Involvement of the "I" domain of LFA-1 in selective binding to ligands ICAM-1 and ICAM-3

To analyze the binding requirements of LFA-1 for its two most homologous ligands, ICAM-1 and ICAM-3, we compared the effects of various LFA-1 activation regimes and a panel of anti-LFA-1 mAbs in T cell binding assays to ICAM-1 or ICAM-3 coated on plastic. These studies demonstrated that T cell binding to ICAM-3 was inducible both from the exterior of the cell by Mn2+ and from the interior by an agonist of the "inside-out" signaling pathway. T cells bound both ICAM ligands with comparable avidity. A screen of 29 anti-LFA-1 mAbs led to the identification of two mAbs specific for the alpha subunit of LFA-1 which selectively blocked adhesion of T cells to ICAM-3 but not ICAM-1. These two mAbs, YTH81.5 and 122.2A5, exhibited identical blocking properties in a more defined adhesion assay using LFA-1 transfected COS cells binding to immobilized ligand. Blocking was not due to a steric interference between anti-LFA-1 mAbs and N-linked carbohydrate residues present on ICAM-3 but not ICAM-1. The epitopes of mAbs YTH81.5 and 122.2A5 were shown to map to the I domain of the LFA-1 alpha subunit. A third I domain mAb, MEM-83, has been previously reported to uniquely activate LFA-1 to bind ICAM-1 (Landis, R. C., R. I. Bennett, and N. Hogg. 1993. J. Cell Biol. 120:1519-1527). We now show that mAb MEM-83 is not able to stimulate binding of T cells to ICAM-3 over a wide concentration range. Failure to induce ICAM-3 binding by mAb MEM-83 was not due to a blockade of the ICAM-3 binding site on LFA-1. This study has demonstrated that two sets of functionally distinct mAbs recognizing epitopes in the I domain of LFA-1 are able to exert differential effects on the binding of LFA-1 to its ligands ICAM-1, and ICAM-3. These results suggest for the first time that LFA-1 is capable of binding these two highly homologous ligands in a selective manner and that the I domain plays a role in this process.     

Structural and ICAM-1-Docking Properties of a Cyclic Peptide from the I-domain of LFA-1: An inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion

Abstract

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)- Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7-Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 α-subunit. We found that cyclic peptide 1 can bind to the D1- domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1- domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable βII'-turn at Asp4-Gly5-Glu6-Ala7 and a βI-turn at Pen1-Ile2-Thr3-Asp4; a less stable βV-turn is found at the C-terminal region. The β-turn at Asp4-Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1- domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

Inhibition of ICAM-1/LFA-1-mediated heterotypic T-cell adhesion to epithelial cells: design of ICAM-1 cyclic peptides

In this work, we have designed cyclic peptides (cIBL, cIBR, cIBC, CH4 and CH7) derived from the parent IB peptide (ICAM-1(1-21)) that are inhibitors of ICAM-1/LFA-1-mediated T-cell adhesion to Caco-2 cell monolayers. Cyclic peptide cIBR has the best activity of any of the peptides evaluated. The active ICAM-1 peptides have a common Pro-Arg-Gly sequence that may be important for binding to LFA-1.     

Phage display mapping for peptide 11 sensitive sequences binding to laminin-1

We utilized a 9-mer random phage display library to identify sequences which bind to laminin-1 and elute with heparan sulfate or peptide 11 (CDPGYIGSR). Laminin-1 derivatized plates were used for biopanning. Three consecutive rounds of low pH elutions were carried out, followed by three rounds of specific elutions, each consisting of a heparan sulfate elution followed by a peptide 11 elution. The random sequence inserts were sequenced for phage populations eluted at low pH, by heparan sulfate and by peptide 11. Specifically eluted phage populations exhibited three classes of mimotopes for different regions in the cDNA derived amino acid sequence of the 67 kDa laminin binding protein (LBP). These regions were (1) a palindromic sequence known as peptide G, (2) a predicted helical domain corresponding to LBP residues 205-229, and (3) TEDWS-containing C-terminal repeats. All elution conditions also yielded phage with putative heparin binding sequences. We modeled the LBP(205-229) domain, which is strongly predicted to have a helical secondary structure, and determined that this region likely possesses heparin-binding characteristics located to one side of the helix, while the opposite side appears to contain a hydrophobic patch where peptide 11 could bind. Using ELISA plate assays, we demonstrated that peptide 11 and heparan sulfate individually bound to synthetic LBP(205-229) peptide. We also demonstrated that the QPATEDWSA peptide could inhibit tumor cell adhesion to laminin-1. These data support the proposal that the 67 kDa LBP can bind the beta-1 laminin chain at the peptide 11 region, and suggest that heparan sulfate is a likely alternate ligand for the binding interactions. Our results also confirm previous data suggesting that the most C-terminal region of the LBP, which contains the TEDWS repeats, is involved in cell adhesion to laminin-1, and we specifically implicate the repeat sequence in that activity.     

