, reduced ketogeneaia ind

(31)

4 .

hyperglyceridenia (8.13.14.15) are observed due to the high energy requirement during elevated body temperature.

The systemic response is charact=rized by elevation of a number of hormones, e.g. insulin, gluoagon, adrenocorticotropic ormone (ACTH). cortisol, catecholamns.

/

growth hormone, thyroxine, thyroid srimulating hornone.

2

vasopressin and aldosterone (15.16.17). The precise role -

of these hornonis in initiating the acute-phase response has yet to be determined.

one o€ the important changes occurring. during the

*

acute-phase response is in t h s levels of some plasm proteins. . Table 1 lists the changes in the levels of some imprtant p l a m - proteins t h a t occur in response to experimental inflamation.

Plasma protein- which increase

following

inflammation are referred t o p s "acute-phase,proteinso8 (181, whereas, proteins such as llbumin which deo'rease in concentration, have been e described as "negative acute-phase proteins" (8). ~t least two common features have be= ascribed t o the acute-phase proteins, i) almoet all are glycoproteina and ii) they are synthesized by the liver (18.19.20.21.22).

(32)

1 5

~ a b l a 1. E f f e c t o f in$,ectian o f cioton a i l i n t o r a t s * . Response

component- analyzed

3

T o t a l P r o t e i n Albumin F i b r i n o g e n

* - G l y c o p r o t e i n Mucoprotein A l p h a - g l o b u l i n s B e t a - g l o b u l i n s Alpha-2-glycoprotain

NO change Decrease Increase Increase I n c r e e e e Increase Increase InETeaSe he i n f o r m a t i o n i n t h i s t a b l e i s t a k e n from Glenn & al (23).

When t h e p r i n c i p a l e v e n t s between t h e occurrenoa of

-

inflammation and t h e appearance oi newly s y n t h a r i e a d a c u t e - p h a s e p ~ o t e i n r in t h e b l o o d afe can i d a r e d , t h e y ' n a y b e (L prementad i n t h e farm of the following chain of events as shown i n Fig. 2.

FDWTION STIMUIATION m C EOF

OF I=- OF THE LIW( APPROTEI

IIEDIATE CELL IN, E X , mRFL

FACMRS

1

1

I

2

I

3 4 5 H-S

-

imw

' .

s y n t h e s i s inhLbited

by a c t i n o w i n D purmycin o r c y c l o h e x i i d e r i g . 2. Ssqvenoe oE events i n t h e arute--phaw p r o t e i n response.

'\

(33)

6

Hormone-like faetors, originating at the site of tissue injury, are believed to be transported by blood to the liver where they stimulate inereasid synthesis of the acute-phase

'

prot~eiin (14.18). Work'by Woloski & al (24) and others (14,25,26,27,28) hare suggested that laucocytaa nay evert an indirect effect an the liver to stimulate acute-phase protein

.

-

synthesis by formation of suoh chemical mediators. an kinins, pyrogen, histamine and cytokiner. Woloski (29) has r h m that monocyte derived factor(s1 are able to'stinulate the pmcers

.

characteristic od the acute-phase response in liver, includinq elevated glysopmtein synthesis. The mechanism by which these chemical mediators stimulate hepatic syntheaia of the acute-phase proteins is unknown. Although cytokines are able to stimulate the synthesis of the acute-phase p;otein,

.

- the response appears to be lower than that found following trauma ( 2 4 , 2 9 ) . ,

Trauma stimulates the pituitary-adrenal system, as well

.

X

as other endocrine glands and it has been suggested that

.

.

hormones; such as oortiooateroids traneported by blood, 'L stimulate the synthesis of acute-phase proteins in the

.

liver. Coeticosteroids have been reported to be involved in st@ilatinq the acute:phasa response resulting in enhanced ,

.

synthesis of glycoproteins in inflammation (30.31). John and

\r

MILLBL. (32) using a liver perfusion system, have shorn that cortisol elevated synthesis of haptoglobin, fibrinogen and - an Gl-globulin. Adrenalectomy caused a pronouncad desreaae

(34)

7

in the synthesis of some of the saute-phase proteins in response to turpentine nduced inflammation. Theae were restored to normal by replacement theeapy with cortisol

