Combinatorial Substitutions Research Articles

Abstract 4980: Design and functional characterization of a first-in-class irreversible inhibitor of HOIL-1-interacting protein (HOIP) with selective antitumor activity against B-cell non-Hodgkin lymphoma

Abstract Activating single-nucleotide polymorphisms of HOIL-1-interacting protein (HOIP), the catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC), have recently been shown to promote myeloid differentiation primary response 88 (MYD88)-mediated B-cell lymphomagenesis. To assess the relevance of targeting HOIP in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) with MYD88 mutation, we used systems biology to build a mathematical model aimed at evaluating the impact of HOIP depletion on DLBCL. From this model, an AI-mediated query demonstrated that HOIP blockade was associated with the suppression of three main pathophysiological motifs in malignant B cells, i.e., cell growth and proliferation, apoptosis evasion and deregulated metabolism. We then undertook a computational study that consisted of a combinatorial substitution of several α-β unsaturated moieties to be used as covalent binding warheads to the catalytic cysteine residue of HOIP. The resulting chemical library was used on subsequent molecular docking to assess the best HOIP binding candidates. Out of the four candidates synthesized from this library, we isolated compound A (Cpd A), a covalent irreversible inhibitor of HOIP with a pyrido[2,3-d]pyrimidine core, which exerted selective antitumor activity in a panel of ABCL-DLBCL cell lines (mean IC50 at 48hours: 90.7± 13.09 µM) while sparing normal B cells. Immunoprecipitation studies demonstrated that Cpd A was able to modulate the interaction between HOIP and the LUBAC component, SHARPIN, leading to the blockade of NF-κB signaling and to the downregulation of several downstream effectors, including CCL3, IL6 and IRF4. Specificity of Cpd A towards HOIP was confirmed by a drug affinity responsive target stability (DARTS) assay based on the immuno-detection of persistent HOIP peptides after Cpd A-mediated blockade of enzymatic proteolysis, and thereafter in a CRISPR-engineered HOIP-knockout ABC-DLBCL model. Finally, efficacy of the compound was confirmed in vivo in a chicken embryo chorioallantoic membrane (CAM)-derived model of ABCL-DLBCL subjected to a twice weekly dosing with Cpd A, in which the compound achieved a 25% tumor growth inhibition, associated with a 60.5% and 89% reduction in brain and bone marrow infiltration by lymphoma cells, respectively. Altogether, our results confirm that HOIP represents a promising therapeutic target for ABC-DLBCL with activating MYD88 mutation, and that a pyrido[2,3-d]pyrimidine derivative can successfully and specifically block HOIP, resulting in NF-κB disruption and selective antitumor activity in this aggressive subtype of B-cell lymphoma. Citation Format: Ana Maria Montagut, Marc Antoni Armengol Cubillos, Gema Gorjón-de-Pablo, Judith Farrés, Laia Huguet Garcia, Núria Profitós-Pelejà, Juliana Carvalho Santos, Marcelo Lima Ribeiro, Miranda Fernández-Serrano, Roger Estrada Tejedor, José I. Borrell, Gaël Roué. Design and functional characterization of a first-in-class irreversible inhibitor of HOIL-1-interacting protein (HOIP) with selective antitumor activity against B-cell non-Hodgkin lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4980.

Identification of responsible amino acid residues in staphylococcal superantigen-like 12 for the activation of mast cells.

Staphylococcal superantigen-like 12 (SSL12) is reported to evoke the degranulation in murine mast cells. The allelic variant of SSL12 in the genome of reference strain NCTC8325 induced the degranulation of murine mast cells, that of MRSA252 strain did not, nevertheless relatively high sequence similarity (82%). To identify responsible amino acid residues of SSL12 for mast cell activation, we created a series of domain swap mutants and amino acid substitution mutants between the active and inactive variants. The mutants that harbored oligonucleotide/oligosaccharide binding (OB)-fold domain of the active variant activated mast cells. The replacement at position 56 (L56F) in the OB-fold domain diminished the mast cell stimulatory activity, and the combinatorial substitutions L56F/K92E, L56F/D95S, and L56F/S100V abolished the stimulatory activities of the mutant that harbored OB-fold domain of the active variant and the intact active variant. These indicate that the responsive elements of SSL12 for mast cell activation are in the OB-fold of SSL12, and L56 would be an essential amino acid residue for the activation of mast cells. The findings would contribute to the understanding of the molecular mechanism of SSL12 for mast cell activation and the development of toxoids preventing allergic inflammations associated with Staphylococcus aureus.

