Jarmila Janatova

Interaction of several complement proteins with gp120 and gp41, the two envelope glycoproteins of HIV-1.

ObjectiveTo study the binding of human complement proteins to gp41 and gp120 of HIV-1. MethodsThe interaction of complement proteins with gp41 and gp120 and their effect on the gp41-gp120 complex in enzyme-linked immunosorbent assays (ELISA) and on stably transfected Schneider-2 cells expressing a gp41-gp120 complex was investigated. The molecular basis of these interactions was analysed by computer-supported sequence analysis. Resultgp41 strongly binds human complement regulatory proteins factors H and properdin, and weakly binds factors I and B. The binding occurs with recombinant soluble (rs) gp41 fixed on ELISA plates as well as gp41-gp120 complex expressed on Schneider-2 cells. The basis for this binding potential might be an amino-acid (aa) sequence of gp41 displaying homologies to sites in human C3. rgp120 also binds C3(H20), a C3b-like form of C3, and C4b. These binding features of gp120 can be explained by homology of constant region (CR) 4 in gp120 to sites in C4b binding protein. Additionally, CR1 in gp120 exhibits a weak similarity to human properdin. Preincubation of rsgp41 with either factor H or properdin, and of rgp120 with C3b or C4b affected the interaction between rsgp41 and rgp120. Incubation of Schneider-2 cells, expressing a functional gp41-gp120 complex, with factor H reduced the detectable amount of gp120. This effect was similar to that induced by soluble CD4. ConclusionThese results strongly suggest that HIV-1 envelope proteins interact with human complement proteins. Additionally, C3b-like features of gp41 and the C3b/C4b binding structures in gp120 may affect the non-covalent association between gp41 and gp120.

Human Complement Proteins C3b, C4b, Factor H and Properdin React with Specific Sites in gpl20 and gp4l, the Envelope Proteins of HIV1

Recently we reported the basic phenomenon of an interaction between the envelope glycoproteins of HIV-1 gp120 and gp41 and components of the human complement system, i.e. activated C4 (C4b) and activated C3 (C3b) and the complement regulator proteins factor H and properdin. In this study we analyze these interactions in detail. Using 46 overlapping peptides of gp120 attached to microtiter plates, binding of activated human C3 to 6 regions in gp120 was found (aa 100-129, 161-190, 231-250, 301-328, 410-449, 470-499). In competition assays with soluble peptides, representatives of four of these regions were capable to partially inhibit C3b binding to immobilized gp120. Activated human C4 interacted only with peptides covering aa 410-449, but both in direct binding assays and fluid phase inhibition studies. The multi-reactivity of gp120 with C3b was also supported by the fact that gp120 agglutinated erythrocytes coated with C3b. Guided by partial aa sequence homology of gp120 and human C4b binding protein (C4bp) as well as human properdin we detected binding of anti-properdin to aa 100-129 in gp120 and of anti-C4bp to aa 410-449 in gp120. This cross-reactivity was also confirmed by a monoclonal antibody directed against aa 416-443 of gp120, which could be shown to bind C4bp. Interestingly, aa 310-328, part of the V3-loop, were found to show an aa sequence similarity to human complement receptor type 3 (alpha-chain). Consequently, of the 4 (or possibly 6) interaction sites of gp120 with activated human C3, 3 may bind due to imitation of either properdin, CR3 or C4bp. In addition to C4b and C3b, we detected interaction of factor H with gp120; it selectively bound to aa 102-129. Using 14 overlapping peptides of gp41 attached to plates, we identified 4 areas in gp-41 (aa 561-585, 587-605, 615-635, 651-675) which bound human factor H. All of them except the first region partially inhibited factor H binding to gp41 in competition assays with soluble peptides. Properdin bound only to 2 regions (aa 584-614, 651-675). The first 3 sites in gp41 were already shown by us to share homology to sites in human C3. The region around aa 651-675 now also turned out to be similar to human C3. These data demonstrate that the interaction of both, gp120 and gp41, with the complement system is polyvalent and complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation and control of complement, inflammation, and infection associated with the use of biomedical polymers.

