The molecular characterisation of pregnancy associated plasma protein A (PAPP A)

The molecular characterisation of pregnancy-associated

plasma protein-A (PAPP-A).

EVANS, Steven.

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EVANS, Steven. (1996). The molecular characterisation of pregnancy-associated plasma protein-A (PAPP-A). Doctoral, Sheffield Hallam University (United Kingdom)..

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The M olecular C haracterisation Of

Pregnancy-A ssociated Plasm a

Protein-A (PAPP-A)

By

Steven Evans

A th esis subm itted

in partial fulfilm ent of the

requirem ents of Sheffield H allam U niversity for the

degree of Doctor of Philosophy.

R esearch Conducted At Sheffield H allam U niversity.

CONTENTS

Title P a g e ... i

C o n ten ts... h List o f Tables ... ix

List o f F ig u r e s... xi

List o f P la te s ... xiv

List o f P lates I n s e ts ...xiv

D e d ic a tio n ...xvi

A ck n ow led gem en ts... xvi

A b b reviation s... xvii

Other activities undertaken as part o f the PhD p rog ram m e xxi A b stra ct... xxii

Chapter O n e ... l.l

1 P regnancy and Pregnancy P r o te in s... 1.2

1.1 Introduction... 1.2 1.2 The Establishm ent of the Hum an Foeto-Maternal In terface 1.2 1.3 Molecules Produced In Response to the Foetal M aternal In tra ­

u terin e Complex ... 1.6 1.4 Pregnancy-Associated Plasm a Protein-A (PAPP-A)... 1.8 1.4.1 The Isolation and Purification of PA PP-A... 1.8 1.4.2 Physico-Chemical Characteristics of PA PP-A ... 1.9 1.4.2.1 Molecular Organisation ... 1.9 1.4.2.2 Electrophoretic Mobility ... 1.13 1.4.2.3 Other Characteristics ... 1.13 1.4.3 PAPP-A Localisation and Control of Its S yn th esis 1.13 1.4.3.1 The Trophoblast... 1.14 1.4.4 Extra-Trophoblastic sites of production for PAPP-A ... 1.15 1.4.4.1 The Endom etrium ... 1.15 1.4.4.2 Other S ite s... 1.15

-1.4.5 Assay Systems For The M easurem ent of PA PP-A ... 1.17 1.4.6 PAPP-A Levels Found In The M aternal Blood During

P regnancy... 1-21 1.4.7 The Diagnostic Usefulness of PAPP-A M easurem ents ... 1.24

1.4.7.1 Threatened M iscarriage/Abortion... 1.24 1.4.7.2 Ectopic Pregnancy... 1.26 1.4.7.3 Pre-eclamptic toxaem ia... 1.27 1.4.7.4 Cornelia-de-Lange Syndrom e...1.28 1.4.7.5 Down Syndrom e... 1.29

1.4.7.5a M aternal Serum Screening for DS in the

2nd T rim ester... 1.30 1.4.7.5b M aternal Serum Screening for DS in the

1st T rim ester... 1.30 1.4.7.6 The use of Biochemical Tests To Assess

Foetal-W ellbeing... 1.32 1.4.8 In-Vitro Biological Roles A ttributed To PA PP-A ... 1.32 1.4.8.1a Im m unosuppression...1.32 1.4.8.1b/c Inhibition of Complement

and Fibrinolytic system s... 1.32 1.4.8.Id Proteolytic Inhibition... 1.34 1.4.8.2 Sperm M otility... 1.35 1.4.8.3 Carrier P ro tein ... 1.36 1.4.8.4 In-Vivo Biological function of PA PP-A ... 1.36 1.5 Aims of the stu d y ... 1.36

2.3 General M ethods... 2.3 2.3.1 The Determ ination of Protein C oncentration... 2.3 2.3.1.1 Bradford Protein A ssay... 2.4 2.3.1.2 BCA Protein A ssay... 2.4 2.3.2 Electrophoretic M ethods... 2.6

2.3.2.1 Horizontal Agarose Slab Gel Electrophoresis

Of D N A ... 2.6 2.3.2.2 SDS-PAGE... 2.6 2.3.2.2a The Laemmli SDS-PAGE S y stem 2.7 2.3.2.2b Modifications to SDS-PAGE system if

sample was to be sequenced... 2.8 2.3.2.2c SDS Linear Gradient Gel Electrophoresis . 2.8 2.3.2.2d Protein detection: CBB Staining of

SDS-PAGE g e ls... 2.12 2.3.2.2e Protein detection: Silver Staining of

SDS-PAGE g e ls... 2.12 2.3.3 M easurem ent of Chitinase A ctivity... 2.12 2.3.4 M easurem ent of N-Acetyl-glucosamine (NAG)... 2.13 2.3.5 Assessment of General Proteolytic A ctivity... 2.14 2.3.6 M easurem ent of Tosyl-arginine methyl ester (TAM E).... 2.15 2.4 Miscellaneous Membrane Associated Techniques... 2.15 2.4.1 Semi-Dry Electroblotting (Western B lotting)... 2.15 2.4.2 Detection of Protein on PVDF B lo ts ... 2.16 2.4.3 Probing W estern b lo ts... 2.17 2.4.4 Lectin Probing... 2.17 2.4.5 Periodate Chemical Treatm ent of Nitrocellulose B lo ts 2.18 2.4.6 Enzyme Digest Treatm ent of Proteins Electroblotted

onto Nitrocellulose ... 2.18 2.4.7 Enzymatic Deglycosylation and Subsequent D etection 2.19 2.5 M aterial and Methods Section for cDNA Screening... 2.20 2.5.1 Screening A Placental cDNA L ib rary ... 2.20

-2.5.1.1 Storage and Preparation of Competent Bacterial

Cells for Phage Infection... 2.20 2.5.1.2 Preparation of Agar Plates Used for Screening .... 2.21 2.5.1.3 Infection of E. coli (strain: Y1090) with

Bacteriophage (X g tll)... 2.21 2.5.1.4 Titration of the L ib rary ... 2.21 2.5.1.5 The Ratio of Recombinant/Vector Phage

in the lib ra ry ... 2.22 2.5.1.6 Plaque Form ation... 2.22 2.5.1.7 Blocking of Nitrocellulose Overlays and

Im m unodetection... 2.22 2.5.1.8 Isolation of Putative Recombinant Phage Plaques .. 2.23 2.6 M aterials and Methods Section for The Quantitative

M easurem ent of PA PP-A ... 2.23 2.6.1 Rocket Im munoelectrophoresis...2.23 2.6.2 Radial Immunodiffusion... 2.24 2.6.3 Sandwich ELISA for PA PP-A ... 2.24 2.6.3.1 Preparation of PAPP-A F(ab’)2 F rag m en ts... 2.24

2.6.3.2 ELISA Coating and Blocking of Solid P h a s e 2.25 2.6.3.3 The ELISA Assay Procedure... 2.28 2.6.3.4 Colorimetric Detection and Reading of R e su lts 2.28 2.6.4 The PAPP-A Radioim m unoassay... 2.28

2.6.4.1 The Chloramine-T Method For Iodination

of PA PP-A... 2.29 2.6.4.2 Affinity Purification of Tracer for use in the

PAPP-A R IA ... 2.29 2.6.4.3 Antibody Titration C urves... 2.30 2.6.4.4 The PAPP-A R IA ... 2.31 2.6.5 D ensitom etry... 2.31 2.7 M aterials and Methods Section For The Isolation of PAPP-A for