J

. (33.34). A amall increase of acute-phase proteins in adrenalestomized rats after turpentine induced inflammation, suggests that a s-rd affector system other than corti-teroid can mediate.ari enhanced eynthesie of these proteins.

r

~ h u s , although hormones may be involved in the sthulation of elevated, hepatic acute-phase protein synthesis, N r r e n t idass suggest that cytoki'nes and other mediators are also rewired for full expression of elevated synthesis of these proteins. Clearly, the acute inelamatory ~ t o o e s s represents a,coordinsted system to limit, modulate or athewire direct

5

host response during of intense inilamafion and . - tieeue destruction.

,

~

.d

1.2. mute-ohasa rotei inn

.

.

This ostegory includes several proteins with diverse phyeicochemical properties (Table 2). Almost all aNte+ase ' proteins oontain significant amounts of cerboydrete and all are synthesized in l.iver parenchymal cells.

.

, When the

.

physicochernieal properties of proteins listed in Table 2 are bompared, it appears that their carbohydzate d-eases witn increasing isoelectrio point. The oligoaaccharide

.

ohains qf the acute-phase proteins are minly of the aspamgine linked (N-1inkid)'cmplex typ; (35) and are synthasisad

x k -

(35)

1 4: 8

able 2. P h y s i c a l and chemical p r o p e r t i e s of t y p i ? a l acute-phase p r o r a i n s . . 8

p he d a t a refer to human plasma e x c e p t o l and a 2 acute-phase g l o b u l i n s f r o m t h e rat and C x - r e a c t i v e p r o t e i n from r a b b i t .

i

- l~oounr i n x s o e l e c r r i c Carbohydrate

normal p ~ a s m a ~ o l e c u l a r PO+ content

p r a t e i n mg/lOO m1 weight P I . 8

y:acld

g l y c o p r o t e i n

\

Haproglobin

(HP. 1-11 /--

OX-AT g l o b u l l n ( r a t ) a l - a n t i t r y p a i n o2-AP g l o b u l i n

( r a t 1 cer$loplasmin

p r o r e i n ( r a b b i t ) F i b inogen S e ~ m u c o i d

\ J'

4 5 000 4 . 5 ,

","

a p p r o r .

160 000

.

4 . 4 ,

138 000 7

120 600 ?

341 000 5 . 8

n e t e r o g e n o u s € f a c t i o n

.

he i n ~ a r m a r i o n l i n t h i s t a b l e was' taken from Koj. A 1181.

P

(36)

/ c 9

dolichol linked intemepiates (36.37.38). Alteration in the car6ohydrate moieties of some acute-phase proteins following an inflammatory challenge has been observed (39).

~cuta-phase proteins

aA

of oonsideraale interest in ,

.

practical and experimental medicine. Changes in their plasma concentration are regarded as a sensitive (although rather non-npeciWc) test for diagnortio and prognostic asaessneqcs. T h e biological activities of some of the acute-phase proteins are well defined. Haptaglobin (HP), combines with haemoglobin (Hb) to give a oomplax (HP.Hb)

% .

thue removing h a w l o b i n from c i d a t i o n (40). The involvement if the adrenal gland in rsgulathg the serum haptoglobin

. .

level in rbsponseto inflammation has been.eatab1i.h-d. The response of s e h m haptoQlobin to ,inflamation ie impaired in

1 .':/

the absence oi the adrenala. (41); Ceruloplsemin is believed to proqdat cells from damage'by ,generating superoxide anion

-

radicals at the -mite of tissue damage (42). ~nfl)mmation, pregnancy, rheumakoid arthritis (43) ,and injections o i estrahiol to chickens (44) a aiqnificant increase in serum ceruloplarmin.

Pibrlnogen ie invplved in localizing infectionsthrough clot fornition (13): al-antichymotrypsin, el-antitrypsin and m 2-macroglobulin'en known as protease inhibitors (45). Some acute-phase prdtains are known t o inhibit certain '

.

lymphocyte responses

in mi.

these inalude C-reacive

, proteins (46). 0 = - a d d glycoprotein (47) and, n-fetoprotein '

(37)

10

-

(48). C-reactive protein (CRP), a member of acute-phase pmteina in humans d n normally undetectable in the plasma

.

but appears to incfe'eqre by as much as lo00 fold in response

1

to inflammation and injury. 1t is interesting ta noto'that CRP in humans and rabbits is a nonqlyoosylated aoute-phase protein whose phyeialogical fudction may be related to host defense and repair (49,;0).