Synergistic inhibitory activity of α- and β-LFA-1 peptides on LFA-1/ICAM-1 interaction

Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the α-subunit and the I-like domain of the β-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.     

A Peptide Derived from LFA-1 Protein that Modulates T-cell Adhesion Binds to Soluble ICAM-1 Protein

Abstract

Leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be critical for adhesion process and immune response. Modulation or inhibition of the interaction between LFA-1/ICAM-1 interactions can result in therapeutic effects. Our group and others have shown that peptides derived from ICAM- 1 or LFA-1 inhibit adhesion in a homotypic T-cell adhesion assay. It is likely that the peptides derived from ICAM-1 bind to LFA-1 and peptides derived from LFA-1 bind to ICAM- 1 and inhibit the adhesion interaction. However, there are no concrete experimental evidence to show that peptides bind to either LFA-1 or ICAM-1 and inhibit the adhesion. Using NMR, CD and docking studies we have shown that an LFA-1 derived peptide binds to soluble ICAM-1. Docking studies using “autodock” resulted in LFA-1 peptide interacting with the ICAM-1 protein near Glu34. The proposed model based on our experimental data indicated that the LFA-1 peptide interacts with the protein via three intermolecular hydrogen bonds. Hydrophobic interactions also play a role in stabilizing the complex.     

Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors.

We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.     

Synergistic inhibitory activity of alpha- and beta-LFA-1 peptides on LFA-1/ICAM-1 interaction.

Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the alpha-subunit and the I-like domain of the beta-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.     

A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins

A Gb₃-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb₃ antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb₃ antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb₃ antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb₃ mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb₃Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb₃ mimic peptide potentially has great promise for use against Stxs.     

Integrin LFA-1 alpha subunit contains an ICAM-1 binding site in domains V and VI.

In order to identify a binding site for ligand intercellular adhesion molecule-1 (ICAM-1) on the beta 2 integrin lymphocyte function-associated antigen-1 (LFA-1), protein fragments of LFA-1 were made by in vitro translation of a series of constructs which featured domain-sized deletions starting from the N-terminus of the alpha subunit of LFA-1. Monoclonal antibodies and ICAM-1 were tested for their ability to bind to these protein fragments. Results show that the putative divalent cation binding domains V and VI contain an ICAM-1 binding site. A series of consecutive peptides covering these domains indicated two discontinuous areas as specific contact sites: residues 458-467 in domain V and residues 497-516 in domain VI. A three-dimensional model of these domains of LFA-1 was constructed based on the sequence similarity to known EF hands. The two regions critical for the interaction of LFA-1 with ICAM-1 lie adjacent to each other, the first next to the non-functional EF hand in domain V and the second coinciding with the potential divalent cation binding loop in domain VI. The binding of ICAM-1 with the domain V and VI region in solution was not sensitive to divalent cation chelation. In short, a critical motif for ICAM-1 binding to the alpha subunit of LFA-1 is shared between two regions of domains V and VI.     

Binding sites of leukocyte beta 2 integrins (LFA-1, Mac-1) on the human ICAM-4/LW blood group protein

The red cell ICAM-4/LW blood group glycoprotein, which belongs to the family of intercellular adhesion molecules (ICAMs), has been reported to interact with CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) beta(2) integrins. To better define the basis of the ICAM-4/beta(2) integrin interaction, we have generated wild-type, domain-deleted and mutated recombinant chimeric ICAM-4-Fc proteins and analyzed their interaction in a cellular adhesion assay with LFA-1 and Mac-1 L-cell stable transfectants. We found that monoclonal antibodies against CD11a, CD11b, CD18, or LW(ab) block adhesion of transfectant L-cells to immobilized ICAM-4-Fc protein and that the ICAM-4/beta(2) integrin interaction was highly sensitive to the presence of the divalent cations Ca(2+) and Mg(2+). Deletion of individual Ig-domains D1 or D2 of the extracellular part of ICAM-4 showed that LFA-1 binds to the first Ig-like domain, whereas the Mac-1 binding site encompassed both the first and the second Ig-like domains. Based on the crystal structure of ICAM-2, we propose a model for the Ig-like domains D1 and D2 of ICAM-4. Accordingly, by site-directed mutagenesis of 22 amino acid positions spread out on all faces of the ICAM-4 molecule, we identified four exposed residues, Leu(80), Trp(93), and Arg(97) on the CFG face and Trp(77) on the E-F loop of domain D1 that may contact LFA-1 as part of the binding site. However, the single and double mutants R52E and T91Q on the CFG face of domain D1, which correspond to the key residues Glu(34) and Gln(73) for ICAM-1 binding to LFA-1, had no effect on LFA-1 binding. In contrast, all mutants on the CFG face of domain D1 and residues Glu(151) and Thr(154) in the C'-E loop of the domain D2 seem to play a dominant role in Mac-1 binding. These data suggest that the binding site for LFA-1 on ICAM-4 overlaps but is distinct from the Mac-1 binding site.     

Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families

Intercellular adhesion molecule 1 (ICAM-1) is a 90 kd inducible surface glycoprotein that promotes adhesion in immunological and inflammatory reactions. ICAM-1 is a ligand of lymphocyte function-associated antigen-1 (LFA-1), an alpha beta complex that is a member of the integrin family of cell-cell and cell-matrix receptors. ICAM-1 is encoded by an inducible 3.3 kb mRNA. The amino acid sequence specifies an integral membrane protein with an extracellular domain of 453 residues containing five immunoglobulin-like domains. Highest homology is found with neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG), which also contain five Ig-like domains. NCAM and MAG are nervous system adhesion molecules, but unlike ICAM-1, NCAM is homophilic. The ICAM-1 and LFA-1 interaction is heterophilic and unusual in that it is between members of the immunoglobulin and intergrin families. Unlike other integrin ligands, ICAM-1 does not contain an RGD sequence.     

Panning of a Phage Display Library against a Synthetic Capsule for Peptide Ligands That Bind to the Native Capsule of Bacillus anthracis

Bacillus anthracis is the causative agent of anthrax with the ability to not only produce a tripartite toxin, but also an enveloping capsule comprised primarily of γ-D-glutamic acid residues. The purpose of this study was to isolate peptide ligands capable of binding to the native capsule of B. anthracis from a commercial phage display peptide library using a synthetic form of the capsule consisting of 12 γ-D-glutamic acid residues. Following four rounds of selection, 80 clones were selected randomly and analysed by DNA sequencing. Four clones, each containing a unique consensus sequence, were identified by sequence alignment analysis. Phage particles were prepared and their derived 12-mer peptides were also chemically synthesized and conjugated to BSA. Both the phage particles and free peptide-BSA conjugates were evaluated by ELISA for binding to encapsulated cells of B. anthracis as well as a B. anthracis capsule extract. All the phage particles tested except one were able to bind to both the encapsulated cells and the capsule extract. However, the peptide-BSA conjugates could only bind to the encapsulated cells. One of the peptide-BSA conjugates, with the sequence DSSRIPMQWHPQ (termed G1), was fluorescently labelled and its binding to the encapsulated cells was further confirmed by confocal microscopy. The results demonstrated that the synthetic capsule was effective in isolating phage-displayed peptides with binding affinity for the native capsule of B. anthracis.     

Human IgA-binding Peptides Selected from Random Peptide Libraries: AFFINITY MATURATION AND APPLICATION IN IGA PURIFICATION*

Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1–A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (K_d = 1.3 μm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (K_d = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.     

Structural and ICAM-1-docking properties of a cyclic peptide from the I-domain of LFA-1: an inhibitor of ICAM-1/LFA- 1-mediated T-cell adhesion

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)-Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7- Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 alpha-subunit. We found that cyclic peptide 1 can bind to the D1-domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1-domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable betaII -turn at Asp4- Gly5-Glu6-Ala7 and a betaI-turn at Pen1-Ile2-Thr3-Asp4; a less stable betaV-turn is found at the C-terminal region. The beta-turn at Asp4- Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1-domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

脓毒症单核巨噬细胞特异性结合肽的筛选

利用噬菌体随机肽库展示技术,筛选出与脓毒症单核/巨噬细胞特异性结合的短肽,探索脓毒症治疗的新方法.分别以经过脂多糖(lipopolysaccharide, LPS)处理的人外周血单核细胞株(THP-1)细胞作为筛选的靶细胞,以未经LPS处理的THP-1细胞作为非特异性噬菌体吸附细胞,对噬菌体随机环七肽库进行4轮“差减"筛选,经过细胞ELISA验证阳性噬菌体克隆,对获得的阳性克隆进行DNA测序及生物信息学分析,并进一步利用免疫荧光实验,鉴定噬菌体克隆与LPS处理THP-1细胞的结合特异性.4轮筛选后,随机挑取的噬菌体克隆,测序后得到可与LPS处理的THP-1细胞特异性结合肽.对去冗余后的七肽进行Clustal W多序列比对分析和BlastP蛋白同源相似性分析,细胞免疫荧光检测确定获得的噬菌体展示七肽可与LPS处理的THP-1细胞特异性结合.噬菌体随机肽库技术为脓毒症单核/巨噬细胞表面靶位的筛选提供了高效、快捷的筛选体系,实验获得的多肽基序具有高度保守性和细胞特异性,这些多肽的生物活性将是下一步的研究内容.     