2.7.2 The Preparation of Source Biological M aterial... 2.32

2.7.3 Ammonium Sulphate Precipitation and D ialysis... 2.32

2.7.4 H eparin Affinity Chrom atography... 2.33

2.7.5 Sulphated Dextran Chrom atography... 2.36

2.7.6 DEAE Ion Exchange C hrom atography... 2.38

2.7.7 Dye Affinity C hrom atography... 2.40

2.7.8 L-Arginine Agarose Affinity Chrom atography... 2.40

2.7.9 Lectin Affinity C hrom atography... 2.42

2.7.10 Metal Chelate C hrom atography... 2.43

2.7.11 Gel F iltratio n ... 2.45

2.7.12 Elution of Proteins From Gel Slices... 2.46

2.8 M aterials and Methods Section For A Molecular Approach:

Towards The PAPP-A G ene-II... 2.47

2.8.1 Assessment of Protein Elution Methods from Blotting

M em branes... 2.47 2.8.2 Solid Phase Cyanogen Bromide (CNBr) D igestion... 2.48

2.8.3 RP-HPLC Separation of P eptides... 2.48

2.8.4 Vapour Phase CNBr Cleavage of PAPP-A in Gel S lices 2.49

2.8.5 Limited Proteolytic Cleavage in-gel Slices (Cleveland) 2.50

2.8.6 N-terminal microsequencing... 2.51

2.8.7 Oligonucleotide Synthesis and Post Synthesis Processing .. 2.51

2.8.8 Polymerase Chain Reaction (PCR )... 2.52 2.9 M aterials and Methods Section for Studies on an interaction

Between Reduced Monomeric PAPP-A and The Endoproteinase

A rg-C ... 2.53

Chapter T h r e e

... 3.1

3 A M olecular Approach: Towards The PAPP-A Gene I 3.2

3.1 Introduction... 3.2

3.2 M aterials and M ethods... 3.6

3.3 Results and Discussion... 3.6

-C h a p t e r F o u r ... 4.1

4 The Q uantitative M easurem ent Of PAPP-A... 4.2 4.1 Introduction... 4.2 4.2 M aterials and M ethods... 4.4 4.3 Results and D iscussion... 4.4 4.3.1 The PAPP-A ELISA ... 4.5 4.3.2 Immunoprecipitation M ethods... 4.10 4.3.3 The PAPP-A R IA ... 4.11 4.3.4 Quantification by D ensitom etry... 4.13

C h a p t e r F i v e ... 5.1

5 The Isolation o f PAPP-A for M icroseq u en cin g... 5.2

5.1 Introduction... 5.2 5.2 M aterials and M ethods... 5.4 5.3 Results and D iscussion... 5.5

5.3.1 Purification Schemes That Were Successful in

Enriching PA PP-A ... 5.5 5.3.2 A Scheme That Significantly Enriched the Undefined

(Group-X) C ontam inant... 5.10

C h a p t e r S i x ... 6.1

6 A M olecular Approach: Towards The PAPP-A G ene I I 6.2

6.1 Introduction... 6.2 6.2 M aterials and M ethods... 6.7 6.3 Results and D iscussion... 6.7 6.3.1 Results Of CNBr Chemical Cleavage of PA PP-A ... 6.7 6.3.2 Limited Proteolytic Digestion of PAPP-A with L ys-C 6.8 6.3.3 Limited Proteolytic Digestion of PAPP-A with G lu-C 6.8

C h a p t e r S e v e n ... 7.1

7.1 In tro d u ctio n ... 7.2 7.2. Studies on The Carbohydrate Com ponent o f P A P P -A 7.2

7.2.1 Introduction... 7.2 7.2.2 M aterials and M ethods... 7.6 7.2.3 Results and discussion... 7.6 7.2.3.1 The Antigenicity of PA PP-A ... 7.6 7.2.3.2 The Carbohydrate Structure of the Glycan

Component of PA PP-A ... 7.8

7.3 Studies on An Interaction B etw een R educed M onom eric

PAPP- A and The E ndoproteinase A rg-C ... 7.14

7.3.1 Introduction... 7.14 7.3.2 M aterials and M ethods... 7.14 7.3.3 Results and D iscussion... 7.14

7.4 The Tertiary Structure Of P A P P-A ... 7.19

C h a p t e r E i g h t ... 8.1

8.1 Sum m ary and G eneral D isc u ssio n ... 8.2 8.2 H ypothesis o f a P ossible F unction for P A P P -A ... 8.5 8.3 C onclusions and P ossible Future Work for P A P P -A ...8.7

C h a p t e r N i n e ...9.1

9.1 R e feren ces... 9.2

A p p e n d i x O n e : M aterials. E quipm ent And S u p p lie r s... A l.l

A l.l M aterials... A1.2

A1.2 E quipm ent...A1.7

A1.3 Suppliers...A1.9

A p p e n d i x T w o : P reparation Of R eagents And B u ffe r s...A2.1

-A ppendix Three:

(D ata fro m serv ices u sin g in th is th e sis) ... A3.1 A III.l A m ino A cid A n a ly s is ... A3.2 AIII.2 P ro te in M ic ro se q u e n c in g ... A3.3 AIII.2.1 Vapour phase CNBr cleavage of PA PP-A ... A3.3 AIII.2.2 Initial N-Terminus obtained for PAPP-A ... A3.4 A III.2.3 Consensus sequence of N-Terminus for PA PP-A ... A3.4 AIII.2.4 PAPP-A N-terminal sequence from Lys-C digestion .... A3.5 AIII.2.5 Other sequencing from Lys-C digestion...A3.12 A III.2.6 O ther sequencing from Lys-C digestion... A3.13 AIII.2.7 Other sequencing from Lys-C digestion... A3.14 A III.2.8 PAPP-A N-terminal sequence from Glu-C digestion .. A3.15 AIII.2.9 Protein Sequencing of Iodination grade PA PP-A A3.15 AIII.2.10 Sequence obtained for design of cPAPP-3 p rim e r A3.16 AIII.2.11 Other sequencing from Glu-C digestion... A3.17 A III.2.12 Protein sequence results from Group-X contam inants A3.17 AIII.3 O lig o n u cleo tid es S y n th e s is e d ... A3.18

AIII.3.1 N-PAPP-1 primer designed from initial N term inus .... A3.18 AIII.3.2 N-PAPP-2 prim er designed from initial N-term inus ... A3.19 A III.3.3 cPAPP-3 internal p rim e r... A3.20 AIII.3.4 PAPP-4 primer designed from consensus N -term inal.. A3.21

List o f Tables

T able 1.1 PAPP-A purification schemes th at have been adopted

by various research groups...1.10 T able 1.2 The yield and purity of PAPP-A obtained by various

investigators... 1.11 T able 1.3 Glycan components of PAPP-A as found by other

Table 1.7 The affect of anti-coagulants on the m easured levels

of PA PP-A ... 1.20

Table 1.8 Clinical Features of D S ... 1.29

Table 1.9 Detection rates for DS in the 2nd Trim ester when

analysing different m aternal serum an aly tes... 1.30

Table 1.10 Comparison of m aternal serum DS screening during

first and second trim ester of pregnancy... 1.31

Table 1.11 In-Vitro Biological Roles A ttributed to PA PP-A ... 1.33

T able 1.12 Type of HGE inhibition observed with PA PP-A ... 1.35

Table 2.1 Composition of Laemmli SDS-polyacrylamide g e ls 2.8

Table 2.2 The standard linear gradient SDS-PAGE gel sy stem ...2.9

Table 2.3 The modified linear gradient SDS-PAGE gel sy stem 2.10

Table 2.4 The relationship of num ber of plaques to Petri dish

diam eter ... 2.21

Table 2.5 Optimisation to yield maximum recovery of PAPP-A

from DEAE ion exchange column using an isocratic

gradient sy stem ... 2.38

Table 2.6 Proteolytic enzymes suitable for limited proteolytic

cleavage in the Cleveland SDS-PAGE gel environm ent.... 2.50

Table 2.7 Oligonucleotides synthesised from PAPP-A sequences

obtained in this study... 2.51

Table 2.8 Ramped PCR program for use with primers listed in

Table 2 .7 ...2.52

Table 3.1 Titration of the placental cDNA lib ra ry ... 3.8

Table 4.1 Features of standard immunoassays th at have

been adapted for use in the m easurem ent of PA PP-A 4.3.