Ths liver is the site of synthesis of acute-phase proteins, and the insreasad concentrations of these plasma proteins have been shown to ba accompanied by their in-sed 'hepatic synthesis (8,18,35.51). During the acuts-phaas

response, inoreased synthesis oi aaute-phase proteilis is accompanied by albrationri in ultrastructural elements-and ' chemical constituents of the liver. T h e most Important '

r

.

.

alterations observed are: proliieration of the Qolqi complex (52). dilation of the rough endoplasnic retioulum. increased amounts of smooth endoplasmic reticulm and inareaad synthesis of plasma menbranis (53.54). mere is also inceeae& synthesis

.

of miorotubulesc (55,56), cytoplasmic actin (57)

,

-RNA,' parti&larly ribosomal FWA (581, cholesterol and other lip,ids by the endoplasmio reticulum (59). These alterations are consistent with increased synthesis, transport and secretion of acute-phaee pioteins.

here is no doubt &at liver is the major organ involved

. .

in acute-phase response. The liver is k n w n to respond in several ways during the aaute-phase response includ4ng '

.

(38)

\

11

activation of the glysoprotein biosynthetic machinery.

This thaaie is mainly concerned with the involvement of liver in the regulation of the synthesis of the aoute-phase

/

proteins.

The presence of oligosaccharide chains covalently attached to the psptide bacKhone is the feature that distinquishes glycoprotains'from non-glycosylated proteins

,

and accounte f m m of their physical and c h d c a l properties.

GlycOpmteins are broadly classified into two types: 0-

.

b

glyoosidically and N-glycosidically-linked g1yr;oproteins.

Virtually. all plasma glycoproteins are N-linkad glyoopmteins and are synthesized by liver. Therefore, only the synthesis of N-linked glycoprotsin will be outlined at length in thls thesis. The synthetic pathway Can be divlded into two paktet (A} tho synthesis of polypeptides and ( 8 ) the synthesis of carbohydrate moieties.

The rough sndoplasmic: reticulum (RER) has been postulated ' t s possess a single translation-coupled translocation system

(in multiple copies) that effects signal sequenca-mediatad translocation of all secretory proteins (such as acute-phase pmteine), lysosomal proteins and all integral membrane proteins whose port of entry is the rough edoplawic retisulm

I

-

(39)

I 12

(60.61.62.631. The fornulation of an hypothesie f o r tho t r a n s f e r of protaino across t h e membranes, i s referred t o as t h e s i g n a l hypothesis.

The a s s ~ n t i a l f e a t u r e of t h e s i g n a l hypatheeia (Pig. a ) IS t h e occurrence of a unlque sequence of codons, located i n m e d ~ a t e l y t o the r i g h t of t h e i n i t i a t i o n codon which i s present only i n those mRNA's whose t r a n s l a t i o n produote are t o be t r a n s f e r r e d across a membrane. N o o t h e r m A ' s contain t h i e unrqua sequence. Translation of t h e sign*; codon *

r e s u l t s i n a unique aaquence of amino acid residues on t h e amino terminal o€ the natlcent chain. Emergense of t h i s s i g n a l sequence of t h e nascent chain i r o n within a space i n the l a r g e ribosomal subunit t r i g g e r s attachment of t h e ribosome t o the membrane, thus providing t h e t o p o l o g i c h

*'

conditions f o r t h e t r a n s f e r of t h e nascent chain across t h e membranw I f the n a e c d f z a i n lacks t h e s i g n a l sequence, attachment of t h e ribosome t o t h e membrane w i l l not occur.

me attachment of I.& t o me endoplamic reticulum membrane i s mediated through an 11s rlbonucleoprotein, t h e so-called s i g n a l recognition p a r t i c l e (SRP), which has s r e c e p t o r on t h e andoplasmic reticulum membrane, termed SRP reoeptor. SRP functions m decoding t h e infornntion contained i n t h e s i g n a l peptide of nascent sacretory p r o t e i n s (62.641 f o r t h e s p e o i f i c attachment of t h e t r a n s l a t i n g ribosome t o I t h e microsornal membrane (62). I n t h e absence of endoplasmic I

r e t i ~ l u m membranes, SRP s p e c i f i c a l l y a r r e s t s t h e elongation

.