Adjusting the α(1[sbnd]6) bond proportion of isomalto-/maltopolysaccharide by regulating the hydrophobicity of the acceptor site of 4,6-α-glucanotransferase

4,6-α-glucanotransferase can synthesize a dietary fiber named isomalto-/maltopolysaccharide (IMMP), and this IMMP has properties of supporting probiotic growth and promoting host health because it is rich in α(16) bonds. It is valuable to understand the transglycosylation mechanism of 4,6-α-GT and modify 4,6-α-GT to synthesize IMMP products with controllable proportions of α(16) bonds. In this study, we docked isomaltose into the active pocket of 4,6-α-GT (GtfBΔN) from Limosilactobacillus fermentum NCC 3057 and obtained a substrate-enzyme docking model. This model indicated that three positions, S346, S348, and L399, were closely related to the α(16)-transglycosylation conformation of the acceptor substrate; thus, 16 mutants were constructed to explore the mechanistic rationale. Among these mutants, the S346T/S348T mutation improved the α(16) bond percentage of glycosidic bonds in the product from 72% (GtfBΔN) to 86%, and combinatorial substitutions at positions S346, S348, and L399 modified the α(16) bond percentage between 6% and 86%. We point out that the hydrophobicity of these three positions affect the α(16) proportion of IMMP product through influencing the acceptor substrate to obtain the α(16)-transglycosylation conformation. This research provides insight into the 4,6-α-GT transglycosylation mechanism.

Viral Evasion of Innate Immune Defense: The Case of Resistance of Pandemic H1N1 Influenza A Virus to Human Mannose-Binding Proteins.

Mannose-binding lectins effectively inhibit most seasonal strains of influenza A virus and contribute to the innate host defense vs. these viruses. In contrast, pandemic IAV strains are largely resistant to these lectins, likely contributing to increased spread and worse outcomes. In this paper, we evaluated the inhibition of IAV by mannose-binding lectins of human, bacterial, and fungal origin to understand and possibly increase activity vs. the pandemic IAV. A modified version of the human surfactant protein D (SP-D) neck and carbohydrate recognition domain (NCRD) with combinatorial substitutions at the 325 and 343 positions, previously shown to inhibit pandemic H3N2 IAV in vitro and in vivo, and to inhibit pandemic H1N1 in vitro, failed to protect mice from pandemic H1N1 in vivo in the current study. We attempted a variety of maneuvers to improve the activity of the mutant NCRDs vs. the 2009 pandemic H1N1, including the formation of full-length SP-D molecules containing the mutant NCRD, cross-linking of NCRDs through the use of antibodies, combining SP-D or NCRDs with alpha-2-macroglobulin, and introducing an additional mutation to the double mutant NCRD. None of these substantially increased the antiviral activity for the pandemic H1N1. We also tested the activity of bacterial and algal mannose-binding lectins, cyanovirin, and griffithsin, against IAV. These had strong activity against seasonal IAV, which was largely retained against pandemic H1N1. We propose mechanisms to account for differences in activity of SP-D constructs against pandemic H3N2 and H1N1, and for differences in activity of cyanovirin vs. SP-D constructs.

Uncovering key residues responsible for the thermostability of a thermophilic 1,3(4)-β-d-glucanase from Nong flavor Daqu by rational design