Table 4.2 Percentage of PAPP-A tracer bound to a heparin affinity

colum n... 4.13

-T able 6.1 Reverse translation methods for the production of

degenerate prim er pools... 6.6 T able 6.2 Microsequencing of PAPP-A m aterial used for iodination . 6.7 T able 6.3 Comparison of PAPP-A N-terminal sequence

information obtained during this thesis against

the published sequence... 6.12

T able 7.1 Specificities of lectins for specific carbohydrate residues ..7.5 T able 7.2 Reduced monomeric PAPP-A’s reactivity

with various lectin s... 7.9 T able 7.3 The esterolytic activity of endoproteinase Arg-C after

pre-incubation for various times with PA PP-A ... 7.19

List of Figures

F ig u re 1.1 Schematic illustration of events leading to im plantation

(a) and development of the chorion and decidua (b )... 1.3 F ig u re 1.2a Schematic illustration of the foetal placental

intrauterine complex ... 1.4 F ig u re 1.2b Enlargem ent of inset from Figure 1 .2 a... 1.5 F ig u re 1.3 The levels of PAPP-A found in m aternal blood during the

the first trim ester of pregnancy ...1.22 F ig u re 1.4 Schematic illustration of m aternal levels of PAPP-A

found in the blood during pregnancy ... 1.23

F ig u re 2.1 The BCA protein micro-assay (+/- alkali addition)...2.5 F ig u re 2.2 Linear gradient SDS-PAGE... 2.11 F ig u re 2.3 Standard calibration curve for N-Acetyl-glucosamine

using DMAB re a g e n t...2.13 F ig u re 2.4 The general protease assay with detection using

F ig u re 2.6 Calibration curve for PAPP-A SR ID ... 2.25 F ig u re 2.7 Determ ination of optimum conditions for coating

PAPP-A F(ab’)2 fragments onto Falcon 3915 p la te s 2.26 F ig u re 2.8 Determ ination of optimum concentration of F(ab’)2 for

coating Falcon 3915 p la te s... 2.27 F ig u re 2.9 PAPP-A antiserum titration cu rv e... 2.30 F ig u re 2.10 The common repeat unit found in h e p a rin ... 2.33 F ig u re 2.11 Elution profile from heparin-Affigel affinity colum n 2.35 F ig u re 2.12 Elution profile from heparin-affinity column (Reactiv) 2.36 F ig u re 2.13 Elution profile from a sulphated dextran affinity m atrix .. 2.37 F ig u re 2.14 Elution profile from a DEAE-Trisacryl ion

exchange colum n... 2.39 F ig u re 2.15 Elution profile from Cibachron-blue affinity colum n 2.41 F ig u re 2.16 Elution profile from a L-arginine affinity colum n... 2.42 F ig u re 2.17 Elution profile from Zinc-MCAC colum n... 2.44 F ig u re 2.18 Calibration curve for the SuperDex-200

gel filtration colum n... 2.45 F ig u re 2.19 A typical elution profile from the superDex 200

gel filtration colum n... 2.46

F ig u re 3.1 Vector/host interactions... 3.4 F ig u re 3.2 Calculated general proteolytic activity present in the

chitinase enzyme preparation ... 3.12 F ig u re 3.3 The calculated chitinase activity of the enzyme

preparation after incubation an inhibitor cocktail 3.13

F ig u re 4.1 The principle of reagent excess (A) and reagent

limited assays (B )... 4.2 F ig u re 4.2 Schematic representation of the PAPP-A ELISA design... 4.6 F ig u re 4.3 Unmodified ELISA +/- 2nd phase PAPP-A antibody with

F ig u re 4.4 Modified PAPP-A ELISA with detection using a

protein-A enzyme conjugate... 4.8 F ig u re 4.5 The modified PAPP-A ELISA ... 4.9 F ig u re 4.6 The PAPP-A SR ID ... 4.11 F ig u re 4.7 A typical PAPP-A RIA calibration cu rv e... 4.12 F ig u re 4.8 A typical calibration curve of protein levels seen

using video densitom etry... 4.14

F ig u re 5.1 Factors involved in the design of a purification scheme

with particular reference to PA PP-A... 5.3 F ig u re 5.2 Purification scheme 1 ... 5.6 F ig u re 5.3 The heparin based purification scheme 2 ... 5.10 F ig u re 5.4 Enrichment scheme for group-X contam inants... 5.11

F ig u re 6.1 Approaches to producing prim ary sequence information with identified outcom es... 6.3 F ig u re 6.2 Principle of Edman degradation... 6.4 F ig u re 6.3 RP-HPLC elution profile of solid-phase CNBr

cleavage of PA PP-A ... 6.9 F ig u re 6.4 Limited proteolytic digestion of PAPP-A with L ys-C 6.10 F ig u re 6.5 Limited proteolytic digestion of PAPP-A with G lu-C 6.11

F ig u re 7.1 Incubation of PAPP-A containing gel slice with

endoproteinase A rg-C ... 7.16 F ig u re 7.2 The calculated activity of the enzyme, endoproteinase Arg-C

incubated with PAPP-A (BSA) in the absence of glycine ... 7.17 F ig u re 7.3 The calculated activity of the enzyme, endoproteinase Arg-C

incubated with PAPP-A (BSA) in the presence of glycine .. 7.18 F ig u re 7.4 Elution profile of 0.3 and 0.6 M heparin eluted fractions

on a SuperDex-200 gel filtration colum n... 7.21

F ig u re 8.1 Observations made for PAPP-A ... 8.6

-List of P lates

Plate 3.1 W estern blots of PAPP-A prepared using DAKO polyclonal

antibody (A) and anti-PAPP-A monoclonal (B )... 3.7

Plate 3.2 An Assessment of Vector/Recombinant Phages present

in the placental cDNA library used in this th e s is ... 3.9

P late 3.3 Positive control of the expression library/screening system ... 3.10

Plate 3.4 W estern blots with PAPP-A immunodetection

(+/- a chitinase enzyme preparation) using a polyclonal

anti-PAPP-A antibody... 3.14

Plate 5.1 A CBB stained 5% SDS-PAGE gel of fractions from heparin

based purification, scheme 2... 5.8

P late 5.2 A CBB stained 5% SDS-PAGE gel illustrating the disulphide

bridged structure of proteins separated by scheme 2 ... 5.9

P late 6.1 PCR products using primers N-PAPP-1/2 and cPAPP-3

(an internal PAPP-A stretch, complementary p rim er)... 6.13

P late 7.1 A chemical assessment of the glycan epitopes detected

by polyclonal anti-PAPP-A p arato p es...7.7

P late 7.2 The reactivity of reduced monomeric PAPP-A with the

LEL lec tin ...7.10

P late 7.3 Enzymatic deglycosylation of control proteins (A,B)... 7.11

P late 7.4 Enzymatic deglycosylation of enriched PAPP-A fractio n ...7.12

List Of Plate Insets

(Note insets were numbered from figures in which they were placed)

P late Inset 2.11 Represented a 5% SDS-PAGE of fractions

from heparin-affigel colum n... 2.35

-P late Inset 2.13 Represented a 5% SDS-PAGE gel of the

fractions indicated... 2.37

P late Inset 2.14 PAPP-A detected by R IE ...2.39

P late In set 2.15 PVDF CBB stained samples from dye affinity column 2.41

P late Inset 2.16 5% SDS-PAGE of bound fraction... 2.42

P late Inset 2.19 Represented a silver stained 5% SDS-PAGE gel of the

fractions indicated... 2.46

P late Inset 5.2 Protein electroblotted onto PVDF m em brane... 5.6

P late Inset 5.3 Pure monomeric chain PA PP-A ... 5.10

P late Inset 5.4 Electroblotted m aterial purified using this schem e 5.11

P late Inset 6.4 PVDF electroblotted sample from Lys-C d ig est 6.10

P late Inset 6.5 PVDF electroblotted sample from Glu-C d ig est 6.11

P late In set 7.4 5% SDS-PAGE of indicated fraction... 7.21

-D edication

This Thesis is dedicated to the memory of my father, Mr T. E. Evans.

A cknow ledgem ents

I would like to extend my deepest gratitude to my supervisor M aria Blair for all the help, encouragement and financial support during this project, also for an inordinate amount of tolerance and patience during the production of the bound thesis.

I would also like to thank everyone within the Universities in Sheffield th at were involved with this project, especially those w ithin the School of Science. For their continued understanding, usually when a piece of equipment miraculously disappeared for th at vital experiment. Thank you all. Special thanks to those th at made me eventually draw a line under things, especially to Di, Anne, Barry and those th at at Langhill.

Lastly thanks to my family for their continuing encouragement as friends were starting to think th at Connie and Alex were a one-parent family.

I wish to acknowledge the financial support of the Medical Research Council and The School of Science, Sheffield Hallam University.

-L ist Of A bbreviations U sed in th e Body of This T hesis

a Alpha.