Identification of a murine ICAM-1-specific peptide by subtractive phage library selection on cells

The ICAM-1 adhesion molecule is expressed selectively at low levels on endothelial cells but is strongly upregulated in dysfunctional endothelial cells associated with inflammation, cancer, and atherogenesis. Using COS-7 cells transfected with murine ICAM-1 (mICAM-1) as a target receptor, a phage display library was screened. Clones were selected by elution with a mAb specific for a functional epitope of ICAM-1 and a novel peptide sequence binding to the extracellular domain of mICAM-1 was identified that can potentially be used as a targeting vector aimed at dysfunctional endothelium. We further showed that the targeting specificity of the peptide was retained following its incorporation at the N terminal end of a large chimeric protein. Moreover, this chimeric protein containing the mICAM-1-specific sequence was found to inhibit ICAM-1-mediated intercellular adhesion during antigen presentation. Taken together, these results demonstrate the potential for improving the cell-selectivity and properties of therapeutical agents toward targeting adhesion molecules involved in cell-cell interactions.     

A Ca2+ binding cyclic peptide derived from the alpha-subunit of LFA-1: inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion.

The objective of this work is to study the conformation of cyclic peptide (1), cyclo (1, 12) Pen1-Gly2-Val3-Asp4-Val5-Asp6-Gln7-+ ++Asp8-Gly9-Glu10-Thr11-Cys12, in the presence and absence of calcium. Cyclic peptide 1 is derived from the divalent cation binding sequence of the alpha-subunit of LFA-1. This peptide has been shown to inhibit ICAM-1-LFA-1 mediated T-cell adhesion. In order to understand the structural requirements for this biologically active peptide, its solution structure was studied by nuclear magnetic resonance (NMR), circular dichroism (CD) and molecular dynamics simulations. This cyclic peptide exhibits two types of possible conformations in solution. Structure I is a loop-turn-loop type of structure, which is suitable to bind cations such as EF hand proteins. Structure II is a more extended structure with beta-hairpin bend at Asp4-Val5-Asp6-Gln7. There is evidence that alterations in the conformation of LFA-1 upon binding to divalent cations cause LFA-1 to bind to ICAM-1. To understand this mechanism, the cation-binding properties of the peptide were studied by CD and NMR. CD studies indicated that the peptide binds to calcium and forms a 1 : 1 (peptide: calcium) complex at low calcium concentrations and multiple types of complexes at higher cation concentrations. NMR studies indicated that the conformation of the peptide is not significantly altered upon binding to calcium. The peptide can inhibit T-cell adhesion by directly binding to ICAM-1 or by disrupting the interaction of the alpha and beta-subunits of LFA-1 protein. This study will help us to understand the mechanism(s) of action of this peptide and will improve our ability to design a better inhibitor of T-cell adhesion.     

A novel LFA-1 activation epitope maps to the I domain

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA- 1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM- 1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA- 1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.     

α1-Antichymotrypsin Binding to Alzheimer Aβ Peptides Is Sequence Specific and Induces Fibril Disaggregation In Vitro

Abstract: The serine protease inhibitor α₁-antichymotrypsin (ACT) consistently colocalizes with amyloid deposits of Alzheimer's disease (AD) and may contribute to the generation of amyloid proteins and/or physically affect fibril assembly. AD amyloid fibrils are composed primarily of Aβ, which is a proteolytic fragment of the larger β-amyloid precursor protein. Using negative-stain and immunochemical electron microscopy, we have investigated the binding of ACT to the fibrils formed by four synthetic Aβ analogues corresponding to the wild-type human 1–40 sequence [H_Wt(1–40)], a 1–40 peptide [H_Du(1–40)] containing the Glu₂₂→ Gln mutation found in hereditary cerebral hemorrhage with amyloidosis of the Dutch type, the N-terminal 1–28 residues [β(1–28)], and an internal fragment of Aβ containing residues 11–28 [β(11–28)]. Each of these peptide analogues assembled into 70–90-Å-diameter fibrils resembling native amyloid and, except for β(11–28), bound ACT, as indicated by the appearance of 80–100-Å globular particles that adhered to preformed fibrils and that could be decorated with anti-ACT antibodies. Under the conditions used, ACT binding destabilized the in vitro fibrils and produced a gradual dissolution of the macromolecular assemblies into constituent filaments and shorter fragments. The internal fragment (11–28) did not exhibit ACT binding or any structural changes. These results suggest that a specific sequence likely contained within the N-terminal 10 residues of Aβ is responsible for the formation of the ACT-amyloid complex. Although the observed fibril disassembly is surprising in view of the notion that ACT contributes directly to the physical process involved in amyloid fibril formation, the induced structural changes may expose new domains in Aβ for additional proteolysis or for interactions with cell-surface receptors.