Hen Egg-white Lysozyme Molecule Research Articles

Mechanism of Hen Egg White Lysozyme Adsorption on a Charged Solid Surface

The mechanism of hen egg white lysozyme (HEWL) adsorption on a negatively charged, hydrophilic surface has been studied using atomistic molecular dynamics (MD) simulation. Sixteen 90 ns trajectories provide adequate data to allow a detailed description of the adsorption process to be formulated. Two distinct adsorption sites have been identified. The main one is located on the N,C-terminal protein face and comprises Arg128 (the crucial one), supplemented by Arg125, Arg5, and Lys1; the minor one is used accidentally and contains only Arg68. Adsorption of this protein is driven by electrostatics, where the orientation of the protein dipole moment defines the direction of protein movement. The diffusion range on the surface depends on protein side-chain penetration through the surface water layers. This is facilitated by the long-range electric field of the charged surface, which can align polar side chains to be perpendicular to the surface. A simulation of adsorption onto a neutral ionic surface shows no such surface water layer penetration. Therefore, protein flexibility is seen to be an important factor, and to adsorb the HEWL has to adjust its structure. Nevertheless, at a flat surface only a slight loss of α-helical content is required. The adsorbed HEWL molecule is oriented between side-on and end-on ways, where the angle between the protein long axis (which mostly approximates the dipole moment) and the surface varies between 45° and 90°. Simulations with targeted mutations confirm the picture that emerges from these studies. The active site is located on the opposite face to the main adsorption site; hence, the activity of the immobilized HEWL should not be affected by the surface interactions. Our results provide a detailed insight into the adsorption mechanism and protein mobility at the surface. This knowledge will aid the proper interpretation of experimental results and the design of new experiments and functional systems.

Analysis of lysozyme-specific immune responses by synthetic peptides. I. Characterization of antibody and T cell-mediated responses to the N-terminal peptide of hen egg-white lysozyme.

The immunological reactivity against the N-terminal region of hen egg-white lysozyme (HEL) has been investigated by a synthetic peptide (PHEL) comprising residue 1-18 of HEL and by an analogue peptide (PREL) in which phenylalanine at position 3 is substituted by tyrosine. Both peptides are immunogenic in (C57BL/10 X DBA/2)F1 mice genetically responder to HEL. In C57BL/6 mice, genetically nonresponder to HEL, PREL induces anti-peptide antibodies that also bind to PHEL whereas PHEL is not immunogenic. Thus, a single amino acid substitution in a synthetic peptide converts a nonresponder mouse strain into a responder one. Anti-PHEL antibodies demonstrate a higher binding to HEL than anti-PREL antibodies, indicating that phenylalanine at position 3 is important for induction of anti-peptide antibodies able to recognize native HEL. At the T cell level the two peptides show very high bidirectional cross-reactivity between themselves and with HEL for interleukin 2 production, antigen-specific proliferation and delayed-type hypersensitivity response, whereas conservation of phenylalanine at position 3 is required for induction of suppressor cells cross-reactive with HEL. This indicates that the N-terminal region of HEL contains epitope(s) able to induce the same level of helper T cell activity as the native HEL molecule. However, helper T cells do not discriminate between PHEL and PREL whereas phenylalanine at position 3 is critical for HEL-specific suppressor T cell induction.

Direct evidence that a class II molecule and a simple globular protein generate multiple determinants.

We have examined the individual contributions of the I-A kappa alpha chain, the I-A kappa beta chain, and the foreign antigen hen egg-white lysozyme (HEL) in the formation of the determinant being recognized by the T cell receptor. As functional probes we have used (a) a panel of 10 HEL-specific T cell hybridomas, (b) a panel of antigen-presenting cells (APC) possessing mutations in either the I-A kappa alpha or I-A kappa beta chains, and (c) proteolytic fragment of HEL and related synthetic peptides. The ability of the I-A kappa beta and I-A kappa alpha mutant cell lines to present antigen to the 10 T cell hybridomas divided these T cells into six distinct groups. These HEL-specific T cells therefore appear to recognize several distinct domains on the I-A kappa molecule. The 10 T cell hybrids were then shown to recognize at least three distinct determinants on the HEL molecule, with 8 of the 10 hybrids recognizing one of two major determinants HEL(46-61) or HEL(34-45). Combining the response patterns to the panel of I-A kappa mutant APC lines with the antigen specificity revealed that the 10 T cell hybrids recognized at least eight unique determinants formed by the I-A kappa alpha chains, I-A kappa beta chains, and HEL peptides. This analysis provides direct evidence that a large number of different determinants or T cell receptor ligands can be generated from a single Ia molecule and a simple globular protein.