0C2M Alpha-2-macroglobulin.

a-CN 4-Chloro-l-Napthol.

A Adenine.

Axxx nm Absorption at xxx nm.

ACS American Chemical Society.

AD Alzheimer’s disease.

AFP Alpha-Fetoprotein.

Arg Arginine.

APP-KD Amyloid p-protein precursor-Kunitz domain.

APS Ammonium Persulphate.

ATZ Anilinothiazoline

P Beta.

BCA Bicinchoninic Acid.

Bp Base pairs.

BPB Bromophenol Blue.

BSA Bovine serum albumin.

C Cytosine.

Ca2+ Calcium

CAPS 3- [cyclohexylamino] - 1-propanesulfonic acid.

CBB Coomassie Brilliant Blue®.

cDNA complementary DNA.

CL Cornelia-de-Lange.

CNBr Cyanogen Bromide.

Con-A Concanavalin - A.

Cong. Congenital.

CPM Counts per minute.

CV Coefficient of variation.

CVS Chorionic villus sample.

d. Density.

DEAE-Trisacryl Diethyl aminoethyl(Trisacryl).

DMAB p-dimethyl aminobenzaldehyde.

DMF Dimethyl formamide.

DMSO Dimethyl sulphoxide.

DNA Deoxyribonucleic acid.

dNTP’s Deoxyribonucleoside Triphosphates

DS Down syndrome.

DTT Dithiothreitol.

EDTA Ethylenediamine tetra-acetic acid.

EEO Electro-endosmosis.

EGF Epidermal growth factor

EGF-BP Epidermal growth factor binding protein ELISA Enzyme Linked Immunosorbent Assay.

EtBr Ethidium Bromide.

EtOH Ethanol.

F(ab')2 Divalent antigen binding region.

-FISH Fluorescence in-situ hybridisation.

FPLC Fast performance liquid chromatography™.

Fig. Figure.

G Guanine.

g- Gramme.

GalNAc N-Acetylgalactosamine

gl. Glacial.

GlcNAc N-Acetylglucosamine.

GPR General Purpose Reagent Grade.

gr. Grade.

hCG hum an Chorionic Gonadotrophin.

HC1 Hydrochloric acid.

HGE Hum an Granulocyte Elastase.

HOAc Acetic acid.

hPL Hum an placental lactogen.

HPLC High performance liquid chromatography.

HRP Horseradish Peroxidase.

HSA Hum an serum albumin.

IPTG P -D -isopropyl-thiogalactopyranoside.

IU International units.

K 103.

kDa 103 daltons.

X Lambda.

LB Luria Broth.

LCA Lentil lectin (Lens culinaris) agglutinin LP4 Size of plastic tube (Denley).

M Molar.

mA 10'3 Amps.

mAb Monoclonal antibody.

Mat. M aternal.

mCi 10'3 Curie.

MEGA-10 Decanoyl-N-Methyl Glucamide

MeOH Methanol.

mg Iq-3 gramme.

ml 10-3 litre.

mm 10'3 Metre.

mM 10'3 Molar.

Mol. Biol. Gr. Molecular Biology Grade chemical. MP Membrane associated placental protein.

mRNA messenger RNA.

MW Molecular weight.

NaCl Sodium Chloride.

NAG N -Ace tylglucos amine.

NaOAc Sodium acetate.

nd. Not determined.

NDS Napthalene-l-5-disulphonic acid-disodium salt. NeuAc Sialic acid (N-Acetyl-neuraminic acid).

nmole 10-9 Mole.

-NSB Non-Specific background.

NT Nuchal translucency.

°C Centigrade.

2-ME 2-mercapto-ethanol.

*X Stock *times concentrate(where *= No.)

[ ] Concentration.

PAPP-A Pregnancy-associated Plasm a Protein-A. PEG Polyethylene Glycol(Average MW 6000).

PET Pre-eclamptic toxaemia.

pfu Plaque forming unit.

Pg 10-12 gramme.

PHA Phytohaemagglutinin.

Pi Iso-electric point.

pmole 10-12 Mole.

PMSF Phenyl methyl sulphonyl fluoride.

PP Placental protein.

PBS Phosphate Buffered Saline.

PCR Polymerase Chain Reaction.

PITC Phenyl isothiocyanate

proMBP Proform of eosinophil major basic protein.

PTC Phenylthiocarbamoyl

PTH Phenylthiohydantoin

PVDF Polyvinylidene difluoride PVP-360 Polyvinylpyrolidone-360.

PZP Pregnancy zone protein.

RCF Relative Centrifugal force.

Rf Relative mobility

RIA Radioimmunoassay.

RID Radial Immunodiffusion.

RIE Rocket Immunoelectrophoresis.

RMM Relative molecular Mass.

RP-HPLC Reverse Phase-HPLC

rpm Revolutions per minute.

RT Ambient Room Tem perature (Approx. 21°C).

SDS Sodium dodecyl sulphate.

ss. Single stranded

Sp. Ac. Specific activity.

SP1 Schwangerschaft spezifische p glycoprotein-1.

T Thymidine.

TAE Tris-Acetate-EDTA buffer.

TAME Na-p-Tosyl-L-Arginine Methyl Ester.

TBE Tris-Borate-EDTA buffer.

TBS Tris Buffered saline.

TCA Trichloroacetic acid.

TEMED N,N,N',N'-tetramethyl ethylene diamine.

TFA Trifluoroacetic Acid.

TMB 3,3',5,5'- Tetramethylbenzidine.

TPCK N-Tosyl-L-Phenylalanine Chloro-Methyl Ketone.

TLCK Na-p-Tosyl--L-Lysine Chloro-methyl Ketone.

TRIS (TRIZMA) Tris(hydroxymethyl)aminomethane. Triton® X-100 Octyl polyethylene ether.

Tween®-20 Polyoxyethylenesorbitan Monolaurate.

gg 10'6 g.

gl 1 0-6 1.

gM 10-6 M.

uE3 Unconjugated Oestriol.

v/v Volume : volume ratio.

V0 Void volume. .

Vt Total volume.

Vc Column volume.

V8 Alternative name for Endoproteinase Glu-C.

w/v W eight: volume ratio.

X-gal 5-Bromo-4-Chloro-3-Indolyl-p-galactopyranoside.

XIE Crossed Immunoelectrophoresis

Coomassie Brilliant Blue® is a registered tradem ark of ICI. Tween® is a registered tradem ark of ICI.

Triton® is a registered tradem ark of Rohm & Haas, USA.

Other Abbreviations are as found and understood by general texts as illustrated by the scientific literature and texts such as th at devoted to the usage of Abbreviations (Baron, 1994).

-Other a ctivities undertaken as part o f the PhD program m e

Post-graduate courses, attendance at conferences, active participation in research sem inars and teaching th at was undertaken as part of the programme of research th at was required as a partial fulfilment of the requirem ents for the degree of PhD.

Attendance at biochemical society meetings and a British Society for

Immunology meeting.

Regular participation in research seminars at: • The school of science.

• Jessop hospital for Women.

• The Institute of Virology, The Royal Hallamshire hospital.

The presentation of a research sem inar on the molecular characterisation of PAPP-A to the school of science. Regular participation in the postgraduate PhD rolling training programme.

Teaching experience at a variety of levels with:

• Lectures on the MSc pathological sciences course and postgraduate DNA technology course.

• Supervision of Practical Classes for the HND, BSc and MSc. • Part-supervision of projects on HND, BSc and MSc.

Actively involved in the development of m aterial th at was used for course m aterial on the HND and BSc courses run in the Biomedical Sciences Division.

-A bstract

PAPP-A is a large glycoprotein with 0C2 - electrophoretic mobility th at is

produced by the placenta during pregnancy. In this thesis a biochemical and molecular characterisation of PAPP-A was performed.

The polyclonal antiserum (DAKO) directed against PAPP-A has been shown to also interact with proteins other than PAPP-A. These non-specific

interactions were abolished by performing W estern blotting

immunodetection at a high salt concentration (0.6M NaCl). At this salt concentration a single band of 195 kDa was immunodetected and this corresponded to the monomeric PAPP-A molecule. It was also discovered th at a subset of paratopes in this antiserum reacted, under the described high salt concentration conditions, with the glycan component of PAPP-A.

