The T ertiary Structure Of PAPP-A

Information obtained during this study yielded data concerning the sub­ unit structure of PAPP-A. Assuming a molecular weight of 205 kDa for

myosin (Appendix 1), the reduced monomeric chain of PAPP-A has an

estim ated molecular weight of 195 +/- 5 kDa in the SDS-PAGE systems

th at were employed in this thesis. This monomer was also disulphide bridged to form a dimer as it was detected in non-reducing conditions by the same gel system (Plate 5.2, Chapter 5).

Assuming th at the monomer was 195 kDa, then a pure dimer not containing any pro-MBP should have a molecular weight of approximately

400 kDa. The proMBP has an estim ated size of 29 kDa (Popken-Harris et

al, 1994) and Oxvig et ah (1993) has suggested an equimolar 2:2 complex of

PAPP-A/proMBP therefore the proposed dimer should have a theoretical weight of at least 440 kDa. Therefore by looking at the PAPP-A separated by gel filtration, an assessm ent of the size and structure of this dimer can be

made.The m aterial th at was eluted at 0.3 and 0 .6 M NaCl from the heparin

affinity m atrix yielded PAPP-A th at eluted in different fractions from the gel filtration column. The 0.3 M heparin eluted fraction had a molecular

weight of approximately 480 kDa whereas the 0 .6 M heparin eluted fraction

had a molecular weight of approximately 400 kDa as determined by gel filtration chromatography (Figure 7.4). This suggests th at the heparin affinity column is possibly separating different forms of PAPP-A. This is interesting in the light of the described proMBP/PAPP-A complex. A similar observation th at PAPP-A purified from different sources is eluted from the heparin affinity m atrix in different ways was made by others (Davey et ah (1983) and Davey and Teisner (1982). PAPP-A isolated from the placenta

was eluted from the heparin affinity m atrix at 0 .6 M NaCl (it was also

shown to have a very different pi to PAPP-A isolated from m aternal serum,

Chapter 1) but did not elute at 0.3 or 0.4 M NaCl. W hereas PAPP-A from

m aternal serum could be eluted from this m atrix at 0.3, 0.4 and 0 .6 M NaCl.

Re-chromatography of the fraction eluted at 0.4 M resulted in a proportion

of the protein which was eluted at 0.6M NaCl, thus suggesting th at this

affinity m atrix allows separation of PAPP-A into fractions w ith different affinities for heparin. This could be due to molecular heterogeneity of PAPP-

A. The differences in the ability of PAPP-A to inhibit HGE which have been

observed between the m aterial purified by Oxvig et al. (1994) and Sinosich

(1990) could also be due to a difference in the tertiary structure of the PAPP-A prepared by these two groups.

E lu a n t (mis) F ig u re 7.4

Elution profile of 0.3 and 0.6 M (Figure overlay) heparin eluted fractions on

a SuperDex-200 gel filtration column (P late in se t o v erlay 7.4: A 5% SDS- PAGE gel of the fraction containing the peak PAPP-A of m aterial eluted at 0.6 M from the heparin affinity matrix).

[Legend: PAPP-A was determined by SDS-PAGE as previously described, the indicated fractions were separated on a 5% SDS-PAGE gel, Plates 5.1 and 5.2). Note: SDS-PAGE of 0.6 M eluted heparin fraction and material separated on the gel filtration column (1): Fractions 4/5. The arrow represented the 205 kDa size marker and the PAPP-A marked was detected by immunodetection with anti- PAPP-A antibodies as previously described.]

The proposed PAPP-A/proMBP complex raises interesting questions about the observed relationship between these two proteins as the affinity of PAPP-A for L-arginine th at is described in this thesis (Figure 2.16,

Chapter 2) demonstrates th at PAPP-A will bind to L-Arginine. proMBP is

rich in arginine (Gleich et al. 1979) and therefore may associate with PAPP- A. Once in close association proMBP has been shown to have a tendency to

form SDS-stable oligomers. Recently Oxvig et al. (1995) have also

demonstrated th at proMBP also forms complexes with other plasma constituents. An understanding as to the specificity and affect of these interactions on the individual components will have to aw ait further studies to characterise this interaction.

A unique feature of the placenta is th at it continues to undergo differentiation throughout the gestation period (Boime et ah 1986). This necessitates the regulated expression of specific genes at different stages, e.g. only 3 of 7 P-hCG genes are expressed in the placenta. The presence of these proteins in extraplacental sites suggest th at they may be involved in other physiological processes such as cell growth. The multigene nature of proteins produced by the placenta has been dem onstrated for other

placental proteins, e.g. S P l (Chan and Qui, 1988). Bonno et al, (1994a,

1994b) demonstrated by in-situ hybridisation th at proMBP and PAPP-A

were synthesised by X-cells of the placental septa. In addition PAPP-A was also synthesised in syncytiotrophoblast cells of the placenta. The PAPP-A

mRNA is present as two distinct species of 8 and 12 kbp (Kristensen et al.

1994). A though a single N-terminus has been found by microsequencing of purified PAPP-A this does not exclude the possibility th at N-terminally blocked chain(s) were also present in this m aterial. Analysis of the proteolytic cleavage products demonstrated the presence of amino acid stretches th at were not present within the published cDNA sequence for PAPP-A (Kristensen et al. 1994). Thus the tertiary structure and forms of

PAPP-A present still rem ain to be resolved. Suggestions from the work in

this thesis are th at different PAPP-A complexes may exist in the m aternal serum but this requires further investigation.

CHAPTER