Fungal 1,3(4)-β-D-glucanases were usually applied in brewing and feedstuff industries, however, the thermostability limits the most their application. The characterized 1,3(4)-β-D-glucanase (NFEg16A) from Chinese Nong-flavor (NF) Daqu showed the highest thermostability among GH16 fungal 1,3(4)-β-D-glucanases, with half-lives of thermal inactivation (t1/2) of 44.9 min at 90 °C, so multiple rational designs were used to identify the key residues for its thermostability. Based on protein sequence and 3D structure analyses around the catalytic regions. Nine site-mutants were constructed, among which N173Y and S187A were identified as the most thermotolerant and thermolabile ones, with t1/2 values of 61 min and 14.0 min at 90 °C, respectively. Therefore, N173 and S187 were then selected as “hotspots” for site-saturation mutagenesis. Interestingly, most of the N173 and S187 variants exhibited a similar thermostability to that of N173Y and S187A, respectively, confirming their different roles in the thermostability of NFEg16A. In addition, each S187A and its surrounding substitutions (D144 N and T164 N) was independently detrimental to the thermostability of NFEg16A, since the t1/2 (90 °C) of S187A, D144 N and T164 N were 14.0 min, 20.6 min and 27.2 min, respectively. Surprisingly, combinatorial substitution of S187A with D144 N or T164 N showed positive effects on the thermostability, with the increase of t1/2 (90 °C) to 30.9 min and 63.5 min for S187A-D144 N and S187A-T164 N, respectively. More importantly, S187A-T164 N showed higher thermostability than that of wild type. In short, we successfully identified two key sites and their surrounding residues in response to the thermostability of NFEg16A and further improved its thermostability by several rational designs. These findings could be used for the protein engineering of homologous 1,3(4)-β-D-glucanases, as well as other enzyme family members with high similarities.

First-principles based machine learning study of oxygen evolution reactions of perovskite oxides using a surface center-environment feature model

Perovskite-type oxide ABO3 catalysts can be used to prepare inexpensive, efficient, durable, and environmentally friendly electrochemical energy conversion devices. The numerous combinatorial substitutions of perovskite oxides provide rich choices of candidates but pose a great challenge to examine all the possibilities both experimentally and computationally. Inspired by the data-driven materials design approach, we proposed a surface center-environment (SCE) feature model and developed first-principles based machine learning (ML) methods to predict the adsorption free energies of intermediate species (HO*, O*, and HOO*) on the surfaces of perovskite oxides and their overpotentials, critical to evaluating the catalytic performance of oxygen evolution reaction (OER). The SCE feature models contain the elementary properties of substitution elements and the structure information of the perovskite surfaces, demonstrated as an effective description of catalytic reactions on surface in ML models. The ML models predicted that the perovskite oxides adsorb the intermediate species most easily when B = Nb, Mo, Ta, W, and Os, and their overpotentials are low when B = Mn, Fe, Co, Ru, Rh, and Ir. This work demonstrated that the machine learning methods based on SCE features can be applied to describe surface chemical reactions and accelerate the screening for potential perovskite catalysts with target properties.

Evidence of combined effect of amino acid substitutions within G-H and B-C loops of VP1 conferring serological heterogeneity in foot-and-mouth disease virus serotype A.

Foot-and-mouth disease virus (FMDV) serotype A exhibits a higher degree of genetic and antigenic diversity resulting in frequent vaccine failure due to serological mismatch between the vaccine and heterologous strains. Currently, knowledge on the molecular basis of antigenic relationships among the FMDVs is limited; nevertheless, intratype antigenic variation due to mutation(s) is widely considered as the main hurdle to appropriate FMD vaccine development. Here, we studied genetic and antigenic variations of four FMDV serotype A isolates, BAN/GA/Sa-197/2013 (BAN-197), BAN/CH/Sa-304/2016 (BAN-304), BAN/DH/Sa-307/2016 (BAN-307) and BAN/DH/Sa-310/2017 (BAN-310) circulating in Bangladesh during 2013-2017. Initially, antigenic relationships (r1 -values) of the field isolates were evaluated by the two-dimensional microneutralization test (2D-MNT) using the hyperimmune antisera raised in cattle against the vaccine strain, BAN-304. Interesingly, the results showed protective serological cross-reactivity (r1 -values>0.4) between the vaccine strain and the field isolates, BAN-307 and BAN-310, except BAN-197 that substantially mismatched (r1 =0.129±0.043) with the BAN-304. Although VP1-based phylogeny grouped all the isolates within the same sublineage C (a subgroup of VP3Δ59 variant) under the lineage A/ASIA/G-VII, strikingly, computational analyses of the viral capsid proteins demonstrated significant deviation at the VP1 G-H loop of BAN-197 from the vaccine strain, while VP(2-4) of both isolates were structurally conserved. To bridge the gap of how the distortion of the G-H loop and consequent antigenic hetergeneity occurred in BAN-197, we performed in silico combinatorial substitutions of the VP1 mutant amino acids (aa) of BAN-197 with the respective residues in BAN-304. Remarkably, our analyses revealed that two substitutions of distantly located aa at B-C (T48I:threonine→isoleucine) and G-H (A143V:alanine→valine) loops, in combination, distorted the VP1 G-H loop. Overall, this work contributes to understanding the molecular basis of antigenic relationships operating in serotype A FMDVs and the selection of suitable vaccine strain(s) for effective prophylaxis of FMD based on VP1-based analyses.