A placental cDNA library was screened using this antibody for the PAPP-A cDNA but this did not yield a clone for PAPP-A. A possible explanation is th at the interaction with this antibody requires carbohydrate components to be present on the PAPP-A molecule. It is known th at proteins expressed in bacterial systems are not post-translationally modified. Therefore another approach to the isolation of the PAPP-A cDNA clone was adopted, but this required some prim ary amino acid sequence of this protein th at was unavailable at the time. To generate this information, PAPP-A was purified using its previously unpublished affinity for L-arginine in combination with the already described procedures of ammonium sulphate precipitation, ion exchange and gel filtration. Final purification of PAPP-A

was achieved by SDS-PAGE electrophoresis. The isolated monomeric PAPP-

A gave a unique single N-terminal amino acid sequence: N-EARGATEEPS.

The N term inal sequence combined with the sequence obtained from limited proteolytic digestion of PAPP-A were used to design oligonucleotide primers specific for PAPP-A. These primers were used in a PCR reaction th at produced 500 and >1200 bp fragments using the cDNA library as DNA template; thus demonstrating th at PAPP-A is synthesised in the placenta.

PAPP-A was shown to have 0 and N-linked carbohydrate chains. Enzymatic deglycosylation demonstrated th at the N-linked chains were 8% (w/w) of the molecule. The O-linked groups were extensively modified with the presence of oligomers of N-acetyl-glucosamine. It was also shown th at it was these groups the PAPP-A antibodies bind to at high salt concentration.

A physical interaction of PAPP-A with endoproteinase Arg-C (EGF-BP) was observed. It was seen th at they form a 1:1 (PAPP-A: endoproteinase) sub-unit complex th at was stable in SDS. A further investigation revealed th at PAPP-A interacted with the endoproteinase Arg-C and this resulted in a 30% inhibition of the esterolytic activity of this enzyme.

Chapter One

P regnancy and P regnancy P roteins

1.1 Introduction

The changes th at are imposed on the m aternal hum an system by pregnancy have led to an interest in the molecules th at are synthesised during pregnancy. The placenta is a highly invasive organ th at is analogous to a locally invasive tumour. It is hoped th at an understanding of the control mechanisms and molecules produced at the trophoblastic interface will lead to a better understanding of how the conceptus in the majority of cases survives to term.

1.2 The E stablishm ent of the Hum an Foeto-M aternal

Interface

After fertilisation of the hum an female oocyte by the male spermatozoan a series of mitotic cleavage divisions occur and two distinctive groups of blastomeres emerge, surrounded by the zona pellucida. One of these groups form the inner cell mass which include the embryogenic cells, while the other consists of a mass of flattened polyhedral cells th at compose the blastocystic trophoblast. At this stage the repeated divisions produce a characteristic mulberry-shaped mass of cells (a morula). By the fifth day following ovulation and fertilisation the zona pellucida is shed. The uterine wall now consists of two main layers, the myometrium (a thick layer of muscles) and the endometrium (an inner mucous membrane, Figure 1.1a). Im plantation of the morula now occurs with the trophoblastic cells adhering to the endometrium. On adhering they exert histiolytic activity on the endometrial epithelium with invasion and destruction of a p art of the m aternal vascular epithelia. The endometrium then undergoes a series of

changes th at results in the decidualisation of the secretory endometrium. These cells acquire a characteristic polygonal shape and this decidual layer thickens and becomes more heavily vascularised (Figure 1.1b)._____________

Corpus luteum

Z o n a p e l l u c i d a \ p r a a ^ a n f o l l i c l e ^ . ^

r~ L ■ / Myometrium

\;\

Fimbria

'

'

Endometrium (progestational stage)

(a)

(b)

Figure 1.1

Schematic illustration of events leading to im plantation (a) and the development of the Chorion and Decidua (b).

(Adapted from Langman, 1975) During this invasive process the trophoblastic cells divide w ith great rapidity and the progeny fuse with each other to form a thin polarized membrane (the syncytio-trophoblast). This places the chorionic (trophoblastic and foetal vascular tissue) attachm ent to the uterus in direct contact with the m aternal blood stream and so this is a haemochorial type of placentation (Bjorkman,1985).

[image:28.619.84.517.78.554.2]

During the described invasive process large lacunar spaces are formed and after enlargem ent become blood filled intervillous spaces th at initially contain a labyrinthine chorionic structure th at develops into a secondary villous structure (Williams and Warick, 1980). The chorion is vascularized by the allantoic blood vessels and so is term ed chorioallantoic. The chorionic attachm ent enlarges in a discoidal p attern to cover approximately 25% of the endometrium at about the third m onth of pregnancy. At this stage this attachm ent is known as the placenta; which is deciduate as m aternal tissue and placental membranes are shed as part of the afterbirth at term (Figures 1.2a and 1.2b).

Uterine cervical canal Internal os uteri

, Amnion

j (c y t o trophoblast) Oecidua capsularls Amniochorion

Virtual uterine lumen -Decidua parietalis " Myometrium ' Amniotic

cavity-P l a c e n t a l disc

Figure 1.2a

Schematic illustration of the foetal placental intrauterine complex

(Adapted from Panigel et al. 1985)

[image:29.614.76.513.291.693.2]

Cbcriai piaca

Syncyciscrccr.coiasz Cycacracnaoias- call islanc-C^

) C y c = c r a c n c = i a s c ( b a s a l p l a c a j

? □ a c i c u a C b a s a l p l a c a ]

rnceracicial cyrai Lina ar* placancal

(paac-parcam) Oaec cecicsja

UPar:

F ig u re 1.2b

Schematic enlargem ent of inset seen in Figure 1.2a

If you consider Mossman's (1987) definition of the placenta as an “ apposition of foetal and parental tissues for the purpose of physiological exchange. ” then it is well suited to this task from a structural point of view as its surface area is equivalent to the calculated absorptive area of the entire adult gastro-intestinal tract.

The placenta also acts as an anchoring device for the foetus, providing it with buoyancy and allowing for freedom of movement and growth in an aquatic environment. Allowing for these described features the placental unit m ust provide for effective synthesis, storage, transport and breakdown of compounds th at are vital for foetal growth. These physiological processes have been reviewed by Bjorkman (1985) and it is not proposed to further cover them here.

gestation survives and proceeds to term. This apparent contradiction is further deepened with the demonstration by Voisin e ta l, (1985) that:

• The immunological capacity of the mother to defend against microbial infections is not significantly impaired.

• The m aternal immune system recognises the foreign paternal antigens. • The haemochorial placentation bathes the paternal antigens present on

the trophoblast in blood and hence one of the major channels for

transport of cells involved in immune surveillance and response.

Therefore it is a surprise th at the immune system does not react as expected to the presence of this semi-allogeneic allograft. It is also likely th at any communication between the foetal and m aternal systems will be endocrine in nature (Bell, 1988). So a more detailed understanding of the trophoblast and related tissues is needed, as they are likely to play a key role in this physiological and immunological exchange th at occurs between the foetal and m aternal systems.

1.3 M olecules Produced In R esponse to th e F oetal

M aternal Intra-U terine Complex

The placenta, foetus and endometrial tissues produce a wide variety of biologically active proteins, which have historically been split into two groups (Bohn and Winkler, 1988).

• Group-A: Molecules th at were identified on the basis of their activity. • Group-B: Molecules th at were identified using an immunochemical

approach.

The first group-A molecule was discovered by Aschheim and Zondek in 1927 who found a hormone with gonadotrophic activity and nam ed it hum an gonado-tropin (later renam ed hum an chorionic gonadotropin, hCG). Some of the molecules identified in this group are identical or analogues to functional proteins th at have been found in other tissues, whilst others are

biologically active substances th at are necessary for the m aintenance of pregnancy.