It is generally acknowledged that artificial biomaterials are much less immunologically active than transplants or tissue derived biomaterials. However, activation of both the coagulation cascade and the complement system is a common occurrence when human blood is exposed to biomaterial surfaces during extracorporeal procedures, such as renal hemodialysis or cardiopulmonary bypass. Both individual and collective activation of these cascades often produce local and systemic effects. A number of complement activation products function as the mediators of inflammation. They serve as ligands for specific receptors on polymorphonuclear leukocytes, monocytes, macrophages, mast cells, and other cells. Such an interaction leads to induction of cellular responses in adhered cells, including release of oxidative products, lysosomal enzymes, or both, which often contribute to a number of pathologic conditions. Most pathogens invading the human body are attacked by the immune system directly following entry, especially when they are in contact with blood. However, bacteria and parasites have developed a large number of specific strategies to overcome immune defense among others by avoiding either recognition or eradication by complement. In this aspect, of concern are several microorganisms responsible for formation of antibiotic resistant biofilms on biomaterial surfaces, namely Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa.

Biomedical polymers differ in their capacity to activate complement.

Conventionally, complement activation by biomedical polymers has been evaluated by determining the C3a concentration in the fluid phase only. According to this criterion, biomaterials such as hemodialysis membranes made from cellulosic or various synthetic polymers were classified as activators or nonactivators of complement. Since certain membranes bind large quantities of C3a from the fluid phase, classification based on fluid-phase C3a concentration has in some instances been inaccurate. As follows from the comparison of complement activation by cuprophane and polyacrylonitrile membranes, the capacity of a biomedical polymer to activate complement is not determined by the number of potential covalent binding sites on its surface. Biomaterial itself may lack hydroxyl and/or amino groups, and yet it may activate C3 in human serum very efficiently. Some of the biomaterials may also bind unactivated/unfragmented C3 whether in the absence or presence of other serum proteins. In addition, binding of factor B (a promotor of C3 activation) and binding of factor H (an inhibitor of C3 activation) to certain biomaterials have been found to be independent of complement activation and unaffected by the presence or absence of C3. Thus, it is becoming apparent that the requirements for the formation and stability of the C3 convertase on artificial surfaces differ from those on biological membranes, and that the relative magnitude of binding of factor B and factor H to the surface per se cannot be used as a reliable indicator of the capacity of the biomaterial to activate complement. Further studies are necessary to elucidate the molecular mechanisms of C3 and C5 activation on the surfaces of biomedical polymers.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption of complement proteins on surfaces with a hydrophobicity gradient

Activation of the complement system is recognized as one of the major problems with respect to biocompatibility of biomaterials. The binding of C3 (central component of complement) and B (factor B, an activator of C3), and H (factor H, an inhibitor of C3 activation) plays a crucial role in the activation of the alternative pathway of complement on the surfaces of biomaterials during extracorporeal procedures. Here we report on the adsorption of C3, B or H on to the silica surface with a hydrophobicity gradient. The amount of native 125I-C3 bound to both hydrophilic and hydrophobic surfaces was very similar (0.8 and 0.9 micrograms/cm2; 4 x 10(-12) mol/cm2). Neither factor H nor factor B was able to displace already adsorbed 125I-C3 from either of the surfaces. The extent of binding of factors B and H to preadsorbed C3 was a function of the surface hydrophobicity: more 125I-B or 125I-H was bound to C3 adsorbed at the hydrophilic end than at the hydrophobic end of the gradient surface. The binding of 125I-B or 125I-H to preadsorbed C3 appeared to be influenced by the availability of their binding sites on adsorbed C3 molecules rather than by the amount of surface-bound C3. At the hydrophobic end of the gradient surface the molar binding ratio of B/C3 was considerably smaller than the molar binding ratio of H/C3. It can be speculated that the hydrophobicity of the surface determines orientation and/or conformation of adsorbed C3 molecule; when adsorbed at the hydrophobic end of the gradient, C3 molecule predominantly exposes the binding site to which only factor H can bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of complement in human serum by some synthetic polymers used for intraocular lenses

Determination of the potential to activate complement can be used as one criterion in testing the biocompatibility of various synthetic polymers that are utilized in the medical field. Intraocular lenses (IOLs) made of poly(methyl methacrylate) (PMMA) with PMMA loops, poly(hydroxyethyl methacrylate) (PHEMA) lenses, silicone lenses, and PMMA lenses with polypropylene loops were examined in this study. The concentrations of the activation peptides C3a, C4a and C5a were measured by radioimmunoassay (r.i.a.) in human serum after incubation with and without IOLs for up to 12 h. The presence of silicone lenses caused an increase in C3a levels. In the presence of polypropylene loops, the concentrations of both C3a and C5a were significantly higher than in serum incubated alone. There was no statistically significant increase in the concentration of C4a caused by any of the materials tested. The results suggest that IOLs made from silicone or lenses with polypropylene loops activate the complement system via the alternative pathway.