The chemical space of B, N-substituted polycyclic aromatic hydrocarbons: Combinatorial enumeration and high-throughput first-principles modeling.

Combinatorial introduction of heteroatoms in the two-dimensional framework of aromatic hydrocarbons opens up possibilities to design compound libraries exhibiting desirable photovoltaic and photochemical properties. Exhaustive enumeration and first-principles characterization of this chemical space provide indispensable insights for rational compound design strategies. Here, for the smallest seventy-seven Kekulean-benzenoid polycyclic systems, we reveal combinatorial substitution of C atom pairs with the isosteric and isoelectronic B, N pairs to result in 7 453 041 547 842 (7.4 tera) unique molecules. We present comprehensive frequency distributions of this chemical space, analyze trends, and discuss a symmetry-controlled selectivity manifestable in synthesis product yield. Furthermore, by performing high-throughput ab initio density functional theory calculations of over thirty-three thousand (33k) representative molecules, we discuss quantitative trends in the structural stability and inter-property relationships across heteroarenes. Our results indicate a significant fraction of the 33k molecules to be electronically active in the 1.5-2.5 eV region, encompassing the most intense region of the solar spectrum, indicating their suitability as potential light-harvesting molecular components in photo-catalyzed solar cells.

Abstract 2562: Affinity-enhanced T-cell receptor (TCR) for adoptive T-cell therapy targeting MAGE-A4

Abstract Melanoma-associated antigens-A (MAGE-A) are members of an extensive family of cancer/testis (CT) antigens. Although emerging studies highlight the potential role of MAGE-A family members in promoting cancer growth, the biological function of MAGE-A4 remains poorly understood. MAGE-A4 is an attractive target because it has a high frequency of expression in multiple solid tumors according to The Cancer Genome Atlas (https://cancergenome.nih.gov/). In an attempt to further characterize its expression in solid tumors, immunohistochemistry analysis was performed in 534 resected non-small cell lung carcinoma (NSCLC) cases, stage I to IV with clinicopathological information including overall survival and recurrence. MAGE-A4 expression was observed in 24% of all NSCLC cases, with higher frequency observed in squamous cell carcinoma (SCC) (51%) versus adenocarcinoma (8%). MAGE-A4 expression was observed in 23% of the primary tumors and 48% of the recurrent tumor samples. Log-rank (Mantel-Cox) analysis showed that histology diagnosis, pathological stage, and MAGE-A4 expression are independent prognostic factors in NSCLC. However, no MAGE-A4 prognostic value was observed when analyzing the SCC subtype. An HLA-A*02-restricted MAGE-A4 TCR specific for MAGE-A4230-239 peptide GVYDGREHTV was identified from a pool of parental TCRs and engineered to optimize specificity and activity (MAGE-A4c1032). TCR fine specificity was assessed initially by mapping the response of the MAGE-A4c1032T-cells to panels of synthetic peptides with combinatorial substitutions at each amino acid with every other possible amino acid (X-Scan), to identify potentially cross-reactive peptides in the human and common pathogen proteomes. Secondly, MAGE-A4c1032T-cells were screened against a wide panel of normal primary cells from multiple organ systems, induced pluripotent stem cell-derived cells (iCells), and autologous whole blood to test for off-target reactivity, and against a panel of EBV-derived B-lymphoblastic cell lines expressing a wide range of HLA molecules to assess the risk of alloreactivity. Alloreactivity was observed in antigen negative cells expressing HLA-A*02:05, and therefore this allele is exclusionary. MAGE-A4c1032T-cell potency was assessed by a variety of in vitro assays, including proliferation, IFN-γ release and cytotoxicity in response to antigen-positive tumor lines in 2D and 3D culture, and cytokine release in response to freshly prepared antigen-positive primary tumor material. In conclusion, MAGE-A4 is a promising target for SPEAR T-cell therapy, and clinical trial opened this year to treat patients with inoperable or metastatic (advanced) NSCLC (SCC, adenosquamous, or large cell carcinoma), ovarian, head and neck SCC, gastric, esophageal (SCC or adenocarcinoma), urothelial tumors and melanoma (NCT03132922). Citation Format: Joana Senra, Pamela Villalobos, Barbara Mino, Luisa Solis, Carmen Behrens, Joseph P. Sanderson, Manoj Saini, Andrew B. Gerry, Nicholas J. Pumphrey, Miguel Maroto, Boris Sepesi, Patrick Hwu, David S. Hong, Elizabeht A. Mittendorf, George S. Blumenschein, Ignacio I. Wistuba, Gwendolyn Binder-Scholl, Rafael Amado. Affinity-enhanced T-cell receptor (TCR) for adoptive T-cell therapy targeting MAGE-A4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2562.

