{"id":{"repo_id":"sheffield-hallam","oai_identifier":"oai:shura.shu.ac.uk:19631"},"canonical_url":"https://search.dev.ndltd.org/etd/sheffield-hallam/oai:shura.shu.ac.uk:19631","repository":{"repo_id":"sheffield-hallam","name":"Sheffield Hallam University","base_url":"https://shura.shu.ac.uk/cgi/oai2"},"display":{"title":"The molecular characterisation of pregnancy-associated plasma protein-A (PAPP-A).","abstract":"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 To generate this information, PAPP-A was purified using its previously unpublished affinity for L-arginine in combination with the already described procedures of ammonium sulphate precipitation, ion exchange and gel filtration.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.","abstract_html":"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.","abstract_has_math":false,"creators":["Evans, Steven."],"institution":"Sheffield Hallam University (United Kingdom).","degree_name":"phd","degree_level":"doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":1996,"date_issued":"1996","date_published":"1996","updated_at":"2026-07-24T06:31:21Z","subjects":[],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":null,"outbound_label":null,"outbound_source":null},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Evans, Steven."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["1996"]},{"key":"dc:date.issued","label":"Date","values":["1996"]},{"key":"dc:publisher.commercial","label":"Dc Publisher Commercial","values":["Sheffield Hallam University,"]},{"key":"dc:publisher.department","label":"Dc Publisher Department","values":["Department not specified."]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["Sheffield Hallam University (United Kingdom)."]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://shura.shu.ac.uk/19631/"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["phd"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://shura.shu.ac.uk/19631/1/10694512.pdf"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["The molecular characterisation of pregnancy-associated plasma protein-A (PAPP-A)."]}]}],"canonical_facts":{"dc:creator":["Evans, Steven."],"dc:date":["1996"],"dc:date.issued":["1996"],"dc:description.abstract":["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."],"dc:format":["application/pdf"],"dc:identifier.uri":["https://shura.shu.ac.uk/19631/1/10694512.pdf"],"dc:language":["en"],"dc:publisher.commercial":["Sheffield Hallam University,"],"dc:publisher.department":["Department not specified."],"dc:publisher.institution":["Sheffield Hallam University (United Kingdom)."],"dc:relation.isreferencedby":["https://shura.shu.ac.uk/19631/"],"dc:title":["The molecular characterisation of pregnancy-associated plasma protein-A (PAPP-A)."],"dc:type":["Thesis"],"dc:type.qualificationlevel":["doctoral"],"dc:type.qualificationname":["phd"]},"updated_at":"2026-07-24T06:31:21Z"}