Immune function and structural analysis of recombinant bovine conglutinin and human lung surfactant protein-DReport as inadecuate

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Reference: Prasad, Alpana., (2000). Immune function and structural analysis of recombinant bovine conglutinin and human lung surfactant protein-D. DPhil. University of Oxford.Citable link to this page:


Immune function and structural analysis of recombinant bovine conglutinin and human lung surfactant protein-D

Abstract: Recognition of sugar moieties on the surface of microorganisms is one ofthe ways the body distinguishes potential pathogens from self-cells. The sugarbindingproteins, lectins, mediate this recognition role of the first line of defenceagainst infections, preceding the antibody-mediated (adaptive) immuneresponse. Collectins are calcium-dependent carbohydrate-binding proteins thathave been implicated in innate immunity. Bovine conglutinin (BC) and lungsurfactant protein-D (SP-D), belong to the family of 'collectins' which arecharacterised by four domains: an N-terminal cysteine-rich region, a collagenlikeregion linked with the carbohydrate recognition domain (CRD) via an ahelicalneck region. BC and SP-D show remarkable similarity in their amino acidsequence (79% identity), function and biological characteristics. They have beenshown to mediate microbial clearance either by directly binding to bacterialeading to phagocytosis or interacting with complement system components.The present study aims to elucidate the biological function of theseproteins more precisely. Recombinant fragments (r) of BC and SP-D consistingof their CRDs and neck regions have been cloned in pET-21a and pMal-c2vectors respectively, for expression in Escherichia coli. Recombinant conglutininwas expressed in BL21(DE3)pLysS and isolated by a denaturation-renaturingprocedure. Binding of rBC(N/CRD) to mannan and complement component,iC3b, was assessed in real-time by BIAcore. The dissociation constants werecalculated by Scatchard analysis.The carbohydrate structures present on the surface of themicroorganisms play an important role in mediating the interactions with theimmune cells. The recombinant molecules showed calcium-dependent bindingto lipopolysaccharides (LPS) from gram-negative bacteria Pseudomonasaeruginosa, Klebsiella pnuemonia and Salmonella typhosa, which was inhibited inpresence of sugars. rBC(N/CRD) also bound to whole bacteria as assessed byELISA and retained its capacity to recognise various complement systemcomponents and the carbohydrate moieties on the surface of various pathogenicmicroorganisms. The recombinant protein retained its ability to bind varioussugar residues, although with lower affinity than that of the native molecule.rBC(N/CRD) is able to bind and aggregate bacteria and cause agglutination ofbacterial cell suspensions.A novel model has been used to describe the interactions of thecollectins at the molecular level based on specificity of carbohydrate-recognitionby the collectins. The pyocin mutant strain 1291 series of Neisseria gonorrhoeaehas sequential deletions of the terminal sugars in their lipooligosaccharides(LOS). Conglutinin showed a preferential high affinity binding to 1291a mutantthat expresses GlcNAc as the terminal hexose, in comparison to other mutants.This provides a unique system to understand the specific cell-surfaceinteractions in relevance to a particular lectin.Further elucidation of the function of CRD and neck region at astructural level is in progress, using X-ray crystallography. Since the submissionof the thesis, the structure of the monomeric CRD has been solved, whichrevealed a remarkable similarity to the SP-D and MBL structure. Trials areunderway to get the structure of the trimeric CRDs.These studies aim to provide a better understanding of the collectinpathogeninteraction at the biological and structural levels. The ultimate aim isto determine if the recombinant forms of these proteins can be usedtherapeutically to enhance the uptake and killing of pathogens.

Type of Award:DPhil Level of Award:Doctoral Awarding Institution: University of Oxford Notes:This thesis was digitised thanks to the generosity of Dr Leonard Polonsky


Prof. Ken ReidMore by this contributor


 Bibliographic Details

Issue Date: 2000Identifiers

Urn: uuid:f9a5ae66-4ed0-4bdf-90eb-c873ca44147d

Source identifier: 603838789 Item Description

Type: Thesis;

Language: eng Subjects: Proteins Lectins Natural immunity Tiny URL: td:603838789


Author: Prasad, Alpana. - institutionUniversity of Oxford facultyFaculty of Biological Sciences - - - - Contributors Prof. Ken Reid More



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