Switch Loop Flexibility Affects Substrate Transport of the AcrB Efflux Pump

The functionally important switch loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11-amino-acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1-proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway toward the deep binding pocket. Two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.

Mutations flanking the carbohydrate binding site of surfactant protein D confer antiviral activity for pandemic influenza A viruses

We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.

Consistency of multidimensional combinatorial substitutions

Multidimensional combinatorial substitutions are rules that replace symbols by finite patterns of symbols in Zd. We focus on the case where the patterns are not necessarily rectangular, which requires a specific description of the way they are glued together in the image by a substitution. Two problems can arise when defining a substitution in such a way: it can fail to be consistent, and the patterns in an image by the substitution might overlap.We prove that it is undecidable whether a two-dimensional substitution is consistent or overlapping, and we provide practical algorithms to decide these properties in some particular cases.

Combinatorial Cysteine Mutagenesis Reveals a Critical Intramonomer Role for Cysteines in Prestin Voltage Sensing

Prestin is a member of the SLC26 family of anion transporters and is responsible for electromotility in outer hair cells, the basis of cochlear amplification in mammals. It is an anion transporting transmembrane protein, possessing nine cysteine residues, which generates voltage-dependent charge movement. We determine the role these cysteine residues play in the voltage sensing capabilities of prestin. Mutations of any single cysteine residue had little or no effect on charge movement. However, using combinatorial substitution mutants, we identified a cysteine residue pair (C415 and either C192 or C196) whose mutation reduced or eliminated charge movement. Furthermore, we show biochemically that surface expression of mutants with markedly reduced functionality can be near normal; however, we identify two monomers of the protein on the surface of the cell, the larger of which correlates with surface charge movement. Because we showed previously by Förster resonance energy transfer that monomer interactions are required for charge movement, we tested whether disulfide interactions were required for dimerization. Using Western blots to detect oligomerization of the protein in which variable numbers of cysteines up to and including all nine cysteine residues were mutated, we show that disulfide bond formation is not essential for dimer formation. Taken together, we believe these data indicate that intramembranous cysteines are constrained, possibly via disulfide bond formation, to ensure structural features of prestin required for normal voltage sensing and mechanical activity.

Combined roles of loops C and D in the interactions of a neonicotinoid insecticide imidacloprid with the α4β2 nicotinic acetylcholine receptor

Neonicotinoid insecticides are widely used for crop protection based on their selective actions on insect nicotinic acetylcholine receptors (insect nAChRs). Loops C and D in insect nAChRs have been shown to possess structural features favorable for neonicotinoid–nAChR interactions. However, it remains to be resolved whether such features serve either co-operatively, or independently, to enhance neonicotinoid sensitivity of nAChRs. We therefore examined using voltage-clamp electrophysiology the effects on the response to imidacloprid of combinatorial substitutions of residues in loops C and D of the chicken α4β2 nAChR by those present in insect nAChRs. The E219P mutation in loop C of the α4 subunit resulted in enhanced responses to imidacloprid of α4β2, whereas E219S and E219T mutations barely influenced its actions. On the other hand, mutations in loop D (T77R; E79V and T77N; E79R) alone shifted the imidacloprid concentration–response curve to the left (lower concentrations). Interestingly, all three mutations did, however, further enhance the agonist efficacy of imidacloprid when combined with the mutations in loop D. Such synergistic effects of the two loops on the interactions with imidaclprid were observed irrespective of subunit stoichiometry. Computational modeling of the ligand binding domain of the wild-type and mutant α4β2 nAChRs using the crystal structure of the acetylcholine binding protein from Lymnaea stagnalis also indicated that interactions with loop F of loops C and D may contribute to determining the response to imidacloprid.