THE THIRD (C3) AND THE FOURTH (C4) COMPONENTS OF COMPLEMENT: LABILE BINDING SITE AND COVALENT BOND FORMATION

Complement is a complex system of a large number of plasma proteins. As a part of the immune system it plays an important role in defending the human body against infections. It operates via two pathways, which are subject to intrinsic and extrinsic controls.’ In the classical pathway, activation of the complement “cascade” follows an antibody-antigen interaction? A wide variety of biological substances can serve as “activators” of the alternative pathway.’ The fourth (C4), the third (C3), and the fifth (C5) components of complement are all synthesized as large single polypeptidechains of M, 180,000-200,000, having the future &chain4, at the N-terminus and the y-chain at its C-end.“ ‘O These proforms are processed into multiple-chain structures,S’ the form in which they are present as a precursor in plasma. Three nonidentical chains, a , @, and y, exist in C4; C3 and C5 have only two subunits each, a and @, of unequal sizes. The chains are held together by disulfide bonds and by noncovalent forces. Their action is related to protein-protein and/or protein-membrane surface interactions. Several recent reviews on chemical and biological properties of these proteins are available’-’ It was shown almost two decades ago by Muller-Eberhard and his collaborators, that both the C4, and then the C3, protein must be activated to be able to attach firmly to sensitized cells or immune complexes.”. At this activation step a single peptide bond in C4 is split by Cli; the multimacromolecular C3 convertase” of either the classical (C4b,C2a) or the alternative (C3b,Bb) pathway cleaves the C3 protein. The resulting activation peptides with anaphylactic activity, C4a or C3a of M, =: 8000-9000, are clipped off the N-terminus of the respective a-chain; the remaining disulfide-bridged protein C4b or C3b must bind via its labile binding site14 to receptors while in the activated state which lasts for only rnicro~econds.2~ The C4b fragment which is bound to a cell-associated IgG m~lecule’~. l6 serves as a noncatalytic subunit of classical C3 converta~e’~; it apparently affects the conformational specificity of the active C2a fragment of a novel type of serine protease, and probably recruits native C3, thus making the latter susceptible to cleavage by C2a. Generated nascent C3b must then bind to a cell membrane “within reach” of C3 convertase to participate in the activation of C5.’*, l9 The cleavage of C5 by C3/C5 convertase into C5a and nascent C5b is the initiating step in the formation of the C5b-9 complex, which is potentially membranocyto1ytic.’-’ In addition, C3b can bind factor B, thus making the latter susceptible to cleavage by factor D. In this function

Apo-transferrin as a potent inhibitor of bacterial adhesion to biomaterials

Methods of inhibiting bacterial adhesion to medical implants and reducing device-associated infection are effectuated by administering an effective amount of apo-transferrin to an individual with such an implant. Preferably the apo-transferrin is administered by controlled release at or near the implant.

Activation and fragmentation of the third (C3) and the fourth (C4) components of complement: Generation and isolation of physiologically relevant fragments C3c and C4c

The degree of the activation and fragmentation of C4 and C3, including chain structure of the activation products, was evaluated by SDS-PAGE analysis of the C4 or C3 antigens that were withdrawn from the reaction media with appropriate immunoadsorbent beads. Full activation of C4 and C3, and subsequent quantitative conversion of C4b into C4c, and C3b into iC3b took place in fresh NHS after the activation of complement with both aggIgG and CVF. For complete conversion of iC3b to C3c erythrocytes carrying the C3b receptor were added to the already activated serum. Both C4c and C3c were isolated by a 2-step procedure involving (i) an adsorption to and (ii) electrophoretic desorption from the respective immunoadsorbent beads.

Multi-analyte bioluminescence-based disposable ChemChips for home-based application: Update

Bio- and chemi-luminescent based biochemical sensors are being developed in a multi-well single use format for multi-analyte applications employing a single step, disposable, easy to use and interpret ChemChip. We briefly review and summarize earlier and ongoing work. We also argue for far more, rather than less or limited, chemical data in all areas, and particularly in education, health, and medicine.