(40)

Pig. 3 . Schemati0 diagqam for co-translational protein p a n s l o o a t i o n awase the rough endoplasnic r e t i a m membrane. Reproduced from Walter and Blobel

,

. .

( 6 2 ) .

-4

(41)

I4

of s e o r e t o r y p r o t e i n synthesis in XL!&LQ j u s t a f t e r t h e s i g n a l p e p t i d e ham emerged from t h s ribosome, t h u s p r e v e n t i n g t h e oompletion of p r e - s e c r e t o r y . p r o t e i n (many of which may

0

,

be p o t e n t i a l l y harmful t o t h e c e l l ) i n t h e cytoplaaraic . ' compartment. novqer, i n t e r a c t i o n of t h e a r r e s t e d riboeornes w i t h the SRP r e c e p t o r on t h e m i c r o s m a 1 membrane r e s q l t s i n U e e l o n g a t i o n ,of t h e n a s c e n t c h a i n which is then translocated

across t h e merbrane. me s i g n a l p a p t i d e is removed by s i g n a l p e p t i d a s e

- .w I

a f t e r +he p r o t e i n i s t r a n s f e r r e d t o t h e endoplasinic , e t i c u l u m , aenbrane.

8. m eand s v n t h e s i s of'N-linked o l i ~ o s a c c h a r i d e e

in

&cauratein.z. ,

Glyoopeoteins are broadly F l a s a i f i e d i n t o tw pes; N- - l i n k e d and O-linked. Most acute-phase p r o t e i n s are

t

- l i n k e d f g l y c o p r o t e i n s i n which the o a r b o h y d r a t e c h a i n i s l i n k e d t d

5 .

t h e p o l y p e p t i d e c h a i n througd an N - g l y c o s i d i c bond. Thie N- g l y c o s i d i o bond i s between t h e C-1 hydroxyl of an N- a c e t y glucosamine r e s i d u e i n t h e c a r b o h y d r a t e c h a i n and t h e . . amiao

7

n i t r o g e n of an abparagine r e s i d u e i n t h e p o l y p e p t i d e I

c h a i n . The & h e r t y p e of l i n k a g e , which is leas cornno? i n serum g l y c o p r b t e i n s ,

d

the O-glycosidic linkage normally found detwaen N - a c e t y l g a l a c t o s a m i n e and e i t h e r e e r i n e or t h r e a n i n e

!esidueb on n u c i n - t y p e g i y c o p r o t e i n s ( 6 5 ) . The D-glyoosidic

.

l i n k a g e also occurs i n c o l l a g e n i n which g a l a c t o s e is l i n k e d J

(42)

'4

>

1 8

to hydroxylysine residues (66). The oligosaccharide of O-

$

glyoasidic linked glycaproteins is formed by direct tradafer of individukl augare from the respective nucleotide sugar.

.

The asparagine linked oligoeaccharides have hetemgeneovs structures but fall into two general classes, i) high loannose type and ii) complex type (15.67.68). Bot\class& have a.

/ common inner care structure at the reducing terminus as shown

d

rig. I .

, -

High mannose type stwctures contain additional a - linked mannose residues, while complex oligosaccharides have sugars such 86 N-acatylglucosamine. galactose, fucose and N- ecetylnsuraminic acid or eialio acid (Fig. 5)

The existence of a c o n o n core structure in many of the N-linked glysoproteins suggests a c-n meohanism of spthesie for at least .the internal region of the saccharide chain.