A primer of substitution tilings of the Euclidean plane

This paper is intended to provide an introduction to the theory of substitution tilings. For our purposes, tiling substitution rules are divided into two broad classes: geometric and combinatorial. Geometric substitution tilings include self-similar tilings such as the well-known Penrose tilings; for this class there is a substantial body of research in the literature. Combinatorial substitutions are just beginning to be examined, and some of what we present here is new. We give numerous examples, mention selected major results, discuss connections between the two classes of substitutions, include current research perspectives and questions, and provide an extensive bibliography. Although the author attempts to represent the field as a whole, the paper is not an exhaustive survey, and she apologizes for any important omissions.

Rational Design of Intercellular Adhesion Molecule-1 (ICAM-1) Variants for Antagonizing Integrin Lymphocyte Function-associated Antigen-1-dependent Adhesion

The interaction between integrin lymphocyte function-associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) is critical in immunological and inflammatory reactions but, like other adhesive interactions, is of low affinity. Here, multiple rational design methods were used to engineer ICAM-1 mutants with enhanced affinity for LFA-1. Five amino acid substitutions 1) enhance the hydrophobicity and packing of residues surrounding Glu-34 of ICAM-1, which coordinates to a Mg2+ in the LFA-1 I domain, and 2) alter associations at the edges of the binding interface. The affinity of the most improved ICAM-1 mutant for intermediate- and high-affinity LFA-1 I domains was increased by 19-fold and 22-fold, respectively, relative to wild type. Moreover, potency was similarly enhanced for inhibition of LFA-1-dependent ligand binding and cell adhesion. Thus, rational design can be used to engineer novel adhesion molecules with high monomeric affinity; furthermore, the ICAM-1 mutant holds promise for targeting LFA-1-ICAM-1 interaction for biological studies and therapeutic purposes.

Reduced Allergenic Potency of VR9-1, a Mutant of the Major Shrimp Allergen Pen a 1 (Tropomyosin)

The major shrimp allergen, tropomyosin, is an excellent model allergen for studying the influence of mutations within the primary structure on the allergenic potency of an allergen; Pen a 1 allows systematic evaluation and comparison of Ab-binding epitopes, because amino acid sequences of both allergenic and nonallergenic tropomyosins are known. Individually recognized IgE Ab-binding epitopes, amino acid positions, and substitutions critical for IgE Ab binding were identified by combinatorial substitution analysis, and 12 positions deemed critical were mutated in the eight major epitopes. The mutant VR9-1 was characterized with regard to allergenic potency by mediator release assays using sera from shrimp-allergic subjects and sera from BALB/c, C57BL/6J, C3H/HeJ, and CBA/J mice sensitized with shrimp extract using alum, cholera toxin, and Bordetella pertussis, as adjuvants. The secondary structure of VR9-1 was not altered; however, the allergenic potency was reduced by 90-98% measuring allergen-specific mediator release from humanized rat basophilic leukemia (RBL) cells, RBL 30/25. Reduced mediator release of RBL-2H3 cells sensitized with sera from mice that were immunized with shrimp extract indicated that mice produced IgE Abs to Pen a 1 and to the same epitopes as humans did. In conclusion, data obtained by mapping sequential epitopes were used to generate a Pen a 1 mutant with significantly reduced allergenic potency. Epitopes that are relevant for human IgE Ab binding are also major binding sites for murine IgE Abs. These results indicate that the murine model might be used to optimize the Pen a 1 mutant for future therapeutic use.

All We Are Saying Is Give Plants a Chance!

This article has no abstract.