Identification, cloning and functional characterisation of lectinsfrom the albumen gland of the Roman snail Helix pomatia
The lectin from the albumen gland of the Roman snail Helix pomatia has been widely used in studies relating glycosylation changes to the metastatic potential of cancer cells. Detailed information related to the sequence, structure and binding site of HPA lectin would assist in identifying the HPA bi...
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University of Westminster
2008
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616.99 Markiv, Anatoliy Identification, cloning and functional characterisation of lectinsfrom the albumen gland of the Roman snail Helix pomatia |
description |
The lectin from the albumen gland of the Roman snail Helix pomatia has been widely used in studies relating glycosylation changes to the metastatic potential of cancer cells. Detailed information related to the sequence, structure and binding site of HPA lectin would assist in identifying the HPA binding partners on cancer cells. Availability of a recombinant form of HPA has the potential to be developed as a diagnostic tool for cancer prognostication. The work embodied in this thesis includes characterisation of the lectin from the albumen gland of Helix pomatia with the goal of obtaining an insight into the identity of the carbohydrate-binding partners on the membrane of cancer cells and producing a recombinant form of this lectin for diagnosis of metastatic cancer. For this purpose the study first identified the amino acid sequence of the HPA lectin, this was cloned into Escherichia coli with a view to obtaining a crystal structure of the agglutinin and its carbohydrate-binding site, to functionally analyse the carbohydratebinding properties of the recombinant lectin and allow comparison with the native material. A comparative analysis of in-house purified HPA and commercially sourced material provided evidence of heterogeneity in the agglutinin preparation. MS/MS analysis of the protein separated by isoelectrofocussing showed that more than one polypeptide sequence was present in the protein sample known as HPA. The cloning and identification of HPA lectin showed that three separate protein sequences were present in the lectin purified from the albumen gland of the Roman snail Helix pomatia. Two of the polypeptides are consistent with GalNAc binding lectins of the H-type family and were termed HPAI and HPAII. The third lectin (HPAIIJ) showed sequence similarity to a sialic acid binding lectin from another snail Cepaea hortensis and structural analysis of the novel protein suggests it will form a C1 q/TNF ' . like fold. The genes for all three polypeptides were cloned and recombinant forms of the proteins were prepared. Different expression plasmid constructs were evaluated. The results provide the first demonstration of successful cloning and expression of all three recombinant forms of the HPA isolectins using an E. coli expression system. A combination of crystal structure analysis and molecular modelling of the GalNAc/GlcNAc binding isolectins (HPAI and HPAII) from Helix pomatia has led to the identification of amino acids participating in direct hydrogen bonding with the carbohydrates and also amino acids involved in lectin trimer formation via noncovalent bonding. Functional analysis of HPAI and HPAII using surface plasmon resonance provided information on the affinity of binding of native and recombinant material to GalNAc/GlcNAc carbohydrates attached to bovine serum albumin. The interactions were observed to be of the same order of magnitude for both native and recombinant material. Glycan chip microarray studies showed that recombinant HPAI and HPAII recognised similar binding partners to native HPA and agglutination studies showed that the recombinant forms of the HPAI and HPAII isolectins were functional in clumping blood group A red blood cells. The recombinant forms of the lectin produced in this study are valuable tools for Glycobiology studies to analyse changes in the posttranslational glycosylation in normal and cancer cells as detected using HPA. The recombinant lectins may also be used in microbiology, serology and other glycome analyses. Structural information on the carbohydrate-binding site of the HPA lectins may later be used to identify their binding partners on the membrane of malignant cells and this might in turn lead to a better understanding of changes in glycosylation that governs the metastatic potential of cancer cells. |
author |
Markiv, Anatoliy |
author_facet |
Markiv, Anatoliy |
author_sort |
Markiv, Anatoliy |
title |
Identification, cloning and functional characterisation of lectinsfrom the albumen gland of the Roman snail Helix pomatia |
title_short |
Identification, cloning and functional characterisation of lectinsfrom the albumen gland of the Roman snail Helix pomatia |
title_full |