Parodi p.t (69) reported in 1972 that a glucose-containing lipid-linkad oligoeaocharide comprising approximately 2 0

_

monosaccharide units could be synthesized and transferred to protein in cell-free preparations from rat liver. Synthesis of the oligosacharide' portion, which is f o m a d dolichol- linked intermediates, is now referred to as the 'dolichdl cyole'. After extensive investigation in different laboratories, it has been found that the oligosaocharide contains three g l u c o ~ i n e mannose and two N-acetylglucosamine residue&!. me oligosaocharide moiety of this complex is

'4

trmsferred to an acceptor protein which undergoes subsequent

4

,

@

(43)

B .

.-

Fig. 4. Core stmcture of asparagine-linked oligosacchayida..

chains.

(44)

W W . W U W

g ?

",

7 2 =?

7 " : N 9 ? f 9 - a a .

ii g ,p

Z.2

Z. &Z&Z

r g g ; g ~ ~

: E & $2

iiii

+,

. 2 7 2 " "

.

T

3 : 2 3

-

39r-V

$g g . $ % % z g . E

,--,,

x 2 . : g;gg -

A ri

$$;$.

,

2, 2 2 . 2 2 2 2 s

Pig. 5. S t r u c t u r e O f N - g l y c O s i d i c a l l y linked

.

oligo~accharide. The high nannoae atruoture (A) shown! is found in bovine thyroglobulin.

Complex type of oligosaccharide ohains with bi-

'

.(B), tri- (C)

aid

tetra (D) dntennary structures have &so been. reported.

,

ReprcdILcad iron . Staneloni and m l o i r (73). me large E represents tho oore, as illustrated in Fig. 4.

. ..

. . ,. . - .

(45)

1

18

m o d i f i c a t i o n s by removal and a d d i t i o n of s u g a r s t o produce e i t h e r high mannose or complex type o l i g o s a c c h a r i d e s . , This . P ~ D C B S S ~ ~ l l e c t i v e l y is r e f e r r e d t o as o l i g o s a c c h a r i d e proc.ssin9.

The involvement

ap

l i p i d i n t h e s y n t h e s i s of complex g l y c a n i n b a c t e r i a had been known f o r some t i m e ( l o ) . - Behrens and L e l o i r ( 7 1 ) were t h e f i r s t t o d e m o n s t r a t e t h e p a r t i c i p a t i o n of a l i p i d n o l e c u l a i n g l y c o p r o t e i n s y n t h e s i s i n e u k a r y o t a r . Other groups (38.70.12) s u b s e q u e n t l y shoved t h a t t h e i n t e r m e d i a t e l i p i d n o i e t y . b e l o n g = t o a f a m i l y of p o l y i a o p r e n o l a l c o h o l s , know? as d o l i c h o l . I n animal t i s s u e s t h e s e compounds are u s u a l l y cornposed gf 16-21 isoprene u n i t s w i t h 2 i n t e r n a l --double bonds. The r e m a i n d e r of t h e i n t e r n a l double bonds are &oriented and the a-isoprene u n i t i n s a t u r a t e d ( F i g . 6 ) .

.

There are two roure,es of d o l i c h o l i n l i v e r , t h e d i e t and de nova s y n t h e s i s ( 7 1 . 7 5 ) . It h a s been determined t h a t d e s y n t h e s i s a c c o u n t s f o r 98I of new d o l i c h a l i n the ,liver -

~ ( 7 5 , 7 6 ) . The major forms o r d o l i c h o l found i n maram.' Ian

!

t i s s u e s are e i t h e r t h e f r e e d o l i o h o l 'or d o l i c h o l e s t e r i led w i t h f a t t y , a o i d s ( 7 7 ) . Furthermore, fiuch of t h e c e l l u l a r

, \

d o l i c h o l is d i s t r i b u t e d i n f r a c t i o n s otherthan the endoplasnio r e t i c u l u m ( 7 8 , 7 9 ) , where t h e enzymes of o l i g o s a c c h a r l d e s y n t h e s i s are predominantly l o c a t e d ( 8 0 ) . It i e t h e dolishol phosphate (Dol-P) and d o l i o h a l pyrophasphata (Dol-P-PI which serve as c a r r i e r s o f eaochpride r e s i d u e s i n t h e ,

-.

(46)

Fig. 6 . --GtFuctUee of dolYchol phosphate. The dolichol c o n s i s t s of a l i n e a r chain of repeating isoprene u n i t s i n which them-isoprene unit i s saturated.

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