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Lipid-restricted recognition of mycobacterial lipoglycans by human pulmonary surfactant protein A: a surface-plasmon-resonance study.

ABSTRACT: The human pulmonary surfactant protein A (hSP-A), a member of the mammalian collectin family, is thought to play a key defensive role against airborne invading pulmonary pathogens, among which is Mycobacterium tuberculosis, the aetiologic agent of tuberculosis. hSP-A has been shown to promote the uptake and the phagocytosis of pathogenic bacilli through the recognition and the binding of carbohydrate motifs on the invading pathogen surface. Recently we identified lipomannan and mannosylated lipoarabinomannan (ManLAM), two major mycobacterial cell-wall lipoglycans, as potential ligands for binding of hSP-A. We demonstrated that both the terminal mannose residues and the fatty acids are critical for binding, whereas the inner arabinosyl and mannosyl domains do not participate. In the present study we developed a surface-plasmon-resonance assay to analyse the molecular basis for the recognition of ManLAM by hSP-A and to try to define further the role of the lipidic aglycone moiety. Binding of ManLAM to immobilized hSP-A was consistent with the simplest one-to-one interaction model involving a single class of carbohydrate-binding site. This observation strongly suggests that the lipid moiety of ManLAM does not directly interact with hSP-A, but is rather responsible for the macromolecular organization of the lipoglycan, which may be necessary for efficient recognition of the terminal mannosyl epitopes. The indirect, structural role of the lipoglycan lipidic component is further supported by the complete lack of interaction with hSP-A in the presence of a low concentration of mild detergent.

SUBMITTER: Sidobre S

PROVIDER: S-EPMC1222659 | biostudies-other | 2002 Jul

REPOSITORIES: biostudies-other

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Project description:Ras GTPases play a crucial role in signal transduction cascades involved in cell differentiation and proliferation, and membrane binding is essential for their proper function. To determine the influence of the nature of the lipid anchor motif and the difference between the active (GTP) and inactive (GDP) forms of N-Ras on partitioning and localization in the lipid membrane, five different N-Ras constructs with different lipid anchors and nucleotide loading (Far/Far (GDP), HD/Far (GDP), HD/HD (GDP), Far (GDP), and HD/Far (GppNHp)) were synthesized. Using the surface plasmon resonance technique, we were able to follow the insertion and dissociation process of the lipidated proteins into and out of model membranes consisting of pure liquid-ordered (l(o)) or liquid-disordered (l(d)) phase and a heterogeneous two-phase mixture, i.e., a raft mixture with l(o) + l(d) phase coexistence. In addition, we examined the influence of negatively charged headgroups and stored curvature elastic stress on the binding properties of the lipidated N-Ras proteins. In most cases, significant differences were found for the various anchor motifs. In general, N-Ras proteins insert preferentially into a fluidlike, rather than a rigid, ordered lipid bilayer environment. Electrostatic interactions with lipid headgroups or stored curvature elastic stress of the membrane seem to have no drastic effect on the binding and dissociation processes of the lipidated proteins. The monofarnesylated N-Ras exhibits generally the highest association rate and fastest dissociation process in fluidlike membranes. Double lipidation, especially including farnesylation, of the protein leads to drastically reduced initial binding rates but strong final association. The change in the nucleotide loading of the natural N-Ras HD/Far induces a slightly different binding and dissociation kinetics, as well as stability of association, and seems to influence the tendency to segregate laterally in the membrane plane. The GDP-bound inactive form of N-Ras with an HD/Far anchor shows stronger membrane association, which might be due to a more pronounced tendency to self-assemble in the membrane matrix than is seen with the active GTP-bound form.

Project description:Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.

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Lipid-restricted recognition of mycobacterial lipoglycans by human pulmonary surfactant protein A: a surface-plasmon-resonance study.

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