Identification, cloning and functional characterisation of lectinsfrom the albumen gland of the Roman snail Helix pomatia |
title_fullStr |
Identification, cloning and functional characterisation of lectinsfrom the albumen gland of the Roman snail Helix pomatia |
title_full_unstemmed |
Identification, cloning and functional characterisation of lectinsfrom the albumen gland of the Roman snail Helix pomatia |
title_sort |
identification, cloning and functional characterisation of lectinsfrom the albumen gland of the roman snail helix pomatia |
publisher |
University of Westminster |
publishDate |
2008 |
url |
http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.502237 |
work_keys_str_mv |
AT markivanatoliy identificationcloningandfunctionalcharacterisationoflectinsfromthealbumenglandoftheromansnailhelixpomatia |
_version_ |
1716818306090926080 |
spelling |
ndltd-bl.uk-oai-ethos.bl.uk-5022372015-09-03T03:30:16ZIdentification, cloning and functional characterisation of lectinsfrom the albumen gland of the Roman snail Helix pomatiaMarkiv, Anatoliy2008The lectin from the albumen gland of the Roman snail Helix pomatia has been widely used in studies relating glycosylation changes to the metastatic potential of cancer cells. Detailed information related to the sequence, structure and binding site of HPA lectin would assist in identifying the HPA binding partners on cancer cells. Availability of a recombinant form of HPA has the potential to be developed as a diagnostic tool for cancer prognostication. The work embodied in this thesis includes characterisation of the lectin from the albumen gland of Helix pomatia with the goal of obtaining an insight into the identity of the carbohydrate-binding partners on the membrane of cancer cells and producing a recombinant form of this lectin for diagnosis of metastatic cancer. For this purpose the study first identified the amino acid sequence of the HPA lectin, this was cloned into Escherichia coli with a view to obtaining a crystal structure of the agglutinin and its carbohydrate-binding site, to functionally analyse the carbohydratebinding properties of the recombinant lectin and allow comparison with the native material. A comparative analysis of in-house purified HPA and commercially sourced material provided evidence of heterogeneity in the agglutinin preparation. MS/MS analysis of the protein separated by isoelectrofocussing showed that more than one polypeptide sequence was present in the protein sample known as HPA. The cloning and identification of HPA lectin showed that three separate protein sequences were present in the lectin purified from the albumen gland of the Roman snail Helix pomatia. Two of the polypeptides are consistent with GalNAc binding lectins of the H-type family and were termed HPAI and HPAII. The third lectin (HPAIIJ) showed sequence similarity to a sialic acid binding lectin from another snail Cepaea hortensis and structural analysis of the novel protein suggests it will form a C1 q/TNF ' . like fold. The genes for all three polypeptides were cloned and recombinant forms of the proteins were prepared. Different expression plasmid constructs were evaluated. The results provide the first demonstration of successful cloning and expression of all three recombinant forms of the HPA isolectins using an E. coli expression system. A combination of crystal structure analysis and molecular modelling of the GalNAc/GlcNAc binding isolectins (HPAI and HPAII) from Helix pomatia has led to the identification of amino acids participating in direct hydrogen bonding with the carbohydrates and also amino acids involved in lectin trimer formation via noncovalent bonding. Functional analysis of HPAI and HPAII using surface plasmon resonance provided information on the affinity of binding of native and recombinant material to GalNAc/GlcNAc carbohydrates attached to bovine serum albumin. The interactions were observed to be of the same order of magnitude for both native and recombinant material. Glycan chip microarray studies showed that recombinant HPAI and HPAII recognised similar binding partners to native HPA and agglutination studies showed that the recombinant forms of the HPAI and HPAII isolectins were functional in clumping blood group A red blood cells. The recombinant forms of the lectin produced in this study are valuable tools for Glycobiology studies to analyse changes in the posttranslational glycosylation in normal and cancer cells as detected using HPA. The recombinant lectins may also be used in microbiology, serology and other glycome analyses. Structural information on the carbohydrate-binding site of the HPA lectins may later be used to identify their binding partners on the membrane of malignant cells and this might in turn lead to a better understanding of changes in glycosylation that governs the metastatic potential of cancer cells.616.99University of Westminsterhttp://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.502237Electronic Thesis or Dissertation |