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

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

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PAPP-A is a large glycoprotein with alpha2-electrophoretic mobility that 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 Western 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 that 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 that the interaction with this antibody requires carbohydrate components to be present on the PAPP-A molecule. It is known that 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 primary amino acid sequence of this protein that 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 To generate this information, PAPP-A was purified using its previously unpublished affinity for L-arginine in combination with the already described procedures of ammonium sulphate precipitation, ion exchange and gel filtration.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 terminal 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 that produced 500 and >1200 bp fragments using the cDNA library as DNA template; thus demonstrating that PAPP-A is synthesised in the placenta.PAPP-A was shown to have O and N-linked carbohydrate chains. Enzymatic deglycosylation demonstrated that 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 that 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 that they form a 1:1 (PAPP-A: endoproteinase) sub-unit complex that was stable in SDS. A further investigation revealed that PAPP-A interacted with the endoproteinase Arg-C and this resulted in a 30% inhibition of the esterolytic activity of this enzyme.

Item Type: Thesis (Doctoral)
Additional Information: Thesis (Ph.D.)--Sheffield Hallam University (United Kingdom), 1996.
Research Institute, Centre or Group - Does NOT include content added after October 2018: Sheffield Hallam Doctoral Theses
Depositing User: EPrints Services
Date Deposited: 10 Apr 2018 17:19
Last Modified: 26 Apr 2021 11:45

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