Studies of the group B proteins were initiated by M acLaren et al, (1959) and their cited work on the protein composition of blood in hum an pregnancy dem onstrated the presence of additional antigenic determ inants, when compared to sera from non-pregnant or male donors. This demonstration led to the suggestion th at a new group of pregnancy-specific proteins exist during pregnancy. Using the immunochemical approach Tatarinov and Masyukevich (cited from 1970) and Bohn (cited from 1971) isolated pregnancy-specific proteins in the early ‘70s. Lin et al, (1974a, 1974b) produced antisera to hum an pregnancy plasma th at had been adsorbed with plasma from males. Using this antisera they showed the presence of four distinct immunoprecipitin lines. They were named alphabetically from the immunoprecipitin pattern seen as pregnancy- associated plasma protein's A, B, C and D. Since then num erous placental proteins have been isolated, with Bohn et al, (1988) isolating more than 50 different antigens over the past 20 years. The initial work utilised whole term placental tissue th at was composed of foetal and m aternal tissue. This led to the identification of a num ber of m aternal proteins initially defined as P ’lacental P ’roteins (PP), e.g. PP2 has subsequently shown to be ferritin (Bohn and Winkler, 1988 cited from Bohn’s initial work, 1973). Subsequent work by other investigators has used a more closely defined tissue source and has led to the characterisation of

:-• Foetal antigens (Fay e ta l, 1989, Price et al. 1995). • Soluble tissue proteins (PP's). and

• Membrane associated placental proteins (MP’s).

The immunochemical approach has led to many proteins being identified,

some of the components have been seen in more th an one tissue compartment and have been shown to be immunochemically identical, e.g. MP2-C shown to be identical to PP21 (Bohn and Winkler, 1988).

An overall class of proteins defined as pregnancy-associated has emerged, several of these proteins have been found outside of pregnancy e.g. in the seminal plasm a and follicular fluid. To keep these proteins as a class it was suggested th at a more appropriate term be Reproductive Proteins (Chard, 1985). One of the proteins whose levels are modulated by pregnancy is term ed pregnancy-associated plasma protein-A (PAPP-A) and is considered further.

1.4 Pregnancy-A ssociated Plasm a Protein-A (PAPP-A)

1.4.1 T h e Iso la tio n a n d P u r ific a tio n o f P A P P-A

Lin et al (1974a) were the first group to purify PAPP-A and raise a polyclonal antiserum against it. Folkersen et al (1979) subsequently isolated a protein they called pregnancy specific protein-4, which was shown using

immunological methods to be identical to PAPP-A. Subsequently Sutcliffe et

al (1979), Bischof (1979a), McIntyre et al (1981), Davey et al (1982) and Oxvig et al. (1994) isolated PAPP-A by a variety of chromatographic techniques (Table 1.1). The described purification procedures were applied to purify PAPP-A from m aternal blood samples, although PAPP-A has also been purified from placental homogenates (Lin and Halbert, 1976 and Davey et ah 1983). In all purification schemes it was found to be difficult to obtain a homogeneous preparation, without resorting to negative (or positive) immuno-affinity chromatography or utilising PAPP-A's interaction with heparin (Sinosich et al. 1981). Techniques th at utilise positive affinity chromatography, i.e. selective binding of PAPP-A to an antibody directed against it require subsequent elution with chaotropic agents (Sutcliffe et al. 1979).

All schemes for the purification of PAPP-A except th at of Oxvig et al, (1994) were published prior to the practical work of this thesis. A guide to the yield and purity of PAPP-A prepared by the various schemes outlined in Table 1.1 is shown in Table 1.2. An assessment of the purity can also be made by looking at specificity of antisera produced by the various investigators, in which they used this m aterial as immunogen for the production of antisera directed against PAPP-A (Table 1.5).

1.4.2 P hysico-C hem ical C h a ra c te ristic s o f PA PP-A

PAPP-A is a glycoprotein containing 19.2-19.4% (Sutcliffe et ah 1980.

Sinosich et al. 1990) carbohydrates with the major sugar residue being glucose (48%). The carbohydrate content has been analysed using lectins which have revealed the presence of sialic acid, a-D-glucose, a-D-mannose, N-Acetyl-glucosamine and N-Acetyl-galactosamine (Sinosich, 1988a). A summary of the various findings is given in Table 1.3

1.4.2.1 M o lecu lar O rg a n isatio n

Prior to the work by Oxvig et al. (1993) it was proposed th at PAPP-A is a homo-tetramer with a Mr of 710 - 820 kDa, (Lin et al. 1974b. Bischof, 1979a. Sinosich et al. 1987) which consists of two dimers held together by van der W aal's forces, each dimer being composed of identical subunits of Mr 200 - 236 kDa covalently linked by disulphide bridges (Bischof, 1979b. Sinosich et al, 1990). This proposed structure was also shown not to contain any thiol-esters. The carbohydrate moiety has also been suggested to play a role in the maintenance of PAPP-A’s structure (Sinosich, 1990). Recently Oxvig et al, (1994) have suggested th at PAPP-A is a heterotetram er composed of two sub-units of PAPP-A with two sub-units of proMBP with a calculated Mr of 474 kDa. The exact tertiary form of PAPP-A present in the m aternal serum is unknown, it has not been established w hether all of the

Tab le 1. 1 PA PP -A pu rif ic at io n sc he m es th at h av e be en ad opt ed by va rio us re se ar ch gr ou ps .

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£ p

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Ta bl e 1.2 Th e y iel d an d pur ity of PA PP -A obt ai ne d by va rio us in ve st ig at or s. _ _ _ So ur ce Fi na l Y ie ld P u ri ty In ve st ig at or (s ) Pr ot ei n C on ce nt ra ti on pu ri fi ca ti on (P er ce nt ag e of {S ub se qu en t co nt am in an ts V ol um e us ed :( m g/ m l) fa ct or st ar t m at er ia l) fo u n d ) n® ON 'd₀ X₀

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fl

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p © « e 03 O 2 ft* 03 fl 73 O Pk io rH rH SH

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'ft g

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• pH

• d *_{© fl} r! 0 e .fl ** fl o

03 03

?■ §

1 §

S o ft P O fl k ft ek 03

73 ,«_{03 £}S

15 g k 03 >fl Po to P CO

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© V p g_{Q 03}

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03 03 & N=> ON oo CS O Pk tH ai <N W) s fl B CO at

£ <0 . N §

T3 _k 00

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fl .

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N?ON d at> a O rfl 03 CO PQ M NO ON SO 73_l-H o Pk io o lO X r—H_o o | ft bJ)_fl fl

fl M

k CO 0CO_in fl ^H k s 0 H o_o

03 N® ON to fl p 03 eg •M pH 03 P fl gg © 00 03 co_k

p_{p .fl}§ §

fl ^

fl *o

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£ S -S_{f t 03}

f l CO 03

ft k -g ■ s.g '_{a> «} S

^ s

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CO NO on_<N O Pk eo 00 rH X

pH ^

f l CO

k 'S ’

g s § I I

fl p0 flJ

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CO y-V_t>

O oo_{fl Ci} O) rH <D ^3 co C 0 Cfl 0 ^3 0 p Jh ^ 0 5-( o 2 a 0 > 0 0 nda₀₃ m be_C3 • T)_d cfl_m 3h o p₀₃ be

• pH_-M

in₀

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• rH p-"H 03 d d • rH 0 rB d o p,_d T3 0 M 03 ^2 0 J-H 0 ts 0 H-S_o

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d ft 'd dd ■d 0 • M 0 ■fl Eh dd 0be 0 fl assa

PAPP-A is in a complex with proMBP or if it also exists as a hom otetram er as described by previous investigators.

T able 1.3 Glycan components of PAPP-A as found by other investigators

PA PP-A p u rifie d C a rb o h y d ra te re sid u e s fo u n d on p u rifie d

an d m e a su re d by: m aterial:

No Galactosamine but contains

Bischof (1979b) 3.1% Glucosamine.

Presence of sialic acid as determined by

Sutcliffe et al. (1980) treatm ent with neuram indase, N-linked groups present as determined by incubation with the

glycosidic enzyme, Endo-H.

% Total carbohydrate: 19.2% (w/w)

3.8% Glucuronic acid 9.4% Glucose

Sinosich et al.(T990) 3.1% Fucose

2.3% Mannose

0.8% Galactose.