Processing and Presentation of Hen Egg-White Lysozyme by Macrophages

The processing and presentation by macrophages of the well-defined protein hen egg-white lysozyme (HEL) was analyzed using two HEL-specific T cell hybridomas. The processing studies revealed that both clones required that native HEL be processed, while neither clone required any processing of a, tryptic digest of lysozyme. A differential requirement for processing was found for the intact, denatured lysozyme (CM-HEL) with one clone (2A11) requiring processing, and a second clone (3A9) did not require any processing. The determinant on the HEL molecule that both clones recognized was localized to a tryptic fragment containing residues 46 to 61. By testing the immunogenicity of fragments of the 46-61 peptide, mouse lysozyme, and human lysozyme, we were able to localize the T cell determinant to either of two residues, Gly-49 or Leu-56.

Immunoregulation of lysozyme-specific suppression. II. Hen egg-white lysozyme-specific monoclonal suppressor T cell factor suppresses the afferent phase of delayed-type hypersensitivity and induces second-order suppressor T cells.

Culture supernatant from a monoclonal T cell lymphoma line (LH8-105) obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme (HEL)-specific suppressor T lymphocytes is able, when injected into mice, to specifically suppress the delayed-type hypersensitivity (DTH) reaction induced by HEL. The suppressor T cell factor (TsF) exhibits fine antigenic specificity since it suppresses the DTH response induced by HEL without affecting the DTH response induced by ring-necked pheasant egg-white lysozyme (REL), a lysozyme closely related to HEL. Conversely, LH8-105 TsF is able to suppress the DTH response induced by human lysozyme, distantly related to HEL but sharing a common epitope critical for induction of suppressive activity. The fine antigenic specificity of LH8-105 TsF for a restricted epitope on the HEL molecule is confirmed by binding to HEL but not to REL immunosorbents. This TsF also bears I-J determinants, as demonstrated by binding to monoclonal anti-I-J immunosorbents, and it suppresses the afferent but not the efferent phase of the DTH response to HEL. The afferent suppression is controlled by genes apparently mapping in the I-J subregion of the H-2 complex since I-J-incompatible mice are not suppressed by LH8-105 TsF injection. This inducer-type TsF induces second-order effector suppressor T cells only in HEL-primed mice indicating the primary role of antigen, in association with H-2 (I-J) products, in the afferent portion of this suppressive circuit.

Immunodominant protein epitopes. II. The primary antibody response to hen egg white lysozyme requires and focuses upon a unique N-terminal epitope.

The antibody response to a defined protein antigen, hen egg white lysozyme (HEL) has been investigated using an aminopeptidase-treated HEL molecule, des-1,2,3-HEL (AP-HEL). Surprisingly, removal of these three N-terminal residues eliminates an epitope which is a dominant B cell determinant recognized in the primary antibody response to HEL. Thus, the initial antibody response focuses on a very small region of the molecule. Even more striking is the observation that removal of this epitope markedly reduces the immunogenicity of HEL. Therefore, the epitope is not only the focus of the primary antibody response, but is essential for the initiation of the response. This report demonstrates that a selective mechanism must be activated during the response to this protein antigen. Of the multitude of B cell determinants present on HEL, only a limited number are focused upon by the immune system.

The fine specificity of regulatory T cells. IV. Idiotypic complementarity and antigen-bridging interactions in the anti-lysozyme response.

The hen egg-white lysozyme (HEL)-specific B cells which are activated in response to HEL share a cross-reactive idiotype as well as submolecular specificity. Helper T cells of two different specificities are implicated in their optimal activation, one recognizing the idiotype and the other, an epitope on the HEL molecule opposite to the one recognized by B cells. Thus, efficient T-B collaboration involves both idiotypic complementary and antigen bridging.

THE HEN EGG-WHITE LYSOZYME MOLECULE

THE HEN EGG-WHITE LYSOZYME MOLECULE