% Total carbohydrate: 19.4% (w/w)

Oxvig et al, (1994) PAPP-A monomer PAPP-A/proMBP

nd... Glucuronic a c id 0.92%

0.42% Fucose...0.41%

3.39% M annose... 3.05%

0.03% G lucose...0.03%

1.87% G alactose... 3.11%

4.52% N-Acetylglucosamine .. 6.34%

0 % N-Acetylgalactosamine.. 0.23%

3.09% .N-Acetylneuraminic acid..3.27%

13.4% Total Carbohydrate (w/w) 17.4%

[Legend: The Carbohydrate composition of PAPP-A, pro-MBP and PAPP-A/pro-MBP complex were as determined by Oxvig et al. (1994) compared with that obtained for PAPP-A (A/proMBP complex) by Sinosich et al. (1990). The 13.4% of glycan present on PAPP-A monomer being equivalent to 26.5kDa carbohydrate and the PPAPP-APP-PAPP-A polypeptide chain,172 kDa as calculated from the cDNA sequence, Kristensen et al. 1994)]

1.4.2.2 E le c tro p h o re tic M obility

PAPP-A has a2 electrophoretic mobility (Lin et ah 1974a. Bischof, 1979b) but its isoelectric point (pi) seems to depend on the body compartment from which it was isolated (Table 1.4).

T able 1.4 The pi of PAPP-A isolated by various investigators

S o u rce of PA PP-A m e a su re d p i

S e r u m ... 4.41 4.52

EDTA p la s m a ... 4.352

Citra te d p la s m a ... 4.3 - 4.53

P la c e n ta l e x tr a c t ... 4.752

Note: Investigators were, halbert and Lin (1979), 2Sinosich (1988a), 3Davey and Teisner (1982) and 4Sinosich et al,(1983)l

1.4.2.3 O th e r C h a ra c te ristic s

It has also been shown th at PAPP-A is a metalloprotein containing zinc

(Sinosich etal,1983) and th at it interacts with heparin (Sinosich et al,1981).

Detection of PAPP-A by immunological means is not affected by repeated

freeze/thaw cycles or exposure to a pH between 4 - 10 at 4°C for 2hrs, but it

is destroyed at pH 2 and 12 (Lin etal,1974b). P art of the PAPP-A molecule

is cleaved during incubations with chondroitinase suggesting th at it is a

proteoglycan (Sinosich, 1990).

1.4.3 P A P P -A L o c a lisa tio n an d C on trol o f Its S y n th e sis

Work published after the completion of the practical aspects of this

thesis by Silahtaroglu et al. (1993) has shown by using a cDNA probe (pPA-

1, Kristensen et al. 1994) and fluorescence in-situ hybridisation (FISH) th at

1.4.3.1 T he T ro p h o b la st

Lin and H albert (1976) demonstrated by immuno-fluoresence th at PAPP-

-A is present in the placenta. W ahlstrom et al. (1981) confirmed this, but found th at it was almost entirely restricted to the apical border of the syncytiotrophoblast. McIntyre et al. (1981) also showed it to be localised in the syncytiotrophoblast but found th at not all tissue sections stained

strongly for PAPP-A. However just because it is present in the

syncytiotrophoblast does not necessarily mean th at this is its site of synthesis, e.g. Isaka and Bischof (1986) demonstrated th at PAPP-A binds to placental subfractions of the trophoblast with a similar binding affinity to

th at of insulin for its GnRH receptor.

Barnea et al. (1986b) examined long term cultures of prim ary cells from the trophoblast to see if they were capable of producing PAPP-A. They demonstrated th at PAPP-A was produced and this process was inhibited by incubating these cells with inhibitors of protein synthesis. Bersinger et al. (1988) also showed th at PAPP-A was produced by a perfused placenta and th at its synthesis was energy dependant.

Recently the identification of PAPP-A cDNA (Kristensen et al. 1994) has allowed the production of PAPP-A RNA probes th at have confirmed th at the site of production of PAPP-A mRNA are the placental X-cells and syncytio­ trophoblast (Bonno et al. 1994b).

Bischof et_al,(1986a) also demonstrated th at trophoblastic production of PAPP-A could be inhibited by RU486 (a progestogen) and th at the inhibition could be overcome by addition of progesterone, with the suggestion th a t

PAPP-A is a progesterone dependant protein. Sorensen et al. (1995) found

th at differing rates of production by the placenta were seen between the

proteins: PAPP-A, SP1, hPL and hCG with the suggestion th a t a more

complicated control mechanism exists for synthesis of PAPP-A and hCG. Work on anim al models by Pepe et_al,(1994) dem onstrated using a Baboon anim al model th at by performing a foetectomy (i.e. removal of the foetus, not the placenta) in mid-gestation resulted in normal delivery of the placenta at term. They also observed continued secretion of PAPP-A but found th at m aternal PAPP-A serum levels did not continue to rise when compared to a

normal control group of baboon pregnancies. This suggests th at PAPP-A is regulated by factors th at regulate placental growth, contradictory findings to th at seen by Bischof et al, (1986a) were observed in th at progesterone was not seen to affect the m aternal serum levels of PAPP-A. The difference in control mechanisms will have to await further molecular studies th at can now be undertaken as the cDNA for PAPP-A has been produced.

1.4.4 E x tra -T ro p h o b la stic site s o f p r o d u c tio n for P A P P -A

1.4.4.1 T he E n d o m e triu m

Duberg et al. (1982) have found th at the endometrium contains 3-4 times more PAPP-A than the trophoblast, with the levels of PAPP-A tending to follow histological staging, they were lowest during the inactive phase and highest in the secretory phase. PAPP-A levels in the endometrium also correlated with the level of steroids during the phases. Duenas et al. (1988) have localised PAPP-A in endometrial (glandular and stromal) tissues during its proliferative and secretory phases. Bischof and Tseng (1986) have also shown th at like the prim ary trophoblast cells, a prim ary cell culture of hum an endometrial cells is capable of producing PAPP-A and th at it is also progesterone dependent. This endometrial link for an extra-trophoblastic site of production is also strengthened by the finding of Ikarashi and Takeuchi (cited from 1987) th at by analysis of PAPP-A and Tissue polypeptide antigen levels it is possible to screen for endometrial cancer.

However in light of the findings by Tornehave et al (1987) it is likely th at

the PAPP-A seen in the endometrium is an artefact of the immunovisualisation technique th at was used. This has subsequently been confirmed at a molecular level by Bonno et al. (1994b).

1.4.4.2 O th e r Sites

head of the epididymis and the seminal vesicles. Duenas et al, (1988) have also found PAPP-A in the stroma of the ovary, cervix and ductal epithelium of the breast. It should however be pointed out th at PAPP-A localised by the immunoperoxidase technique is very dependant upon the specificity of the prim ary antibody and the technique used for immunovisualisation (Tornehave et al. 1986).

Tornehave et ah (1987) have already demonstrated th a t the PAPP-A

antisera available are not monospecific. Kuhajda et ah (1989) for example

identified a cDNA clone for a haptoglobulin related protein using the commercially available antisera. It has also been shown th at PAPP-A

specific staining of tissues from Stage I breast cancer could be abolished by

pre-incubating the prim ary antibody with haptoglobulin. It is therefore clear th at care is needed in interpreting those studies th at have localised PAPP- A using an immunovisualisation technique.

Recently Chemnitz et al, (1986) have also shown th at the antisera developed by Bischof and used in many localisation studies is not monospecific for PAPP-A but contains specificities for at least 6 other antigens. In their study they compared four different polyclonal antisera for PAPP-A and showed th at if Bischof s antisera was adsorbed with hum an serum and foetal/connective tissue then they could abolish the detection of PAPP-A on the decidualised endometrium. They found th at PAPP-A was only found in the cytoplasm of the syncytiotrophoblast, suggesting th at PAPP-A is localised in the trophoblast and th at other immuno-localisations in other tissues may be due to using a polyspecific antisera. W estergaard et al, (1988) have also confirmed these findings, but have also noted th at by setting up a competitive system between PAPP-A added and the tissue for the antisera it is possible to demonstrate th at PAPP-A is associated with the apical rim of the syncytiotrophoblast, confirmation at a molecular level using anti-sense probes confirmed th at PAPP-A mRNA was produced by syncytio-trophoblast cells (Bonno et al, 1994b). PAPP-A mRNA was however

demonstrated in the testes and follicular cells (Kristensen et al, 1994). The

use of anim al models (Pepe et al. 1994) also allows an in - vivo model to be

used to look at the affect of hormones on regulating the placental production of PAPP-A.

1.4.5 Assay Systems For The Measurement of PAPP-A

The immunoassays which have been used by various investigators are listed in Table 1.5. Investigators term pregnancy pools are compared to an

International World Health Organisation term serum reference pool

(W.H.O. 78/610) which consists of a pool of late pregnancy serum allowed the comparison of PAPP-A levels th at have been found by different investigators and revealed very disparate levels for PAPP-A with over a 400% difference seen in the concentrations determined ((3) and (4a), Table 1.5).

Various antisera have been raised against PAPP-A (Table 1.6) but as observed by Chemnitz et al (1986) not all of the antisera produced had epitopes directed solely against PAPP-A. This variability in the quality of one of the prim ary reagents will ultim ately have an affect on assays for PAPP-A th at utilise an immunochemical m easurem ent system e.g. the immunoassays and the subsequent levels found for PAPP-A using assays will also be affected by the quality of the prim ary reagents th at have been used.

A variety of non-specific effects on the described assay systems for

PAPP-A (Table 1.5) have been observed, see Table 1.7. The ELISA described

suffered from non-specific m atrix effects with markedly increased (by 50%) concentrations of PAPP-A found in haemolysed plasm a when compared to matched non-haemolysed controls, suggesting th at a red cell component can

affect the levels of PAPP-A found when using this ELISA system.

T able 1.5 PAPP-A immunoassays used by other investigators.

M ethod aS e n sitiv itv bT erm c f Ref.

C rossed Im m u n o electro p h o resis* Mid-2nd trim ester 156

R o ck et Im m u n o electro p h o resis^ 2nd trim ester 123

R ad io ro c k et lin e e lectro p h o resis^ 30 pg/1. 65

R ad io im m u n o assay

a) - (Bischof et al, 1981b) 33 pg/1. 258

h) - (Sinosich et al, 1982b) 3 pg/1.

c) - (Anthonv et al,1983) _{20 pg/1.}

d) - (Pinto-Furtado et al,1984) _{5 pg/1.} 110

R ad io im m u n o m etric4 5 pg/1. 110

E nzym eim m unoassayS 2-3rd trim ester 90

[Legend: a The sensitivity quoted were the own investigators measure of the minimum levels that the listed assay was capable of measuring, if no figure was quoted then it was given as an estimate of the minimum level able to be measured during pregnancy. b Term cf Ref: This referred to the investigators own late third trimester serum pool compared to the W.H.O. standard.]

(^Lin et al. 1976b. ^Bischof et al. 1979c. ^Folkersen et al. 1981.4Mowles et al. 1986. ^Pledger and Bellfield, 1983.)

T able 1.6 Antisera to PAPP-A produced by various investigators

Investigator

(designation) aTvoe raised in:Antisera Immunadsorbedwith: bSoecificitv

Lin et a l 1

(Miami) P Rabbits HNPPP

Bischof et al 2

(Aberdeen) P Rabbits HNPPPO c at least 6. others

Sutcliffe et al 3

(Glasgow) P Sheep HMS

Folkersen et

al4 (Odense) P Goats HNPPP+FCS dSpecific

DAKO5

(Commercial) P Rabbits HNPPP specificitiese>f Other

Sinosich et al6

(Sydney) P Rabbits MHS d Specific

Mowles et al7

(London) M Mice -

-[LEGEND: aType: (P) Polyclonal. (M) Monoclonal. bSpecificity: Cross - reactivity, also see text. c :PZP, (X2-M, P-lipoprotein, AT-III, SP1 plus 1 undefined specificity. d:Sinosich

(1988a). e : Kuhajda et al (1989) antibody shares epitopes with haptoglobulin related protein. f : also binds SPl (Sinosich, 1988c). HNPPP: Human Non-Pregnant Plasma Proteins. HNPPPO: Human Non-Pregnant Plasma Proteins from women on Oestrogen therapy. MHS: Male Human Serum. FCS: Foetal Connective Tissue.]

OLin et al. (1974a), 2Bischof et al. (1979c), 3Sutcliffe et al. (1980), Folkersen et al. (1979), 5DAKOPATTS (Appendix 1), 6 Sinosich et al. (1987) and 7Mowles et al. (1986)).

Two RIA procedures (Table 1.5) gave markedly different results depending on the type of anti-coagulant th at was used to collect the blood samples. The difference in PAPP-A levels th at were found when different anti-coagulants were used was most marked in this type of assay when

et al, (1982b) noted a 100% increase in levels). The RIA described by Pinto- Furtado et al. (1984) did find a statistically significant difference in the

levels found in serum compared to EDTA plasm a but this RIA procedure

had a far less pronounced difference (20% increase) in the levels th at were seen. The exact nature and cause of these observed differences is unknown.

T able 1.7 The affect of anti-coagulants on the m easured levels of PAPP-A

Type of Assay Type of Anticoagulant Used Effect of

Haemolysis

dACD Citrate Oxalate Heparin EDTA

-RIE1 - nc - aInc. -

-RIE2 Dec. - - Inc. Inc.

-RIA3 Dec. nc nc nc Inc.

-RIA4 - - - aInc. bInc.

-ELISA5 - - - - - Clnc.

[Legend: Levels compared against its serum value in that assay. (-): Not determined, (nc): No significant change in levels observed. (Inc.): Increase in levels observed in relation to that seen in serum. (Dec.): Decrease in levels observed in relation to that seen in serum. a(Action reversed by protamine sulphate). b(Increase neutralised if chelation affect abolished). c(Higher values observed in haemolysed samples from males). d (Acid Citrate Dextrose). ] OWestergaard et al (1983a), 2Toop and Klopper (1983),3Pinto-Furtado et al (1984), 4Sinosich et al (1985) and 5Pledger and Bellfield (1983)).

It has been demonstrated th at it is the nature of the PAPP-A molecule used for the RIA standard and tracer in these assays th at was responsible for these observed differences and not due to the antisera th at was used (Bischof and Meisser, 1988); these investigators re-evaluated their RIA in term s of tracer, PAPP-A standard and antibody used and concluded th at the

PAPP-A isolated from late pregnancy EDTA plasm a had a component th at

was recognised by an antiserum against hum an serum proteins. They also noted th at irrespective of the source of antiserum used (Table 1.6) th at coagulation also changed the level of PAPP-A detected, therefore some form of immunological heterogeneity has been observed for PAPP-A. It has been recently shown (Oxvig et al, 1993) th at PAPP-A is linked by disulphide bonds to pro-MBP. It is unknown however w hether all the PAPP-A found in

the m aternal serum of pregnant women is complexed with pro-MBP or w hat

effect this complex has on the different assay systems. Sinosich et al, (1991)

have also dem onstrated th at PAPP-A levels when m easured by crossed

immuno-electrophoresis are reduced when Hum an Granulocyte Elastase

(HGE) was included in an interm ediate gel, thus suggesting an effect on the

m easured level of PAPP-A when PAPP-A was interacting with HGE. It is

not known whether a similar effect is seen for the suggested PAPP- A/proMBP complex.

PAPP-A is a metallo-protein (Sinosich et al, 1983) and was

dem onstrated to bind Zinc. This has been confirmed at a structural level as the PAPP-A cDNA (Kristensen et al. 1994) contains a Zinc binding region.

Therefore it is possible th at the effect observed by Sinosich et al. (1985) th at

changes in the PAPP-A levels could be removed, if the chelating affect of the EDTA was neutralised immediately by the addition of an excess of m etal ions. It is interesting to speculate th at as this reversible change in PAPP-A levels may be due to structural changes in the conformation of PAPP-A th at

are effected by the chelation of Zinc with EDTA which is reversed when this

affect was abolished. Similar changes in the PAPP-A levels seen when

heparin was present were reversed if the heparin antagonist, protamine sulphate was used. This indicates th at the tertiary structure of PAPP-A affects the PAPP-A levels measured in the blood using the described immunoassays

1.4.6 PAPP-A Levels Found In The Maternal Blood During

Pregnancy

The affects of the PAPP-A immunoassays described in this Chapter may explain the different biochemical profiles th at were obtained for PAPP-A when different investigators have followed the levels of PAPP-A found in the m aternal blood during pregnancy (Figures 1.3 and 1.4).

Bischof and Meisser (1988) have also observed th at depending upon